DOI: 10.5061/dryad.9kd51c5ng
title: Data for: The cerebellum regulates fear extinction through thalamo-prefrontal cortex interactions in male mice

description: Fear extinction is a form of inhibitory learning that suppresses the
 expression of aversive memories and plays a key role in the recovery of
 anxiety and trauma-related disorders. Here, using male mice, we
 identify a cerebello-thalamo-cortical pathway regulating fear extinction.
 The cerebellar fastigial nucleus (FN) projects to the lateral subregion of
 the mediodorsal thalamic nucleus (MD), which is reciprocally connected
 with the dorsomedial prefrontal cortex (dmPFC). The inhibition of FN
 inputs to MD in male mice impairs fear extinction in animals with high
 fear responses and increases the bursting of MD neurons, a firing pattern
 known to prevent extinction learning. Indeed, this MD bursting is followed
 by high levels of the dmPFC 4 Hz oscillations causally
 associated with fear responses during fear extinction,
 and the inhibition of FN-MD neurons increases the coherence of MD
 bursts and oscillations with dmPFC 4 Hz oscillations. Overall,
 these findings reveal a regulation of fear-related
 thalamo-cortical dynamics by the cerebellum and its contribution to fear
 extinction.
description: 1. Animals Adult male C57BL/6N mice (Charles
 River, France,MSR Cat# CRL_27,RRID:IMSR_CRL:27 ) were housed in groups of
 4 mice per cage, with free access to food and water, maintained at
 21–22°C, with a 12 h : 12 h light/dark cycle. Adult male wild-type mice
 8–12 weeks of age were used for all the experiments. Male and female mice
 are equally suitable for fear conditioning analysis, but male mice are
 bigger and have therefore more strength to carry the implant and
 preamplifier in electrophysiological experiments. Animal care and
 experimental procedures followed the European Community Council Directives
 (authorization number APAFIS#1334-2015070818367911 v3 and APAFIS
 #29793-202102121752192). 2. Stereotactic
 surgeries Surgeries for tracing, chemogenetic,
 optogenetic, and electrophysiological experiments were performed in 7–8
 weeks-old mice, placed in a stereotaxic frame (Kopf Instruments) after
 buprenorphine administration, and anaesthetized with isoflurane 2% along
 the whole procedure. In addition, a local analgesia was administered
 subcutaneously above the skull with 0.02% of lidocaine. The body
 temperature was monitored and maintained at 36°C with a heating pad and a
 rectal thermometer. For viral injections and electrodes
 and optic fiber implantations, the stereotaxic coordinates used for FN
 (AP: -6.37, ML: ±0.70, DV: -2.12), MD (AP: -1.12, ML: ±0.65, DV: -3.15)
 and dmPFC (AP: +2.34, ML: ±0.25, DV: -1.5) were taken relative to the
 bregma, and the depth was considered from the brain surface. Glass
 capillaries used for viral infusions were kept at the injection site for 5
 minutes after viral infusion before being slowly withdrawn. 
 3. Neuroanatomical tracing and histology For
 neuroanatomical tracing, a group of mice were injected with 100 nl of
 anterograde AAV1-CB7-Cl-mCherry-WPRE-rBG (Upenn Vector) in the FN (n = 6),
 100 nl CTB-alexa 488 (Invitrogen) in the dmPFC (n=6), or retrograde
 AAV-Syn-eGFP (Addgene) in the MD (n = 6). For trans-synaptic labelling, a
 group of mice was injected with 75 nl of AAV9-CMV-PI-Cre-rBG (Addgene) in
 the FN and 100 nl of AAV1-CAG-Flex-tdTomato-WPRE-bGH in the MD (n = 3),
 and MD sections were used to Calbindin immunostaining.
 Mice were deeply anesthetized with ketamine 80 mg
 kg<sup>-1</sup> and xylazine 10 mg kg<sup>-1</sup>
 i.p., and transcardially perfused with formalin (Sigma) 3–4 weeks after
 the surgery to assess the viral expression. Brains were dissected and kept
 overnight in formalin at 4°C and then placed in PBS solution until
 slicing. Coronal brain sections of 50 or 90 μm were made using a vibratome
 (Leica VT 1000S), then dried and mounted with Mowiol (Sigma) or
 Fluoroshield with DAPI medium (Sigma). The slices were analyzed and imaged
 using a confocal microscope (Leica TCS Sp8). The same
 procedure was used to verify electrode and optic fiber implantation sites
 and viral expressions for each experiment, mice were perfused as detailed
 above, and mice with no viral expression or misplacement of the viral
 infusion, optic fiber or electrode were excluded from the analysis.
