DOI: 10.5061/dryad.bcc2fqzsz
title: Data from: A brainstem circuit integrating reflexive and anticipatory salivation

description: Salivation supports oral health, taste sensation, swallowing, and
 digestion, yet the brain mechanisms that control it remain largely
 unknown. Here, we identify a salivatory center in the mouse brainstem that
 integrates sensory and learned anticipatory signals to control salivation.
 We show that activating choline acetyltransferase-expressing neurons in
 the inferior salivatory nucleus (IS) is sufficient to trigger saliva
 secretion. Using fiber photometry, we monitor real-time IS neural
 responses to mechanosensory and gustatory stimulation and find tight
 correlation with salivatory output. We further demonstrate that IS neurons
 receive input from local brainstem circuits, mediating rapid hardwired
 responses to taste, as well as direct cortical projections. Notably,
 gustatory cortex input is required for salivatory responses to predictive
 sensory cues in a Pavlovian conditioning paradigm. Together, our findings
 define the circuit underlying taste-evoked and anticipatory salivation and
 provide a foundation for dissecting this autonomic response in health and
 disease.
description: <strong>Submandibular gland ganglion
 injection</strong>  Animals were anesthetized
 with ketamine and xylazine (100 mg/Kg and 10 mg/Kg, intraperitoneal) and
 given a nonsteroidal anti-inflammatory drug (carprofen 5 mg/Kg,
 subcutaneous). After surgery, animals received sustained release
 formulation pain relief (Buprenorphine ER 1 mg/Kg, subcutaneous). Mice
 were placed on a custom-made platform in the supine position, and the body
 temperature was maintained between 34 °C and 36 °C. A small incision was
 made along the midline of the cervical skin, which was gently retracted
 laterally to expose a submandibular gland duct. A 2 % Fluorogold
 (Fluorochrome) solution was loaded into a pulled glass capillary needle
 (length 9 cm, I.D. 0.53 mm, O.D. 1.14 mm) and unilaterally injected into
 the largest visible SMG ganglion on the salivary duct using a
 nano-injector system (Drummond, Nanoject III) at 5 nL/sec to a total
 volume of 100 nL. The incision was closed with a polyglycolic acid suture
 (Stoelting, Visorb 4-0, 50496) and 7 days later, animals were sacrificed
 for histology.  <strong>Stereotaxic
 injection</strong> All stereotaxic surgery
 procedures were carried out under aseptic environments and all tools for
 surgery were autoclaved before use. Mice were anesthetized with a mixture
 of ketamine and xylazine (100 mg/Kg and 10 mg/Kg, intraperitoneal) and
 received carprofen (5 mg/Kg, subcutaneous). After surgery, animals
 received sustained release formulation pain relief (Buprenorphine ER 1
 mg/Kg, subcutaneous). Mice were then placed onto a stereotaxic frame
 (Stoelting) with a closed-loop heating system to maintain body temperature
 between 34 °C and 36 °C. A small incision was made to expose the skull and
 a craniotomy (&lt; 1 mm) was made at the injection site using a drill
 (Roboz, RS-6300A). The viral vectors were loaded into pulled glass
 capillaries and injected using nano-injector system (Drummond) at 5 nL/sec
 to the total of 90 nL (<em>AAV-Syn-Flex-GCaMP6s</em>), 150 nL
 (<em>AAV-EF1a-hChR2(H134R)-mCherry</em>) or 300 nL
 (<em>AAV-CaMKIIa-hM4D(Gi)-mCherry</em>). Injections were
 performed unilaterally for fiber photometry and optogenetic stimulation
 experiments, and bilaterally for the gustatory cortex silencing
 experiments (total 4 spots). The coordinates were: inferior salivary
 nucleus (bregma −5.88 mm; lateral 1.1 mm; ventral 3.8 mm); two bilateral
 cortical fields for gustatory cortex silencing (bregma - 0.3 mm; lateral
 +/- 4.25 mm; ventral 2.6 mm and bregma 1.7 mm; lateral +/- 3.1 mm; ventral
 1.75 mm). For fiber photometry and optogenetic stimulation, a commercially
 prepared optical fiber from Doric Lenses (400 μm O.D., NA = 0.48) was
 implanted ~100 μm above the GCaMP or ChR2 virus injection site. All
 implants were secured onto the skull using dental cement (C&B
 Metabond, S380). A custom-made head-post was placed to facilitate handling
 during set up for experiments. After surgery, the animals were returned to
 their home cages and allowed to recover for at least 14 days before any
 experiments were performed. In this manuscript, we use ‘Flex’ as a unified
 term to refer to both double-floxed inverted orientation (DIO) and
 Flip-Excision (FLEx) configurations for Cre-dependent reporters.
