The pivotal role of the periaqueductal grey (PAG) in fear learning is reinforced by the identification of neurons in male rat ventrolateral PAG (vlPAG) that encode fear memory through signalling the onset and offset of an auditory-conditioned stimulus during presentation of the unreinforced conditioned tone (CS+) during retrieval. Some units only display CS+ onset or offset responses, and the two signals differ in extinction sensitivity, suggesting that they are independent of each other. In addition, understanding cerebellar contributions to survival circuits is advanced by the discovery that (i) reversible inactivation of the medial cerebellar nucleus (MCN) during fear consolidation leads in subsequent retrieval to (a) disruption of the temporal precision of vlPAG offset, but not onset responses to CS+, and (b) an increase in duration of freezing behaviour. And (ii) chemogenetic manipulation of the MCN-vlPAG projection during fear acquisition (a) reduces the occurrence of fear-related ultrasonic vocalisations, and (b) during subsequent retrieval, slows the extinction rate of fear-related freezing. These findings show that the cerebellum is part of the survival network that regulates fear memory processes at multiple timescales and in multiple ways, raising the possibility that dysfunctional interactions in the cerebellar-survival network may underlie fear-related disorders and comorbidities. Anxiety disorders are a cluster of mental health conditions characterised by persistent and excessive amounts of fear and worry. They affect millions of people worldwide, but treatments can sometimes be ineffective and have unwanted side effects. Understanding which brain regions are involved in fear and anxiety-related behaviours, and how those areas are connected, is the first step towards designing more effective treatments.A region known as the periaqueductal grey (or PAG) sits at the centre of the brain’s fear and anxiety network, regulating pain, encoding fear memories and responding to threats and stressors. It also controls survival behaviours such as the ‘freeze’ response, when an animal is frightened.A more recent addition to the fear and anxiety network is the cerebellum, which sits at the base of the brain. Two-way connections between this region and the PAG have been well described, but how the cerebellum might influence fear and anxiety-related behaviours remains unclear.To explore this role, Lawrenson, Paci et al. investigated whether the cerebellum modulates brain activity within the PAG and if so, how this relates to fear behaviours. Rats had electrodes implanted in their brains to record the activity of nerve cells within the PAG. A common fear-conditioning task was then used to elicit ‘freeze’ responses: a sound was paired with mild foot shocks until the animals learned to fear the auditory signal. In the rats, a subset of neurons within the PAG responded to the tone, consistent with those cells encoding a fear memory. But when a drug blocked the cerebellum’s output during fear conditioning, the timing of the PAG response was less precise and the rats’ freeze response lasted longer.Lawrenson, Paci et al. concluded that the cerebellum, through its interactions with the brain’s fear and anxiety network, might be responsible for coordinating the most appropriate behavioural response to fear, and how long ‘freezing’ lasts.In summary, these findings show that the cerebellum is a part of the brain’s survival network which regulates fear-memory processes. It raises the possibility that disruption of the cerebellum might underlie anxiety and other fear-related disorders, thereby providing a new target for future therapies. The periaqueductal grey (PAG) lies at the core of central networks that coordinate survival, including coping behaviours mediating defensive and fear-evoked responses. Neurons in the functionally distinct longitudinal columns of the PAG coordinate different aspects of survival behaviours. Of particular relevance to the current study, fear-evoked freezing is mediated by neurons in its ventrolateral sector vlPAG; . Fear-reCentral nervous system survival networks involving the PAG are well documented . Until rThe cerebellum is reciprocally connected to many brain regions associated with survival networks , and in An emerging concept is that the cerebellar vermis and its output nucleus MCN are involved in the control of an integrated array of fear-related functions, including fear memory and fear-induced behaviours such as freezing and their extinction . ImportaThe experiments reported here used a combination of electrophysiological, behavioural, and interventionist approaches in an auditory cued fear conditioning paradigm in rats to interrogate the role of MCN interactions with the PAG in the expression of fear-related behaviours. Our main findings are that (i) PAG encodes temporally precise information about the onset and offset of a fear-conditioned auditory stimulus and that these two neural signals may be generated by independent mechanisms; (ii) both onset and offset responses correlate with fear-related freezing behaviour; (iii) MCN is part of a larger circuit that regulates the temporal accuracy of PAG encoding of fear-conditioned stimulus offset but not onset during retrieval of a conditioned response; and (iv) MCN is involved in distinct aspects of survival behaviour at different times during fear conditioning: during acquisition, the