Additions for Review

Accepted Publication Froemke.pdf

PI: FROEMKE, ROBERT CROOKS Title: Synaptic basis of perceptual learning in primary auditory cortex

Received: 07/05/2016 Opportunity: PA-16-160 Council: 01/2017

Competition ID: FORMS-D FOA Title: NIH Research Project Grant (Parent R01)

2R01DC012557-06 Dual: MH,LM Accession Number: 3954637

IPF: 5998304 Organization:

Former Number: Department: Otolaryngology

IRG/SRG: ZRG1 AUD-Z (90)S AIDS: N Expedited: N

Subtotal Direct Costs

(excludes consortium F&A)

Animals: Y 

Humans: N 

Clinical Trial: N

Current HS Code: 10 

HESC: N

New Investigator: N

Early Stage Investigator: N

Year 6:

Senior/Key Personnel: Organization: Role Category:

Robert Froemke PD/PI
OMB Number: 4040-0001

Expiration Date: 06/30/2016

APPLICATION FOR FEDERAL ASSISTANCE

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Synaptic basis of perceptual learning in primary auditory cortex

12. PROPOSED PROJECT

Start Date*

04/01/2017

13. CONGRESSIONAL DISTRICTS OF APPLICANT

Ending Date*

03/31/2022

Funding Opportunity Number: PA-16-160 . Received Date:

2016-07-05T15:19:02.000-04:00

Tracking Number: GRANT12208477
Contact PD/PI: Froemke, Robert C

SF 424 (R&R) APPLICATION FOR FEDERAL ASSISTANCE Page 2

14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION

First Name*: Robert 

Assistant Professor

Prefix: Middle Name: C Last Name*: Froemke

Position/Title:

Suffix:

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Department: Otolaryngology

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Street2: 

City*: 

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EXECUTIVE ORDER 12372 PROCESS?*

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AVAILABLE TO THE STATE EXECUTIVE ORDER 12372

PROCESS FOR REVIEW ON:

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❍ PROGRAM HAS NOT BEEN SELECTED BY STATE FOR 

REVIEW

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Prefix: First Name*: Anthony Middle Name: R Last Name*: Carna

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07/05/2016

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Funding Opportunity Number: PA-16-160 . Received Date:

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Tracking Number: GRANT12208477
Contact PD/PI: Froemke, Robert C

424 R&R and PHS-398 Specific 

Table Of Contents 

SF 424 R&R Cover Page----------------------------------------------------------------------------------------- 

Table of Contents------------------------------------------------------------------------- 

Performance Sites--------------------------------------------------------------------------------------------- 

Research & Related Other Project Information------------------------------------------------------------------ 

Project Summary/Abstract(Description)----------------------------------------------------- 

Project Narrative------------------------------------------------------------------------- 

Facilities & Other Resources-------------------------------------------------------------- 

Equipment--------------------------------------------------------------------------------- 

Research & Related Senior/Key Person-------------------------------------------------------------------------- 

Research & Related Budget Year - 1---------------------------------------------------------------------------- 

Research & Related Budget Year - 2---------------------------------------------------------------------------- 

Research & Related Budget Year - 3---------------------------------------------------------------------------- 

Research & Related Budget Year - 4---------------------------------------------------------------------------- 

Research & Related Budget Year - 5---------------------------------------------------------------------------- 

Budget Justification------------------------------------------------------------------------------------------ 

Research & Related Cumulative Budget-------------------------------------------------------------------------- 

PHS398 Cover Page Supplement---------------------------------------------------------------------------------- 

PHS 398 Research Plan----------------------------------------------------------------------------------------- 

Specific Aims----------------------------------------------------------------------------- 

Research Strategy------------------------------------------------------------------------- 

Progress Report Publication List---------------------------------------------------------- 

Vertebrate Animals------------------------------------------------------------------------ 

Bibliography & References Cited----------------------------------------------------------- 

Letters of Support------------------------------------------------------------------------ 

Resource Sharing Plan(s)------------------------------------------------------------------ 

Authentication of Key Biological and/or Chemical Resources-------------------------------- 

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Table of Contents Page 3
Contact PD/PI: Froemke, Robert C

OMB Number: 4040-0010

Expiration Date: 06/30/2016

Project/Performance Site Location(s)

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Additional Location(s)

Page 4

Funding Opportunity Number: PA-16-160. Received Date:

2016-07-05T15:19:02.000-04:00

Tracking Number: GRANT12208477
Contact PD/PI: Froemke, Robert C

OMB Number: 4040-0001

Expiration Date: 06/30/2016

RESEARCH & RELATED Other Project Information

Page 5

Funding Opportunity Number: PA-16-160. Received Date:

2016-07-05T15:19:02.000-04:00

Tracking Number: GRANT12208477

1. Are Human Subjects Involved?* ❍ Yes ● No

1.a. If YES to Human Subjects

Is the Project Exempt from Federal regulations? ❍ Yes ❍ No

If YES, check appropriate exemption number: 1 2 3 4 5____ 6

If NO, is the IRB review Pending? ❍ Yes ❍ No

IRB Approval Date:

Human Subject Assurance Number

2. Are Vertebrate Animals Used?* ● Yes ❍ No

2.a. If YES to Vertebrate Animals

Is the IACUC review Pending? ❍ Yes ● No 

IACUC Approval Date: 06-09-2016

Animal Welfare Assurance Number A3435-01

3. Is proprietary/privileged information included in the application?* ❍ Yes ● No

4.a. Does this project have an actual or potential impact - positive or negative - on the environment?* ❍ Yes ● No

4.b. If yes, please explain:

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environmental assessment (EA) or environmental impact statement (EIS) been performed?

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collaborators?*

6.a. If yes, identify countries:

6.b. Optional Explanation:

Filename

7. Project Summary/Abstract* project_summary_final.pdf

8. Project Narrative* health_relevance_final.pdf

9. Bibliography & References Cited references_cited_final.pdf

10.Facilities & Other Resources facilities_final.pdf

11.Equipment equipment_final.pdf
Project Summary

The brain has the remarkable capability to change in response to experience. This plasticity is essential for 

learning and memory, and is an important feature of the auditory cortex, especially for learning the significance 

of sensory signals such as speech, for the use of devices such as cochlear implants, and for recovery after shortterm deafness. These changes are thought to occur primarily at synapses, basic units of information processing 

and plasticity. Long-term synaptic plasticity requires sensory experience and activation of neuromodulatory 

systems which convey behavioral context to local cortical circuits. However, little is known about the interactions 

between synaptic inputs and release of neuromodulators in vivo, making it challenging to relate perceptual 

learning to plasticity in the auditory cortex or other brain areas. Recently we developed an approach to measuring 

dynamics of synaptic modifications for hours, coupled with imaging techniques enabling us to monitor the same 

cells over days during training, directly monitoring and manipulating activity in behaving mice. These approaches 

allow for a close examination of links between modulation, cortical plasticity and auditory perceptual learning.

Specifically, we will study how auditory perceptual training activates the cholinergic vs noradrenergic 

modulatory systems. These two modulators are principally involved in selective attention towards behaviorallyimportant stimuli, general arousal, and learning. However, there may be important functional differences in these 

systems in terms of when they are active during different phases of training or consequences of cholinergic and

noradrenergic modulation on auditory neurons for contextual information processing. This proposal describes a 

series of imaging, recording, optogenetic, and behavioral experiments that will compare and contrast the effects 

of locus coeruleus activation and norepinephrine release vs the effects of nucleus basalis activation and 

cholinergic modulation on the primary auditory cortex of behaving mice. Many studies have highlighted the 

importance of recording in awake animals during behavior, and we will first examine how ensembles of excitatory 

and inhibitory neurons are affected by learning over the entire duration of training, as animals go from naïve and 

poor performers, to having reliable performance on an auditory detection and recognition task we have used in 

the lab for years. Next, we determine when and how cholinergic and noradrenergic modulation affect behavioral 

and neural responses. Finally, we will make some of the first direct measurements of modulatory neuron 

responses, asking how these systems are activated by task-relevant variables such as sounds linked to reward. 

In summary, here we use in vivo recording and imaging methods to ask how behavioral training engages 

and modifies noradrenergic and cholinergic systems, to collectively affect auditory cortical processing and

persistently improve auditory perceptual abilities in behaving mice. 

Contact PD/PI: Froemke, Robert C

Project Summary/Abstract Page 6
Project Narrative

Neuroplasticity- the ability of the brain to change in response to experience- is an essential feature of the auditory 

cortex, especially for speech and language learning as well as the successful use of devices such as cochlear 

implants. However, it is unclear how motivational state and behavioral training drive plasticity within the central 

auditory system. The experiments to be performed in this proposal provide essential data on basic mechanisms

of neuromodulation and plasticity in the auditory cortex, required for improvement of prosthetic design and 

therapeutic strategies for treatment of deafness and language disorders. 

Contact PD/PI: Froemke, Robert C

Project Narrative Page 7
Contact PD/Pl: Froemke, Robert C 

Facilities and Other Resources 

Laboratory Space 

Our main 

School of 

laboratory 

Medicine. 

space 

Both the 

is a

li

oximate

Institute 

ly 1500 

and 

square 

my laboratory 

feet on the 

are 

fifth floor 

well equipped

of the -torelectrophysInstitute 

io

of_

lo

imaging, histological, optogene Ic, ana behavioral experiments. The laboratory has space for 10-15 people to 

have their own benches and desks. We have two large walk-in acoustic isolation chambers within the main lab 

space (each 8' x 6') fully equipped for in vivo extracellular and whole-cell electrophysiological experiments, and 

two brain slice rig for fluorescence micropscopy and quadruple whole-cell recordings also within the main lab 

space. In a dedicated room down the hall from the main lab, we have a 2-photon in vivo imaging system with a 

Mai Tai Deepsee laser (Spectra-Physics), built with a Movable Objective Microscope (Sutter) for imaging from 

auditory cortex in awake animals. Each rig is dedicated full-time to one student or postdoc. Each in vivo recording

rig has a computer, a preamplifier, a digitizer and whole-cell recording amplifier (Molecular Devices MultiClamp), 

a digital signal processor for acoustic stimulus generation (TOT), a small tabletop laser or LED system for 

optogenetic stimulation, and a current generator for electrical stimulation (Grass). The lab is outfitted with a 

workbench for electronics and small parts machining, and two chemical fume hoods. I have two quiet, isolated 

annexes for behavioral experiments (50-100 square feet), one for operant conditioning in rats, the other for 

behavioral studies in mice as described in the current application. We have one multielectrode system for chronic 

electrophysiological recordings and stimulation in freely-moving animals (BlackRock). 

I designed the lab space and renovations were complete by the time I arrived at-on 5/1/10. The main lab 

space is an open area, with rigs and desks along the walls, facilitating and maximizing interactions, discussions,

and my ability to oversee and mentor trainees. I received additional space in 2015. 

Animal Facilities 

The Institute is particularly well-suited for physiological and behavioral studies in rodents. Our animals are 

housed in the Medical Center and supervised by a full-time veterinarian and her staff. Each animal is inspected 

daily, food and water provided, and bedding is changed 2/week. Vet staff are on-call and monitor implanted 

animals daily to ensure their health. We have authorization to perform surgeries, electrophysiological recordings,

and behavioral experiments all within our own lab space, and the Pl and all lab members receive annual training

in the ethical care and use of animals in research. Consequently, we will carry out our studies in an environment 

with great technical expertise and experience. 

Computers

Each desk and rig has a PC dedicated for electrophysiological data acquisition, analysis, and routine laboratory

use. All computers have ethernet access to a lab printer and network services, including licensed software such 

as Matlab. The Skirball Institute has excellent computer support via an in-house IT support staff. 

Office Space 

The Pl's office is approximately 120 square feet, located directly within the main laboratory space. I have a desk,

a networked PC, a black-and-white printer and a color scanner/printer, and a table and extra chairs for meetings

with other faculty and students. 

Clinical 

The Pl's primary departmental ap

active cochlear im lant center at 

, r mg in psy , 

oss an coc ear imp an s me u Ing Tom, Susan, and Mario), and rehabilitation medicine, to discuss issues in 

potential therapeutic approaches, training programs, adaptation and plasticity. 

-

Other 

Medical provides an optimal environment to conduct the proposed studies, both intellectually and in terms oTTlie available resources. The-Institute of Biomolecular Medicine at- Medical Center consists of

~30 laboratories, and-Medicailiou'ses a large number of laboratories dedicated to neuroscience in general

and cortical physiology in particular. Importantly, a new initiative- the-Neuroscience Institute- has recently

Facilities & Other Resources Page 8 
Contact PD/Pl: Froemke, Robert C 

ii 

started at-School of Medicine. This new opportunity has made-an even more e

to 

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work, 

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have collaborated on 

projects involving 2-photon imaging as well as neuromodulation and inhibitory plasticity in the forebrain. Our 

laboratory is also on the same floor as laboratory, so we have meetin arding 2-photon 

imaging in behaving mice, cortical organization, plasticity, and rodent behavior with his rou . 

Likewise, we have re ular discussions (and a few direct collaborations) with 

, experts on cortical circuit development and organization, in vivo whole-cell 

recording, cortical neuromodulation, and behavioral experiments in rodents, respectively. We also have close 

-

contact with colleagues at the Center for Neural Science, located about a mile from the Medical Center, including 

who we have collaborated with on electron microscopic studies of neuromodulatory receptor

expression in the cortex.

-· 

The Pl has a faculty mentoring committee, 

We meet annually to discuss career progress, gran I eas, an

The --Institute and - Medical have core facilities for microscopy, histology, solution preparation,

statistics, andthe Division of Laboratory Animal Resources performs a number of services for 

ordering, housing, and carin laboratory animals. Our histological experiments to verify electrode positions 

have been performed in the-Medical histology core. 

Facilities & Other Resources Page 9 
Contact PD/PI: Froemke, Robert C

Equipment

Almost all of the major equipment required for these experiments has been obtained and is already operational 

in the lab. This includes:

Two in vivo recording rigs for extracellular and whole-cell recordings in awake, head-fixed animals. We have two 

8' x 6' double-walled acoustic isolation chambers within the main lab space. Inside each is an air table for 

mechanical isolation (TMC), a surgical microscope (Zeiss), a stereotax (Kopf), a micromanipulator (Sutter MP285), a fiber-optic lamp for illumination (Fisher), and a pre-amplifier (WPI). The pre-amp is connected to the data 

collection system outside the sound booth, consisting of a digitizer (Molecular Devices Digidata), a multichannel 

whole-cell recording amplifier (Molecular Devices MultiClamp), a PC with terabyte external hard drive for data 

storage, a current generator for electrical stimulation (Grass), and a digital signal processor for acoustic stimulus 

generation (TDT RZ6) connected to a speaker within the sound booth. We have several custom-built clamp 

systems for training and recording from awake, head-fixed mice, and a tabletop laser for optogenetic stimulation 

(CrystaLaser).

One 2-photon imaging system for imaging Ca2+ signals in awake, head-fixed animals, with a Movable Objective 

Microscope for imaging from auditory cortex (Sutter) and a Mai Tai Deepsee laser (Spectra-Physics). This 

imaging system is in an environmentally controlled annex down the hall from the main lab space. Our 2-photon 

microscope is dedicated for use to only our lab members, and contains a whole-cell recording setup for in vivo 

electrophysiology (as above) combined with 2-photon imaging.

One system for multielectrode recording from behaving animals (Blackrock). This system is designed for 

simultaneous extracellular recording with 16- or 32-channel chronically-implanted multielectrode arrays and optetrodes containing optical fibers. This system includes a PC for data analysis and storage, and a commutator 

for recording from animals within the behavioral testing chambers.

Three additional systems for stereotaxic in vivo surgeries and injections. Given the large number of surgeries, 

implantations, and injections we perform, we have three additional setups involving minimal recording but 

enabling users to stereotaxically inject or implant animals as needed, including acoustic stimulation (sound 

delivery) and extracellular multi-unit recording systems for verification of primary auditory cortex in head-fixed 

mice. 

We have an electrode puller (Sutter P1000) in the main lab space. We have two refrigerators, a freezer, and two

fume hoods for chemical and solution storage, and an analytical balance (Fisher) to aid in making internal 

solution for whole-cell recording. We have two isoflurane vaporizers (VetEquip).

We have access to the histology core, containing a cryomicrotome for tissue sectioning, and microscopes 

for imaging fixed tissue on slides.

For these reasons, we are asking only for a limited amount of funding for equipment in this proposal. We collected 

pilot data on targeted whole-cell recordings under 2-photon control with amplifiers and manipulators taken from 

other rigs, and so we are requesting funds in year 2 to purchase additional manipulators to perform these key 

experiments. Additionally, while the scan mirrors in our 2-photon system have been sufficient to obtain 

preliminary data for this proposal, our imaging experiments- particularly axonal imaging- would benefit greatly 

by using resonant galvo scanners to increase the temporal resolution. Thus in year 3 we are requesting funds 

for these scanners to improve our data collection rates.

Equipment Page 10
Contact PD/PI: Froemke, Robert C

OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date:

2016-07-05T15:19:02.000-04:00

RESEARCH & RELATED Senior/Key Person Profile (Expanded)

PROFILE - Project Director/Principal Investigator

Prefix:  First Name*: Robert Middle Name C Last Name*: Froemke Suffix: 

Position/Title*: Assistant Professor

Organization Name*:

Department: Otolaryngology

Division:

Street2:

City*:

Province:

Country*: USA: UNITED STATES

Zip / Postal Code*:

Phone Number*:  Fax Number:  E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  PD/PI Other Project Role Category: 

Degree Type:  Ph.D. Degree Year:  2004

File Name

Attach Biographical Sketch*: Froemke_Renewal_Biosketch.pdf

Attach Current & Pending Support:

 Page 11
Contact PD/PI: Froemke, Robert C

BIOGRAPHICAL SKETCH

Provide the following information for the Senior/key personnel and other significant contributors.

Follow this format for each person. DO NOT EXCEED FIVE PAGES.

NAME: Froemke, Robert Crooks

eRA COMMONS USER NAME (credential, e.g., agency login): 

POSITION TITLE: Assistant Professor of Otolaryngology, Neuroscience & Physiology, Skirball Institute, NYU 

School of Medicine; Center for Neural Science, NYU

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, 

include postdoctoral training and residency training if applicable. Add/delete rows as necessary.)

INSTITUTION AND LOCATION

DEGREE

(if 

applicable)

Completion 

Date

MM/YYYY

FIELD OF STUDY

Tufts University, Medford MA B.A. 11/1998 Computer Science

University of California, Berkeley CA Ph.D. 05/2004

Molecular & Cell 

Biology

University of California, San Francisco CA Postdoctoral 04/2010 Neuroscience,

Otolaryngology

A. Personal Statement

We study neuromodulation, cortical plasticity, and auditory perception in rats and mice. I have a broad

background in systems neuroscience, performing my undergraduate work at Tufts on machine learning and

building modeling tools for complex systems analysis. For my PhD work with at UC Berkeley, I

examined spike-timing-dependent plasticity induced by natural spike trains in cortical networks. My postdoctoral

research with at UCSF focused on synaptic plasticity in vivo as related

to auditory perception and behavior. As PI or co-Investigator on several university- and NIH-funded grants

(including a K99/R00 and an R01), I have performed studies on auditory perception and synaptic plasticity in

rodents for 12+ years. I have successfully administered the projects, trained several students (graduating three

PhDs and one MD/PhD), postdocs, and technicians, collaborated with many other researchers, and produced

several peer-reviewed publications from each project. This includes several studies examining the relation

between excitatory-inhibitory balance, cholinergic/noradrenergic modulation, long-term synaptic plasticity, and

auditory perception in rodents that the proposed studies directly extend. As a result of these experiences, I am

aware of the importance of frequent communication with lab members and collaborators for constructing a

realistic research plan and timeline, working towards our scientific goals. The current application builds logically

on my prior work, and uses cutting-edge approaches to examine neuromodulation and synaptic plasticity in the

context of controlled auditory behavior and psychophysical testing in mice.