 Electrode placements are summarized in Supplementary Figure 6.
 4. Immunohistochemistry For the
 immunostaining, 50 µm slices containing MD were washed with PBS-0.3%
 Triton X-100, and blockage of nonspecific sites was assessed by 2 h of
 incubation with 3% normal donkey serum (NDS). Sections were then incubated
 in a solution containing monoclonal mouse anti-Calbindin-D-28K (1:300,
 Sigma, C9848) in PBS-0.3% Triton X-100 with 1.5% NDS at 4°C for 24 h.
 After first antibody incubation, slices were rinsed and sections were
 incubated at room temperature for 2 h with secondary antibody, donkey
 anti-mouse IgG conjugated to Alexa Fluor 488 (1:300, Invitrogen). Slices
 were mounted with Mowiol and analyzed with the confocal
 microscope. 5. Fear conditioning and extinction
 protocol Previous to behavioral experiments, mice were
 handled daily during one week. Fear conditioning and extinction protocol
 were performed in two different chamber configurations (Context A or B),
 placed inside of a sound-attenuating box (Ugo Basile). The fear
 conditioning was performed in Context A, which consisted of a
 rectangular-shaped Plexiglas chamber, 17x17x25 cm (L x W x H) dimension,
 with a grid floor and black and white checker walls, scented with
 peppermint soup. On fear conditioning day (day 1), mice were left for 5
 min to habituate to context A, then they were exposed to 5 presentations
 of a tone (30 s, 80 dB, 2.7 kHz, representing the CS), that co-terminated
 with a mild foot-shock (0.5 sec, 0,4 mA, representing the US), and with an
 interval between CS-US presentations of 120 sec. During the following 3
 days, mice underwent the fear extinction training, which consisted in 3
 days of extinction sessions (EXT1-3). Extinction sessions were performed
 in Context B, composed of a yellow cylindrical chamber (17 cm diameter, 25
 cm H), grey Plexiglas floor, and vanilla scented.  Each extinction session
 started with 5 min of habituation period in the novel context B, followed
 by 25 consecutive un-reinforced 30s-long CS presentations, with an
 interval between CS of 30 s. Mice were video-tracked
 using a high-definition video camera, positioned above the testing
 chambers. Stimuli administration was controlled by the EthoVision XT 14
 software (Noldus Information Technology), which assessed also the freezing
 behavior during the experiments as inactive periods (the threshold of
 inactivity was set to not detect the breathing movements as active
 periods). All mice were returned to their home cage after the experiments.
 Analysis of freezing levels in each session was performed by comparisons
 between groups of the first CS and CS5 for FC, and averaged of CS1-CS5
 representing “early” EXT, CS10-CS14 as “middle” EXT, and CS21-CS25 as
 “late” EXT. 6. Anxiety tests Open
 field, Elevated plus maze and Dark-light box tests were performed in order
 to analyze anxiety-like behavior. One week before subjecting the mice to
 fear conditioning, they were subjected on successive days to anxiety
 tests, starting with the less anxiogenic, open field test, followed by
 Elevated plus maze and Dark-light box. Open field test consisted in a 38
 cm diameter circular arena (Noldus), under 40-50 lux luminosity. Each
 mouse was placed in the center of the arena and allowed to explore freely
 for 10 min. Frequency of entries to the center of the arena, time spent in
 center, and distance moved in center were measured. Elevated plus maze
 test was realized using an elevated (52 cm above the floor), plus-shaped
 apparatus (Noldus) with 2 open arms (36x6 cm L x W) and 2 closed arms
 (36x6x25 cm L x W x H), under 40–50 lux luminosity in the open arms. Mice
 were left to freely explore the arena for 5 min, while assessing the
 frequency of entries in open and closed arms, time spent in open arms, and
 total distance moved. In the dark-light box test, each mouse was placed in
 the light zone (luminosity 500–600 lux, 40x20x20 cm L x W x H dimension)
 of the apparatus (Noldus) and left to freely explore for 5 min, while
 assessing the frequency of entries in light zone, time spent in light
 zone, latency to enter in dark zone (0 lux, 20x20x20 cm L x W x H
 dimension), and total distance moved. Mice were video tracked, and
 behavior was analyzed using EthoVision XT 14. 7. Hot
 plate and tail immersion tests Pain sensitivity was
 analyzed using hot plate and tail immersion assays performed two weeks
 after the end of the other experiments. Mice were placed on a hot plate
 (55°C) (Harvard apparatus), until they jumped or licked their hind paw.