emission of USVs and subsequent rate of extinction of conditioned freezing during retrieval. While during consolidation its influence on survival circuits affects the duration of conditioned freezing in subsequent retrieval. In summary, the present study provides evidence that the cerebellum through its interactions with survival circuits regulates the ability of the PAG to encode a fear memory trace with temporal precision and also regulates the timing of appropriate patterns of behaviour during fear acquisition and retrieval.As a first step, an auditory cued fear conditioning paradigm  was usedA total of 32 vlPAG units  were recorded during habituation . The majWe recorded the activity of 50 vlPAG units  during acquisition where the CS tone was paired with the unconditioned stimulus (US footshock at tone offset ). Some oDuring retrieval and extinction of the conditioned response, we recorded a total of 55 vlPAG units . Retrieval is defined here as early extinction (EE) training when the unreinforced CS+ reliably elicits a conditioned response. In retrieval, 29 units  displayed a transient increase in activity during presentation of the unreinforced conditioned stimulus CS+, . In keepOf the remainder of the sample of vlPAG units recorded in control animals during EE, 22 units (40%) demonstrated no significant change in firing rate and were therefore defined as type 2 . Four unt-test; type 1 offset units, t-test). For type 1 onset responses, this reduction was not evident for mean peak z-score (t-test) but was evident for type 1 offset responses (t-test). This distinction between onset and offset responses provides additional evidence that they may be evoked by separate mechanisms.Since the majority of responsive vlPAG units recorded in control experiments were type 1 (i.e. displayed an increase in firing rate), the following analysis is confined mainly to consideration of their activity during fear-conditioned retrieval and extinction. The majority of type 1 onset units  and type 1 offset units  showed a significant reduction in responsiveness during extinction training, as measured by the integrated area of response during CS+ trials in EE versus late extinction . These results are therefore consistent with vlPAG activity signalling the conditioned tone offset rather than a prediction of the timing of the US.For the results described above, a delay classical conditioning paradigm was used where the US was timed to occur at CS offset . We alsoIt was also of interest to consider any changes in neural population activity during extinction training. To assess such changes, auditory event-related potentials (ERPs) were recorded simultaneously from the MCN and vlPAG, following a delay conditioning paradigm . However, latency to peak was not significantly different  = 1.418, p=0.215, paired t-test). At CS+ offset, the ERP in the vlPAG was also significantly shorter in latency than the ERP in MCN  = 2.991, p=0.0304; paired t-test), while latency to peak was similar  = 1.746, p=0.141, paired t-test). The difference in onset latency between ERP responses recorded at CS+ onset and offset within both vlPAG and MCN was statistically significant  = 4.720, p=0.0008, unpaired t-test; MCN onset vs. offset, t(10) = 4.319, p=0.0015, unpaired t-test). These findings indicate that both MCN and vlPAG receive auditory inputs that convey information about conditioned tone onset and offset. Given the likely differences in central pathways involved, the disparity in latency of the ERPs recorded within the two brain regions is perhaps unsurprising  = 4.33, p=0.007, paired rprising . Howevert-test; vlPAG: EE ERP 501.1.2 ± 104.7 mV vs. LE ERP 418 ± 84.08 mV, t(5) = 1.122, p=0.313, paired t-test). By comparison, ERPs recorded at CS+ offset in the vlPAG (t-test), while in the MCN there was no significant decrease  = 2.258, p=0.074, paired t-test). The differences found in vlPAG between onset and offset ERPs provide further evidence that they are generated and regulated by different neural pathways.During extinction training, the peak-to-peak amplitude of the ERPs recorded in the MCN and vlPAG at CS+ onset did not show a statistically significant difference .To assess if changes in ERP amplitude during extinction covaried between MCN and vlPAG, we compared average ERP amplitude at CS+ onset  and CS+ Taken together, these ERP data therefore suggest that population activity in MCN and vlPAG broadly parallels the changes that occur in vlPAG single-unit peak activity at onset and offset of the CS+ during extinction of a fear-conditioned response.In eight additional animals, a delay conditioning paradigm was used see , and must-test). In keeping with the findings from control animals, no significant difference was found for mean peak z-score (t-test). There was also no statistically significant difference between control and muscimol animals for type 1 CS+ onset responses in EE or LE for either measure of response  = 0.949, p=0.351, unpaired t-test; mean peak z-score in EE, t(26) = 0.103, p=0.919, unpaired t-test; mean peak z-score in LE, t(26) = 0.454, p=0.654, unpaired t-test), nor was there a difference in overall firing rates for all units during extinction training  = 0.407, p=0.685, unpaired t-test; In the four muscimol animals with tetrodes, a total of 14 type 1 vlPAG units were recorded from three animals during extinction. Units were recorded from the fourth animal but were not type 1 responses