DOI: 10.5061/dryad.qrfj6q5c4
title: Corticothalamic gating of population auditory thalamocortical transmission in mouse

description: The mechanisms that govern thalamocortical transmission are poorly
 understood. Recent data have shown that sensory stimuli elicit activity in
 ensembles of cortical neurons that recapitulate stereotyped spontaneous
 activity patterns. Here, we elucidate a possible mechanism by which gating
 of patterned population cortical activity occurs. In this study,
 sensory-evoked all-or-none cortical population responses were observed in
 the mouse auditory cortex in vivo and similar stochastic cortical
 responses were observed in a colliculo-thalamocortical brain slice
 preparation. Cortical responses were associated with decreases in auditory
 thalamic synaptic inhibition and increases in thalamic synchrony.
 Silencing of corticothalamic neurons in layer 6 (but not layer 5) or the
 thalamic reticular nucleus linearized the cortical responses, suggesting
 that layer 6 corticothalamic feedback via the thalamic reticular nucleus
 was responsible for gating stochastic cortical population responses. These
 data implicate a corticothalamic-thalamic reticular nucleus circuit that
 modifies thalamic neuronal synchronization to recruit populations of
 cortical neurons for sensory representations. 
description: <b>Animals:
 </b> C57BL/6J (Jackson Laboratory,
 stock # 000664), C57BL/6J-Tg (Thy1-GCaMP6s) GP4.3Dkim/J a.k.a. GCaMP6s
 mice (Jackson Laboratory, stock # 024275), BALB/c (Jackson Laboratory,
 stock # 000651), Gad2-IRES-Cre (Jackson Laboratory, stock # 010802),
 NTSR1-Cre (MMRRC, 017266-UCD) RBP4-Cre Mice of
 both sexes were used. All applicable guidelines for the care and use of
 animals were followed. All surgical procedures were approved by the
 Institutional Animal Care and Use Committee (IACUC). Animals were housed
 in animal care facilities approved by the American Association for
 Assesment and Accreditation of Laboratory Animal Care (AAALAC).
 <b><i>In
 vivo</i></b><b> imaging:</b> The detailed procedures have been
 described before (81). In brief, adult GCaMP6s mice were used for
 transcranial <i>in vivo</i> imaging of evoked calcium signals
 from the left auditory cortex (AC). For each experiment, the mouse was
 anesthetized with a mixture of ketamine and xylazine (100 mg/kg and 3
 mg/kg, respectively) delivered intraperitoneally. The animal’s body
 temperature was maintained within the range of 35.5 and 37
 <sup>o</sup>C using a DC temperature controller (FHC, ME,
 USA). Mid-sagittal and mid-lateral incisions were made to expose the
 dorsal and lateral aspects of the skull along with the temporalis muscle.
 The temporalis muscle was separated from the skull to expose the ventral
 parts of the underlying AC. The site was cleaned with sterile saline, and
 the surface of the skull was thinned by a surgical drill. A small amount
 of dental cement (3M ESPE KETAC) was mixed to a medium level of viscosity
 and added to the head of the bolt just enough to cover it. A head-bolt was
 bonded to the top of the skull, and the dental cement was allowed to
 set. An Imager
 3001 integrated data acquisition and analysis system (Optical Imaging
 Ltd., Israel) was used to image the cortical responses to sound in mice. A
 macroscope consisting of 85 mm f/1.4 and 50 mm f/1.2 Nikon lenses was
 mounted to an Adimec 1000m high-end CCD camera (7.4 x 7.4- µm pixel size,
 1004 X 1004 resolution), and centered above the left AC, focused
 approximately 0.5 mm below the surface of the exposed skull. Acoustic
 stimuli were generated using a TDT system 3 with an RP 2.1 enhanced
 real-time processor and delivered via an ES1 free field electrostatic
 speaker (Tucker-Davis Technologies, FL, USA), located approximately 8 cm
 away from the contralateral ear. All imaging experiments were conducted in
 a sound-proof chamber and images were obtained at 10 frames per second.
 Each trial of sound presentation was composed of two conditions (10
 seconds each); Condition 0 (C0), where there is no sound and condition 1
 (C1), where there is a 500 ms sound presentation that comes after 5
 seconds from the onset of the C1. For imaging, the blue light exposure was
 only on during the 10 seconds of each trial, and there was a 5 second
 interval between the two conditions during which the blue light was off.
