Corticothalamic gating of population auditory thalamocortical transmission in mouse
Ibrahim, Baher et al. (2021), Corticothalamic gating of population auditory thalamocortical transmission in mouse, Dryad, Dataset, https://doi.org/10.5061/dryad.qrfj6q5c4
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.
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).
In vivo imaging:
The detailed procedures have been described before (81). In brief, adult GCaMP6s mice were used for transcranial in vivo 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 oC 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, 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.
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 - 5.7X1012, 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.
For all in vitro 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 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 10 MgCl2, 0.5 CaCl2. 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 NaHCO3, 2.5 KCl, 10 glucose, 126 NaCl, 1.25 NaH2PO4, 3 MgCl2, and 1 CaCl2). 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 NaHCO3, 2.5 KCl, 10 glucose, 126 NaCl, 1.25 NaH2PO4, 2 MgCl2, and 2 CaCl2. 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 NaHCO3, 20 HEPES, 1.8 KCl, 1.05 CaCl2, 2.8 MgSO4, 1.2 KH2PO4, and 10 glucose] at 0–4°C. Slices were maintained in cACSF at 24°C for >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 NaHCO3, 20 HEPES, 1.8 KCl, 2.1 CaCl2, 1.4 MgSO4, 1.2 KH2PO4, and 10 glucose]. All of the solutions were bubbled with 95% oxygen/5% carbon dioxide and all experiments were done at room temperature.
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).
For calcium imaging in vitro, 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.
To inhibit GABAA receptors globally, GABAA-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 GABAA 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.
Electrophysiology and photoinhibition:
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 MgCl2, 0.07 CaCl2, 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 MgCl2*6H2O, 0.07 CaCl2, 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 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.
Histology and Confocal imaging:
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).
Imaging analysis and statistics:
Using customized MATLAB codes (attached to the source data file), all the pseudocolor images (jet colormap) were produced showing the tonotopic map of AC in vivo 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 < 0.05. Power analyses were run in G*Power (www.gpower.hhu.de). Using an effect size of 50% and alpha < 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.
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.
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.
National Science Foundation, Award: 1515587
National Institute on Deafness and Other Communication Disorders, Award: R01DC013073
National Institute on Deafness and Other Communication Disorders, Award: R21DC014765