so were not analysed further. Similar to the control results described above, the majority  of available type 1 onset units  showed at-test). There was also a reduction in mean peak z-score at EE (t-test). In LE, the mean integrated area of response was also significantly different. On average, muscimol experiments had a significantly increased area of response than control animals . Similar to CS+ onset, the mean peak z-score in LE was not significantly different between muscimol and control experiments (t-test).By comparison to controls (t-test) nor latency to peak (2(4) = 2.50, p=0.645, chi-square), with both groups having about 85% of peaks occurring in the 200 ms immediately following CS+ onset.To investigate further the difference in pattern of neural response between muscimol and control animals, we plotted for each unit the number of significant peaks of activity in the first 500 ms following CS+ onset and CS+ offset . After CS+ onset, there was no significant difference in total number of peaks between muscimol and control animals (t-test) and latency (2(4) = 13.31, p<0.01, chi-square) of peaks significantly increased during EE in muscimol animals. In LE, there was no increase in the number of peaks (t(22) = 0.019, p=0.98). In control animals, 82% of peaks occurred in the 200 ms immediately following the CS+ offset, while only 20% of muscimol animals had peaks during the same time period.By contrast, following CS+ offset both the number , extinction rate during the CS+ and ITI, and the duration of freezing and movement epochs.t-tests; for trial-by-trial variation, see t-test), nor was there a difference detected in rate of extinction of freezing (t-test). In terms of freezing behaviour during the ITI, both control and muscimol animals showed extinction learning similar to that found during presentation of the CS+ . Nor was there a significant difference in the rate of extinction between groups (t-test).During presentation of the CS+, both control and muscimol animals showed extinction of the conditioned freezing response between EE vs. LE , there was no difference in the duration of freezing or movement epochs between control and muscimol animals  s, median ± interquartile range [IQR], n = 8 and muscimol 4.5  s, median ± IQR, n = 8, U = 19, p=0.195, Mann–Whitney test; movement duration for control 7.3  s, median ± IQR, n = 10, and muscimol 7.7  s, median ± IQR, n = 8, U = 38, p=0.877, Mann–Whitney test). However, upon presentation of the CS+, there was a significant increase in the duration of freezing epochs in muscimol compared with control animals in both EE and LE . A significant positive correlation was also present between unit response at CS+ offset and percentage time spent freezing during ITI for both control and muscimol animals .To investigate the extent to which changes in unit activity correlate with fear behaviour during extinction training, the percentage of time spent freezing was compared to the average integrated area of unit responses after CS+ onset and also for the ITI following CS+ offset. Consistent with previous reports , at CS+ To investigate whether the extent of change in freezing behaviour and unit response during extinction training were related, a comparison was made between the absolute change in freezing for each animal with the mean absolute change in unit response. No significant relationship was found between freezing and unit response area at CS+ onset, nor for freezing during the ITI and unit response area at CS+ offset . Taken tTo investigate whether MCN inactivation during consolidation had an effect on other defence-related behaviours, rearing activity and USVs were monitored during presentation of the CS+ and ITI during extinction training. However, by comparison to controls, no statistically significant effects were observed in either type of behaviour in muscimol animals .Given that during extinction training (i) population activity in MCN resembles changes in vlPAG population and unit activity, and (ii) the finding that global inactivation of MCN during consolidation can lead to changes in the fear network that disrupt encoding in vlPAG, it was of interest to determine whether the behavioural effects described above were dependent on a direct projection between the two brain structures . As a fiTo investigate the function of this direct MCN-PAG projection, a viral vector encoding DREADD hM4D(Gi) -mCherry, termed DREADD, see Materials and methods) or a control virus  was injected bilaterally into the MCN. Terminal projections containing either virus were targeted by implanted cannulae in the PAG to deliver clozapine N-oxide (CNO)  and the ITI (t-test); for trial-by-trial variation, see t-test) and the ITI  = 3.950, p=0.004; DREADD, t(9) = 8.679, p<0.0001, paired t-tests). However, the rate of extinction during the CS+ was significantly slower in DREADD animals (t-test), but not during the ITI (t-test). The latter finding suggests that the effect is not a general disruption of the expression of freezing behaviour.During extinction training, control and DREADD animals showed similar levels of freezing during presentation of the CS+ (t-test), nor across all extinction training (t-test). Similarly, there was also no significant difference in the total number of USVs per animal in DREADD versus control groups across all extinction training  were also tested on the following tasks after CNO infusion into vlPAG: elevated