 The latency to the first reaction was measured. After the hot plate test,
 tail immersion test was performed. The mice tale tips were immersed in hot
 water (50°C) and the latency of the tail flick or withdrawal was measured.
 All experiments were video tracked. 8.
 Chemogenetics Specific chemogenetic inhibition of the
 FN neurons projecting to MD was carried out by bilateral injection of 250
 nl of a retrograde virus expressing a cre-recombinase in the MD
 (CAV2-cre-GFP, from Platforme de Vectorologie de Montpellier) in
 combination with the infusion of 200 nl of inhibitory cre-dependent DREADD
 (AAV‐hSyn‐DIO-hM4Di-mCherry, Addgene) bilaterally in FN, adapted from
 previous works in our team, which demonstrated a strong reduction in
 cerebellar nuclei firing rate upon injection of CNO (1mg/kg). The
 behavioral experiments started 10–14 days after the surgery, to ensure the
 expression of the receptors and the recovery of the mice. Transient
 inhibition of the FN MD-projecting neurons was assessed by intraperitoneal
 administration of the clozapine N-oxide (CNO, Tocris Bioscience) dissolved
 in saline solution (1.25 mg/kg), while a control group received only
 saline administration, 30 min before the first 2 extinction sessions (EXT1
 and EXT2) or 30 min before anxiety or nociceptive tests.
 Additional controls were performed to verify the specificity of
 the pathway inhibition and the DREADD-modulation effect. To corroborate
 that the CNO does not have an effect <em>per se</em> in our
 experimental conditions, a batch of mice (named “Sham” mice) underwent the
 same surgical procedure with the injection of AAV‐hSyn‐mCherry instead of
 the DREADD vector. During behavioral experiments, the Sham mice received
 either an intraperitoneal injection of saline or CNO solution (1.25
 mg/kg), 30 min before the tests, and freezing levels were analyzed in
 absence of DREADD expression. On the other hand, to verify that the effect
 observed under DREADD inhibition was specific of the FN terminals in MD,
 mice were implanted with 30G cannulas bilaterally in the MD to allow the
 local infusion of 200 nl of CNO or saline intracranially. During
 extinction sessions, mice expressing inhibitory DREADD received either
 sterilized filtered PBS or CNO (0.5 mM) infusion, and another group of
 Sham mice received CNO (0.5 mM). The intracranial infusions were performed
 10 min before the beginning of each session, by using a pump at an
 infusion speed of 100 nl/min, and a total volume of 250 nl. Freezing
 levels were analyzed and the positions of the cannulas were corroborated
 histologically. 9. Electrophysiological
 recordings Extracellular recordings were assessed
 using standard 16 channels electrode interface boards (EIB-16; Neuralynx),
 to which attached 2 cannulas were attached, one for each recorded region,
 MD and dmPFC. The electrode bundles consisted of nickel chrome wires (16μm
 diameter, Coating ¼ Hard PAC, KANTHAL Precision Technology) twisted in
 groups of 6, gold-plated to 100–400 kΩ (cyanure-free gold solution,
 Sifco). Two bundles were inserted per cannula (stainless steel, 30 Gauge,
 Phymep). The recording electrodes were then progressively lowered until
 they reached the targeted brain structures and the electrode interface
 boards were cemented to the skull (Dental Parkell Adhesive Resin Cement
 Super-Bond C&B). Miniature stainless steel screws were implanted
 on the left parieto-occipital suture, serving as electrical reference and
 ground. The skin ridges were sutured and mice were allowed to recover in
 their home cage for at least 10 days. 10. Optogenetics
 and electrophysiological recordings The neural
 activity in the MD and dmPFC was recorded during the optogenetic
 activation of the contralateral FN neurons. Expression of channelrhodopsin
 2 (ChR2) was assessed by injection of an anterograde
 AAV8-Syn-ChR2-H134R-EYFP (Addgene) in the FN, and an optical was fiber
 implanted above (0.22 aperture, fixed in a stainless steel ferula, Thor
 Labs) and contralateral to the recording sites. Electrodes were placed
 through cannulas in the MD and dmPFC and the EIB were fixed to the skull.