 This intermittent schedule of the blue light exposure was done to lower
 the likelihood of photobleaching. 500 ms pure tones of 5 kHz, at 37, 44, 50, 55,
 60, 65, 70, 75, and 80 dB SP. The response window was set as one second
 starting from the 5th second for C0 or from the onset of sound for C1. The
 Δf/f of the response window was computed as the difference between Δf/f0
 (C0) and Δf/f1 (C1) using a custom MATLAB code. <b>Virus injection:
 </b> To
 modulate specific cell types, Cre recombinase-expressing mice were used to
 provide an expression of opto- or chemo-genetic probes in those specific
 cells 11 days after viral injection at P4. The detailed procedures have
 been described before (82). For all neonates, cryoanesthesia was induced
 after five to ten minutes. A toe pinch was done to confirm that the mice
 were fully anesthetized. A small animal stereotaxic instrument (David Kopf
 Instruments, Tujunga, CA) was used with a universal syringe holder (David
 Kopf Instruments, Tujunga, CA) and standard ear bars with rubber tips
 (Stoelting, Wood Dale, IL) were used to stabilize the head. The adaptor
 stage was cooled by adding ethanol and dry ice to an attached well. A
 temperature label (RLC-60-26/56, Omega, Norwalk, CT) was attached to the
 stage to provide the temperature of the stage during cooling. The
 temperature was kept above 2°C to prevent hypothermia or cold-induced skin
 damage of the neonatal mice and below 8°C to sustain cryoanesthesia. Glass
 micropipettes (3.5-inches, World Precision Instruments, Sarasota, FL) were
 pulled using a micropipette puller (P-97, Sutter Instruments, Novato, CA)
 and broken back to a tip diameter between 35-50μm. The micropipette was
 filled with mineral oil (Thermo Fisher Scientific Inc., Waltham, MA) and
 attached to a pressure injector (Nanoliter 2010, World Precision
 Instruments, Sarasota, FL) connected to a pump controller (Micro4
 Controller, World Precision Instruments, Sarasota, FL). The AC of
 NTSR1-Cre (43, 83-85) or RPB4-Cre (86, 87) neonates was injected with
 eNpHR3.0 AAV1 (AAV-EF1a-DIO-eNpHR3.0-YFP with titer equal to 4.7 -
 5.7X10<sup>12</sup>, referred to hereafter as
 halorhodopsin-AAV) constructs from UNC Vector Core (Chapel Hill,
 NC), AAV1-Ef1a-DIO EYFP (control for halorhodopsin), Gi-coupled hM4Di
 DREADDs AAV8 (AAV8-DIO-hSyn-hM4Di-mCherry, referred to here as
 DREADDs-AAV), AAV8-hSyn-DIO-mCherry (control for DREADDs), or
 AAV9-FLEX-EGFP (for the histology done on RPB4-Cre mouse) constructs from
 Addgene (Cambridge, MA). The micropipette carrying the
 viral particles was first located above the AC in the left hemisphere at
 1.5 mm anterior to lambda and just at the edge of the skull’s flat
 horizon. The tip was lowered to 1.2 mm from the brain surface and was then
 pulled back to 1.0 mm for the first injection where 200 nL of
 halorhodopsin-AAV or DREADDs-AAV was injected at 200 nL/min. After the
 injection was finished, the micropipette was left in the brain for 1
 minute before removing to allow the injectate to settle into the brain.