plus maze, open field, and beam walking. By comparison to controls, no statistically significant difference was found in any of these behaviours  Thus, not-test).In all available animals , we therefore made an intraperitoneal injection of CNO in order to test for non-specific effects, including general action on MCN and its projections. Consistent with the muscimol experiments, in every DREADD case, the CNO produced ataxia, although this was generally less severe and shorter lasting (~1 hr) than was observed in the muscimol animals. Impairments in motor behaviour were assessed by testing the animals on the beam walking task; DREADD animals were significantly slower than control animals at traversing the beam (We have shown that vlPAG neurons (type 1 units) encode temporally precise information about both the onset and offset of a fear-conditioned auditory stimulus and that these two neuronal signals may be generated by independent mechanisms. This is because during extinction (i) some vlPAG units only respond to CS+ onset or only to CS+ offset; (ii) unit onset and offset responses exhibited different characteristics during extinction training; (iii) MCN inactivation during consolidation disrupted the vlPAG pattern of unit activity at CS+ offset but not onset; (iv) the latency of the ERP in vlPAG at CS+ onset was significantly shorter than the ERP recorded at CS+ offset; and (vi) the ERP recorded at CS+ onset remained similar in amplitude over extinction training while the ERP at CS+ offset showed a significant reduction.Importantly, vlPAG units displayed little or no response to an auditory tone during habituation but displayed robust activity at tone onset and/or offset when the same tone was classically conditioned. This provides evidence that the responses were related to the associative conditioning rather than the sensory stimulus. Moreover, disruption of the temporal precision of the conditioned tone offset neural response following MCN inactivation during consolidation suggests a role of the cerebellum in the subsequent timing, but not the generation, of the vlPAG offset response. However, the possibility remains that at other times during conditioning  the cerebellum is also involved in generating the response in vlPAG, but this requires further study.More generally, our study applied novel approaches to address fundamental questions about the role of cerebellar-PAG interactions in survival behaviour. As such, consideration of the methods used is critical to the interpretation of the data and is included in the Materials and methods section.Previous inactivation studies in rats using TTX have indicated that the cerebellar vermal cortex is involved in the consolidation of associatively conditioned cue and context-dependent fear memories . This suThe present results are also consistent with In regard to our electrophysiological findings, similar responses to CS+ onset in vlPAG have been reported previously by ourselves  and otheFear-conditioned CS+ onset responses in vlPAG are thought to be generated, at least in part, as a result of preceding activity in the amygdala  and may In terms of previous reports of CS+ offset responses in vlPAG, An unanswered question concerns the origin of CS+ offset responses in vlPAG. We estimate that they occur approximately 30 ms after the end of the CS+ tone. Such a delay provides ample opportunity for many possible pathways to generate them. Detecting the offset (and onset) of sensory events is a fundamental requirement of sensory processing by the CNS. Given the importance in the present experiments of the auditory system in the initial processing of the tone signals used for fear conditioning, one possibility is a route from the auditory cortex to the PAG. Both onset and offset responses have been reported in the auditory cortex elicited by tones and other sounds in a range of species . Also, cA further important question relates to what information CS+ offset responses encode. Learning theory proposes that Pavlovian fear conditioning is instructed by an error signal that encodes the difference between actual and expected intensity of the US . The vlPWe also show that inactivation of the cerebellar output nucleus MCN during consolidation disrupts but does not abolish CS+ type 1 offset responses in vlPAG upon retrieval of the fear memory. This is in line with MCN modulating vlPAG activity , and advA role of MCN in temporal patterning is in good agreement with the timing hypothesis of cerebellar function . This hyCerebellar manipulations can affect emission of USVs . Taken tIn summary, MCN is part of a survival circuit network that regulates the precise temporal encoding of fear memory within vlPAG and also regulates the expression of different survival behaviours depending on the phase of Pavlovian fear conditioning. Inactivation of MCN output during consolidation increases the duration of freezing and movement epochs during extinction, while a direct pathway to the vlPAG appears to be important in eliciting fear-related USVs during acquisition, and the rate of expression of freezing during EE. The cerebellum, through its interactions with the survival network, might therefore be coordinating the most appropriate behavioural response at the most appropriate time.All animal procedures were performed in accordance with the UK Animals (Scientific Procedures) Act of 1986 and were approved by the University of Bristol Animal