1. Marlin BJ, Mitre M, D'amour JA, Chao MV, Froemke RC. Oxytocin enables maternal behaviour by

balancing cortical inhibition. Nature 2015; 520:499-504. PMID: 25874674 PMC: 4409554

2. Martins ARO, Froemke RC. Coordinated forms of noradrenergic plasticity in the locus coeruleus and

primary auditory cortex. Nature Neuroscience 2015; 18:1483-1492. PMID: 26301326 PMC: 4583810

3. D'amour JA, Froemke RC. Inhibitory and excitatory spike-timing-dependent plasticity in the auditory cortex.

Neuron 2015; 86; 514-528. PMID: 25843405 PMC: 4409545

4. Froemke RC, Carcea I, Barker AJ, Yuan K, Seybold B, Martins ARO, Zaika N, Bernstein H, Wachs M,

Levis PA, Polley DB, Merzenich MM, Schreiner CE. Long-term modification of cortical synapses improves

sensory perception. Nature Neuroscience 2013; 16:79-88. PMC: 3711827

Biosketches Page 12
B. Positions and Honors

Positions and Employment

2010- Assistant Professor, New York University School of Medicine, Skirball Institute Program in 

Molecular Neurobiology; Neuroscience Institute; Departments of Otolaryngology, Neuroscience

& Physiology; Kimmel Center for Stem Cell Biology; Center for Neural Science.

Other Experience and Professional Memberships

1996-1998 Senior research staff, Center for Connected Learning and Computer-Based Modeling

2000 Neural Systems & Behavior Course, Marine Biological Laboratory, Woods Hole, MA

2000- Member, American Association for the Advancement of Science

2000- Member, Society for Neuroscience

2004 Grass Fellow, Marine Biological Laboratory, Woods Hole, MA

2005- Member, Association for Research in Otolaryngology

2007 Okinawa Computational Neuroscience Course, Okinawa, Japan

2008 Methods in Computational Neuroscience, Marine Biological Laboratory, Woods Hole, MA

2009 Teaching Assistant, Biology of Memory Course, Cold Spring Harbor Laboratory, NY

2010 Kavli Institute for Theoretical Physics, UC Santa Barbara

2013 Co-Director, Biology & Disorders of Learning & Memory Course, Cold Spring Harbor Lab, NY

2013-2015 Co-Chair, Cosyne Workshop Committee (2 year appointment)

Honors

1998 Benjamin Brown Prize in Computer Science, Tufts University

2001 Howard Hughes Medical Institute Predoctoral Fellowship

2002 Outstanding Graduate Student Instructor, University of California, Berkeley

2004 First place, General Scientific Meeting presentation, Marine Biological Laboratory

2005 Jane Coffin Childs Postdoctoral Fellowship 

2006 Sandler Translational Research Postdoctoral Fellowship 

2008 K99/R00 Career Award, NIDCD

2011 Whitehead Fellowship 

2012 Alfred P. Sloan Research Fellowship Award

2012 Pew Scholar Award

2012 Klingenstein Fellowship Award

2013 NYU Grand Challenge Award

2013 Hirschl/Weill-Caulier Career Award

2014 McKnight Scholar Award

2015 NYU “Next Gen Stars” Inaugural Speaker

2016 HHMI Faculty Scholars Award

C. Contribution to Science

1. We study the organization and plasticity of cortical synapses, and how cortical modulation and plasticity can 

improve auditory perception and behavior, in the context of behavioral training and perceptual learning. We 

have a particular emphasis on conducting well-controlled and parametric studies of auditory psychophysics, 

combined with mechanistic studies of neuromodulation and long-term plasticity of excitatory and inhibitory 

synapses. We have conducted a series of studies examining how manipulations of modulatory systems- the 

cholinergic attentional system of the nucleus basalis and the noradrenergic arousal system of the locus 

coeruleus - lead to synaptic plasticity and produce behavioral changes in adult rats. We assessed baseline 

auditory abilities to determine which stimuli were difficult to perceive, and leveraged the cholinergic system 

to boost up the strengths of synapses at these thresholds. We found that auditory perception was improved 

for at least hours afterwards, indicating that direct cortical modifications can be useful for enhancing sensory 

perception and behavior. We have contrasted these changes with the action of noradrenalin and stimulation 

(electrical or optogenetic) of the rat locus coeruleus, identifying how cholinergic and noradrenergic 

modulation differentially affect plasticity and perception. This is some of the first work identifying inhibitory 

synapses and circuits as central targets of neuromodulators, and our findings that acetylcholine disinhibits 

auditory cortex has been replicated by several other labs, in rats and in mice, and other sensory systems. 

Contact PD/PI: Froemke, Robert C

Biosketches Page 13

Contact PD/Pl: Froemke, Robert C 

B. Positions and Honors 

Positions and Employment 

2010- Assistant Professor, New York University School of Medicine, Skirball Institute Program in 

Molecular Neurobiology; Neuroscience Institute; Departments of Otolaryngology, Neuroscience 

& Physiology; Kimmel Center for Stem Cell Biology; Center for Neural Science. 

Other Experience and Professional Memberships 

1996-1998 Senior research staff, Center for Connected Learning and Computer-Based Modeling 

2000 Neural Systems & Behavior Course, Marine Biological Laboratory, Woods Hole, MA 

2000- Member, American Association for the Advancement of Science 

2000- Member, Society for Neuroscience 

2004 Grass Fellow, Marine Biological Laboratory, Woods Hole, MA 

2005- Member, Association for Research in Otolaryngology 

2007 Okinawa Computational Neuroscience Course, Okinawa, Japan 

2008 Methods in Computational Neuroscience, Marine Biological Laboratory, Woods Hole, MA 

2009 Teaching Assistant, Biology of Memory Course, Cold Spring Harbor Laboratory, NY 

2010 Kavli Institute for Theoretical Physics, UC Santa Barbara 

2013 Co-Director, Biology & Disorders of Learning & Memory Course, Cold Spring Harbor Lab, NY 

2013-2015 Co-Chair, Cosyne Workshop Committee (2 year appointment) 

Benjamin Brown Prize in Computer Science, Tufts University 

Howard Hughes Medical Institute Predoctoral Fellowship 

Honors 

1998 

2001 

2002 

2004 

2005 

2006 

2008 

2011 

2012 

2012 

2012 

2013 

2013 

2014 

2015 

2016 

Outstanding Graduate Student Instructor, University of California, Berkeley 

First place, General Scientific Meeting presentation, Marine Biological Laboratory 

Jane Coffin Childs Postdoctoral Fellowship 

Sandler Translational Research Postdoctoral Fellowship 

K99/R00 Career Award, NIDCD 

Whitehead Fellowship 

Alfred P. Sloan Research Fellowship Award 

Pew Scholar Award 

Klingenstein Fellowship Award 

NYU Grand Challenge Award 

Hirschl/Weill-Caulier Career Award 

McKnight Scholar Award 

NYU "Next Gen Stars" Inaugural Speaker 

HHMI Faculty Scholars Award 

C. Contribution to Science 

1. We study the organization and plasticity of cortical synapses, and how cortical modulation and plasticity can 

improve auditory perception and behavior, in the context of behavioral training and perceptual learning. We 

have a particular emphasis on conducting well-controlled and parametric studies of auditory psychophysics, 

combined with mechanistic studies of neuromodulation and long-term plasticity of excitatory and inhibitory 

synapses. We have conducted a series of studies examining how manipulations of modulatory systems- the 

cholinergic attentional system of the nucleus basalis and the noradrenergic arousal system of the locus 

coeruleus - lead to synaptic plasticity and produce behavioral changes in adult rats. We assessed baseline 

auditory abilities to determine which stimuli were difficult to perceive, and leveraged the cholinergic system 

to boost up the strengths of synapses at these thresholds. We found that auditory perception was improved 

for at least hours afterwards, indicating that direct cortical modifications can be useful for enhancing sensory 

perception and behavior. We have contrasted these changes with the action of noradrenalin and stimulation 

(electrical or optogenetic) of the rat locus coeruleus, identifying how cholinergic and noradrenergic 

modulation differentially affect plasticity and perception. This is some of the first work identifying inhibitory 

synapses and circuits as central targets of neuromodulators, and our findings that acetylcholine disinhibits 

auditory cortex has been replicated by several other labs, in rats and in mice, and other sensory systems. 

Biosketches Page 13 
a. Froemke RC, Merzenich MM, Schreiner, CE. A synaptic memory trace for cortical receptive field 

plasticity. Nature 2007; 450:425-429. PMID: 18004384 PMC in process

b. Froemke RC, Carcea I, Barker AJ, Yuan K, Seybold B, Martins ARO, Zaika N, Bernstein H, Wachs 

M, Levis PA, Polley DB, Merzenich MM, Schreiner CE. Long-term modification of cortical synapses 

improves sensory perception. Nature Neuroscience 2013; 16:79-88. PMC: 3711827

c. Froemke RC. Plasticity of cortical excitatory-inhibitory balance. Annual Review of Neuroscience

2015; 38:195-219. PMID:25897875 PMC:4652600

d. Martins ARO, Froemke RC. Coordinated forms of noradrenergic plasticity in the locus coeruleus and 

primary auditory cortex. Nature Neuroscience 2015; 18:1483-1492. PMID:26301326 PMC: 4583810

2. Excitatory-inhibitory balance is an important property of mature neural circuits, ensuring that excitability is 

carefully controlled for information processing without seizure generation or propagation failure. How are 

inhibitory inputs calibrated during development and adjusted throughout life, to ensure that inhibition 

balances excitation? Inhibitory maturation is believed to determine critical periods for cortical development, 

and changes to excitatory synapses related to learning must be matched by coordinated changes in co-tuned 

inhibitory inputs. Our lab specializes in examining combined forms of excitatory and inhibitory long-term 

synaptic plasticity with whole-cell recordings and 2-photon imaging in vivo, monitoring the dynamics by which 

excitation and inhibition are adjusted after changes in patterns of electrical activity or sensory experience. 

We have shown how these processes occur during a critical period for frequency tuning in the rodent auditory 

cortex. We are also the first and perhaps only group to show how, after transplantation, inhibitory progenitor 

cells integrate into the existing network to open a new critical period in adult visual cortex, using paired 

recordings from host and transplanted cells to show that these new neurons make and receive synaptic 

connections in the local circuit.

a. Dorrn A, Yuan K, Barker AJ, Schreiner CE, Froemke RC. Developmental sensory experience 

balances cortical excitation and inhibition. Nature 2010; 465:932-936. PMID:20559387 PMC2888507

b. Southwell DG, Froemke RC, Alvarez-Buylla A, Stryker MP, Gandhi SP. Cortical plasticity induced by 

inhibitory neuron transplantation. Science 2010; 327:1145-1148. PMID:20185728 PMC:3164148

c. Southwell DG, Paredes MF, Galvao RP, Jones DL, Froemke RC, Sebe JY, Alfaro-Cervello C, GarciaVerdugo JM, Baraban SC, Alvarez-Buylla A. Intrinsically determined cell death of developing cortical 

interneurons. Nature 2012; 491:109-113. PMID:23041929 PMC:3726009

d. Cohen S, Ma H, Kuchibhotla K, Watson BO, Buzsáki G, Froemke RC, Tsien RW. Excitationtranscription coupling in parvalbumin-positive interneurons employs a novel CaM Kinase-dependent 

pathway distinct from excitatory neurons. Neuron 2016; 90:292-307. PMID:27041500 PMC:4866871

3. Our studies of neuromodulation and behavior also examine circuit dynamics and the control of social 

cognition, with a particular emphasis on oxytocin. It has historically been difficult to determine how 

modifications of specific synapses relate to changes in behavior. We have examined how neurons in the 

rodent hypothalamus affect synaptic transmission in the cortex and elsewhere to produce behavioral 

changes in adult rats and mice. This is some of the first work using cortical plasticity to persistently enhance

sensory perception and cognition. Furthermore, we provided the first direct evidence that oxytocin transiently 

reduces synaptic inhibition in the cortex, increasing the salience of incoming sensory inputs. We have used 

optogenetics and pharmacological approaches to examine how oxytocin can enable newly-maternal mice to 

recognize the significance of infant vocalizations and distress calls. As part of our work on oxytocin, in 

collaboration with Moses Chao’s lab we generated the first specific antibodies to the mouse oxytocin 

receptor, which we have shared with many international laboratories (including the Grinevich and Stoop 

labs). We have also worked to understand the functional anatomy and circuit logic by which oxytocin neurons 

and other hypothalamic cell types project to target areas. 

a. Marlin BJ, Mitre M, D'amour JA, Chao MV, Froemke RC. Oxytocin enables maternal behaviour by 

balancing cortical inhibition. Nature 2015; 520:499-504. PMID: 25874674 PMC: 4409554

b. Mitre M, Marlin BJ, Schiavo JK, Morina E, Norden S, Hackett TA, Aoki C, Chao MV, Froemke RC. A 

distributed network for social cognition enriched for oxytocin receptors. Journal of Neuroscience

2016; 36:2517-2535. PMID: 26911697 PMC: 4764667

c. Eliava M, Melchior M, Knobloch-Bollmann S, Wahis J, da Silva Gouveia M, Tang Y, Ciobanu AC, del 

Rio RT, Roth LC, Althammer F, Chavant V, Goumon Y, Gruber T, Petit-Demoulière M, Busnelli M, 

Chini B, Tan L, Mitre M, Froemke RC, Chao MV, Giese G, Sprengel R, Kuner R, Poisbeau P, Seeburg 

PH, Stoop R, Charlet A, Grinevich V. A new population of parvocellular oxytocin neurons controlling 

Contact PD/PI: Froemke, Robert C

Biosketches Page 14

Contact PD/Pl: Froemke, Robert C 

a. Froemke RC, Merzenich MM, Schreiner, CE. A synaptic memory trace for cortical receptive field 

plasticity. Nature 2007; 450:425-429. PMID: 18004384 PMC in process 

b. Froemke RC, Carcea I, Barker AJ, Yuan K, Seybold B, Martins ARO, Zaika N, Bernstein H, Wachs 

M, Levis PA, Polley DB, Merzenich MM, Schreiner CE. Long-term modification of cortical synapses 

improves sensory perception. Nature Neuroscience 2013; 16:79-88. PMC: 3711827 

c. Froemke RC. Plasticity of cortical excitatory-inhibitory balance. Annual Review of Neuroscience 

2015; 38:195-219. PMID:25897875 PMC:4652600 

d. Martins ARO, Froemke RC. Coordinated forms of noradrenergic plasticity in the locus coeruleus and 

primary auditory cortex. Nature Neuroscience 2015; 18:1483-1492. PMID:26301326 PMC: 4583810 

2. Excitatory-inhibitory balance is an important property of mature neural circuits, ensuring that excitability is 

carefully controlled for information processing without seizure generation or propagation failure. How are 

inhibitory inputs calibrated during development and adjusted throughout life, to ensure that inhibition 

balances excitation? Inhibitory maturation is believed to determine critical periods for cortical development, 

and changes to excitatory synapses related to learning must be matched by coordinated changes in co-tuned 

inhibitory inputs. Our lab specializes in examining combined forms of excitatory and inhibitory long-term 

synaptic plasticity with whole-cell recordings and 2-photon imaging in vivo, monitoring the dynamics by which 

excitation and inhibition are adjusted after changes in patterns of electrical activity or sensory experience. 

We have shown how these processes occur during a critical period for frequency tuning in the rodent auditory 

cortex. We are also the first and perhaps only group to show how, after transplantation, inhibitory progenitor 

cells integrate into the existing network to open a new critical period in adult visual cortex, using paired 

recordings from host and transplanted cells to show that these new neurons make and receive synaptic 

connections in the local circuit. 

a. Dorrn A, Yuan K, Barker AJ, Schreiner CE, Froemke RC. Developmental sensory experience 

balances cortical excitation and inhibition. Nature 201O; 465:932-936. PMID:20559387 PMC2888507 

b. Southwell DG, Froemke RC, Alvarez-Buylla A, Stryker MP, Gandhi SP. Cortical plasticity induced by 

inhibitory neuron transplantation. Science 2010; 327:1145-1148. PMID:20185728 PMC:3164148 

c. Southwell DG, Paredes MF, Galvao RP, Jones DL, Froemke RC, Sebe JY, Alfaro-Cervello C, GarciaVerdugo JM, Baraban SC, Alvarez-Buylla A. Intrinsically determined cell death of developing cortical 

interneurons. Nature 2012; 491:109-113. PMID:23041929 PMC:3726009 

d. Cohen S, Ma H, Kuchibhotla K, Watson BO, Buzsaki G, Froemke RC, Tsien RW. Excitationtranscription coupling in parvalbumin-positive interneurons employs a novel CaM Kinase-dependent 

pathway distinct from excitatory neurons. Neuron 2016; 90:292-307. PMID:27041500 PMC:4866871 

3. Our studies of neuromodulation and behavior also examine circuit dynamics and the control of social 

cognition, with a particular emphasis on oxytocin. It has historically been difficult to determine how 

modifications of specific synapses relate to changes in behavior. We have examined how neurons in the 

rodent hypothalamus affect synaptic transmission in the cortex and elsewhere to produce behavioral 

changes in adult rats and mice. This is some of the first work using cortical plasticity to persistently enhance 

sensory perception and cognition. Furthermore, we provided the first direct evidence that oxytocin transiently 

reduces synaptic inhibition in the cortex, increasing the salience of incoming sensory inputs. We have used 

optogenetics and pharmacological approaches to examine how oxytocin can enable newly-maternal mice to 

recognize the significance of infant vocalizations and distress calls. As part of our work on oxytocin, in 

collaboration with Moses Chao's lab we generated the first specific antibodies to the mouse oxytocin 

receptor, which we have shared with many international laboratories (including the Grinevich and Stoop 

labs). We have also worked to understand the functional anatomy and circuit logic by which oxytocin neurons 

and other hypothalamic cell types project to target areas. 

a. Marlin BJ, Mitre M, D'amour JA, Chao MV, Froemke RC. Oxytocin enables maternal behaviour by 

balancing cortical inhibition. Nature 2015; 520:499-504. PMID: 25874674 PMC: 4409554 

b. Mitre M, Marlin BJ, Schiavo JK, Morina E, Norden S, Hackett TA, Aoki C, Chao MV, Froemke RC. A 

distributed network for social cognition enriched for oxytocin receptors. Journal of Neuroscience 

2016; 36:2517-2535. PMID: 26911697 PMC: 4764667 

c. Eliava M, Melchior M, Knobloch-Bollmann S, Wahis J, da Silva Gouveia M, Tang Y, Ciobanu AC, del 

Rio RT, Roth LC, Althammer F, Chavant V, Goumon Y, Gruber T, Petit-Demouliere M, Busnelli M, 

Chini B, Tan L, Mitre M, Froemke RC, Chao MV, Giese G, Sprengel R, Kuner R, Poisbeau P, Seeburg 

PH, Stoop R, Charlet A, Grinevich V. A new population of parvocellular oxytocin neurons controlling 

Biosketches Page 14 
magnocellular neuron activity and inflammatory pain processing. Neuron 2016; 89:1291-1304. PMID:

26948889

d. Wong LC, Wang L, Yumita T, D’amour JA, Chen G, Chang B, Bernstein H, You X, Feng J, Froemke 

RC, Lin D. Effective modulation of male aggression through the lateral septum to medial 

hypothalamus projection. Current Biology 2016; 26:593-604. PMID: 26877081 PMC: 4783202

4. Neural activity can be complex. We have examined spike-timing-dependent plasticity (STDP), focusing on 

synaptic modifications induced by naturalistic patterns of pre- and postsynaptic spikes recorded in vivo. From 

hundreds of experiments we could predict the sign and magnitude of long-term synaptic plasticity induced 

by complex spike trains, and continue to work with computational neuroscientists to understand how STDP 

might enable neural networks to store and recall information. Some experiments examine how synaptic 

integration and dendritic properties affect NMDA receptor activation to control induction of synaptic plasticity. 