 The optical stimulations were performed three or four weeks after the
 surgery (to ensure the ChR2 expression) in freely moving mice. After a 5
 min habituation period in an open field (38 cm diameter), the activation
 of the FN projections was done using light pulses of 100 ms, at 0.25 Hz, 1
 mW, for 30 min (with a 2 min break every 10 min block). The choice of
 light intensity to stimulate FN neurons was based on the calibration
 performed in our previous work. The activation of MD and dmPFC neurons by
 optogenetic stimulation of FN neurons was assessed by computing the
 Peri-Stimulus Time Histogram (5 ms bins) around the optogenetic
 stimulation. This PSTH was then normalized using a classical Z-score
 (subtracting by the mean value of the PSTH during the 100 ms before the
 stimulation and dividing it by its standard deviation). Normalized PSTH
 reaching an absolute Z-score value superior to 3 during the 100 ms
 stimulation were considered responsive. In another
 group of mice, the specific activation of the FN-MD pathway was assessed
 by injecting a cre-dependent anterograde adenovirus expressing ChR2
 (AAV-Dio-ChR2-EYFP, Addgene) in the FN, together with a retrograde
 adenovirus expressing cre-recombinase (AAV-Cre-mCherry, Addgene) in the
 contralateral MD (left side). The implantation and surgical procedures
 were identical to the ones described above. The FN MD-projecting neurons
 were stimulated by pulses of 10 ms, at 0.5 Hz and 1 mW, during 10 min.
 Luminous stimuli were administered and neural signals were recorded using
 Tucker Davis Technology System 3 acquisition system (25 Hz sampling rate,
 RZ2, RV2, Tucker-Davis Technologies) and the spike sorting was performed
 using Matlab scripts. Viral infusions and implant positions were confirmed
 histologically when the experiment ended. Analysis of the spike-sorted
 data was performed using Python. The specific activation of MD and dmPFC
 neurons by optogenetic stimulation of FN MD-projecting neurons was
 assessed by computing the Peri-Stimulus Time Histogram (5 ms bins) around
 the optogenetic stimulation. This PSTH was then normalized using a
 classical Z-score (subtracting by the mean value of the PSTH during the
 100 ms before the stimulation and dividing it by its standard deviation).
 Normalized PSTH reaching an absolute Z-score value superior to 3.5 were
 considered responsive. In order to assess the validity
 of the optostimulation experiments, we performed a control experiment to
 verify that FN illumination in absence of ChR2 expression does not affect
 spike firing in the areas recorded. We implanted an optic fiber in the FN
 together with electrodes in FN, MD and dmPFC, in mice injected with
 retrograde AAV-GFP (without the expression of ChR2) in the MD, and
 performed 100 ms illuminations at 0.25 Hz, 1 mW, for 30 min (with a 2 min
 break every 10 min block), while recording in the FN, MD and dmPFC
 (Supplementary Fig. 2A-B). There was no variation of firing rate at the
 population level in the FN, MD and dmPFC (Supplementary Fig. 2C), although
 we observed 1 neuron in the FN and 3 neurons in the MD reaching the
 threshold of significant variation of firing rate during the 100 ms of
 illumination (Supplementary Fig. 2D). However, the transient nature of the
 mild increase in firing rate suggests a coincidental classification as
 responsive cells. Since the wireless transmitter used
 in the electrophysiological experiments represented a significant
 hindrance for the movement and was associated with atypical immobile
 postures (nose down and/or tilted head to rest the preamplifier on the
 floor or against the wall) which could indifferently reflect freezing or
 resting, we could not score freezing in these mice.
 11. Chemogenetics and electrophysiological recordings
 Mice expressing cre-dependent inhibitory DREADDs in
 FN MD-projecting neurons (see chemogenetics section) were implanted with
 electrodes in the MD and dmPFC (as described in Electrophysiological
 recordings section). The neuronal activity of the MD and dmPFC was
 examined during FC and extinction learning, under chemogenetic inhibition
 of the FN-MD projections during EXT1 and without manipulation during EXT3.
 After experiments were concluded, viral expression and electrode positions
 were confirmed by histology. 12. Quantification and
 statistical analysis Behavioral data
 analysis For freezing analysis, the data were analyzed
 by repeated-measure ANOVA computed with R (<em>lme</em>
 package version 3.1-3), and posthoc tests were performed with the package
 <em>emmeans</em> (version 1.7.0). During extinction, Early,
 Middle, and Late values of freezing respectively correspond to the average
 of the 5 first, 5 middle, and 5 last CS freezing scores. For other
 behavioral measures, t-test were used (Graph Pad Prism® version 5). All
 tests used are two-sided (when applicable).