Welfare and Ethical Review Body (PPL number: PA26B438F). A total of 47 adult male Sprague–Dawley rats  were used in this study. They were housed under normal environmental conditions in a normal 12 hr dark/light cycle and provided with food and water ad libitum. Animals were single housed after surgery to prevent damage to implants.Rats were anaesthetised initially with gaseous isoflurane, followed by intraperitoneal injections with ketamine and medetomidine . Each animal was mounted in a stereotaxic frame with atraumatic ear bars, and surgery was performed under aseptic conditions. Depth of anaesthesia was monitored regularly by testing corneal and paw withdrawal reflexes with supplementary doses of ketamine/medetomidine given as required. A midline scalp incision was made, and craniotomies were performed to gain access to the cerebellum and/or the PAG as required in each line of experiment. Microdrives and/or cannulae were implanted and/or viral injections were made as described below depending on the experiment. At the end of every surgery, the rat was administered the analgesic Metacam  and the medetomidine antidote Atipamezole . Animals were handled for 1 week prior to surgery and during recovery before any behavioural paradigms or electrophysiological recordings were undertaken.Dual microdrive experiments (n = 6 rats). Two in-house-built microdrives, designed to slot closely next to each other, were positioned over craniotomies to allow tetrodes to be independently advanced into the right MCN  and contralateral vlPAG . The microdrives were attached to the skull with screws and dental acrylic cement. Each microdrive contained 3–4 tetrodes for local field potential (LFP) and single-unit recording . (2) Single microdrive experiments (n = 12 rats). These implants were the same as described above except that only one microdrive was implanted to record single units from the vlPAG. In eight of these animals, infusion cannulae were implanted bilaterally to target the MCN  during delay conditioning experiments (details below). The remaining four animals were used in trace conditioning experiments. Single units were recorded from three of these animals.(1) Since similar electrophysiological data see  were recEffects of the muscimol infusion on general motor coordination were carefully monitored. Immediately after the infusion all animals (n = 8 rats) displayed mild to moderate ataxia, providing a positive control that the muscimol was disrupting cerebellar activity. The severity of the ataxia gradually reduced over several hours, and the animals were behaviourally normal after 24 hr.http://n2t.net/addgene:59462; RRID:Addgene59462). Injections of the AAV were made following previously published methods  vector expressing tdTomato under the CAG promoter was injected unilaterally into the MCN . pAAV-CAG-tdTomato (codon diversified) was a gift from Edward Boyden ; and active DREADD, pAAV-hSyn-hM4D(Gi)-mCherry (AAV5) . Using the same techniques as outlined above, animals were injected bilaterally into MCN with 350 nl of either the control (n = 12) or the DREADD (n = 12). In the same surgery, bilateral cannulae  were also chronically implanted with tips  located just above the vlPAG .To assess the proportion of PAG projecting neurons transfected with the DREADD virus, four animals were injected with the DREADD virus in the MCN as described above and with a rAVV-CAG-GFP (AAVr) retrograde virus into vlPAG . Survival time was 3 weeks prior to terminal perfusion and histology (see below).The delay conditioning paradigm was based on This test was used to assess general motor coordination and balance . AnimalsThis test was used to assess both general motor behaviour and anxiety levels. Animals were exposed for the first time to the circular arena on the test day . They were placed at the perimeter of the arena and were allowed to explore for 10 min. Videos were recorded of exploratory behaviour for the whole session, and DeepLabCut  was usedThis test was used as an additional assessment of anxiety . AnimalsMultisite electrophysiological data were recorded using a Blackrock Microsystems (UT) data capture system synchronised with OptiTrak software. Raw data were processed offline to extract single-unit activity and LFPs . Neural data for single units were sampled at 30 kHz and band-pass filtered between 300 Hz and 6 kHz. LFPs were extracted from the data by downsampling to 1 kHz and band-pass filtered at 1–32 Hz.Video recording of animal behaviour during the fear conditioning paradigm was captured with an OptiTrak camera and software, allowing synchronisation with neural data. Fear-related freezing behaviour was manually scored using Solomon Coder software . Freezing behaviour was identified as periods in which the animal had an absence of movement  connected to the Blackrock Microsystems. Although USVs were recorded as an aliased signal , we were able to identify USV events. For analysis, USVs were visualised using Spike7 software (Cambridge Electronic Design Limited) and individual USV emissions manually identified and the total number counted during each recording session.Beam balance, open field, and elevated plus maze tests were recorded via standard webcams linked to OBS .In eight animals, muscimol   was infused via indwelling cannulae at a rate of 0.3 µl/min to target MCN. The infusion was made immediately after acquisition (i.e. during early consolidation). Extinction training