More recently, we are examining how multiple synapses are co-modified, and we were the first group to show 

that excitatory and inhibitory STDP can be coordinated and induced together. We found that spike pairing 

can normalize the strength of inhibition relative to the strength of co-activated excitation, providing a natural 

mechanism by which excitatory-inhibitory balance can be established and maintained.

a. Froemke RC, Dan Y. Spike-timing-dependent synaptic modification induced by natural spike trains. 

Nature 2002; 416:433-438. PMID: 11919633

b. Froemke RC, Poo MM, Dan Y. Spike-timing-dependent plasticity depends on dendritic location. 

Nature 2005; 434:221-225. PMID: 15759002

c. Ponte Costa R, Froemke RC, Sjöström PJ, van Rossum MC. Unified pre- and postsynaptic longterm plasticity enables reliable and flexible learning. eLife 2015; 4. PMID: 26308579 PMC:4584257

d. D'amour JA, Froemke RC. Inhibitory and excitatory spike-timing-dependent plasticity in the auditory 

cortex. Neuron 2015; 86:514-528. PMID: 25843405 PMC: 4409545

5. Studies of plasticity in the auditory cortex have the potential to inform and transform training procedures and 

use of neuroprosthetic devices such as cochlear implants. Our lab has pioneered a new rat model of multichannel cochlear implant use, behaviorally and physiologically validated. This is being combined with μECoG recordings for stable, long-term monitoring of cortical changes with use of the implants.

a. Wang J, Trumpis M, Insanally M, Froemke R, Viventi J. A low-cost, multiplexed electrophysiology 

system for chronic μECoG in rodents. Conf Proc IEEE Eng Med Biol Soc 2014; 2014:5256-5259.

PMID: 25571179 PMC: in process

b. King J, Insanally M, Jin M, Martins ARO, D'amour JA, Froemke RC. Rodent auditory perception: 

critical band limitations and plasticity. Neuroscience 2015; 296:55-65. PMID: 27281743 

c. Insanally M, Trumpis M, Wang C, Chiang CH, Woods V, Bossi S, Froemke RC, Viventi J. A low-cost, 

multiplexed μECoG system for long-term, reliable high-density recordings in rodents. Journal of 

Neural Engineering 2016; 13:026030. PMID: 26975462, PMCID: in process.

d. King J, Shehu I, Roland JT, Svirsky MA, Froemke RC. A physiological and behavioral system for 

hearing restoration with cochlear implants. Journal of Neurophysiology 2016; in press.

Complete List of Published Work in MyBibliography: 

http://www.ncbi.nlm.nih.gov/sites/myncbi/robert.froemke.1/bibliography/44917017/public/?sort=date&direction=

ascending

D. Research Support

Ongoing Research Support

HHMI Faculty Scholars Award 2016-2021

Cortical plasticity and control of social behavior

This career award supports our research on the mammalian oxytocin system, and cortical plasticity for learned 

maternal behavior. Role: PI

NYU CTSI Collaborative Translational Pilot Project Program 2016-2017

Optimizing cochlear implant use with neural recordings 

The goal of this study is to understand the neural basis of cochlear implant use in trained, deafened rats. Role: 

Co-PI (with Mario Svirsky, NYU) 

Contact PD/PI: Froemke, Robert C

Biosketches Page 15

Contact PD/Pl: Froemke, Robert C 

magnocellular neuron activity and inflammatory pain processing. Neuron 2016; 89:1291-1304. PMID: 

26948889 

d. Wong LC, Wang L, Yumita T, D'amour JA, Chen G, Chang B, Bernstein H, You X, Feng J, Froemke 

RC, Lin D. Effective modulation of male aggression through the lateral septum to medial 

hypothalamus projection. Current Biology 2016; 26:593-604. PMID: 26877081 PMC: 4783202 

4. Neural activity can be complex. We have examined spike-timing-dependent plasticity (STOP), focusing on 

synaptic modifications induced by naturalistic patterns of pre- and postsynaptic spikes recorded in vivo. From 

hundreds of experiments we could predict the sign and magnitude of long-term synaptic plasticity induced 

by complex spike trains, and continue to work with computational neuroscientists to understand how STOP 

might enable neural networks to store and recall information. Some experiments examine how synaptic 

integration and dendritic properties affect NMDA receptor activation to control induction of synaptic plasticity. 

More recently, we are examining how multiple synapses are co-modified, and we were the first group to show 

that excitatory and inhibitory STOP can be coordinated and induced together. We found that spike pairing 

can normalize the strength of inhibition relative to the strength of co-activated excitation, providing a natural 

mechanism by which excitatory-inhibitory balance can be established and maintained. 

a. Froemke RC, Dan Y. Spike-timing-dependent synaptic modification induced by natural spike trains. 

Nature 2002; 416:433-438. PMID: 11919633 

b. Froemke RC, Poo MM, Dan Y. Spike-timing-dependent plasticity depends on dendritic location. 

Nature 2005; 434:221-225. PMID: 15759002 

c. Ponte Costa R, Froemke RC, Sjostrom PJ, van Rossum MC. Unified pre- and postsynaptic longterm plasticity enables reliable and flexible learning. elife 2015; 4. PMID: 26308579 PMC:4584257 

d. D'amour JA, Froemke RC. Inhibitory and excitatory spike-timing-dependent plasticity in the auditory 

cortex. Neuron 2015; 86:514-528. PMID: 25843405 PMC: 4409545 

5. Studies of plasticity in the auditory cortex have the potential to inform and transform training procedures and 

use of neuroprosthetic devices such as cochlear implants. Our lab has pioneered a new rat model of multichannel cochlear implant use, behaviorally and physiologically validated. This is being combined with μECoG recordings for stable, long-term monitoring of cortical changes with use of the implants. 

a. Wang J, Trumpis M, lnsanally M, Froemke R, Viventi J. A low-cost, multiplexed electrophysiology 

system for chronic μECoG in rodents. Conf Proc IEEE Eng Med Biol Soc 2014; 2014:5256-5259. 

PMID: 25571179 PMC: in process 

b. King J, lnsanally M, Jin M, Martins ARO, D'amour JA, Froemke RC. Rodent auditory perception: 

critical band limitations and plasticity. Neuroscience 2015; 296:55-65. PMID: 27281743 

c. lnsanally M, Trumpis M, Wang C, Chiang CH, Woods V, Bossi S, Froemke RC, Viventi J. A low-cost, 

multiplexed μECoG system for long-term, reliable high-density recordings in rodents. Journal of 

Neural Engineering 2016; 13:026030. PMID: 26975462, PMCID: in process. 

d. King J, Shehu I, Roland JT, Svirsky MA, Froemke RC. A physiological and behavioral system for 

hearing restoration with cochlear implants. Journal of Neurophysiology 2016; in press. 

Complete List of Published Work in MyBibliography: 

http://www.ncbi.nlm.nih.gov/sites/myncbi/robert.froemke.1 /bibliography/44917017/public/?sort=date&direction=

ascending 

D. Research Support 

Ongoing Research Support 

HHMI Faculty Scholars Award 2016-2021 

Cortical plasticity and control of social behavior 

This career award supports our research on the mammalian oxytocin system, and cortical plasticity for learned 

maternal behavior. Role: Pl 

NYU CTSI Collaborative Translational Pilot Project Program 2016-2017 

Optimizing cochlear implant use with neural recordings 

The goal of this study is to understand the neural basis of cochlear implant use in trained, deafened rats. Role: 

Co-Pl (with Mario Svirsky, NYU) 

Biosketches Page 15 
NYU Global Seed Grant for Collaborative Research 2016-2018 

Neuromodulatory influences on economic preferences 

The goal of this study is to examine the neurobiological mechanisms for impulsivity behaviors in trained rats.

Role: Co-PI (with Jeff Erlich, NYU Shanghai) 

Simons Foundation Explorer Grant 2016-2017 

Synthetic infrared nanosensors for real-time monitoring of oxytocin release 

The goal of this study is to screen carbon nanotube-based sensors for specific detection of oxytocin in real 

time. Role: Co-PI (with Markita Landry, UC Berkeley) 

McKnight Scholar Award 2014-2017

Neural circuitry and plasticity for control of mammalian social behavior

The goal of this study is to determine the 'social receptive field' of oxytocin neurons in the hypothalamus, and 

relate activation of the oxytocin system and cortical plasticity to learned maternal behavior. Role: PI

Hirschl/Weill-Caulier Career Award 2014-2018

Neural basis of long-term memory formation

The goal of this study is to examine how neuromodulatory circuits of the locus coeruleus are modified by

sensory experience and neural activity. Role: PI

R01 NIDCD DC012557 2012-2017 

Synaptic basis for perceptual learning in primary auditory cortex

The goal of this study is to directly examine the relation between adult cortical synaptic plasticity and 

perceptual learning via the noradrenergic modulatory system. Role: PI

Completed Research Support

K99/R00 NIDCD DC009635 2008-2015 

Synaptic basis for perceptual learning in primary auditory cortex

The goal of this study was to directly examine the relation between adult cortical synaptic plasticity and 

perceptual learning via the cholinergic modulatory system. Role: PI

Pew Scholarship 2012-2016

Neural basis of learned social behavior

The goal of this study was to determine how oxytocin affects responses to pup calls in the auditory cortex. 

Role: PI

Klingenstein Fellowship 2012-2015

Plasticity of excitatory-inhibitory balance in the auditory cortex

The goal of this study was to determine the network dynamics and mechanisms that calibrate and balance 

excitation and inhibition in the developing auditory cortex. Role: PI

Alfred P. Sloan Research Fellowship 2012-2014

Synaptic plasticity in the cerebral cortex

The goal of this study was to examine the functional consequences of long-term synaptic plasticity in cortical 

networks for behavioral modification. Role: PI

NYU Provost's Grand Challenge Grant 2013-2015

Smart neuroprosthetics: brain-machine interfaces for the 21st century

The goal of this study was to generate brain-machine interface devices for improving sensory, motor, and 

cognitive behavioral performance in rodents and primates. Role: Co-PI (with Michael Long, Bijan Pesaran, Dan 

Sanes, and Jonathan Viventi, NYU) 

NYU Provost's Office Mega-Grants Initiative 2013-2014

Creating a neuroengineering core facility at NYU

The goal of this study was to seed construction of a core facility at NYU for fabrication of neuroprosthetic 

devices. Role: Co-PI (with Michael Long, Dan Sanes, John Simspon, and Jonathan Viventi, NYU) 

Contact PD/PI: Froemke, Robert C

Biosketches Page 16

Contact PD/Pl: Froemke, Robert C 

NYU Global Seed Grant for Collaborative Research 2016-2018 

Neuromodulatory influences on economic preferences 

The goal of this study is to examine the neurobiological mechanisms for impulsivity behaviors in trained rats. 

Role: Co-Pl (with Jeff Erlich, NYU Shanghai) 

Simons Foundation Explorer Grant 2016-2017 

Synthetic infrared nanosensors for real-time monitoring of oxytocin release 

The goal of this study is to screen carbon nanotube-based sensors for specific detection of oxytocin in real 

time. Role: Co-Pl (with Markita Landry, UC Berkeley) 

McKnight Scholar Award 2014-2017 

Neural circuitry and plasticity for control of mammalian social behavior 

The goal of this study is to determine the 'social receptive field' of oxytocin neurons in the hypothalamus, and 

relate activation of the oxytocin system and cortical plasticity to learned maternal behavior. Role: Pl 

Hirschl/Weill-Caulier Career Award 2014-2018 

Neural basis of long-term memory formation 

The goal of this study is to examine how neuromodulatory circuits of the locus coeruleus are modified by 

sensory experience and neural activity. Role: Pl 

R01 NIDCD DC012557 2012-2017 

Synaptic basis for perceptual learning in primary auditory cortex 

The goal of this study is to directly examine the relation between adult cortical synaptic plasticity and 

perceptual learning via the noradrenergic modulatory system. Role: Pl 

Completed Research Support 

K99/R00 NIDCD DC009635 

Synaptic basis for perceptual learning in primary auditory cortex 

2008-2015 

The goal of this study was to directly examine the relation between adult cortical synaptic plasticity and 

perceptual learning via the cholinergic modulatory system. Role: Pl 

Pew Scholarship 2012-2016 

Neural basis of learned social behavior 

The goal of this study was to determine how oxytocin affects responses to pup calls in the auditory cortex. 

Role: Pl 

Klingenstein Fellowship 2012-2015 

Plasticity of excitatory-inhibitory balance in the auditory cortex 

The goal of this study was to determine the network dynamics and mechanisms that calibrate and balance 

excitation and inhibition in the developing auditory cortex. Role: Pl 

Alfred P. Sloan Research Fellowship 2012-2014 

Synaptic plasticity in the cerebral cortex 

The goal of this study was to examine the functional consequences of long-term synaptic plasticity in cortical 

networks for behavioral modification. Role: Pl 

NYU Provost's Grand Challenge Grant 2013-2015 

Smart neuroprosthetics: brain-machine interfaces for the 21st century 

The goal of this study was to generate brain-machine interface devices for improving sensory, motor, and 

cognitive behavioral performance in rodents and primates. Role: Co-Pl (with Michael Long, Bijan Pesaran, Dan 

Sanes, and Jonathan Viventi, NYU) 

NYU Provost's Office Mega-Grants Initiative 2013-2014 

Creating a neuroengineering core facility at NYU 

The goal of this study was to seed construction of a core facility at NYU for fabrication of neuroprosthetic 

devices. Role: Co-Pl (with Michael Long, Dan Sanes, John Simspon, and Jonathan Viventi, NYU) 

Biosketches Page 16 
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date: 2016-07-05T15:19:02.000-04:00

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  1

ORGANIZATIONAL DUNS*:   

Budget Type*: ● Project ❍ Subaward/Consortium

Enter name of Organization: 

Start Date*: 04-01-2017 End Date*: 03-31-2018 Budget Period: 1

A. Senior/Key Person

Prefix First Name* Middle

Name

Last Name* Suffix Project Role* Base

Salary ($)

Calendar

Months

Academic

Months

Summer

Months

Requested

Salary ($)*

Fringe

Benefits ($)*

Funds Requested ($)*

1 . Robert C Froemke PD/PI 5 0 0

Total Funds Requested for all Senior Key Persons in the attached file

Additional Senior Key Persons: File Name: Total Senior/Key Person

B. Other Personnel

Number of

Personnel*

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)*

2 Post Doctoral Associates 15 0 0

Undergraduate Students

Secretarial/Clerical

1 Technician 12 0 0

Total Salary, Wages and Fringe Benefits (A+B)

RESEARCH & RELATED Budget {A-B} (Funds Requested)

Contact PD/PI: Froemke, Robert C

 Page 17
Contact PD/PI: Froemke, Robert C

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date:

2016-07-05T15:19:02.000-04:00

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: 

Start Date*: 04-01-2017 End Date*: 03-31-2018 Budget Period: 1

C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment Item Funds Requested ($)*

Total funds requested for all equipment listed in the attached file

Total Equipment

Additional Equipment: File Name:

D. Travel Funds Requested ($)*

1. Domestic Travel Costs ( Incl. Canada, Mexico, and U.S. Possessions)

2. Foreign Travel Costs

Total Travel Cost

E. Participant/Trainee Support Costs Funds Requested ($)*

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other: 

 Number of Participants/Trainees Total Participant Trainee Support Costs

RESEARCH & RELATED Budget {C-E} (Funds Requested)

 Page 18
Contact PD/PI: Froemke, Robert C

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date:

2016-07-05T15:19:02.000-04:00

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: 

Start Date*: 04-01-2017 End Date*: 03-31-2018 Budget Period: 1

F. Other Direct Costs Funds Requested ($)*

1. Materials and Supplies

0.00

G. Direct Costs Funds Requested ($)*

Total Direct Costs (A thru F)

H. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)*

1 . Research (MTDC)

Cognizant Federal Agency

(Agency Name, POC Name, and POC Phone Number)

DHHS, 

I. Total Direct and Indirect Costs Funds Requested ($)*

Total Direct and Indirect Institutional Costs (G + H)

J. Fee Funds Requested ($)*

K. Budget Justification* File Name: budget_justification_final.pdf 

(Only attach one file.)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

 Page 19
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date: 2016-07-05T15:19:02.000-04:00

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  2

ORGANIZATIONAL DUNS*:   

Budget Type*: ● Project ❍ Subaward/Consortium

Enter name of Organization: 

Start Date*: 04-01-2018 End Date*: 03-31-2019 Budget Period: 2

A. Senior/Key Person

Prefix First Name* Middle

Name

Last Name* Suffix Project Role* Base

Salary ($)

Calendar

Months

Academic

Months

Summer

Months

Requested

Salary ($)*

Fringe

Benefits ($)*

Funds Requested ($)*

1 . Robert C Froemke PD/PI 5 0 0

B. Other Personnel

Number of

Personnel*

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)*

1 Post Doctoral Associates 12 0 0

Undergraduate Students

Secretarial/Clerical

1 Technician 12 0 0

RESEARCH & RELATED Budget {A-B} (Funds Requested)

Contact PD/PI: Froemke, Robert C

 Page 20
Contact PD/PI: Froemke, Robert C

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date:

2016-07-05T15:19:02.000-04:00

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 2

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: 

Start Date*: 04-01-2018 End Date*: 03-31-2019 Budget Period: 2

C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment Item Funds Requested ($)*

Total funds requested for all equipment listed in the attached file

Total Equipment

Additional Equipment: File Name:

D. Travel Funds Requested ($)*

1. Domestic Travel Costs ( Incl. Canada, Mexico, and U.S. Possessions)

E. Participant/Trainee Support Costs Funds Requested ($)*

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other: 

 Number of Participants/Trainees Total Participant Trainee Support Costs

RESEARCH & RELATED Budget {C-E} (Funds Requested)

 Page 21
Contact PD/PI: Froemke, Robert C

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date:

2016-07-05T15:19:02.000-04:00

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 2

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: 

Start Date*: 04-01-2018 End Date*: 03-31-2019 Budget Period: 2

F. Other Direct Costs Funds Requested ($)*

1. Materials and Supplies

G. Direct Costs Funds Requested ($)*

Total Direct Costs (A thru F)

H. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)*

1 . Research (MTDC)

Cognizant Federal Agency

(Agency Name, POC Name, and POC Phone Number)

DHHS, 

I. Total Direct and Indirect Costs Funds Requested ($)*

Total Direct and Indirect Institutional Costs (G + H)

J. Fee Funds Requested ($)*

K. Budget Justification* File Name: budget_justification_final.pdf 

(Only attach one file.)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

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OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date: 2016-07-05T15:19:02.000-04:00

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  3

ORGANIZATIONAL DUNS*:   

Budget Type*: ● Project ❍ Subaward/Consortium

Enter name of Organization: 

Start Date*: 04-01-2019 End Date*: 03-31-2020 Budget Period: 3

A. Senior/Key Person

Prefix First Name* Middle

Name

Last Name* Suffix Project Role* Base

Salary ($)

Calendar

Months

Academic

Months

Summer

Months

Requested

Salary ($)*

Fringe

Benefits ($)*

Funds Requested ($)*

1 . Robert C Froemke PD/PI

B. Other Personnel

Number of

Personnel*

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)*

1 Post Doctoral Associates 12 0 0

Undergraduate Students

Secretarial/Clerical

1 Technician 12 0 0

RESEARCH & RELATED Budget {A-B} (Funds Requested)

Contact PD/PI: Froemke, Robert C

 Page 23
Contact PD/PI: Froemke, Robert C

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date:

2016-07-05T15:19:02.000-04:00

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 3

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: 

Start Date*: 04-01-2019 End Date*: 03-31-2020 Budget Period: 3

C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment Item Funds Requested ($)*

Total funds requested for all equipment listed in the attached file

Additional Equipment: File Name:

D. Travel Funds Requested ($)*

1. Domestic Travel Costs ( Incl. Canada, Mexico, and U.S. Possessions)

E. Participant/Trainee Support Costs Funds Requested ($)*

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other: 

 Number of Participants/Trainees Total Participant Trainee Support Costs

RESEARCH & RELATED Budget {C-E} (Funds Requested)

 Page 24
Contact PD/PI: Froemke, Robert C

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date:

2016-07-05T15:19:02.000-04:00

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 3

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: 

Start Date*: 04-01-2019 End Date*: 03-31-2020 Budget Period: 3

F. Other Direct Costs Funds Requested ($)*

1. Materials and Supplies

G. Direct Costs Funds Requested ($)*

Total Direct Costs (A thru F)

H. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)*

1 . Research (MTDC)

Cognizant Federal Agency

(Agency Name, POC Name, and POC Phone Number)

DHHS, 

I. Total Direct and Indirect Costs Funds Requested ($)*

Total Direct and Indirect Institutional Costs (G + H)

J. Fee Funds Requested ($)*

K. Budget Justification* File Name: budget_justification_final.pdf 

(Only attach one file.)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

 Page 25
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date: 2016-07-05T15:19:02.000-04:00

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  4

ORGANIZATIONAL DUNS*:   

Budget Type*: ● Project ❍ Subaward/Consortium

Enter name of Organization: 

Start Date*: 04-01-2020 End Date*: 03-31-2021 Budget Period: 4

A. Senior/Key Person

Prefix First Name* Middle

Name

Last Name* Suffix Project Role* Base

Salary ($)

Calendar

Months

Academic

Months

Summer

Months

Requested

Salary ($)*

Fringe

Benefits ($)*

Funds Requested ($)*

1 . Robert C Froemke PD/PI

B. Other Personnel

Number of

Personnel*

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)*

1 Post Doctoral Associates 12 0 0

Undergraduate Students

Secretarial/Clerical

1 Technician 12 0 0

RESEARCH & RELATED Budget {A-B} (Funds Requested)

Contact PD/PI: Froemke, Robert C

 Page 26
Contact PD/PI: Froemke, Robert C

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date:

2016-07-05T15:19:02.000-04:00

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 4

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: 

Start Date*: 04-01-2020 End Date*: 03-31-2021 Budget Period: 4

C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment Item Funds Requested ($)*

Total funds requested for all equipment listed in the attached file

Additional Equipment: File Name:

D. Travel Funds Requested ($)*

1. Domestic Travel Costs ( Incl. Canada, Mexico, and U.S. Possessions)

E. Participant/Trainee Support Costs Funds Requested ($)*

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other: 

 Number of Participants/Trainees Total Participant Trainee Support Costs

RESEARCH & RELATED Budget {C-E} (Funds Requested)

 Page 27
Contact PD/PI: Froemke, Robert C

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date:

2016-07-05T15:19:02.000-04:00

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 4

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: 

Start Date*: 04-01-2020 End Date*: 03-31-2021 Budget Period: 4

F. Other Direct Costs Funds Requested ($)*

1. Materials and Supplies

G. Direct Costs Funds Requested ($)*

Total Direct Costs (A thru F)

H. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)*

1 . Research (MTDC)

Cognizant Federal Agency

(Agency Name, POC Name, and POC Phone Number)

DHHS, Darryl W. Mayes, 212-264-2069

I. Total Direct and Indirect Costs Funds Requested ($)*

Total Direct and Indirect Institutional Costs (G + H)

J. Fee Funds Requested ($)*

K. Budget Justification* File Name: budget_justification_final.pdf 

(Only attach one file.)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

 Page 28
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date: 2016-07-05T15:19:02.000-04:00

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  5

ORGANIZATIONAL DUNS*:   

Budget Type*: ● Project ❍ Subaward/Consortium

Enter name of Organization: 

Start Date*: 04-01-2021 End Date*: 03-31-2022 Budget Period: 5

A. Senior/Key Person

Prefix First Name* Middle

Name

Last Name* Suffix Project Role* Base

Salary ($)

Calendar

Months

Academic

Months

Summer

Months

Requested

Salary ($)*

Fringe

Benefits ($)*

Funds Requested ($)*

1 . Robert C Froemke PD/PI

B. Other Personnel

Number of

Personnel*

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)*

1 Post Doctoral Associates 12 0 0

Undergraduate Students

Secretarial/Clerical

1 Technician 12 0 0

RESEARCH & RELATED Budget {A-B} (Funds Requested)

Contact PD/PI: Froemke, Robert C

 Page 29
Contact PD/PI: Froemke, Robert C

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date:

2016-07-05T15:19:02.000-04:00

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 5

ORGANIZATIONAL DUNS*: 121911077

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: New York University School Of Medicine

Start Date*: 04-01-2021 End Date*: 03-31-2022 Budget Period: 5

C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment Item Funds Requested ($)*

Total funds requested for all equipment listed in the attached file

Additional Equipment: File Name:

D. Travel Funds Requested ($)*

1. Domestic Travel Costs ( Incl. Canada, Mexico, and U.S. Possessions)

E. Participant/Trainee Support Costs Funds Requested ($)*

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other: 

 Number of Participants/Trainees Total Participant Trainee Support Costs

RESEARCH & RELATED Budget {C-E} (Funds Requested)

 Page 30
Contact PD/PI: Froemke, Robert C

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date:

2016-07-05T15:19:02.000-04:00

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 5

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: 

Start Date*: 04-01-2021 End Date*: 03-31-2022 Budget Period: 5

F. Other Direct Costs Funds Requested ($)*

1. Materials and Supplies

G. Direct Costs Funds Requested ($)*

Total Direct Costs (A thru F)

H. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)*

1 . Research (MTDC)

Cognizant Federal Agency

(Agency Name, POC Name, and POC Phone Number)

DHHS, Darryl W. Mayes 212-264-2069

I. Total Direct and Indirect Costs Funds Requested ($)*

Total Direct and Indirect Institutional Costs (G + H)

J. Fee Funds Requested ($)*

K. Budget Justification* File Name: budget_justification_final.pdf 

(Only attach one file.)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

 Page 31
Contact PD/PI: Froemke, Robert C

Budget Justification

The high cost of living necessitates salaries that are higher than elsewhere in the US. This project 

requires our participation, and the full-time effort of lab members in the lab. These salaries alone, 

together with fringe benefits, would almost be the maximum budget for a modular grant. We are requesting 

minimal equipment to perform the Aims of this study, but costs for essential supplies and mice increase the total 

budget to As this is our renewal application of our sole R01, we have no other support to study how 

cholinergic and noradrenergic neuromodulation enable behaviorally-relevant plasticity in the auditory system. 

Personnel

Dr. Robert C. Froemke (PI, 5 months effort) will be responsible for administrating this project, determining the 

directions, establishing priorities for experiments and coordinating research efforts with lab members. He will 

perform some in vivo recording and behavioral experiments, especially at the outset, and train personnel.

 a postdoctoral associate (3 months effort in year 1, zero salary) will be responsible 

for training the graduate students and technicians on surgical procedures, recording and two-photon imaging in 

awake head-fixed mice. has extensive experience in 2-photon imaging of cortical activity in awake headfixed mice, having earned his Ph.D. under the supervision of 

His thesis work showed how visual cortical processing is disrupted by pathologies such as epilepsy and 

Alzheimer’s disease, and thus he is highly trained in performing surgeries; building and using a 2-photon 

microscope; generating, calibrating, and controlling presentation of sensory stimuli; and data analysis with 

Matlab and other programs. In graduate school, was supported by an NIH NRSA Predoctoral Fellowship, 

a James Mills Pierce Scholarship from Harvard Medical School, and won the Best Paper in Neuroimaging Award 

at the International Alzheimer’s Disease conference. He had a first-author paper in Science and several other 

publications as a graduate student, and has built our 2-photon system. As shown by our preliminary data and 

our co-authored publication in Neuron (Cohen et al., Neuron 2016), we are already acquiring stable imaging data 

from awake head-fixed mice and performing 2-photon guided whole-cell recordings. He joined the Froemke lab 

in 2012; his salary is , but we are requesting no salary support as has a K99/R00 award.

TBD, a postdoctoral associate (12 months effort), will be trained on imaging and recording procedures 

in head-fixed mice.

 a PhD graduate student (9 months effort), will perform most of the behavioral 

studies and in vivo recordings, focusing on cortex. got her BS in Industrial Biotechnology from the 

 where she founded an undergraduate program in neuroscience (NeuroRUM) across multiple UPR campuses. will begin as a graduate student Fall 2016 and been working in 

the Froemke lab as a research assistant during the summers for the past 2 years. She is already working in our 

lab collecting pilot data for this proposal. Her salary is 

a technician (12 months effort), will perform some behavioral experiments (including 

implantation of head-posts and training mice to head fix), help with anatomical and immunohistological studies, 

assist with recovery surgeries, and monitor implanted animals. has been trained in the Froemke lab in 

these techniques since Spring 2015. She recently graduated from Teachers College with 

a MS in Neuroscience and Education. Her salary is 

Equipment

To perform stereotaxic injections and implantations, as well as 2-photon targeted whole-cell recordings in awake 

animals, we are requesting funds to purchase additional manipulators in year 2 This equipment is

required for this study, given the importance of whole-cell recordings and viral injections for GCaMP6s or opsin 

expression. We also request funds to purchase resonant galvo scanners in year 3 , which will greatly 

increase the temporal resolution of imaging, especially important for the 2-photon imaging of axon fibers.

Supplies 

We are requesting funds to purchase and house animals, and order viral constructs, anesthetic agents and other 

pharmacological reagents, and surgical supplies and electrodes. 

Budget Justification Attachment Page 32
Travel

We are requesting funds to support the PI’s travel to two meetings each year (the Society for Neuroscience 

annual meeting and either Cosyne or the Association for Research in Otolaryngology midwinter meeting), as 

well as support the attendance of the postdocs and graduate students at one meeting each year.

Contact PD/PI: Froemke, Robert C

Budget Justification Attachment Page 33
Contact PD/Pl: Froemke, Robert C 

RESEARCH & RELATED BUDGET-Cumulative Budget 

Section A, Senior/Key Person 

Total Number Other Personnel 

Total Salary, Wages and Fringe Benefits 

(A+B) 

1. Domestic

-

Section E, Participant/Trainee Support 

Costs 

1. Tuition/Fees/Health Insurance 

2. Stipends 

3. Travel 

4. Subsistence 

5. Other 

6. Number of Participants/Trainees 

Section F, Other Direct Costs 

1. Materials and Supplies 

2. Publication Costs 

3. Consultant Services 

4. ADP/Computer Services 

5. Subawards/Consortium/Contractual 

Costs 

6. Equipment or Facility Rental/User 

Fees 

8. Other 1 

9. Other 2 

10. Other 3 

Section G, Direct Costs 

(A thru F) 

-

Totals($) 

16 

-

• 

0.00 

0.00 

0.00 

0.00 

-

0.00 

0 

0.00 

0.00 

-

0.00 

0.00 

-

0.00 

0.00 

0.00 

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•

0.00 

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Page 34 

Track ng Number: GRANT12208477 Funding Opportunity Number: PA 16-160. Received Date: 

2016 07 05T15:19:02.000 04:00 
PHS 398 Cover Page Supplement

OMB Number: 0925-0001

Expiration Date: 10/31/2018

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date:

2016-07-05T15:19:02.000-04:00

1. Human Subjects Section

Clinical Trial? ❍ Yes ● No

*Agency-Defined Phase III Clinical Trial? ❍ Yes ❍ No

2. Vertebrate Animals Section

Are vertebrate animals euthanized? ● Yes ❍ No

If "Yes" to euthanasia

Is the method consistent with American Veterinary Medical Association (AVMA) guidelines?

● Yes ❍ No

If "No" to AVMA guidelines, describe method and proved scientific justification

3. *Program Income Section

*Is program income anticipated during the periods for which the grant support is requested?

❍ Yes ● No

If you checked "yes" above (indicating that program income is anticipated), then use the format below to reflect the amount and

source(s). Otherwise, leave this section blank.

*Budget Period *Anticipated Amount ($) *Source(s)

Contact PD/PI: Froemke, Robert C

 Page 35
Contact PD/PI: Froemke, Robert C

PHS 398 Cover Page Supplement

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160 . Received Date:

2016-07-05T15:19:02.000-04:00

4. Human Embryonic Stem Cells Section

*Does the proposed project involve human embryonic stem cells? ❍ Yes ● No

If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the

following list: http://grants.nih.gov/stem_cells/registry/current.htm. Or, if a specific stem cell line cannot be referenced at this time,

please check the box indicating that one from the registry will be used:

Specific stem cell line cannot be referenced at this time. One from the registry will be used.

Cell Line(s) (Example: 0004):

5. Inventions and Patents Section (RENEWAL)

*Inventions and Patents: ❍ Yes ● No

If the answer is "Yes" then please answer the following:

*Previously Reported: ❍ Yes ❍ No

6. Change of Investigator / Change of Institution Section

❏ Change of Project Director / Principal Investigator

Name of former Project Director / Principal Investigator

Prefix:

*First Name:

Middle Name:

*Last Name:

Suffix:

❏ Change of Grantee Institution

*Name of former institution:

Page 36
 

PHS 398 Research Plan

OMB Number: 0925-0001

Expiration Date: 10/31/2018

 

Tracking Number: GRANT12208477 Funding Opportunity Number: PA-16-160. Received Date:

2016-07-05T15:19:02.000-04:00

Introduction

1. Introduction to Application

(Resubmission and Revision)

Research Plan Section

2. Specific Aims aims_final.pdf

3. Research Strategy* research_strategy_final.pdf

4. Progress Report Publication List progress_report_publication_list.pdf

Human Subjects Section

5. Protection of Human Subjects

6. Data Safety Monitoring Plan

7. Inclusion of Women and Minorities

8. Inclusion of Children

Other Research Plan Section

9. Vertebrate Animals animals_final.pdf

10. Select Agent Research

11. Multiple PD/PI Leadership Plan

12. Consortium/Contractual Arrangements

13. Letters of Support support_letters_final2.pdf

14. Resource Sharing Plan(s) resource_sharing_final.pdf

15. Authentication of Key Biological and/or

Chemical Resources resource_authentication_final.pdf

Appendix

16. Appendix

Contact PD/PI: Froemke, Robert C

 Page 37
I. Specific Aims

Synaptic basis of perceptual learning in primary auditory cortex

The goal of this project is to determine how cholinergic and noradrenergic modulatory systems collectively 

promote synaptic plasticity in primary auditory cortex (AI), to improve auditory perceptual abilities in behaving 

mice. Modulation and plasticity are important features of AI, especially for forming representations of sensory 

signals such as speech, music, or other behaviorally-relevant sounds1-10. Changes in neural circuits and behavior

can be incredibly long-lasting after auditory conditioning11-14, but the mechanisms by which AI networks are 

persistently modified and affect auditory perception are unclear. In particular, it remains challenging to connect 

perceptual learning to plasticity in AI or elsewhere in the brain. Impaired cortical processing and plasticity are 

believed to occur in disorders such as hearing loss, language impairments, and tinnitus15-17; conversely, enabling

plasticity by training programs and devices such as hearing aids and cochlear implants may improve outcomes

in pathological conditions and hearing loss18-22. Successful completion of this project will result in a significantly 

improved mechanistic model of auditory learning and AI plasticity, critical for studies of language learning, 

speech processing, deafness, and the design of medical devices and training procedures for hearing restoration. 

Adult cortical plasticity requires sensory experience and neuromodulation, which relays global behavioral context 

to local cortical circuits. Many modulatory systems, including the noradrenergic system of the locus coeruleus23-

30 and the cholinergic system of the basal forebrain1,2,31-40, are recruited by surprising or arousing stimuli, and 

promote plasticity in target circuits including rodent AI. What are the functional differences between acetylcholine 

and norepinephrine for perceptual learning and auditory behavior? Specifically, here we will ask how training 

affects AI circuits during behavior (Aim 1); if cholinergic and noradrenergic systems are recruited at distinct times, 

to differentially modulate AI during auditory training for enhanced perception (Aim 2); and finally, when and how

neuromodulatory neurons become responsive to task-relevant sounds and other cues during training (Aim 3).

Previously we studied cholinergic or noradrenergic modulation separately in behaving rats, measuring

physiological changes under anesthesia10,28,35,36

. Here we will relate behavior to neural activity with in vivo wholecell recording and 2-photon imaging from AI and modulatory neurons in behaving head-fixed mice. These studies

take advantage of our past work on AI modulation and plasticity28,35,36,41-45, using a novel approach we developed 

to document synaptic plasticity in multiple cells over days, in behaving mice performing an auditory task. This 

allows us to measure and manipulate modulatory systems on a trial-by-trial basis while monitoring responses 

over the entire time-course of learning in individual mice. We hypothesize that initially, norepinephrine reduces

tonic inhibition to increase excitability to all stimuli; this may increase detection and behavioral responses but at 

the cost of false alarms. As mice learn the task over days, acetylcholine then selectively enhances a subpopulation of excitatory and inhibitory AI neurons for reliable task performance. Our goal is a unified model of 

cholinergic and noradrenergic modulation for AI excitatory and inhibitory plasticity important for behavior.

Aim 1. To determine how AI excitatory/inhibitory cells and synapses are modified by auditory training 

We will train adult mice on a go/no-go auditory task, to examine perceptual learning by measuring detection 

thresholds and sound recognition abilities. We will use in vivo 2-photon imaging (Subaim 1a) and whole-cell 

recordings (Subaim 1b) from AI neurons in behaving mice to determine how excitatory/inhibitory inputs and 

outputs are modified by learning, and assess if these changes are required for task performance. 

Aim 2. To ask how cholinergic and noradrenergic modulation affect auditory learning and AI plasticity

We will record from AI during behavior, using optogenetics and pharmacology to ask if and when the cholinergic 

vs noradrenergic systems are necessary for initial learning and maintained peak performance (Subaim 2a). We 

will ask if pairing task-relevant sounds with cholinergic and/or noradrenergic modulation can accelerate learning 

or enhance peak performance (Subaim 2b). We will determine the synaptic mechanisms by which cholinergic 

and noradrenergic modulation enable AI excitatory/inhibitory plasticity during learning. 

Aim 3. To test the hypothesis that noradrenergic and cholinergic neurons respond to task variables

How are the noradrenergic and cholinergic systems recruited when mice are engaged in the task? We will ask if 

there is a sequence of AI modulation over task learning; specifically if the noradrenergic system is recruited 

transiently during the first day, while the cholinergic system comes on-line over subsequent days and is needed 

for steady-state performance. We will record from locus coeruleus and nucleus basalis, and perform 2-photon 

imaging of modulatory axons within AI in trained animals and over the duration of perceptual learning. We also 

recently found that tone-evoked responses with short latency are induced in locus coeruleus neurons after 

pairing tones with locus coeruleus stimulation28. Here we will ask if plasticity in nucleus basalis and/or locus 

coeruleus is important for auditory learning and task performance. 

Contact PD/PI: Froemke, Robert C

Specific Aims Page 38
II. Research Strategy

A. Significance

The auditory cortex is modulated and modified after significant experiences, learning meanings of words or other 

sounds, sensory deprivation, or use of devices such as cochlear implants1-22. Synaptic plasticity is thought to be 

a fundamental mechanism for long-term changes in network function. However, relations between modulation,

plasticity, network reorganization, and changes in perception and behavior are complex and remain unclear. 

This proposal describes a series of in vivo imaging and recording experiments in AI of behaving mice to reveal: 

1) when and how auditory input interacts with the neuromodulators acetylcholine and norepinephrine (the main 

modulators of sensory attention) to modify AI responses, and 2) under what conditions these changes improve 

auditory perception. The main hypothesis is that the cholinergic and noradrenergic systems have different 

temporal dynamics and distinct effects on AI responses and perception (Fig. 1a). Specifically, we hypothesize 

that on the first training day, norepinephrine boosts AI and behavioral responses to all stimuli. This is important 

for animals to begin responding, to learn task variables and stimulus-response contingencies. As mice learn the 

task, cholinergic modulation then refines AI representations for reliable performance (in high-performers). We 

aim to document this process over the training duration with imaging and recordings in behaving mice (Fig. 1b).