 <strong>Intraoral cannulation</strong>
 Tastant solutions were delivered via an intraoral cannula. The
 cannula implantation was based on an original procedure on rats since
 adapted for mice. A curved needle (Miltex, Regular Surgeon’s Needle ½
 circle, Size 17, MS192-17) attached to silicon tubing (Braintree
 Scientific, Microrenathane Tubing O.D. 0.037", I.D. 0.023")
 pierced the gum near the upper left 4th molar. This catheter tubing then
 exited through the lateral surface of the skull. The mouth-end of catheter
 tubing was trimmed and the loose top end (~1”) was secured onto the skull
 surface with dental cement. For the 2-3 days following implantation,
 animals were given daily antibiotic ointments to prevent
 infection. <strong>Retrograde monosynaptic
 tracing</strong> Stereotaxic injections were
 performed with 100 nL mixture (1:1) of
 <em>AAV8-CAG-FLEX-TVA-mCherry</em> and
 <em>AAV8-CAG-FLEX-oG-WPRE-SV40-PA</em>, which was injected
 into the IS coordinates of ChAT-Cre animals. 4 weeks later, 50 nL of
 <em>EnvA-pseudotyped RabiesΔG-eGFP</em> (N2c) was injected
 into the same locale. The animals were sacrificed 14 days later for
 histological analysis. <strong>Photostimulation
 and saliva collection</strong> For
 channelrhodopsin-2 (ChR2) stimulation experiments, 473 nm light pulses
 were delivered using an Optogenetics-LED system (Prizmatix) through a
 single optogenetics fiber with a rotary joint. Stimulation was controlled
 through Pulser software (Prizmatix), and laser intensity was maintained
 between 8.87 mW at the fiber tip. To measure saliva flow during
 optogenetic photostimulation, we anesthetized animals with a
 ketamine/xylazine mixture (100 mg/Kg and 10 mg/Kg, intraperitoneal) and
 placed pre-weighed pH test papers (Micro Essential Lab MES445) under the
 tongue, and saliva was wicked for 10 minutes without light stimulation.
 The paper was then returned to the pre-weighed tube for post-collection
 weighing. pH of mouse saliva is typically between 7.0 and 8.7, but
 immediately after salivation, it can rise to a range of 9.0 to 10.0. This
 change in pH can be observed instantly by the color change on the pH paper
 upon contact with the saliva. After a 30-minute interval, a new
 pre-weighed pH test paper was placed under the tongue for the light
 stimulation condition. During the subsequent 10 minutes, 473 nm light was
 delivered to the inferior salivatory nucleus via the implanted optical
 fiber. Saliva was again collected on the pH test paper and weighed. Saliva
 volume for each photostimulation condition was calculated based on the
 weight difference before and after the collection. To ensure that
 anesthesia does not affect saliva secretion, we alternated the order of
 control and illumination sessions.  <strong>Fiber
 photometry and stimuli presentation</strong> We
 measured the activity of IS neurons in freely-moving animals using fiber
 photometry during intraoral stimulation. Bulk GCaMP fluorescence signals
 were measured as described previously. Briefly, for fiber photometry, a
 real-time processor from Tucker Davis Technologies (RZ5P) modulated 465 nm
 and 405 nm LEDs at 200 and 330 Hz, respectively, and were offset by 7 mA.