 Following the first injection, the tip was pulled back stepwise in 0.1 mm
 increments, and 200 nL of the injectate was injected at every step until
 the tip reached 0.3mm from the surface. In total, 1600 nL of AAV was
 injected into the AC. The incision was sutured using 5/0 thread size,
 nylon sutures (CP Medical, Norcross, GA). To target the GABAergic cells of
 the inferior colliculus (IC), the IC of GAD2-Cre (88-90) neonatal mice was
 injected with halorhodopsin-AAV following the same procedures showing
 above, but the micropipette loaded by halorhodopsin-AAV was located over
 the IC at the left hemisphere at 2.0 mm posterior to lambda and 1.0 mm
 laterally from the midline. The neonates were transferred back onto a
 warming pad to recover. After 5-7 minutes, their skin color was returned
 to normal, and they started
 moving. After recovery, all neonates were returned to their nest with the
 parents.   <b>Brain slicing:
 </b> For
 all <i>in vitro</i> experiments, 15-18 day old mice were
 initially anesthetized with ketamine (100 mg/ kg) and xylazine (3 mg/kg)
 intraperitoneally and transcardially perfused with chilled (4°C)
 sucrose-based slicing solution containing the following (in mM): 234
 sucrose, 11 glucose, 26 NaHCO<sub>3</sub>, 2.5 KCl, 1.25
 NaH<sub>2</sub>PO<sub>4</sub>, 10
 MgCl<sub>2</sub>, 0.5 CaCl<sub>2</sub>. After the
 brain was removed from the skull, it was cut to obtain auditory
 colliculo-thalamocortical brain slice (aCTC) as shown (Fig. S1) and as
 described before (20, 21). 600 μm thick horizontal brain slices were
 obtained to retain the connectivity between IC, MGB, TRN and AC. All
 slices were incubated for 30 min at 33 °C in a solution composed of (in
 mM: 26 NaHCO<sub>3</sub>, 2.5 KCl, 10 glucose, 126 NaCl, 1.25
 NaH<sub>2</sub>PO<sub>4</sub>, 3
 MgCl<sub>2</sub>, and 1 CaCl<sub>2</sub>). After
 incubation, all slices were transferred to a perfusion chamber coupled to
 an upright Olympus BX51 microscope, perfused with artificial cerebrospinal
 fluid (ACSF) containing (in mM) 26 NaHCO<sub>3</sub>, 2.5 KCl,
 10 glucose, 126 NaCl, 1.25
 NaH<sub>2</sub>PO<sub>4</sub>, 2
 MgCl<sub>2</sub>, and 2 CaCl<sub>2</sub>. Another
 set of experiments was done in a different laboratory to exclude any
 experimental factors related to our laboratory environment, chemicals, or
 anesthesia. As reported previously (91), following full anesthesia by
 isoflurane, a C57BL/6J mouse was immediately decapitated without cardiac
 perfusion, the animal’s brain was extracted and immersed in cutting
 artificial CSF [cACSF; composed of (in mM) 111 NaCl, 35
 NaHCO<sub>3</sub>, 20 HEPES, 1.8 KCl, 1.05
 CaCl<sub>2</sub>, 2.8 MgSO<sub>4</sub>, 1.2
 KH<sub>2</sub>PO<sub>4</sub>, and 10 glucose] at
 0–4°C. Slices were maintained in cACSF at 24°C for &gt;1 h before
 transfer to the recording chamber, which was perfused at 3–6 ml/min with
 ACSF [composed of (in mM) 111 NaCl, 35 NaHCO<sub>3</sub>, 20
 HEPES, 1.8 KCl, 2.1 CaCl<sub>2</sub>, 1.4
 MgSO<sub>4</sub>, 1.2 KH2PO<sub>4</sub>, and 10
 glucose]. All of the solutions were bubbled with 95% oxygen/5% carbon
 dioxide and all experiments were done at room temperature. <b>Electrical stimulation:
 </b> All
 the electrical stimulation protocols in the IC evoked similar cortical
 response patterns. One second electrical train pulses of (250 µA, 40Hz,
 1ms pulse width) to IC was the main stimulating protocol as described
 before (92). However, one second durations of electrical stimulation were
 not suitable for electrophysiology experiments because responses were
 intermingled with the stimulus artifact. Therefore, for whole-cell and LFP
 recording, the stimulation of IC was done with a single 3 ms pulse
 (300-500 mA) or 100 ms long trains of pulses (250uA, 40 Hz, 1ms pulse
 width) were used. The electrical stimulation was done using a concentric
 bipolar electrode (Cat#30201, FHC) every 10-20s. The parameters of the
 electrical pulses were adjusted by a B&K precision wave generator
 (model # 4063) and World Precision Instruments stimulation isolator
 (A-360). In another stimulation set up (Our collaborator’s laboratory), a
 biphasic current pulse (200 µA, 5 ms; STG4002 stimulator, Multichannel
 Systems, Reutlingen, Germany) was delivered to IC at 0.05 Hz using bipolar
 tungsten electrodes (100KΩ, FHC Inc., Bowdoin, ME). <b>Imaging:
 </b> For
 calcium imaging <i>in vitro</i>, slices from GCaMP6s mice or
 wild-type mice loaded with CAL-520, AM (Abcam, ab171868) calcium dye were
 used. For CAL-520, AM calcium dye loading, the aCTC slices were incubated
 in 48 µl of DMSO dye solution + 2 µl of Pluronic F-127 (Cat# P6866,
 Invitrogen) in 2.5 ml of the incubating solution at 35-36 °C for 25-28
 minutes according to (93, 94). The slices then were incubated in normal
 incubating solution (shown above) for 30 minutes to wash the extra dye.