The overall goal of our research is to understand the circuit mechanisms of auditory perceptual learning. Longterm plasticity requires two factors: sensory experience and activation of neuromodulatory systems, which relay 

global behavioral context to local cortical circuits1,2,10,11,46-51. Sensory stimulation alone does not usually lead to 

positive long-term changes in adults without context-defining modulatory signaling. Conversely, modulation 

alone can have negligible effects or be potentially pathological without sensory input to guide plasticity3,10,52-54. 

Our lab has studied the complex interactions between these variables for years in the rodent auditory system, 

with behavioral training, electrophysiological recordings in vivo and in vitro, two-photon imaging, computational

modeling, and anatomical/biochemical studies of modulatory systems28,35,36,41-45,55-62. We have examined how 

sensory input (e.g., tones, frequency modulated (FM) sweeps, vocalizations) interact with brain state (e.g., 

encoded by norepinephrine or acetylcholine), to induce long-term synaptic plasticity and affect perception. We 

previously examined cholinergic or noradrenergic systems separately, studying how these modulators improve 

(or degrade) auditory perception in behaving rats, while making whole-cell recordings in anesthetized animals to 

examine how modulation paired with sounds changes AI representations of paired or unpaired input28,35,36. 

In this proposal, we take the next logical steps, studying modulation, plasticity, and learning in the same animals. 

Specifically, in Aim 1 we will perform 2-photon imaging and whole-cell recording from AI neurons in behaving 

mice to measure changes to synaptic input and spiking output. In Aim 2 we ask if noradrenergic or cholinergic 

modulation of AI excitatory/inhibitory plasticity are required to learn and improve perceptual abilities. In Aim 3, 

we image and record from cholinergic and noradrenergic modulatory neurons during auditory training. We will 

relate spatio-temporal patterns of modulatory activity to the time course of auditory learning, and examine 

changes to neuromodulatory systems directly. Previous results23,24,28,38,63-65 suggest modulatory cells can begin 

responding to sounds after conditioning, possibly important for prolonging AI plasticity and perceptual changes. 

Auditory processing is context specific, reflecting the importance of perceptual salience and behavioral relevance

for sensory perception66-69. Among other purposes, this allows humans and animals to focus on vocalizations or 

other behaviorally-important sounds to learn their meanings, recognize such sounds in complex and noisy 

acoustic environments, and increase arousal level during surprising or hazardous experiences. Studying auditory 

plasticity in AI is particularly important because contextual modulation of auditory processing is stronger in AI as 

compared to subcortical stations36,70-73, and because it has been linked to the activity of neuromodulatory nuclei 

Figure 1. General hypotheses of this proposal. a, Left, 

experiment schematic- in vivo 2-photon imaging and wholecell recording in mice performing an auditory detection and 

recognition task for water reward. Right, we predict that 

norepinephrine (NE) release is initially high immediately at 

start of training, but decays rapidly over days as acetylcholine 

(ACh) modulation becomes important for task performance. b,

Network hypothesis: AI cells (triangle, excitatory; circle, 

inhibitory) are not initially positively (green) or negatively (red) 

modulated by task before learning begins. At Day 1, NE leads 

to large-scale activation of AI during task. By Day 10, ACh 

activates a sub-ensemble, but silences many other cells.

Contact PD/PI: Froemke, Robert C

Research Strategy Page 39
that project to AI28,29,30-36. Electrical or optogenetic stimulation of these subcortical regions enables plasticity of 

AI tuning curves and auditory responses, influences perception, and modifies behavior, making neuromodulation 

a valuable tool for therapeutic purposes and for identifying mechanisms of plasticity in vivo1,2,5,10,14,20,30-36,42,43,47. 

Many neuromodulators affect cortical responses in 

some way, such as social salience cues provided by 

oxytocin42,43,74 or reward prediction errors signaled by 

dopamine75-78. However, the two main modulators for 

attentional control of sensory processing are 

acetylcholine (released specifically into cortex from 

the nucleus basalis; Fig. 2a-c) and norepinephrine

(from locus coeruleus and other brainstem centers; 

Fig. 2d-f). Nucleus basalis contains a distribution of 

cholinergic and non-cholinergic cells, important for

selective attention to behaviorally-relevant stimuli31-

33,37-39,79. Nucleus basalis projects mainly to cortex, 

with inputs from limbic areas and prefrontal 

cortex31,39. In contrast, locus coeruleus is a small 

brainstem area (~1-2k and 20-50k cells in rodents 

and humans, respectively) with a primary role in 

general arousal and selective attention to some 

degree. Locus coeruleus inputs include brainstem, 

limbic system and prefrontal cortex, and outputs are 

widely targeted throughout the brain, including AI and 

thalamus25,26. Both systems are implicated in many 

physiological and pathological aspects of brain 

function, including learning and memory, arousal, 

and addiction26,29,32-36,47,64,,65,80. Both systems 

degenerate early in Alzheimer’s disease, possibly 

leading to the ‘cognitive fog’ in this condition81,82

. 

Despite some similarities between cholinergic and 

noradrenergic systems, there are anatomical and 

functional distinctions perhaps important for contextdependent modulation and auditory learning. Locus 

coeruleus is hypothesized to convey 'top-down' 

uncertainty between actual and expected input, or 

'bottom-up' alerting signals to surprising events. 

Cholinergic signaling might also convey this type of

mismatch, but may be more important for ‘expected 

uncertainty’ (i.e., steady-state attention to learned

environmental/behavioral contingencies). In contrast, 

norepinephrine signals ‘unexpected uncertainty’23-

26,64,83,84 (i.e., at start of training or dramatic changes to environments or behavioral outcomes).

How do cholinergic and noradrenergic modulation affect AI synaptic responses? Many studies in vivo and in vitro

show that cholinergic or noradrenergic agonists affect neuronal excitability, synaptic transmission, and enable 

plasticity; in contrast, receptor blockers prevent plasticity28,36,47-51,85-94. Over the last decade we have 

systematically studied cholinergic or noradrenergic systems in vivo (Fig. 2), examining how modulation affects 

spontaneous excitatory and inhibitory postsynaptic currents (E/IPSCs, Fig. 2b,e), tone-evoked responses and 

AI receptive fields at synaptic (Fig. 2c,f) and spiking levels (Fig. 3), and examined behavioral consequences of 

neuromodulatory pairing (electrically or optogenetically) and plasticity of AI frequency or intensity tuning (Fig. 4).

Both modulators reduce inhibition, but in different ways. Nucleus basalis stimulation in adult rats increased 

spontaneous IPSCs measured with in vivo whole-cell recordings (Fig. 2b, orange); while tone-evoked IPSCs 

were reduced (Fig. 2c), replicating past work1

. This depends on muscarinic receptors as it was blocked by 

atropine, and induced NMDA receptor-dependent long-term potentiation (LTP) of evoked EPSCs (Fig. 2c).

Importantly, excitation and inhibition are co-tuned and correlated (Fig. 2c, gray), or ‘balanced’ in adult AI10,41,44,95-

98. Cholinergic disinhibition transiently disrupts excitatory-inhibitory balance, promoting tuning curve plasticity 

before inhibition re-balanced excitation after hours35,36. Synaptic changes induced by 5 min nucleus basalis 

Figure 2. AI modulation. a, Acetylcholine is released 

into AI from nucleus basalis (NB). b, In vivo voltageclamp recording in rodent AI; NB stimulation increased

spontaneous IPSCs. c, NB stimulation paired with 2 

kHz tone repetitively reduced tone-evoked IPSCs and 

enhanced paired tone EPSCs. d, Norepinephrine is 

released by locus coeruleus (LC). e, In contrast to NB, 

LC stimulation reduced spontaneous IPSCs. f, LC 

pairing enhanced tone-evoked EPSCs and IPSCs 

together. Note magnitude of increase (refs. 28,35).

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pairing shifted tuning curves towards paired tones (Fig. 

3a). However, >6 hrs without additional nucleus basalis 

stimulation, responses shifted back to the original tuning 

in register with AI tonotopy (Fig. 3b). In contrast, locus 

coeruleus pairing lowered spontaneous ISPCs (Fig. 

2e). Reduced spontaneous inhibition greatly increased 

excitability for all frequencies28, leading to large 200-

1000% increases of tone-evoked excitation and 

inhibition together (Fig. 2f,3c). This nonspecific

increase recovered, and tuning curves remained shifted 

to paired frequencies for 12+ hours, essentially 

persisting as long as measured (Fig. 3d). We measured 

perceptual effects of AI plasticity by performing

behavioral studies28,36, conditioning rats on a ‘go/no-go’ 

tone detection-recognition task, rewarding them for 

responding to 4 kHz targets (Fig. 4a). Nucleus basalis 

pairing selectively but transiently enhanced behavior

(Fig. 4b,c). Locus coeruleus pairing initially impaired or 

did not affect performance, but after a few hours, 

detection and recognition were improved for days (Fig. 

4d,e), similar to AI changes. Moreover, just one episode 

of locus coeruleus pairing accelerated learning rate

when target frequency was switched28. 

Thus the major effects of cholinergic modulation are: 

reduced stimulus-evoked inhibition, producing hourslong shifts in tuning curve peaks while conserving total 

excitability (i.e., x-axis translations in AI tuning). In 

comparison, noradrenergic modulation leads to: reduced spontaneous inhibition but increased stimulus-evoked 

EPSCs/IPSCs, first leading to a large response gain to all stimuli (i.e., y-axis translation and broadening of AI 

tuning), before excitability recovers and long-lasting changes emerge. Pairing-induced changes in AI tuning are 

directly reflected in auditory behaviors, in terms of enhanced detection, improved (or worse) target recognition, 

and duration of these behavioral changes. These results suggest that cholinergic and noradrenergic modulation 

play a crucial role underlying auditory plasticity and learning. We hypothesize that even when these forms of 

modulatory pairing are not experimentally imposed (as in our past studies28,35,36), they are naturally engaged by 

perceptual training, which we will confirm by recording and imaging in behaving mice in Aims 1 and 2. 

Figure 3. AI frequency tuning plasticity. a, Recording 

in 1 kHz region of AI (arrowhead); NB stim paired 

with 4 kHz (arrow). Tuning shifts lasted 1-3 hrs, but 

retuned to 1 kHz after ~6 hrs; tuning shift was x-axis 

peak translation. b, Summary of NB pairing shifts. c, 

LC pairing increased responses to all tones by ~10X, 

producing y-axis increase in response gain. After ~1-

3 hrs responses recovered, but peak shift (16 kHz) 

endured. d, LC pairing effect duration (ref. 28).

Figure 4. NB and LC pairing improve 

auditory behavior. a, Food-deprived rats 

were operantly conditioned to respond for 

food reward to target 4 kHz tones (any 

intensity), withholding responses to foils. 

We can systematically vary sound level to 

probe psychophysical detection thresholds 

(background noise level ~30 dB SPL), or 

adjust frequency range of foils to assess 

recognition abilities. b, NB pairing 

improved target recognition for hours 

(gray, baseline; orange, 1-2 hrs after 

pairing), but performance returned on the 

next day. c, NB pairing selectively 

improved detection of quiet tones at the 

paired intensity, but changes lasted only a 

few hrs. d, LC pairing initially had little 

effect on the same session, but led to 

selective and enduring changes thereafter. 

e, LC pairing greatly enhanced detection 

for 5-30 days (refs. 28,36).

Contact PD/PI: Froemke, Robert C

Research Strategy Page 41
We examined circuit mechanisms of persistent changes in AI responses and behavior after locus coeruleus 

pairing28. We recorded from locus coeruleus cells (Fig. 5a) and found these neurons were also plastic; shortlatency tone responses (~50 msec from onset) emerged after pairing tones with locus coeruleus stimulation or 

foot shock. In vivo whole-cell recordings showed subthreshold responses developing in initially-unresponsive 

cells; plasticity was prevented with APV (NMDA receptor antagonist) infused into locus coeruleus (Fig. 5b). 

Preventing locus coeruleus plasticity reduced duration of AI tuning curve shifts (Fig. 5c) and perceptual 

improvement after pairing (Fig. 5d). This replicated and extended past studies showing locus coeruleus 

responses to conditioned stimuli after 

training23,24. Now in this proposal, we will ask 

in Aim 3 if this ‘plasticity of neuromodulation’ 

naturally occurs in nucleus basalis and locus 

coeruleus during training, and how this 

control of modulation impacts AI plasticity

and auditory perceptual learning in mice. 

Neuromodulation is widespread in the 

auditory system, important for attention, 

arousal, plasticity, and learning. There is a 

large literature on modulation of AI

processing and plasticity1-14, but direct 

connections between synaptic plasticity and 

perceptual learning are lacking. Our studies

provide foundational data towards a general 

theory of cortical modulation and plasticity, 

examining cortical-subcortical interactions, 

and how training drives multiple modulatory

systems to synergistically enhance sensory 

processing for improved perception. We 

expect these methods and results will apply 

to other neural systems, and contribute to 

studies of language learning and the role of 

attention vs. arousal in auditory processing. 

These studies will also inform training 

programs and rehabilitation strategies15-22 for

disorders such as hearing loss or tinnitus, 

and use of hearing aids or cochlear implants. 

B. Innovation

This project is innovative in four main ways: 1) Connecting synaptic plasticity to behavioral changes is a central 

challenge in neuroscience. While training and experience affect neural circuits, we have shown how AI synaptic 

modifications causally improve behavior for hours, days, or weeks28,35,36,42

. We are one of the few groups studying 

long-term excitatory/inhibitory plasticity in vivo with whole-cell recording (Fig. 2) and optogenetics. Here we 

extend this approach for head-fixed mice (Fig. 6), to leverage genetic advantages (particularly for imaging). 

2) Perceptual learning can take days, but the duration of whole-cell recordings is much shorter. We are one of 

the only labs combining whole-cell recordings with 2-photon in vivo imaging to examine long-term synaptic 

plasticity in behaving mice, relating AI synaptic and spiking changes to task performance in behaving animals

(Figs. 2-5). We refined methods for in vivo recordings from multiple cells over hours and days28,35,36,41,42,61, and 

use 2-photon imaging45,61 to monitor the same cells for weeks during behavior (Fig. 7) or image neuromodulatory 

axons within AI. This enables us to record and monitor behavioral changes as animals learn and task 

performance improves, and relate single-trial neural responses to behavioral performance in the same animals. 

3) Our task is parametric, enabling us to perform careful psychophysical studies. We can control consequences 

of nucleus basalis and locus coeruleus pairing, by over- or under-representing particular paired or unpaired 

sounds28,36. This allows us to ask more directly how the neurometric functions relate to psychophysical abilities. 

4) We are one of the only labs to record directly from modulatory neurons, including in vivo whole-cell recordings 

in locus coeruleus28. These experiments reveal that these neuromodulatory neurons rapidly develop sensory 

responses after pairing even at the subthreshold synaptic level, and govern auditory plasticity and perception. 

Figure 5. LC plasticity affects AI plasticity and learning. a, 

Recording schematic. b, In vivo whole-cell recordings from 

adult rat LC. Shown are membrane potential responses to 

paired tone. Top, tone-evoked responses increased from ~0.1 

mV (noise level) to ~1.0 mV. Inset, EPSPs. Bottom, APV (1 

mM in LC) prevents LC plasticity. c, Synaptic and spiking 

tuning shift duration is reduced to 1-3 hrs when LC plasticity is 

blocked w/ APV. d, Long-lasting behavioral changes are also 

prevented by APV infused into LC (c.f. Fig. 4c,e) (ref. 28).

Contact PD/PI: Froemke, Robert C

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C. Approach

Responses of central auditory neurons are changed by experience and training, but mechanisms and functional 

relations of synaptic plasticity to auditory perception are unclear. Here we will clarify how AI plasticity relates to 

enhanced auditory perception during training by in vivo imaging and recording in head-fixed behaving mice. Mice

offer many advantages including optogenetics and ease of intracellular recordings in awake animals. The overall 

goal is to gain insight into functional differences between the cholinergic and noradrenergic modulatory systems, 

and how behavioral context interacts with experience to change auditory circuits for improved perception. The 

central hypothesis is that perceptual training refines AI representations through plasticity of local inhibition (Aim 

1), driven initially by noradrenergic modulation before cholinergic tone increases to support task performance 

(Aim 2). Task variables also come to be represented by neuromodulatory neurons in order to appropriately 

regulate cortical modulation during initial learning, and then for continued reliable behavioral responses (Aim 3).

Aim 1. To determine how AI excitatory/inhibitory cells and synapses are modified by auditory training 

We will train adult mice on a go/no-go auditory task, to examine detection thresholds and sound recognition 

abilities. We will use in vivo 2-photon imaging (Subaim 1a) and whole-cell recordings (Subaim 1b) from AI 

neurons in behaving mice to determine how excitatory/inhibitory inputs and outputs are modified by learning. 

Subaim 1a. To determine the dynamics of AI excitatory and inhibitory cell responses during auditory training. 

Our first goal is to adapt our go/no-go auditory detection/recognition task for head-fixed mice. We will use 2-

photon imaging to monitor excitatory cells and inhibitory cell subclasses over the course of learning. 

Methods: We will train male and female adult C57Bl6 mice (~20-30 g) on a go/no-go auditory detectionrecognition task (Fig. 6a) where correct responses are licks after a target sound and withholding licking to other 

non-target/foil sounds. For some mice, tones-in-noise will be used, in other animals we will use FM sweeps. 

Stimuli will be 100-500 msec duration, 2-7 s inter-trial intervals. This design gives us a high level of control over 

task difficulty, varying signal-to-noise level or spectral similarity of targets vs foils. Mice will be water-restricted 

and trained in daily sessions of ~2 hrs each. Performance will 

be measured by hit, miss, correct withhold, and false alarm

rates, and number of trials/time to criterion. We will calculate 

learning rates, discrimination (d’), and bias of each animal. 

Successful learning will be animals that achieve a d’ of >1.75 

(Fig. 6b). In pilot studies, we have previously successfully 

trained animals on this task when the target sound is a pure 

tone. Results will be analyzed with t-tests; power analyses and 

past work28,36,61 indicate we need 5-10 mice per condition. We 

found this behavior requires auditory cortex; bilateral infusion 

of the GABA agonist muscimol reduced performance (Fig. 6c).

For imaging, we will inject AAV1-SYN-GCAMP6s into AI for 

GCaMP6s expression45,61,99. Before training, we will implant

headposts and cranial windows. Mice are acclimated to head 

fixation over 2 weeks for viral expression. We will perform 2-

photon imaging of Ca2+ signals from head-fixed mice during 

training (Fig. 7a-c). Our data (Fig. 7d) show we can image the 

same cells over days in behaving mice. Fluorescence changes 

are quantified relative to background (dF/F) for each cell, with 

strict criteria for motion correction and artifact exclusion45,61,100. 

For excitatory cell imaging, we use Thy1-GCaMP6 mice101. To 

selectively image interneurons, some C57Bl/6 mice are PVtdTomato, VIP-tdTomato, or SST-tdTomato45,61,102-104 (Fig. 8). 

A postdoc in the lab Kishore Kuchibhotla (key personnel) has 

extensive experience with 2-photon imaging45,61,105-107. Stimuli 

will be selected based on local AI frequencies. For tones, 

targets and foils will be within one octave; this will be starting 

frequency for FM sweeps with downsweeps for target, 

upsweeps for foil. In the animal shown in Figure 7a, median 

best frequency was 6 kHz; 8 kHz was chosen as target and 4.7 

kHz as foil (Fig. 7b). Background noise from the 2-photon is 

~40 dB SPL; stimuli are played at 70-80 dB SPL. 