 LED currents were adjusted between 200 and 220 mV from the LEDs driver
 (four ports mini-cube, Doric) for each signal and excited a through fiber
 optic patch cord (400 µm diameter, Doric Lenses). Taste delivery timing
 was synchronized with the photometry system via TTL signals input into the
 RZ5P processor. Emitted fluorescence was collected and focused onto a
 Doric photoreceiver, then amplified and digitized by the RZ5P for further
 analysis. Experiments were performed at least 14 days after surgical
 procedures to give animals ample time for recovery. After at least 7 days
 of habituation in the fiber photometry arena, stimuli were delivered via
 implanted intraoral cannula using a pressurized perfusion system (AutoMate
 Scientific). A typical panel consisted of a 7-minute session of tastants
 presented in the following order: 30 mM acesulfame potassium (AceK;
 sweet), 3 mM quinine (bitter), 50 mM citric acid (sour), 30
 mM monopotassium glutamate and 1 mM inosine monophosphate (MPG+IMP) mix
 (umami), and 100 mM NaCl (salty). Each infusion trial consisted of a 20
 second delivery of a single taste solution using a pressure-based system
 (3–5 psi). Between taste deliveries, a 50 second flow of artificial saliva
 (6 mM NaHCO<sub>3</sub>, 6 mM KHCO<sub>3</sub>, 4
 mM NaCl, 10 mM KCl, 3.6 mM HCl, 0.5 mM MgCl<sub>2</sub>
 6H<sub>2</sub>O, 0.24 mM
 K<sub>2</sub>HPO<sub>4</sub>, 0.24 mM
 KH<sub>2</sub>PO<sub>4</sub>, 0.5 mM
 CaCl<sub>2</sub> 2H<sub>2</sub>O) at the same
 pressure was used to wash out residual taste stimuli. Pressurized
 perfusion device used for tastant presentation was set at a high flow rate
 (23 µL/sec) to discount post-ingestive effects. Under these perfusion
 settings, animals are unable swallow the solutions (data not shown),
 therefore we expect minimal exposure of tastants at the back of the
 tongue. For slower infusion conditions in Figure S2D, flow rate was set to
 7 µL /sec, at which none of the solutions dripped out of the
 mouth. Animals underwent at least 5 days of daily training sessions with
 all five taste solutions prior to the test date when fiber photometry
 recordings were made. Additional conditions, including air infusion and
 standard chow presentation, were included to assess
 mechanosensation-induced salivation. The collected
 photometry data were analyzed by custom python code and Fiber Photometry
 Modular Analysis Tool (pMAT) software. Z-scored ΔF/F signals were
 calculated in pMAT by normalizing to the median of a 5-second baseline
 window (–5 to 0 s), using a bin size of 100 ms. The resulting data were
 exported as a CSV file and imported into MATLAB, where the average trace
 across animals was plotted. Shaded regions around the trace represent the
 standard error of the mean (SEM). The area under the curve (AUC) was
 calculated by integrating fluorescence signals under identified calcium
 transients. <strong>Chemogenetic silencing of the
 gustatory cortex</strong> For chemogenetic
 silencing of the gustatory cortex, animals were trained with five taste
 (sweet, bitter, sour, umami and salty) deliveries before initiating fiber
 photometry recording experiments. On the test day, fiber photometry was
 performed with taste solution trials after DMSO injection (500 µL/Kg, 5 %
 DMSO). Subsequently, on the same day, fiber photometry was performed on
 animals after clozapine-n-oxide (CNO) injection (Tocris, 49-361-0, 1 mg/Kg
 in 5 % DMSO, intraperitoneal) with the same taste regimen. 
 <strong>Classical (Pavlovian)
 conditioning</strong> Animal was placed in a
 behavior arena inside a sound-isolation chamber (Med Associates). Each
 animal received 3 days of water infusion to acclimate with the behavior
 arena and with the solution delivery via the intraoral cannula. Each day
 consisted of 3 test sessions of 5 trials, with a 30-minute rest between
 sessions. Each trial consisted of 10 cycles of 2 seconds of taste delivery
 and 58 seconds of rest.  For classical conditioning, we
 trained animals for 7 days to associate a taste solution (30 mM AceK for
 the sweet group, or 3 mM quinine for the bitter group) with the
 conditioned stimuli. The cues consisted of a 1 second-long tone (0.25 Hz,
 1 second later taste solutions were delivered for 2 seconds. This was
 repeated for 10 cycles with a 1 minute rest between each trial.