was carried out 48 hr after the infusion (day 3).A total of 24 rats were randomly assigned to either a saline control (n = 12) or a DREADD experimental group (n = 12) and coded so the experimenter was blinded. A total of 9 controls and 10 DREADD animals were included in the analysis. The remainder  were excluded because postmortem histology showed off-target cannulae placement.Unblinding occurred once all procedures and analysis were completed. Six weeks after viral transfection (see above), animals were tested in the following behavioural paradigms: auditory cued fear conditioning, beam balance, open field, and elevated plus maze. One animal was excluded from the study after the fear conditioning test because of poor health; a further three animals were excluded from the open field analysis because of technical problems with the video recording. In every animal, a volume of 500 nl of CNO  was infused at a rate of 0.5 µl/min to target the PAG, 15 min prior to each behavioural test . Since the effect of CNO is estimated to last 60–90 min , this meTo verify the physiological activity of hM4D(Gi) receptors following MCN transfection with DREADD, we carried out whole-cell current-clamp recordings from MCN neurons in cerebellar slices obtained from n = 4 rats. Four weeks after transfection, we investigated how CNO altered spontaneous firing rate in a sample of n = 4 neurons. CNO application to the bath caused an increase in the spontaneous firing rate in three neurons (data not shown). The one additional neuron was located in the interpositus nucleus where there was no DREADD expression, and no change in activity was detected. In transfected neurons, the hM4Di-DREADD might be expected to cause a decrease in firing rate by hyperpolarising the membrane potential via G-protein inward rectifying potassium channels. However, To examine the behavioural effect of hM4D(Gi) receptors in the CNS following MCN transfection with DREADD, in awake rats (n = 17) CNO was injected i.p. (2.5 mg/kg) 20 min before the beam balance test. This resulted in temporary ataxia consistent with DREADD expression having a functional effect see .m phosphate buffer) and the brains extracted. After post-fixation, the brains were cryoprotected in 30% sucrose solution. The cerebellum was cut sagittally and the midbrain including the PAG cut coronally into sections of 40 or 60 μm thickness, respectively. To aid verification of electrode and cannula brain loci, sections were stained with cresyl violet.At experimental end points, all animals were deeply anaesthetised  and terminated by transcardial perfusion  was performed. No signal amplification was required for the eGFP controls. Sections were visualised on an Axioskop 2 Plus microscope (Zeiss) and photomicrographs captured using AxioVision software, or with a widefield microscope .Offline spike sorting was carried out using MClust software in MATLAB. Clustering was classified as single unit if L ratio < 0.35, ID > 15, and < 1% of interspike intervals was >2 ms . For an For habituation, acquisition, and extinction training sessions, peri event time histograms  of the activity of individual units were created in NeuroExplorer, with tone onset and offset as time 0. During acquisition, the footshock caused electrical interference, so it was not possible to record unit activity during the 0.5 s period of stimulus delivery. The following analysis was performed in MATLAB. In all experimental groups, PETHs of unit activity to the unreinforced conditioned tone (CS+) during extinction training were constructed for individual units over all trials (1–35) and also separately for EE  and LE trials (21–35). PETHs were z-score normalised to a 5 s baseline recording of unit activity immediately before CS+ onset and data grouped according to response type and averaged. A significant response was defined as one or more consecutive 40 ms time bins where the z-score was ±2 SD from baseline mean in the first 500 ms following the tone. The area of the response was calculated as the trapezoidal numerical integration of the first 1 s after tone onset or offset (arbitrary units). Peak z-score was measured for each unit at the time where the average peak response occurred. Total peak number was calculated as the sum of all (non-consecutive) 40 ms time bins where the z-score was ±2 SD from baseline mean within the first 500 ms after tone onset. Any single units in EE or LE that had peaks occurring after 500 ms were excluded from this analysis. The first 500 ms period after tone onset was also divided into 100 ms time bins and the proportion of units with maximal peak response within each bin expressed as a percentage of total number of maximal peaks. Maximal peak per unit was identified as the response with the largest increase relative to baseline mean.Auditory ERPs were extracted by averaging LFP activity in relation to tone onset and offset using MATLAB . The tetrode recording site yielding the largest mean amplitude peak to trough response was identified in each animal and used to calculate group average data of peak amplitude of LFP response recorded in the PAG and cerebellum.t-tests or Wilcoxon test were used for within-group comparisons, while unpaired t-tests or Mann–Whitney test and ANOVA were used to compare groups. Differences were considered significant at p<0.05. Pearson r correlation was used to compare the absolute change in freezing per animal from EE to LE