Figure 6. Auditory task and training of headfixed mice. a, Mice lick to target sound for 

water reward. b, Training time; includes 

preparing mice for 2-photon imaging. Mice 

trained to criterion (d’ > 1.75) in 3-10 days. c, 

Task requires auditory cortex; bilateral 

muscimol infusion reduced d’ and hit rates.

Contact PD/PI: Froemke, Robert C

Research Strategy Page 43
Experiments: We will perform six studies in behaving mice to show how auditory training refines AI. 

1) We will image AI responses in untrained vs. fully-trained mice, comparing responses to targets and foils from 

the same cells during behavior (‘active context’) vs. passively during head-fixation without licktube (‘passive 

context’). We hypothesize that AI responses differ in trained mice in active vs passive contexts. We expect that 

after switching to the active context (simply by adding the licktube), most neurons will be suppressed, but a subensemble will have enhanced responses to targets and foils. This is supported by our pilot data: many cells

respond to the target in the passive context, but decreased responses in the active context (Fig. 8). This taskengaged suppression replicates recent studies of AI and other cortical areas104,108-113. We also observed a subnetwork of active-preferring cells encoding targets and foils after learning (Fig. 8). Context-dependent responses

were stimulus-specific and task-dependent, not due to reward expectation or delivery or from motor signals 

during licking, because of context-dependent changes to foil responses when trained mice do not lick. 

2) We will image longitudinally from the same cells in each mouse over the entire training duration (Fig. 7d). We 

hypothesize that we can observe construction of the ‘active ensemble’ over days. Specifically, many cells will be 

first enhanced during day 1 in the active context, but as mice improve in performance (and learn to suppress 

licking to foils), the majority of cells become suppressed (compare day 0 and days 8-9, Fig. 7d). 

3) We will image AI inhibitory cells, examining PV+, 

VIP+, and SST+ interneurons45,61,102-104 in different

mice during training. Our pilot data show that inhibitory 

cells are generally enhanced during behavior in trained 

mice (n=19/31 or 61% PV+, 12/20 or 60% SST+ cells

significantly increased activity, compared to only 8/36

or 22% VIP+ and 142/620 or 33% excitatory cells that 

increased activity; Fig. 8). We predict PV+ and SST+ 

cells are ‘first-responders’ when training begins.

4) We will examine how stimuli statistics affect AI 

responses and behavior. We will compare mice trained

to detect/recognize FM sweeps vs. tones, asking if this

produces differential dynamics by which excitatory or 

inhibitory cells are modified by training. After reaching

criterion, we will adjust stimuli to make the task more 

challenging (e.g., foils more spectrally similar to target). 

5) We showed that excitatory and inhibitory plasticity in 

mouse AI requires NMDA receptors44. We will ask if AI infusion of APV (NMDA receptor blocker, 0.1 mM) slows 

learning when given over days during training, or impairs performance when given to highly-trained animals. 

6) We will relate single-trial performance of AI network activity to behavior, asking if AI responses differ on correct 

vs error trials. We are collaborating with Ken Miller (see letter of support) to build a single-trial decoder based on

AI excitatory and inhibitory responses, both in terms of single neurons as well as population dynamics.

Figure 7. 2-photon imaging from AI during behavior. a, Example field-of-view of tone-evoked AI activity. Color,

best frequency of each cell. Note low-kHz tuning. b, Best frequency histogram of all cells for this mouse. 

Target was 8 kHz, foil 4.7 kHz. c, Behavior of 23 mice. d, Imaging from same cell over nine training days.

Figure 8. Context-dependent AI responses. Top,

imaging from all three cortical interneuron types. 

Bottom, % of cells activated during behavior in trained 

mice (purple), vs suppressed (blue).

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Research Strategy Page 44
Discussion/Alternatives: Many studies have highlighted state-dependent responses4,104,108-118, especially in AI.

Here we ask how such state-dependent processing emerges with training. To understand how AI plasticity

relates to auditory learning, it is vital to record from cell populations during behavior. Imaging is advantageous 

as we can track the same cells over days, clearly separating inhibitory from excitatory cells, and simultaneously 

image dozens of cells per trial. This enables us to try single-trial decoding at individual cell and network levels. 

If dF/F signals have poor temporal resolution for decoding, we will use deconvolution119 to predict single spikes. 

If learning rates in initially-naïve mice are slow, we will try ‘reversal learning’28, first training mice on one target 

before switching to target and foils represented in the imaging window. Our pilot data and past studies45,61,120-124

indicate that background noise from the microscope does not preclude imaging auditory-evoked responses, but 

if needed we will use insertable speakers and earplugs to reduce noise and effectively control stimuli. If changes 

to AI lag or are uncoupled from behavioral improvement, we will image from cortical ‘belt’ areas around AI125. 

Subaim 1b. To examine plasticity of AI excitatory and inhibitory synaptic inputs during auditory training.

Next we examine the synaptic basis for changes to AI spiking responses, by making whole-cell recordings in 

behaving mice. We hypothesize that inhibitory inputs are more context-dependent than excitatory inputs. There 

are two co-existing phenomena we will try to explain: that training leads to stable long-term enhancement of AI 

representations of task-relevant stimuli3-11, but also that during behavior, most AI cells are suppressed108-113. 

Methods: In mice from Subaim 1a, we will drill holes through the 

coverslip for whole-cell voltage-clamp recording during behavior. We 

will measure target- and foil-evoked excitatory/inhibitory postsynaptic 

currents (E/IPSCs) by holding cells at different potentials (Fig. 9a). 

To monitor synaptic changes over multiple cells and days, we take 

advantage of co-tuning of E/IPSCs, computing IPSC/EPSC ratios for 

single stimuli or E:I correlation over frequencies, reliable measures of 

hours-long changes in AI. Analysis will be with ANOVAs or MannWhitney tests. In vivo whole-cell recordings are lower-yield than 

imaging, but from power analyses and our past studies28,36,41,42,62, we 

expect to need ~30-50 cells per study, feasible in this project timeline. 

Experiments: We will perform four studies of AI synaptic changes. 

1) We will record from neurons over different days of training, asking 

how EPSCs and IPSCs are modified by auditory training. We can 

record from different cells in the same animal over days (Fig. 9b). In 

our pilot data, voltage-clamp recordings from trained mice during 

behavior showed that IPSCs were modulated by behavioral context 

more than EPSCs; some cells had stronger tone-evoked inhibition 

during the active behavioral context vs passive (Fig. 9a; ΔI/ΔE=2.1), 

and other cells have less inhibition relative to excitation in the active 

context. We predict that EPSCs and IPSCs evoked by targets and 

foils are first enhanced during behavior (via noradrenergic lowering 

of inhibitory tone, to be examined in Aim 2), together with increased 

hits and false alarms. However, behavioral improvement (by reducing

false alarms) over days is paralleled by AI inhibitory plasticity, leaving 

EPSCs generally intact but leading to learned context-dependent 

modulation of inhibition (via acetylcholine, also tested in Aim 2). 

Specifically, we hypothesize that most cells have higher IPSC/EPSC 

ratios during behavior following training, while a fraction (the active 

ensemble) will have much lower I/E ratios (Fig. 9c).

2) In some cells, we will first measure spiking in cell-attached mode 

before break-in. We use simulations to predict spikes from synaptic 

input41,42,62, relating synaptic to spiking changes in Subaim 1a.

3) We will make 2-photon targeted recordings from excitatory or

inhibitory cells (Fig. 9d), from cells modulated by context over learning. We predict decreased I/E ratios in PV+ 

and SST+ cells, increasing activity during behavior (Fig. 8), but VIP+ cells are suppressed due to high I/E ratios.

4) Imaging experiments in Subaim 1a necessarily focus on superficial layers. With cell-attached and whole-cell 

recordings, we will record from cells from each cortical layer, asking if cells across lamina show similar patterns 

of context-dependent modulation (in highly-trained animals) and are modified over learning at similar rates.

Figure 9. AI whole-cell recordings in 

behaving mice. a, Recording spikes & 

currents from same cell during active 

behavior vs passive state. b, Two cells 

recorded in same mouse on days 1, 7 

of training. c, Active context modulates 

IPSCs more than EPSCs. d, Targeted 

recording of SST+ interneuron in vivo.

Contact PD/PI: Froemke, Robert C

Research Strategy Page 45
Discussion/Alternatives: Here we monitor synaptic plasticity during auditory learning. Successful completion of 

these experiments will provide new data directly relating AI changes to learning and behavioral performance, in 

real time as both neural and behavioral changes occur. We will carefully examine synaptic and spiking responses 

during early phases of learning, asking if there are intracellular correlates of ‘eureka’ moments (e.g., bursts or 

NMDA spikes)10,126-132. If targeted recordings or recordings in mice for imaging in Subaim 1a are too low-yield, 

we will make blind whole-cell recordings, which we previously used to study synaptic plasticity in vivo28,36,41,42.

Aim 2. To ask how cholinergic and noradrenergic modulation affect auditory learning and AI plasticity

What mechanisms convey behavioral context to AI, and how are these mechanisms recruited during learning? 

We will record from AI during behavior, using optogenetics and pharmacology to ask if and when the cholinergic 

vs noradrenergic systems are necessary for learning and peak performance (Subaim 2a). We will ask if pairing 

task-relevant sounds with cholinergic and/or noradrenergic modulation accelerates learning or enhances peak 

performance (Subaim 2b). We will determine synaptic mechanisms by which cholinergic and noradrenergic 

modulation enable AI plasticity during learning. Our pilot data (Fig. 10) show that atropine impairs contextdependent modulation of behavioral and neural responses in highly-trained mice, motivating these studies.

Subaim 2a. To examine when noradrenergic and cholinergic modulation of AI is required for auditory behavior.

We will examine behavioral performance on the auditory task, using optogenetic and pharmacological methods 

to suppress noradrenergic or cholinergic neuromodulation at different phases of learning. 

Methods: We will virally express halorhodopsin in 

nucleus basalis of ChAT-Cre mice37,38,133 or locus 

coeruleus of TH-Cre or DBH-Cre mice134-136

, via 

stereotaxic injection of AAV1.EF1a.DIO.eNpHR3.0, 

and optical fibers implanted in these nuclei. We will 

inject/implant some wild-type mice. We will implant 

cannulas in some mice bilaterally in AI for local drug 

infusion. In some mice we image and record in vivo 

as in Subaims 1a,1b. These studies each require ~4-

8 mice and cells, analyzed with t-tests or ANOVAs.

Experiments: We perform seven studies to ask if/when modulation is necessary for learning. 

1) We will pharmacologically block noradrenergic receptors each day during training. We will first try α- and βreceptor blockers together. Previously we found that phentolamine (α-noradrenergic receptor antagonist, 0.1 

mM) prevented AI plasticity after locus coeruleus pairing28. We will also try propranolol (β-receptor antagonist) 

in a few mice. As α1 and α2 have opposite effects on excitability depending on site of action26,51,137, we will use 

progressively more selective receptor blockers to determine specific subtypes for learning and performing this 

task. We hypothesize that blocking noradrenergic receptors impairs performance only early in training rather 

than later. We may be able to assess blockade daily in the same mice, training them 2/day with a saline-infusion 

control session followed by drug to ask how noradrenergic blockade impacts behavior day-by-day. 

2) AI atropine reduces behavior in highly-trained mice (Fig. 10a,b). We will ask if atropine (to block muscarinic 

receptors) or nicotinic blockade slows learning rate, impairing performance later rather than earlier in training. If 

needed, drugs will be given on only one day; but as above, we will also try drug infusion every day per mouse.

3) We will pharmacologically block noradrenergic and cholinergic receptors together. After determining temporal 

sensitivity and receptor identity (muscarinic vs nicotinic, α- vs β –noradrenergic), we will ask if noradrenergic and 

cholinergic systems act together linearly, if there are periods early or late in training when these systems act 

together synergistically (as in past studies of ocular dominance plasticity47,85), or if with both systems blocked 

there is still some residual performance (e.g., possibly supported by dopaminergic-based reinforcement).

4) We will perform 2-photon imaging of excitatory and inhibitory cell populations. Informed by results from the 

previous experiments in wild-type mice, we will block noradrenergic or cholinergic receptors during training, to 

measure effects on specific inhibitory cell populations. We will ask if there is temporal sensitivity in inhibitory 

responses (e.g., interneurons of each type are affected more by noradrenergic blockade early in training, but 

more impacted by cholinergic blockade later in training), and/or if there is cell-type specificity (e.g., PV+ cells are 

more affected by cholinergic antagonists while SST+ cells more affected by noradrenergic antagonists). Some 

cell-attached and whole-cell recordings will be made to confirm imaging results at the intracellular level.

5) We will optogenetically suppress activity in locus coeruleus noradrenergic neurons during training. We predict 

that silencing locus coeruleus on the first day of training will completely prevent learning, as animals will 

recognize the presence of the context cue (i.e., licktube). Silencing on days 1-3 will also slow learning rates.

Figure 10. Atropine in AI during behavior. a, Setup. b, 

Reduced hit rate. c, No contextual modulation of IPSCs.

Contact PD/PI: Froemke, Robert C

Research Strategy Page 46
However, later in training we hypothesize that locus coeruleus inactivation is less deleterious. Specifically, we 

predict that as false alarm rate to foils begins to decrease, locus coeruleus norepinephrine is no longer needed.

6) We will optogenetically suppress activity in nucleus basalis cholinergic neurons. In contrast to locus coeruleus, 

we hypothesize that early in training (e.g., day 1) the cholinergic system is dispensable. However, the effects of 

optogenetic suppression will be greater with each subsequent day of training, especially for peak performance.

7) We will image AI cell populations during episodes of optogenetic silencing. We will compare optogenetic 

suppression to pharmacology, to control for potential issues related to lack of specificity or drug delivery in vivo. 

Discussion/Alternatives: We hypothesize that locus coeruleus controls the initial increase in performance from 

zero in untrained animals due to increased licking (hits and false alarms), while nucleus basalis is involved in AI 

inhibitory control, maintaining hit rate and reducing false alarms over days. Previous studies and our own 

work13,23-26,28,36-40,63-65 indicate that cholinergic and noradrenergic systems are important for sensory tasks such 

as the auditory behavior used here. However, there may be other mechanisms we need to consider, including 

nicotinic receptor control of auditory thalamocortical excitability91. We are using a reward-based paradigm, and 

so dopaminergic modulation also might be important as suggested by past studies75-77. If needed we will examine 

the VTA dopamine system, or modify our task to use aversive reinforcement as in other studies of AI4,138,139. We 

have successfully used 0.1 mM doses to block effects of nucleus basalis and locus coeruleus stimulation28,36, 

but if needed we will measure dose-response curves or cellular mechanisms in mouse AI brain slices as we 

have done previously43. There are many opto- and pharmacogenetic compounds now available; if halorhodopsin 

expression is poor or does not provide sufficient silencing, we will try archaerhodopsin or DREADD-based 

inactivations, as we have done previously58. We will perform calibration experiments in vivo (and in brain slices 

if needed) to ensure that optical stimulation effectively hyperpolarizes neurons to prevent them from firing.

Subaim 2b. To ask how norepinephrine and acetylcholine separately and synergistically affect learning rate. 

We will optogenetically stimulate the noradrenergic system of the locus coeruleus and/or the cholinergic system 

of the nucleus basalis, pairing optical release of endogenous neuromodulation with auditory stimuli. These 

studies directly extend our past work showing that neuromodulatory pairing leads to long-lasting improvements 

in rat auditory detection and recognition (Figs. 4,5); moreover, one episode of locus coeruleus pairing on the 

first day of stimulus reversal was found to accelerate learning rate and enhanced behavior days later (Fig. 11).

Methods: Methods are similar to Subaim 

2a, but we express channelrhodopsin228,42,43,140 ‘ChETA’ variant in nucleus 

basalis of ChAT-Cre or locus coeruleus 

of TH-Cre or DBH-Cre mice, via 

stereotaxic injection of pAAV5-Ef1aDIO-ChETA-EYFP. Some animals will 

have optical fibers implanted in nucleus 

basalis or locus coeruleus, other animals 

have fibers bilaterally implanted in AI. 

Experiments: We will perform six studies 

to determine how AI modulation can 

improve learning and affect behavior. 

1) We will pair target sounds with locus 

coeruleus stimulation during training. Some animals will be optically stimulated in locus coeruleus, other animals 

will receive optical stimulation only in AI; we expect results to be similar. Some animals will receive only a single 

episode of pairing (5 min, sound concurrent with 20 Hz optical stimulation; Fig. 11), other mice will receive 

stimulation on every training session. We hypothesize that learning will be accelerated by a single episode of 

pairing early in training; repetitive locus coeruleus stimulation may instead impair performance28,141. 

2) We will pair target sound presentation with nucleus basalis stimulation during training. As above, some animals 

will receive a single episode of pairing; other mice will receive daily pairing. We hypothesize that a single episode 

will not have lasting gains in performance; this is supported by our past studies of nucleus basalis pairing and 

auditory behavior in rats (Fig. 4b,d). However, we expect that multiple rounds of pairing will lead to lasting gains. 

3) We will pair targets with locus coeruleus and nucleus basalis stimulation together. We will express ChETA 

both in locus coeruleus and nucleus basalis in the same animals (with a non-floxed ChETA via synapsin 

promoter). Although we lose neurochemical specificity for one modulator, this provides information as to whether 

effects of pairing add linearly, are synergistic, or occlude each other depending on when pairing is performed.

Figure 11. One pairing episode (5 min on day 0) of optogenetic 

locus coeruleus stimulation accelerated reversal learning. a, Unpaired animal, switching target on day 0 from 4 kHz (arrowhead) to 

new 16 kHz target. b, Paired animal. c, Summary (ref. 28).

Contact PD/PI: Froemke, Robert C

Research Strategy Page 47
4) We will image and record during training and optogenetic stimulation of the locus coeruleus and/or nucleus 

basalis. This can be done in many of the same animals used above in experiments 1-3. We have two goals herefirst, to see if optogenetic stimulation accelerates or disrupts changes in IPSC/ESPC ratios expected to emerge 

over training in these stimulated animals. Second, to record during pairing target sounds with nucleus basalis 

and/or locus coeruleus stimulation, to ask how optogenetic neuromodulatory release affects EPSCs, IPSCs, and 

spiking in excitatory or inhibitory cells in awake animals. As we hypothesize that modulatory effects will be similar 

as we previously described under anesthesia (Fig. 2), this reduces the total number of experiments required.

5) We will pair non-targets with locus coeruleus or nucleus 

basalis stimulation. We predict that off-target pairing with 

locus coeruleus stimulation impairs performance, while 

pairing foils with nucleus basalis stimulation may instead

accelerate learning (as also predicted for target pairing).

6) We will examine effects of unpaired stimulation, asking 

if activation of either modulator system can lead to 

behavioral responses outside of behavioral context (i.e., 

when stimuli are passively presented in absence of

licktube). Our data show that highly-trained mice lick after 

nucleus basalis stimulation (Fig. 12).

Discussion/Alternatives: While studies of Subaim 2a ask about the necessity of neuromodulation for perceptual 

learning and task performance, here we ask if stimulation of these systems can enhance learning or suffice to 

provide behavioral context. If viral expression is problematic, we can try using ChAT-ChR2 mice, expressing 

channelrhodposin-2 in cholinergic neurons. While these animals are reported to have some behavioral 

problems142, we found on our task they perform similar to wild-types (Fig. 12). Past studies have found good 

correspondence between AI plasticity induced by pairing sounds (generally pure tones) with neuromodulator 

agonists and stimulation (generally electrical) of modulatory centers1,2,10,28,35,42,43,138. If initial learning is 

complicated by procedural aspects of the task (learning to head-fix, lick for reward) and confounds these studies, 

we will study reversal learning in trained mice, with tone and foil stimuli reversed as we did previously (Fig. 11). 