 Imaging was done under ACSF perfusion as described before. Depending on
 the experiment, the evoked calcium or flavoprotein autofluorescence (FA)
 signals following IC stimulation (92, 95-97) were measured using a stable
 DC fluorescence illuminator (Prior Lumen 200) and a U-M49002Xl E-GFP
 Olympus filter cube set (excitation: 470–490 nm, dichroic 505 nm, emission
 515 nm long pass, 100 ms exposure time for FA and 5 ms for calcium
 signals). All data were collected using Retiga EXi camera at a frame rate
 of 4 Hz for FA and 10 Hz. The time trace of the FA or calcium population
 signals were obtained by placing regions of interest (ROI) over brain
 regions (IC, MGB, TRN or AC). The collected time traces were used to
 compute the Df/f. For the calcium signals obtained from cortical or
 thalamic cells, the ROIs were manually made around the cell body. A strong
 correlation between voltage and calcium signals validated the calcium
 signals obtained from thalamic cells containing CAL-520 dye after
 simultaneous whole-cell recording and calcium imaging following the
 injection of a positive current to the cell (Fig. S7). The average
 background value was calculated by drawing an ellipse with radii 2.25
 times that of the original ROI and subtracting all cell ROI from that
 ellipse to eliminate overlap. Finally, the Df/f was computed after a 40%
 of the background value was subtracted from the cell's average value
 for neuropil correction. Df/f was computed using the average signal of a 2
 second period before the stimulus onset. As illustrated in Fig. S8, the
 ON-cortical responses were determined following two criteria. Each signal
 required a rising and falling phase as well as a z-score greater than
 3.  <b>Pharmacological
 intervention:   </b> To inhibit
 GABA<sub>A</sub> receptors globally,
 GABA<sub>A</sub>-receptor antagonist (98), gabazine (Cat#
 1262, Tocris) was added to the bath ACSF solution (200 nM, which is near
 the synaptic IC50 for this compound (99). To specifically inhibit the
 GABA<sub>A</sub> receptors in either MGB or AC, a continuous
 flow of gabazine 400 nM) was injected into MGB or AC in a counterbalanced
 manner through a glass pipette (broken tip, 35 µm) which was connected to
 a picospritzer (Toohey Company, New Jersey, USA). The pipette was filled
 by a solution composed of 1 ml ACSF+10 µL Alexa Fluor 594 hydrazide,
 sodium salt dye (, Cat#A10438,
 Invitrogen) to. Visualize the flow of the solution and to ensure that it
 was restricted to the site of injection (Fig. S9). The injection was done
 under 10 psi pressure for 5 minutes and continuously during imaging. As
 reported before, to block TRN activity (33, 34), the AMPA receptor blocker
 NBQX (20 mM, Cat# 0373, Tocris) was injected into TRN of the aCTC slice
 following the same described procedures. The chemical inhibition of
 corticothalamic layer 6 cells was conducted by bath perfusion of
 clozapine-n-oxide (CNO, 5uM, Cat# 4936, Tocris), the chemical actuator of
 the chemogenetic probe, hM4Di (100) that was solely expressed in
 corticothalamic layer 6 of NTSR1-Cre mouse after viral injection. CNO was
 also perfused to the imaging chamber containing a slice with a layer 6
 expressing m-cherry only as a control for DREADDs. <b>Electrophysiology and
 photoinhibition: </b> Whole-cell recording of cortical
 layer 4, TRN, or MGB cells was performed using a visualized slice setup
 outfitted with infrared-differential interference contrast optics.
 Recording pipettes were pulled from borosilicate glass capillary tubes and
 had tip resistances of 2–5 MΩ when filled with potassium gluconate based
 intracellular solution (in mM: 117 K-gluconate, 13 KCl, 1.0
 MgCl<sub>2</sub>, 0.07 CaCl<sub>2</sub>, 0.1
 ethyleneglycol-bis(2-aminoethylether)- N,N,N′,N′-tetra acetic acid, 10.0
 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid, 2.0 Na-ATP, 0.4
 Na-GTP, and 0.5% biocytin, pH 7.3, 290 mOsm) for current-clamp mode.