 Electrophysiological data analysis Burst
 analysis We quantified the occurrence of bursts in MD,
 detected using the Robust Gaussian surprise algorithm 1. Spike trains were
 processed in the following way: Inter-Spike Intervals (ISI) were
 calculated, then transformed to log (ISI). A central set of ISI was
 defined as the portion of this distribution lying under [E-1.64*MAD;
 E+1.64*MAD], where E is the midpoint of top and bottom 100*p percentile of
 the log (ISI)s with p = 0.05. The central location of this distribution
 was defined as the median of the central set previously mentioned, and the
 distribution of log (ISI) was then normalized by subtracting the central
 location. This normalization process was performed on the entire spike
 train using a sliding window of half-width 0.2*N/2, where N is the number
 of spikes in the spike train. The burst-threshold is set as 0.5 percentile
 of the central distribution. Bursts seeds were defined as normalized ISI
 being below the burst-threshold. Those seeds were then extended by
 recursively trying to add the previous and the next normalized ISI; if the
 addition led to an increase of the associated p-value, the concatenation
 process was stopped, otherwise, it was continued. Overlapping bursts
 strings were cleaned by keeping the strings with the lowest associated
 p-value, thus making them mutually exclusive. Burst occurrence was
 computed as the number of bursts strings divided by the duration of the
 recorded spike trains. The average burst firing rate was calculated as the
 number of spikes contained within a burst divided by the total duration of
 burst strings in the spike train. Local field
 potential, spectral analysis and phase locking In
 order to process the LFP, the raw electrophysiological traces sampled at
 25 KHz were filtered between 1 Hz and 200 Hz, then downsampled to 1 kHz.
 All signals were filtered using the <em>filtfilt</em> function
 from the package <em>scipy</em>, with zero-phase distortion
 5th order Butterworth filters. Power Spectral Densities (PSD) were
 computed using the welch function from the <em>scipy</em>
 package. The fraction of the PSD representing 4Hz oscillations was defined
 the integral of the PSD in the 4Hz range (2-6Hz) divided by the integral
 of the PSD from 1 to 100 Hz. To assess the relationship between LFP in the
 dmPFC and in the MD, Generalized Partial Directed Coherence analysis was
 performed on the processed LFP traces using the package
 spectral_connectivity from Eden Kramer Lab (https://github.com/Eden-Kramer-Lab/spectral_connectivity). For the specific analysis of 4 Hz LFP, the processed LFP traces were filtered in the desired frequency band (2-6 Hz) using the <em>filtfilt</em> function from the <em>scipy</em> package, with zero-phase distortion 5th order Butterworth filters. To study the relationship between the timing of MD neuronal activity and the dmPFC 4 Hz oscillations, Hilbert’s transform was performed using the function <em>hilbert</em> from the package <em>scipy</em>, allowing the extraction of the instantaneous phase and amplitude of envelope estimated at every sample point of the signal. Phases were expressed in radians, with a phase of 0 corresponding to a negative peak in the 4 Hz LFP. Phase locking analysis of bursting activity was performed by considering the first spike of each bursts. Considering spikes occurring in periods associated to weak 4 Hz oscillations would result in the inclusion of uninformative phases in the analysis. So, in order to prevent this bias, only burst occurring in periods of high 4 Hz were considered. Periods of high 4 Hz were defined as periods were the amplitude of envelope was superior to the median amplitude of envelope during the baseline before the Extinction session plus one median absolute deviation (MAD). In other words, we considered the periods where the Robust Z-score of the amplitude of envelope was superior to 1, creating a spike train corresponding to MD bursting during periods of high 4 Hz oscillations in dmPFC. Von Mises distributions fit on phase distributions were performed using the function <em>vonmises</em> from the package <em>scipy</em>, allowing the computation of the parameter Kappa, indicative of the concentration of a circular distribution. Rayleigh tests and circular V-tests with a preferred direction of pi were performed using the package <em>pingouin</em>, respectively using the functions <em>circ_rayleigh</em> and <em>circ_vtest</em>. In order to assess the relationship between MD bursting and the coherence between dmPFC and MD LFP, we used the function <em>spectral_connectivity</em> included in the package <em>mne</em>, For this, LFP snippets of 2000ms centered on the bursts of each individual MD neurons were extracted from the channels displaying the highest fraction of the PSD corresponding to 4 Hz oscillations in the MD and in the dmPFC, and the coherence between MD and dmPFC LFP was calculated using Morlet wavelets of the first order, yielding a time frequency coherence analysis centered on MD bursts.