Our goal is to provide mechanistic insights for changes 

in behavioral performance, as related to AI excitatory 

and inhibitory inputs (via voltage-clamp recordings) and 

spiking output (via 2-photon imaging and cell-attached 

recording). Our data show that muscarine in mouse AI 

depolarizes all cells, but inhibitory cells more than 

excitatory cells (Fig. 13). Consistent with work from 

other labs33,138,143, neuromodulation depolarizes cortical

interneurons to increase spontaneous GABA release 

and IPSC frequency, depleting the available pool of 

GABA available. Alternatively, modulation might 

decrease transmitter release directly144. In either case, stimulus-evoked IPSC amplitude is reduced (Fig. 2).

These interactions between multiple cell types lead to complex AI network dynamics on single trials. We are 

collaborating with Ken Miller (see letter of support) to build network models that describe these dynamics in 

terms of the lower-level cellular responses. We are also collaborating with Susan Sara, world expert on locus 

coeruleus, cortical responses, and behavioral control; Dr. Sara has an office at NYU and has been helping us 

target locus coeruleus for recordings and injections (see letter of support), to help ensure success of this project.

Aim 3. To examine when sensory stimuli and behavioral context activate modulatory systems

What aspects of training and behavioral context activate modulatory systems and promote AI plasticity? We will 

ask if there is a sequence of AI modulation over task learning; specifically if the noradrenergic system is recruited 

transiently during the first day, while the cholinergic system comes on-line over subsequent days and is needed 

for steady-state performance. We will record from locus coeruleus and nucleus basalis, and perform 2-photon 

imaging of modulatory axons within AI in trained animals and over the duration of learning. Recently we found 

that tone-evoked responses with short latency are induced in locus coeruleus neurons after pairing tones with 

locus coeruleus stimulation28. Here we ask if plasticity in nucleus basalis and/or locus coeruleus is important for 

learning or task performance. We test the hypothesis that the noradrenergic system is initially active early in 

training (e.g., on day 1), while cholinergic activity increases gradually over days in parallel with performance. We 

note that the studies in Aim 3 do not depend strongly on particular findings or details of studies in Aims 1 or 2. 

Figure 12. Unpaired nucleus basalis stimulation 

in trained mice can induce licking without licktube 

(behavioral context) if tone is passively played.

Figure 13. Muscarine depolarizes AI interneurons 

significantly more than AI excitatory cells.

Contact PD/PI: Froemke, Robert C

Research Strategy Page 48
Methods and Experiments: We will use five approaches to measure the responses of modulatory neurons in 

behaving mice. As shown by our preliminary data (Figs. 5,14,15), each of these techniques- in vivo axonal 

imaging, op-tetrode recording, fiber photometry, and whole-cell recordings- are working in the lab. 

1a) We will image cholinergic axons from nucleus basalis projecting within AI during behavior. 2-photon imaging 

will be performed as in Subaim 1a, but we will express GCaMP6s in cholinergic neurons via stereotaxic nucleus 

basalis injection of AAV1-SYN-GCAMP6s in ChAT-Cre mice. After ~2 weeks for expression, we will image Ca2+

signals in cholinergic axons in AI. Our pilot data (Fig. 14a-c) and previous results demonstrate feasiblility61,145; 

we will be careful to correct for motion artifacts. We will play tones and FM sweeps of varying frequencies and 

intensities, measuring axonal Ca2+ signals both during auditory training and passive presentation. Our pilot 

results show that in highly-trained animals, cholinergic axons are much more responsive during correct trials 

than during passive presentation (Fig. 14a-c). We will monitor the same axons over the course of training to 

determine when responses occur, and if axonal responses are also evoked by task variables such as licktube 

insertion. We hypothesize that cholinergic axon activation gradually increases over learning and is required for 

successful task performance in experienced animals. For these studies we expect to need ~5-8 animals.

1b) We will image from locus coeruleus 

noradrenergic axons in AI, injecting AAV for 

GCaMP6s expression in TH-Cre or DBH-Cre

mice. Our pilot data in a naïve untrained 

animal shows large locus coeruleus axon 

responses in AI to surprising sounds (Fig. 

14d); we expect these responses to diminish 

with exposure or experience. 

1c) We will perform simultaneous imaging of 

cholinergic and noradrenergic axons within AI

of the same animal during behavior (Fig. 14e), 

to directly compare responses of modulatory 

fibers over days as mice learn the task.

GCaMP6s will be expressed in one area, the 

red-shifted indicator RCaMP146 in the other.

2) We will use multi-optrode array recording, 

implanting electrodes with fibers for optical

stimulation147 in nucleus basalis of ChAT-Cre

or ChAT-ChR2 mice with channelrhodopsin-2 

in cholinergic neurons. In other TH-Cre or 

DBH-Cre mice we will implant op-tetrodes in 

locus coeruleus. We will identify specific 

modulatory cells by optical stimulation148; and 

analysis done on light-sensitive units (Fig. 

14f), which are cholinergic or noradrenergic

cells tagged by opsin expression. Histological 

methods will verify ChETA expression and electrode placement. We will compare local modulatory signals in AI 

(measured axonally in studies 1a-c) with activation of more global signals by recording from cell bodies. We will 

ask if modulatory neurons respond to task variables, and if stimulus specificity changes during auditory training. 

3) We will perform fiber photometry in nucleus basalis or locus coeruleus of transgenic mice expressing 

GCaMP6s only in modulatory neurons. Fiber photometry149 is essentially an ‘optical LFP’, measuring bulk 

GCaMP6s signal representing integrated firing of GCaMP6s-expressing cells. Although this decreases our 

temporal resolution somewhat and we lose single-unit specificity, photometry signals are straightforward to 

measure and quite stable. We will compare photometry signals (Fig. 14g) to axonal responses in AI (experiment 

1) and single-units in experiment 2. If modulatory projections to AI are topographic and it proves challenging to 

obtain single-unit tetrode recordings from neurons activated during behavior, photometry may provide suitable 

data on the bulk response of cholinergic vs noradrenergic cells to behavioral context early vs late in training.

4) We will perform in vivo whole-cell recordings from nucleus basalis or locus coeruleus neurons. We have 

successfully obtained whole-cell recordings from rat locus coeruleus in vivo (Fig. 5a,b). In our previous study, 

we found that about half of all locus coeruleus cells developed auditory responses after conditioning or pairing28. 

Whole-cell pipettes contain biocytin and we will recover cells histologically, co-staining for TH or ChAT to 

Figure 14. Imaging and recording from modulatory neurons. 

a-c, In vivo 2-photon imaging of GCaMP6s in cholinergic 

axons of behaving mouse AI. Axonal responses are strongly 

enhanced by behavioral context. d, In vivo imaging of LC

axons within AI; bottom, sound-evoked Ca2+ responses. 

e, Two-color imaging in AI with RCaMP in LC and GCaMP6s 

in NB. f, Multi-tetrode recording in vivo from optically-tagged 

LC noradrenergic units. g, In vivo fiber photometry in LC.

Contact PD/PI: Froemke, Robert C

Research Strategy Page 49
Contact PD/Pl: Froemke, Robert C 

detennine cell identity (Fig. 15). While it will be challenging to find 

responsive nucleus basalis cells, this is a 'high-risk, high-reward' 

experiment, not critical to other goals of this project, and even a few 

cells (~4-5 required for statistical power) may be informative. We are 

routinely able to obtain ~5-10 recordings per session, including cellattached recordings, allowing us to screen many cells. 

Figure 15. lmmunohistochemistry: LC 5) Finally, we aim to determine if these changes in modulatory neural 

whole-cell recordin and bioc tin fill. responses are due to local plasticity in subcortical areas, and if 

preventing this plasticity affects learning. Modulatory neurons are activated by task context, and develop 

24 28 responses to conditioned stimuli after training23• • . Emergence of auditory responses in nucleus basalis or locus 

coeruleus (with short latency and present even under anesthesia; Fig. 5) are potentially essential components

by which previously-innocuous stimuli develop meaning, and thus it is critical to understand the mechanisms and 

behavioral relevance of these changes for auditory learning. Here we perform two studies, asking first if infusion 

of APV (to block NMDA receptors) or modulatory receptor antagonists prevent auditory responses or responses 

to context cues (e.g., licktube) from arising in these nuclei. We will then infuse these blockers into either locus 

coeruleus or nucleus basalis on the first day of training (in some animals) or each day of training (in other animals) 

to detennine how behavioral performance and learning is impacted when modulatory plasticity is blocked. 

Discussion/Alternatives: We aim to obtain a multi-dimensional view of how behavioral context and task variables 

differentially engage cholinergic vs. noradrenergic modulatory systems. This entails examining subthreshold vs 

suprathreshold responses, and local vs more global signals over training. We hypothesize that the presence of 

the licktube (which signals active behavioral context) initially activates locus coeruleus. before these neurons 

begin responding to task-relevant sounds (both targets and foils; Fig. 14d,g). As performance improves. locus 

coeruleus activation sharply declines. and instead cholinergic neurons begin responding (shown by our 

preliminary data in Fig.14a-c). We will examine the behavioral importance of the emergence of these responses. 

Our past work (Fig. 5) showed that NMDA receptors were critical for this plasticity, but we will examine other 

candidate mechanisms (e.g., modulatory autoreceptors) if necessary. In future studies beyond the scope here,

we will map the functional anatomy that provides potential auditory information to modulatory centers. 

These are technically-challenging methods, but we have experience and success with each approach. Past 

studies showed that surprising events or contextual cues increase cholinergic or noradrenergic tone, often 

measured with amperometry in rodent and primate cortex64•

65•150. However, this is indirect with low spatiotemporal 

resolution, making it difficult to relate degree of modulatory activation with single-trial neural or behavioral 

responses. Instead, here we take advantage of mouse lines for modulatory control, for optical identification of 

cell-types (Fig. 14). This is important, as locus coeruleus to some degree and nucleus basalis to a potentially 

39 65 high degree are neurochemically heterogeneous29• • •. In vivo whole-cell recordings might reveal if even in na'ive 

(untrained) animals. these neurons receive sensory-evoked synaptic inputs. but these inputs might be 

subthreshold or masked by inhibition to keep modulatory cells from spiking. It may be technically infeasible to 

make in vivo whole-cell recordings from cholinergic cells given their sparsity; we and others have already made 

151 in vivo whole-cell recordings from locus coeruleus noradrenergic neurons28• . Conversely, it may be difficult to 

perform tetrode recording from locus coeruleus. For these reasons we use multiple methods to ask the same 

questions, and provide new data on spatial and temporal patterns of Al neuromodulation during auditory training. 

Summary: Neuromodulation is important for auditory plasticity and perceptual learning. However, it is unclear 

how two of the main modulators involved in these processes- acetylcholine and norepinephrine- are naturally 

recruited during auditory training to differentially modulate target circuits. Our studies will provide new insight into 

these mechanisms, reveal how cortical plasticity is involved in auditory behavior, and suggest how modulatory 

systems are activated and modified by training for effective control of auditory perception. In Aim 1, we ask how 

auditory training relates to Al plasticity measured in behaving mice. We perform in vivo whole-cell recordings 

and imaging to determine dynamics of response changes. In Aim 2, we examine distinctions between cholinergic 

and noradrenergic modulation at different phases of learning. In Aim 3, we record from these systems and ask 

if they are plastic, relating auditory responses in modulatory axons and neurons to auditory perceptual learning. 

AIMS YR 1 YR 2 YR 3 YR 4 YR 5 Feasibility: All Aims are feasible 

1 a. Al ima in durin behavior KK,EM,RCF in 5 rs. 

1 b. In vivo whole-cell recordings KK,NL,RCF

2a. Necessity of Al neuromod KK,NL,RCF

2b. Does Al mod aid learning? KK,NL

3. Plasticity of neuromodulation KK,NL, EM,RCF 

Research Strategy Page 50 
Progress Report Publication List

For the original R01 proposal, we published several papers related to rodent auditory perception and cortical 

plasticity, including one major study (Martins and Froemke, Nature Neuroscience 2015) that constitutes Aim 1a, 

1b, and almost all of the proposed studies in Aims 2 and 3. We are examining loci of plasticity both in vitro and 

in vivo (as relates to Aim 1c). We have two manuscripts that were favorably reviewed, now being revised for 

resubmission, involving recordings in rat primary auditory cortex, frontal cortex, and locus coeruleus during the 

auditory behavior used in this application (Carcea et al., Nature Communications under revisions; Insanally et 

al., Nature under revisions). 

Froemke RC, Carcea I, Barker AJ, Yuan K, Seybold B, Martins ARO, Zaika N, Bernstein H, Wachs M, Levis PA, 

Polley DB, Merzenich MM, Schreiner CE. Long-term modification of cortical synapses improves sensory 

perception. Nature Neuroscience 2013; 16:79-88. PMC: 3711827

Carcea I, Froemke RC. Cortical plasticity, excitatory-inhibitory balance, and sensory perception. Progress in 

Brain Research 2013; 207, 65-90. PMID: 24309251 PMC: 4300113

Martins ARO, Froemke RC. Coordinated forms of noradrenergic plasticity in the locus coeruleus and primary 

auditory cortex. Nature Neuroscience 2015; 18:1483-1492. PMID:26301326 PMC: 4583810

Marlin BJ, Mitre M, D'amour JA, Chao MV, Froemke RC. Oxytocin enables maternal behaviour by balancing 

cortical inhibition. Nature 2015; 520:499-504. PMID: 25874674 PMC: 4409554

D'amour JA, Froemke RC. Inhibitory and excitatory spike-timing-dependent plasticity in the auditory cortex. 

Neuron 2015; 86; 514-528. PMID: 25843405 PMC: 4409545

Ponte Costa R, Froemke RC, Sjöström PJ, van Rossum MC. Unified pre- and postsynaptic long-term plasticity 

enables reliable and flexible learning. eLife 2015; 4. PMID: 26308579 PMC: 4584257

Froemke RC. Plasticity of cortical excitatory-inhibitory balance. Annual Review of Neuroscience 2015; 38, 195-

219. PMID: 25897875 PMC: 4652600

Froemke RC, Schreiner CE. Synaptic plasticity as a cortical coding scheme. Current Opinion in Neurobiology 

2015; 35, 185-199. PMID: 26497430 PMC: 4641776

King J, Insanally M, Jin M, Martins ARO, D'amour JA, Froemke RC. Rodent auditory perception: critical band 

limitations and plasticity. Neuroscience 2015; 296, 55-65. PMID:25827498 PMC: 4426073

Mitre M, Marlin BJ, Schiavo JK, Morina E, Norden S, Hackett TA, Aoki C, Chao MV, Froemke RC. A distributed 

network for social cognition enriched for oxytocin receptors. Journal of Neuroscience 2016; 36:2517-2535. PMID: 

26911697 PMC: 4764667

Insanally M, Trumpis M, Wang C, Chiang CH, Woods V, Bossi S, Froemke RC, Viventi J. A low-cost, multiplexed 

μECoG system for long-term, reliable high-density recordings in rodents. Journal of Neural Engineering 2016; 

13:026030. PMID: 26975462, PMCID: in process.

King J, Shehu I, Roland JT, Svirsky MA, Froemke RC. A physiological and behavioral system for hearing 

restoration with cochlear implants. Journal of Neurophysiology 2016; in press.

Cohen S, Ma H, Kuchibhotla K, Watson BO, Buzsáki G, Froemke RC, Tsien RW. Excitation-transcription 

coupling in parvalbumin-positive interneurons employs a novel CaM Kinase-dependent pathway distinct from 

excitatory neurons. Neuron 2016; 90:292-307. PMID: 27041500 PMC: 4866871

Contact PD/PI: Froemke, Robert C

Progress Report Publication List Page 51
Animal Subjects Justification

1. Proposed Use of Animals: All experimental procedures used in this study are currently approved under an 

NYU School of Medicine IACUC protocol: "Synaptic Plasticity in Rodents", protocol number 160611-01, 

approved on 6/13/2016. 

Mice: Experiments in mice are necessary for these proposed studies. For most experiments, we will use adult 

male and female C57Bl/6 mice 6-12 weeks old and weighing 20-30 g. We have verified that our animals hear at 

these ages, and our experiments are not compromised by hearing loss. Some animals will be ChAT-Cre mice 

expressing Cre recombinase in cholinergic neurons (ChAT: choline acetyltransferase), other animals will be THCre or DBH-Cre mice expressing Cre recombinase in noradrenergic neurons (TH: tyrosine hydroxylase; DBH: 

dopamine-β-hydroxylase). In a few experiments we will use ChAT-ChR2 mice expressing channelrhodopsin-2 

in cholinergic neurons. For imaging inhibitory neurons in PV-tdTomato, SST-tdTomato, and VIP-tdTomato mice, 

PV-Cre, SST-Cre and VIP-Cre animals have been crossed with the Ai9 tdTomato-flox reporter line (Allen 

Institute). We will also use Thy1-GCaMP6 mice for imaging excitatory neurons. As shown by our preliminary 

data, we have already obtained all of these mouse lines and are actively maintaining breeding colonies at NYU.

For optogenetic control of cholinergic or noradrenergic neurons, we will perform stereotaxic viral 

injections in ChAT-Cre, TH-Cre, or DBH-Cre mice, similar to our previous studies in mice and rats28,42,43,45,58-61. 

Mice will be anesthetized with 0.7-2.5% isoflurane, placed into a stereotaxic apparatus, and a craniotomy 

performed over nucleus basalis (from bregma: 0.5 mm posterior, 1.85 mm lateral, 4.5 mm ventral) or locus 

coeruleus (from bregma: 5.5 mm posterior, 0.85 mm lateral, 3.65 mm ventral). Injections will be performed with 

a 5 μL Hamilton syringe and a 33 gauge needle. Cre-inducible pAAV5-EF1α:-DIO-ChETA-EYFP virus53 will be 

injected at 0.1 nl/s for a final injection volume of 1.2-1.5 μl. An optical fiber ferrule will be implanted either 

subcortically or in electrophysiologically-identified AI (100 μm ventral). Some wild-type animals will be cannulated 

in a similar manner for drug delivery. The craniotomy and implant will be sealed with dental cement, and the 

virus given a minimum of two weeks to express.

Overall, we plan on using approximately ~295-540 animals for the experiments described in this 

proposal (see Section 2 below for breakdown of animal use by specific Aims).

Behavioral training: All behavioral events (imaging time-stamping, stimulus delivery, water delivery and lick 

detection) will be monitored and controlled by custom-written programs in MATLAB interfacing with an RZ6 

auditory processor (Tucker-Davis Technologies). After at least 7 days of water restriction, training would 

commence. Training takes place during the day and begins with habituation to head-fixation, immediately 

followed by water-sampling sessions. Mice typically learn to lick for 3-5 μl droplets of water within 1-2 sessions. 

Once the animals lick reliably, they are placed in the complete behavioral task with minimal shaping. We utilize 

a go/no-go behavior task with the target and foil chosen based on the two-photon targeted imaging. A behavioral 

block typically consists of 100 trials and with 4 blocks of training per day (total of ~400 trials per day). Target 

versus foil trials were pseudo-randomly ordered, each of which consisted of scan onset (imaging), pre-stimulus 

period (1.25 s), stimulus period (100-500 ms), delay (250 ms), a response period (1.75 s), and an inter-trial 

interval (ITI with variable duration as described below). Mice only receive water for correct responses to the 

target during the response period. Incorrect licks after the foil presentation (a false alarm) are mildly punished 

by increasing the ITI. Animals are not punished if they licked during any other time epoch (i.e., if animal licks in 

the pre-stimulus period, tone presentation or delay period). This enables us to confirm that animals actively 

increase lick rate for targets during hit trials and reduce lick rate for foil tones during correct rejects (from baseline 

lick rate to zero during response window). This measurement confirms that both the target and foil tone have

behavioral effects on the animal; without this, animals could take a single-tone strategy (i.e., learn to lick only for 

the target tone or withhold licking for the foil tone). 