versus the absolute change in unit area response from EE to LE. To investigate the relationship within animals between unit response size and freezing activity and the relationship between MCN and vlPAG ERPs, repeated-measures correlation , while we used the DREADD method to specifically modulate the direct pathway from MCN to vlPAG. Our experimental design therefore allowed a comparison between blocking all MCN pathways and a specific pathway targeting vlPAG. The muscimol was delivered during consolidation, while in the DREADD experiments CNO was delivered just prior to acquisition so was likely to include both a period during and after acquisition  to fear-associated behaviors. The manuscript extends a prior, albeit incomplete, understanding of how the cerebellum mediates aversive conditioning via the PAG. In particular, the authors show that vPAG neurons respond both to conditioned stimulus onset and offset, but that there are likely 3 populations of neurons that participate in this response, Type 1, Type 1 onset, and Type 1 offset. They show that blocking cerebellar nuclear signaling with muscimol delivered to the medial cerebellar nucleus during consolidation preferentially effects the only vPAG signaling at offset. The correlate to this in terms of fear behavior was seen to be an increase in rearing during early extinction, with no changes seen in USV or freezing behaviors. The authors then extend their electrophysiology analysis by showing that at the population level, neurons in both the fastigial nucleus and the vPAG show alterations in firing amplitude at CS offset, but not onset, in late vs early extinction, further strengthening the case of involvement of the cerebellum in this process. Finally, the authors use inactivation of the fastigial to vPAG circuit during the acquisition phase using DREADD technology and show effects on freezing and USV behaviors. The manuscript does an elegant job of defining a key cerebellar pathway involved in fear and succeeds in broadening the role of the cerebellum beyond basic motor function. The main weaknesses of the results are related to the presentation of certain control data and in some cases a need for additional clarity in how the data were interpreted and presented.(1) Experimental design and paradigm: Please include the timing of both DREADD injection and muscimol injection in the experimental design schematic in Figure 1a. Alongside this, there is some concern regarding the apparent break required in the timing of muscimol injection and washout in the experimental paradigm. This extends the paradigm from the 3 days described in methods  to 5 days , day 2 ). Please clarify.(2) There are some discrepancies in the figure legends and the methods regarding the number of controls. In the methods it is stated that there are 4 saline injected controls, while in the figure this number is stated as 8.(3) As the pooled control electrophysiology data (as stated on page 4 lines 5-8) reflects both the 3 and 5 day paradigm, are similar changes seen in EE vs LE in all three groups ? Adding PETH panels similar to Figures2/3 A & B to supplementary figure 2 would be helpful in this regard.(4) vPAG offset responses during trace conditioning: While this is a well-designed control, these data appear too preliminary in their current form. Please increase the sample size.(5) Muscimol effect on rearing behavior: Given the very low number of rearings observed in control animals, saline controls, DREADD, and vehicle animals I am left to wonder whether muscimol itself has an effect on rearing independent of the conditioning paradigm used. Is such data published and could that be cited? If not, what is the explanation for this? Clarification of this point would be useful especially as it is the only behavioral outcome associated with the muscimol paradigm.(6) Please discuss and justify why only male animals were used.(7) Please discuss why there is such a large amount of variability in the control condition as shown in Figure 5B for the inter-CS-US interval graph (bottom right).(8) What is the level of inhibition achieved with the DREADD experiments? That is, after CNO, what does inhibition actually look like at the single cell level and/or at the local population level? What is the efficiency of inhibition after drug delivery?(9) For the supplementary figures , for example supplementary figure 1, please show the actual histology and not just the schematized data. There seems to be some ambiguity in the locations and it is much more informative of the reader has the actual data to examine alongside the functional data.Specific items:1. Line 14: change "rat ventral (vPAG)" to "rat ventral PAG (vPAG)"2. Please clarify whether the vPAG is a subsection of the longitudinal columns of the PAG – some readers may not have a detailed knowledge of this structure.3. Figure 1C: Please make the red bar translucent, it is difficult to tell whether there is a peak being obscured or not.4. Please refrain from using qualitative language where statistics are not done. Instead refer to the actual numbers  use "75% (n=24/32)" or simply "majority"). Similarly regarding "small proportion (18.5%)…".Reviewer #2:This is an interesting study describing the interactions between the cerebellum and periaqueductal grey (PAG) in fear conditioning behavior in rats. To this end the authors first studied the PAG cellular responses during an auditory cued fear conditioning