 Upon completion of training, animals underwent test sessions
 consisting of a 1-second auditory beep followed by a 59-second rest
 period, repeated three times. Each animal received two test sessions: one
 following infusion of vehicle and the other following administration of
 clozapine-N-oxide (1 mg/kg in 5 % DMSO). The order of drug administration
 was counterbalanced across animals.
 <strong>Immunohistochemistry</strong><br>
 Animals were sacrificed by CO<sub>2</sub> inhalation and
 perfused with phosphate-buffered saline (PBS) and then 4 %
 paraformaldehyde (PFA, Electron Microscopy Sciences, 15714). After
 perfusion, brains were post-fixed in 4 % PFA overnight at 4 °C and
 sectioned coronally at 40 µm thickness. For the Fluorogold antibody
 staining, brain sections were permeabilized and blocked with 10 % normal
 donkey serum (Millipore, S30-100ML) in PBS with 0.5 % Triton X-100 (Sigma)
 at room temperature for 2 hr. Brain tissues were stained overnight at 4 °C
 with anti-Fluorogold primary antibody (Sigma Aldrich, AB153-I, 1: 1000
 dilution), and labeled with fluorescence tagged Alexa fluor 488 secondary
 antibody (Jackson ImmunoResearch, 711-545-152, 1:1000 dilution) at room
 temperature for 1 hr. Tissues were then washed with PBS and mounted with
 Fluoromount-G (SouthernBiotech, OB100-01). Images were collected using a
 Leica DM6B Widefield epifluorescent microscope in the in the Biological
 Imaging Facility at Northwestern University. Cell numbers were counted
 manually. <strong>QUANTIFICATION AND STATISTICAL
 ANALYSIS</strong> Statistical tests were
 performed using unpaired t test or paired t test when appropriate. Applied
 statistical methods are indicated in the figure legends.
 <em>p</em> &lt; 0.05 was considered to be statistically
 significant.
description: # README: A Brainstem Circuit Integrating Reflexive and Anticipatory
 Salivation Dataset
 DOI: [10.5061/dryad.bcc2fqzsz](https://doi.org/10.5061/dryad.bcc2fqzsz) ##
 Description of the data and file structure These files contain the data
 used to construct the figures in the associated published manuscript in
 *Cell Reports*. Data files contain processed data from GCaMP fiber
 photometry recordings (in .csv format).  The raw data have been processed
 as described in the manuscript, and are provided as z-score against time.
 Time-zero corresponds to the moment stimulus (specified by folder name)
 was presented to the animals. Each column corresponds to different data
 points. In some cases, there are multiple recordings from an individual
 animal collected at successive sessions; these do not have column headers.
 The data specifically align to figures presented in the paper. Detailed
 information about animals, such as genetic background, sex, and age are
 found in the associated manuscript.  Please contact the authors if you
 would like the data in any other format ### Files and variables File:
 Fiber_photometry.zip **Description:** Fiber photometry data provided as
 z-scores against time (seconds). File: Optogenetic_stim_for_salivation.zip
 **Description:** CSV files containing salivation measurements during
 optogenetic stimulation. Salivation_amounts.csv measured saliva secreted
 (grams) in 15 minutes with/without optogenetic stimulation of the Inferior
 Salivatory Nucleus from 6 mice. Laser_power_calibration.csv measured
 saliva secreted (grams) at varying laser powers, measured from 3 mice.  
 ### Code/software #### Fiber photometry analysis * Anaconda: any version
 (24.11.3) * Jupiter notebook (7.2.2) * Matlab: any version (R2024b) *
 pMAT: Matlab
 version, [https://github.com/djamesbarker/pMAT](https://github.com/djamesbarker/pMAT) (v1.3) #### Image analysis * imageJ (2.17.0) * LAS X Life Science Microscope Software Platform