 Voltage was clamped at -60 mV or +10 mV to measure either the excitatory
 or inhibitory currents, respectively, using cesium-based intracellular
 solution (in mM: 117.0 CsOH, 117.0 gluconic acid, 11.0 CsCl, 1.0
 MgCl<sub>2</sub>*6H<sub>2</sub>O, 0.07
 CaCl<sub>2</sub>, 11.0 EGTA, 10.0 HEPES, pH 7.3, 290 mOsm).
 Series resistance was not compensated for. Local field potential (LFP)
 recordings were performed using glass pipette with a broken tip 5-10 µm).
 LFP signals were filtered offline using Clampfit 10.7 software under
 Gaussian low pass frequency at 300 Hz as well as a notch filter at 60Hz. A
 Multiclamp 700B amplifier and pClamp software (Molecular Devices) were
 used for data acquisition (20 kHz sampling). To analyze the distribution
 of the membrane potential of layer 4 cells during the UP state, a time
 period starting from the stimulation offset and including a similar time
 of both the cortical upstate event and the basal membrane potential was
 used. The sampling rate was reduced from 20kHz to 2kHz, and spikes and
 stimulus artifacts were excluded from the analysis. For photoinhibition, the
 halorhodopsin eNpHR3.0 probe was expressed selectively in either
 corticothalamic layer 6, layer 5, or IC-GABAergic cells and was activated
 by illuminating a yellow light (565 nm) obtained from DC fluorescence
 illuminator (Prior Lumen 200) and Olympus filter cube (U-MF2, Olympus,
 Japan). The same light was used to illuminate the brain slice with a layer
 6 expressing eYFP only as a control for halorhodopsin.The light was set to
 illuminate the whole field of the LFP recording chamber using a 4X objective for
 three seconds extending from one second pre-stimulus and two seconds after
 the onset of IC stimulation. Based on the initial results related to the
 peri-stimulus dynamics of corticothalamic layer 6 cells and the
 post-stimulus cortical activity, three seconds of illumination was chosen
 to cover the time period one second before the onset of the stimulus as
 well as the post-stimulus period.  <strong>Histology and
 Confocal imaging:</strong> After collecting the aCTC slice
 from RPB4-Cre mouse injected with AAV9-FLEX-EGFP into the AC, the aCTC
 slice was placed in a 4% paraformaldehyde (PFA) solution for fixation.
 After one day, the aCTC slice was removed from the PFA and moved to graded
 sucrose solution (10, 20, and 30%). The aCTC slice was sectioned using a
 Leica cryostat as 50 mm sections. The sections were imaged using Leica SP8
 confocal microscope (excitation: 488 nm & emissions: 515-550
 nm). <b>Imaging analysis and
 statistics: </b> Using customized MATLAB codes
 (attached to the source data file), all the pseudocolor images (jet
 colormap) were produced showing the tonotopic map of AC <i>in
 vivo</i> and the activated brain regions in the aCTC slice.
 Origin-Pro 2017 software was used to run the statistical tests and
 generate the graphs. The normal distribution of the data was examined
 based on Kolmogorov-Smirnov test. Accordingly, the suitable parametric
 (paired t-test or RM-One Way ANOVA test followed by Bonferroni post hoc
 test) or non-parametric tests (Paired Wilcoxon signed-rank test or
 Kruskal- Wallis test followed by Dunn's Test) were used. Differences
 were deemed significant when p-value &lt; 0.05. Power analyses were
 run in G*Power (www.gpower.hhu.de). Using an effect size of 50% and alpha
 &lt; 0.05, sample sizes in all experiments provided power to detect
 significant differences of at least 80%. The animals were randomly
 allocated in each experimental group.   <b>Work art:
 </b> All
 figures were designed and made using Adobe Illustrator (Adobe, San Jose,
 CA). To keep working within Adobe environment to avoid losing the
 resolution of the figures, Adobe Photoshop (Adobe, San Jose, CA) was used
 to crop the borders of some images to save space, draw scale bars, and
 adjust brightness and gamma balance of grayscale images showing the
 electrophysiological traces. All manipulations in
 brightness/contrast/gamma were uniform across the entire image.
description: The data are used to make
 the manuscript titled "Corticothalamic gating of population auditory
 thalamocortical transmission in mouse" under the review process by
 Life journal.