Hit trial ITIs are 4-5 s (to enable full licking of reward), miss trials are not punished and have an ITI of 2-

3 s, false alarm trials are punished with an ITI lasting 7-9 s, and correct rejects immediately moved to the next 

trial with an ITI of 2-3 s. Lick tube removal is automated using a micromanipulator and licks are detected with an 

infrared beam. Licking is also monitored using an infrared camera or beam to determine whether presentation 

of sound stimuli elicited a licking movement from the animal. We have confirmed that the animals are still thirsty 

immediately after training ends, i.e., the animals would lick for ad-libitum access to water. This confirms that any 

changes in neuronal responses were not due to “satiety” indicating the animal was no longer thirsty.

For pharmacological experiments, animals will be injected bilaterally with either saline vehicle or receptor 

antagonists (1 μL of 1 mg/mL) at a depth of ~600 μm below the pial surface. The drug will be allowed to diffuse 

while the animals were headfixed for 10-15 minutes before behavior was initiated. 

Contact PD/PI: Froemke, Robert C

Vertebrate Animals Page 52
Two-photon imaging: Imaging will be performed as in our recent studies45,61. Mice will be prepared for cranial 

window implantation by isoflurane anesthesia (2.0% during induction and surgery, 0.75% during physiology). A 

craniotomy will be performed over the temporal lobe and the auditory cortex exposed (3 mm craniotomy, 

centered 1.75 mm anterior to the lambda suture on the ridge line). An adeno-associated virus (AAV) vector 

encoding the calcium indicator GCaMP6s (AAV1-SYN-GCAMP6s, U. Penn Vector Core) will injected for 

expression in AI layer 2/3 neurons. Above the injection coordinates, a cranial window will be implanted replacing 

a circular piece of skull by a glass coverslip (diameter of 3 mm, Warner Instruments) secured in place using a 

mix of dental cement and Krazy glue. A custom-made stainless-steel headpost (Ponoko) will be affixed to the 

skull using C&B Metabond dental cement (Parkell). A small burr hole will be drilled anterior to bregma and a 

ground wire placed under the skull above the pial surface. Each animal is allowed to recover for at least 2-3 

weeks, and then habituated to head-fixation for ~1 week. For experiments imaging the activity of axonal 

projections, we will carefully level the head and drill a small burr hole targeting nucleus basalis or locus coeruleus. 

We will inject 1.5 μl of AAV1-SYN-FLEX-GCAMP6s (U. Penn vector core) into ChAT-Cre mice to specifically 

target cholinergic neurons or into TH-Cre or Dbh-Cre mice to target noradrenergic neurons. We will inject at a 

rate of 100 nl/min and wait 15 minutes before extracting the injection needle. We will fill the burr hole with bone 

wax and seal it with dental cement.

For imaging, 2‐photon fluorescence of GCaMP6s will be excited at 900 nm using a mode-locked 

Ti:Sapphire laser (MaiTai, Spectra‐Physics, Mountain View, CA) and detected in the green channel (GFP 

emission). Imaging will be performed on a multiphoton imaging system (Moveable Objective Microscope, Sutter 

Instruments) equipped with a water immersion objective (20X, NA=0.95, Olympus) and the emission path 

shielded from external light contamination. Images will be collected using ScanImage (HHMI). To image auditory 

cortex, the objective is tilted to an angle of 50-60 ̊. To record time courses of auditory-evoked neuronal activity, 

awake animals are head-fixed under the microscope and a speaker placed adjacent to the microscope 

(microphone-ear distance ~10 cm). During auditory stimulation, ~300 μm2 areas in layer 2/3 of A1 containing 

multiple GCaMP6s expressing neurons will be selected and imaged (scan rate ~4 Hz, 0.26 s/frames, laser power 

≤40 mW). 

For motion artifacts, we use a multi-step approach to compensate for x-y motion due to movements of 

the awake mouse. In brief, we perform a frame-by-frame, rigid registration of the imaging frames that accounts 

for x-y changes in displacement, rotation and translation. In animals with a structural marker (tdTomato in PV+, 

SST+, and VIP+ interneurons) we separate the functional channel (green, GCaMP6s) from the structural channel 

(red, tdTomato). Negative deflections in the fluorescent traces are assumed to be due to motion out of the zplane. To remain conservative in our data inclusion criteria, any imaging planes that exhibited greater than 5% 

of cells with statistically-defined negative deflections (pairwise t-test of baseline versus negative peak) will be 

excluded from the analysis. At all stages of motion correction process, manual inspection is performed to identify 

motion out-of-the-plane that we are unable to correct by the above approach. In these rare cases, these data 

will be excluded from further analysis.

For auditory stimulation, we will use pure tones (4-96 kHz at 0.5-1 octave intervals, 100-500 ms duration, 

3 ms cosine ramped onsets, 10-80 dB SPL intensity, 0.5-1.0 Hz rate) or FM sweeps (upsweeps from 4-96 kHz 

or downsweeps from 96-4 kHz, at rates of 1,2,4,8,16,32, and 64 octaves/s, 500 msec in duration), in background 

white noise at a level of 40 dB SPL. Stimuli will be presented in pseudo-random sequence delivered by a 

calibrated speaker. These procedures and stimuli are similar to our published studies28,36,41-43,45,61,62. 

In vivo whole-cell recordings: In vivo whole-cell recordings will be performed in awake, head-fixed mice in a 

sound-attenuating chamber, and recordings obtained from AI neurons with a Multiclamp 700B amplifier 

(Molecular Devices). For voltage-clamp recordings, pipettes will contain: 130 Cs-methanesulfonate, 1 QX-314, 

4 TEA-Cl, 0.5 BAPTA, 4 MgATP, 20 phosphocreatine, 10 HEPES, pH 7.2. Data will be filtered at 5 kHz, digitized 

at 20 kHz, and analyzed with Clampfit 10 (Molecular Devices). Recordings will be excluded from analysis if Rs 

or Ri changes >30% relative to baseline. For 2-photon targeted recordings, we will utilize a preparation which 

enables access to auditory cortex via a ~200 μm hole in a 3 mm piece of round glass, providing the stability 

required to hold cells across conditions. 

For release of endogenous neuromodulators via optogenetic stimulation, blue light will be delivered to 

the brain surface via a 200 μm fiber (Thorlabs) positioned ~0.5 mm above the surface, coupled to a 473 nm laser 

(CrystaLaser). During whole-cell recordings, blue light pulses will be triggered 100 ms before stimulus onset and 

last for 1 s (473 nm wavelength, 2-2.5 mW) at the surface of the brain. Changes in synaptic responses will be 

compared by Student's paired two-tailed t-tests assuming data passes Kolmogorov-Smirnov normality tests.

Contact PD/PI: Froemke, Robert C

Vertebrate Animals Page 53
Chronic in vivo multi-electrode recordings: Mice will be implanted with microdrive arrays (Versadrive-8 

Neuralynx) in either nucleus basalis or locus coeruleus. For surgery, animals will be anesthetized with ketamine 

(40 mg/kg) and dexmedetomidine (0.125 mg/kg). A craniotomy will be performed, and the location of primary 

auditory cortex will first be determined by mapping multiunit responses 500-700 μm below the surface using 

tungsten electrodes in a sound-attenuating chamber. Stainless steel screws and dental cement will be used to 

secure the microdrive to the skull, and one screw used as ground. Each drive consists of 8 independently 

adjustable tetrodes. Tetrodes are made by twisting and fusing four polyimide-coated nichrome wires (Sandvik 

Kanthal; wire diameter 12.5 μm). The tip of each tetrode will be gold-plated to an impedance of 200-500 kOhms 

at 1 kHz (NanoZ, Neuralynx). After animal recover from surgery (~7 days), recordings will begin after testing 

baseline retrieval abilities. Animals will be monitored daily by NYU veterinary staff and the member of my lab 

who performed the surgery, with analgesics and antibiotics provided as necessary.

Tetrodes will be advanced ~60 μm 12 hours prior to each recording session. For recording, signals are 

first amplified onboard using a small 16-bit unity-gain preamplifier array (CerePlex M, Blackrock Microsystems) 

before reaching the acquisition system. Spikes are sampled at 30 kS/sec and bandpass filtered between 250 Hz 

and 5 kHz. Data are digitized and all above-threshold events with signal to noise ratios > 3:1 stored for offline 

spike sorting. Single units will be identified on each tetrode using OfflineSorter (Plexon Inc.) by manually 

classifying spikes projected as points in 2D or 3D feature space. The parameters used for sorting include the 

waveform projection onto the first two principal components, energy, and nonlinear energy. Artifacts are rejected 

based on refractory period violations (< 1 msec). Clustering quality will be assessed based on the Isolation 

Distance and Lratio sorting quality metrics. Spontaneous average firing rate will be established by averaging 

spikes in a 500 msec time window immediately prior to stimulus onset on each trial. To quantify stimulus-evoked 

responses, we will calculate z-scores of changes in spike count during stimulus presentation: z= μ/σ, where μ 

is the mean change in spike count and σ is the standard deviation of the change in spike count. 

Histology: We will perform immunohistochemistry to confirm specific expression of transgenes. Mice will be

transcardially perfused using ~45 mL PBS followed by ~ 45 mL 4% PFA under deep anesthesia. Brains are 

extracted from the skull and post-fixed in 4% PFA for ~2 hours at 4o C, then cryoprotected overnight in 30% 

sucrose solution and sliced coronally with 16 μm thickness on a cryostat. Fixed brain sections are washed with 

PBS, then incubated for 2-3 hours at room temperature in blocking buffer containing PBS with 0.5% v/v Triton 

X-100 (Sigma), 2% v/v normal goat serum (Life Technologies) and 50 mg/mL BSA. The blocking solution will be

aspirated and slices incubated in primary antibody (1:1000 dilution of chicken anti-GFP IgY, catalog # ab13970 

(Abcam); 1:200 rabbit anti-ChAT IgG, cat. # AB143 (Millipore); 1:1000 dilution of VIP antibody IgG, product. 

#20077 (Immunostar); 1:1000 dilution of anti-parvalbumin IgG, code #235 (Swant); 1:500 dilution of antisomatostatin IgG, clone YC7, cat. #MAB354 (Millipore)) overnight at 4oC. Afterwards, slices are washed and 

incubated for 1-2 hours at room temperature in secondary antibody (1:1000 dilution, goat anti-chicken IgY Alexa 

Fluor 488, cat. # A11039; goat anti-rabbit IgG Alexa Fluor 555, cat. # A21430, both from Life Technologies; 

1:1000 dilution of goat anti-mouse IgG Alexa Fluor 555, cat. #A21422 (Life Technologies); 1:500 dilution of goat 

anti-rat IgG, Alexa Fluor 555, cat. #A21434 (Life Technologies)). Finally, slides are washed, incubated at room 

temperature in DAPI/Hoechst solution (1:1000 in PBS), washed, then coverslipped using Fluoromount (Sigma). 

Images are acquired with a 20x air objective on a Zeiss LSM 710 Confocal Microscope (Carl Zeiss Inc.) and cell 

counts were performed in Image J (NIH).

2. Animal Use Justification: The ultimate goal of these experiments is a deep understanding of how the brain 

responds and changes during auditory learning, due to acoustic input and cholinergic/noradrenergic modulation 

interacting within auditory cortex. However, our knowledge of these processes is rather limited, and thus 

research involving animal tissue is unfortunately still necessary. As we are investigating the effects of direct 

sensory experience and the modulation of local networks of excitatory and inhibitory neurons by subcortical 

attentional modulators, cell culture systems cannot be used. Computer simulations and mathematical models 

will be developed in parallel with our experiments, to help inform future directions and limit the overall number of 

animal studies that are required. Our collaborative studies with other labs- Claudia Clopath, Tim Vogels, Ken 

Miller, Mark von Rossum, Jason Hunzinger, and others- attest to these efforts. We also aim to develop more 

powerful methods for studying these important issues in progressively more efficient animal model experiments. 

We will work to improve the efficiency and effectiveness of experimental methods to still further reduce the 

numbers of animal experiments that must be conducted.

These experiments will be conducted in mice, which are ideal experimental animals in which to study the 

circuitry and plasticity of the auditory cortex. The PI is very experienced in conducting behavioral and 

Contact PD/PI: Froemke, Robert C

Vertebrate Animals Page 54
Contact PD/PI: Froemke, Robert C

electrophysiological studies in rodents. Mice are widely used for studies of neural circuit function, as it is 

straightforward to express opsins, DREADDs, fluorophores and Ca2+ indicators in vivo for optogenetic stimulation 

or 2-photon imaging, in combination with electrophysiological and behavioral experiments. Importantly, there is 

also an extensive existing literature and methodological basis for this research. This means that many 

experimental questions and technical issues have already been solved, minimizing the amount of animals 

required for our purposes. Mice serve as excellent models for studies of both cortical processing and plasticity. 

Results from from mouse studies are widely applicable to studies in other mammals such as carnivores, 

primates, and humans. 

We will minimize the number of animals needed for these studies. First, each study has been designed 

to minimize the number of animals required for statistical significance. A considerable amount of data can be 

obtained from a single in vivo whole-cell recording, and multiple recordings can be obtained from each animal. 

Thus multiple studies can be carried out at once. In addition, the PI has a background and training in statistics. 

This knowledge will be used to design experiments with the goal of reaching statistical significance in the minimal 

amount of time and using a minimal number of animals. All statistical analyses are performed in MATLAB or 

GraphPad Prism 6. Datasets will be tested for normality, and appropriate statistical tests applied (e.g., t-test for 

normally distributed data, Fischer’s exact test for categorical observations, Mann Whitney U-test for nonparametric data, Friedman test with Dunn post-hoc for non-parametric data with repeated measurements).

From prior experience and power analysis calculations performed for prior studies28,36,41-43,45,61,62, we 

estimate that to obtain statistically reliable results requires approximately 4-8 animals for experiments involving 

2-photon imaging (as hundreds of cells can be imaged per animal), 15-50 animals for experiments involving 

whole-cell recordings, 4-5 animals for multielectrode recording studies in behaving animals, and another 10-15

animals serving as a control group. In total then, we expect to require 295-540 mice. We breed many of our 

animals in our own facility to save on animal costs and numbers. Numbers reported here are ~10-20% higher 

than those reported for statistical purposes in the research strategy section, to account for animals lost during 

surgery or post-surgical monitoring, poor imaging windows, etc. Given that our lab can perform in vivo recordings

and imaging from 1-3 animals/day, we believe that the scope of these experiments and this amount of animal 

usage is entirely feasible within the five year project duration. Subaim 1a contains six major experiments. The 

first two experiments will use the same 10-20 animals. Experiment three looks specifically at three interneuron 

subtypes vs excitatory cells, requiring 40-60 mice. Experiment four requires 10-20 mice. Experiment five requires 

5-10 mice. Experiment six uses data from previous experiments. Subaim 1b contains four major experiments. 

The first two experiments will use animals from Subaim 1a, but we expect to also require another 20-40 mice for 

whole-cell and cell-attached recordings. The third experiment also uses excitatory and inhibitory neuron mice 

from Subaim 1a, but we expect to also require another 20-40 mice. The fourth experiment (recording in different 

layers) we expect to require 20-30 mice. Subaim 2a consists of seven behavioral studies each requiring 5-10 

mice, except for the first two studies determining receptor sensitivity, which we expect to each require 10-20 

mice. Subaim 2b consists of six experiments; five are behavioral and require 5-10 mice each; the recording and 

imaging experiments of experiment four will require an additional 10-20 mice. Experiments in Aim 3 involve more 

technically challenging in vivo imaging and recording experiments from neuromodulatory neurons. Axonal 

imaging experiments will require 20-40 mice, tetrode recordings will require 10-20 mice, fiber photometry studies 

will require 10-20 mice, whole-cell recordings will require 30-50 mice, and behavioral studies 20-30 mice.

We will use all appropriate parametric and non-parametric statistical tests for assessing statistical 

significance. In previous experiments we have conducted, we can usually achieve adequate statistical power 

(i.e., p < 0.05) with the number of animals given above. During an experiment, we will continually evaluate the 

achieved statistical reliability, and if possible, we will reduce the number of animals used in individual experiments 

if appropriately high reliability (e.g., p < 0.01) is obtained.

3. Veterinary Care: Veterinary care of all animals will be provided by vigilant in-house veterinarians and 

technicians of the Division of Laboratory Animal Resources (DLAR) at Medical. Animals will be monitored 

daily, including weekends, to ensure that they have enough food and water. Bedding will be changed twice per 

week. Animals will be provided with toys and objects for environmental enrichment. Medical DLAR staff will 

individually examine animals for signs of weight loss and pathological signs, and alert the PI and ACUC in 

these cases. 

4. Procedures for Minimizing Discomfort: Animals will be deeply anesthetized with isoflurane or ketamine/

dexmedetomidine before beginning surgery. Animals will be monitored continuously during recording sessions 

to assess their general well-being, and with a more specific assessment of any possible problems that might 

Vertebrate Animals Page 55
Contact PD/PI: Froemke, Robert C

arise from foreign body reaction or infection. All animals are maintained at a surgical level of anesthesia 

throughout surgical and electrophysiological procedures. Reflexes and vital signs such as respiration and 

temperature are monitored and logged throughout these procedures at 15-30 minute intervals, and fluids infused 

to maintain hydration and blood sugar levels. We will routinely administer prophylactic antibiotics in acute 

electrophysiological studies, and conduct them using clean surgical procedures throughout to minimize the 

possibility of having a limited time for data acquisition due to later-developing infection. Each member of the lab 

will first receive training from veterinary staff in the responsible use of animals and correct ways of 

conducting procedures before beginning experiments. Furthermore, the PIs will personally oversee the initial 

experiments and animal usage of each scientist in my laboratory, to ensure that procedures are correctly and 

ethically performed. 

5. Method of Euthanasia: After in vivo experiments, animals will be anesthetized and euthanized by overdose 

of pentobarbital anesthetic, followed by intracardiac perfusion of saline and paraformaldehyde for histological 

study. All of the experimental procedures proposed here are in accordance with an existing protocol approved 

by the Medical IACUC, and these methods are consistent with the recommendations of the American 

Veterinary Medical Association Guidelines on Euthanasia.

Vertebrate Animals Page 56
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Resource Sharing 

We are committed to sharing the data and other resources that are generated in our laboratory. There are three 

major ways in which we share resources, techniques, and data: 

1) We have welcomed several intra- and extramural colleagues to the lab to watch our in vivo whole-cell 

recording and 2-photon imaging experiments in awake or behaving animals, hel in them learn to erform this 

techni ue themselves, includin students and ostdocs from the labs 

2) The Pl has a long history of performing parametric experiments on the requirements for long-term synaptic 

plasticity, and sharing them with other investigators and laboratories. In articular, we re ularl send our results 

to theoretical neuroscientists for model testin 

e wI ma e our a a easI y avaI a e o 

o er expenmen an eore Ica Inves Iga ors, In or er o acI 1 a e scientific discourse and ease the speed and 

reliability of replication. We have an NIH supplement from the National Library of Medicine to work with 1111 

library staff (R01 NIDCD DC12557-S), enabling us to rapidly distribute our published data soon after publication 

to international collaborators or others who are interested in our results. These published data are now easily 

available on our lab website, along with Matlab code we have used for simulations in these published studies. 

3) We also regularly attend conferences (at least 3/year) to disseminate our new results, and the Pl has 

organized workshops, conference symposia, and frequently gives seminars to extramural institutions (18 invited 

talks and conference presentations in the 2015-2016 academic year). These include the Society for 

Neuroscience annual meeting, the International Society for Developmental Psychobiology meeting, Cosyne, and 

the Association for Research in Otolaryngology midwinter meeting. 

Resource Sharing Plan(s) Page 65 
Authentication of Key Resources Plan

All resources, chemicals, and reagents used for the experiments in this proposal are standard and supported by 

numerous publications (e.g., APV to block NMDA receptors, pharmacological reagents, the ChETA variant of 

channelrhodopsin-2, tdTomato interneuron transgenic mouse lines).

Contact PD/PI: Froemke, Robert C

Authentication of Key Biological and/or Chemical Resources Page 66