paradigm. Then, they performed global cerebellar inactivation with muscimol after fear conditioning, during consolidation period and they revealed ataxia in all animals and no difference during extinction training except for rearing behavior. They also performed chemogenetic experiments to inhibit cerebello-PAG pathway during acquisition and early consolidation and show effects on extinction rate of fear related freezing and ultrasonic vocalizations. The main findings are (1) a change in PAG response to the offset of the CS and an increase solely in rearing  during the extinction phase following cerebellar inhibition immediately after the conditioning, (2) a reduction of vocalization and rate of extinction during the extinction phase following the selective inhibition of cerebellar terminals in the PAG. The introduction and discussion are clear and well written, the experiments seem carefully performed; yet the results seem a bit descriptive in the current state of the manuscript and the different elements that compose the manuscript are still a bit too loosely connected. There are notably several weaknesses which need to be addressed:(1) In the experiments with muscimol in MCN (page 11), the infusion induced ataxia. This is a severe condition that likely induces intense stress and involves many structures. The interaction with the fear learning could thus result from an interference between the newly-formed memory and a muscimol-induced fear/stress. An effort to control for this effect would be needed to discard this alternate cause .(2) The results of figures 2 and 3 are presented in parallel, in a way that needlessly complicates the reading. The CS+ onset and CS+ offset neuronal responses are presented together with respectively the fear responses during and between the CS in separate figures but the link between the electrophysiological and behavioral responses is correlative (see point 4 below). A better presentation would be to provide separate figures for the electrophysiology and the behavior as in the text (or change the text).(3) The behavioral analysis with n=4 rats (muscimol) does not have much sense given the variability of the behavior(s). More animals are needed for the behavior.(4) The presence of a correspondence between the changes in electrophysiological and behavioral responses in each animal (explored by pairwise correlations) would reinforce the observations: are the animals with the weakest extinction showing weaker changes in the electrophysiological responses? .(5) The physiological relevance of the neuronal discharge at the CS offset (where a cerebellar contribution is suspected) remains undefined. The experiments for vPAG offset responses during trace conditioning (page 11) is very interesting in light of the characterization of the offset response. Yet, this result is too anecdotal  and not fully analyzed (no quantification for the EE and LE). The manuscript would benefit greatly from a proper demonstration of this response.6) The ERP experiment is suffering from two limitations: (1) it is unclear how the LFP relates to the neuronal activity vs afferent activity; it would be much helpful to show using the available PAG recordings how the LFP relates to the multiunit activity. (2) the number of animals is too limited for the analysis ; testing the covariance of MCN and PAG ERPs for each rat could help circumvent the limited number of animals . More animals should be added otherwise.7) It is difficult to judge from the specificity of the action of CNO; some cannulas  seem off-target : were these experiments included in the analysis? Terminal labeling has been found in the region bordering the vlPAG: how can the authors be sure that the behavioral effects are not due to an action on these terminals outside of the PAG? The diffusion of CNO needs to be documented to interpret these experiments.8) If I understand correctly, the extended response to the CS-offset  is interpreted as poor temporal patterning (discussion p22-23); the average trace indicates longer responses to CS+ offset after muscimol during consolidation; are these responses less precisely associated to the end of the CS ? Because the traces are only presented as a grand average, it is not possible to see whether some responses become poorly correlated to the end of the CS.Reviewer #3:The authors investigated the role of projections from the medial cerebellar nucleus (MCN) to the ventral periaqueductal gray (vPAG) in a conditioned fear learning task in rats, measuring vPAG neuronal activity and behavior before and after inhibiting MCN activity or the MCN-vPAG pathway. Inactivation of the MCN and the MCN – vPAG projections both affect fear learning related neuronal activity and behavior but in different ways. MCN inactivation results in a reduced neuronal response at the end of the conditioned tone stimulus, and also more frequent rearing and more frequent ultrasonic vocalizations during extinction. Neither manipulation changed the expression of fear as measured by freezing behavior, or the rate of fear extinction. Several statements in the manuscript are not supported by the results, such as the statement that the timing of neuronal activity signaling the end of the conditioned stimulus is altered by inhibition of the MCN or its projections.The differences between the observed effects of the two manipulations (MCN or pathway inhibition) on fear learning may be due to the differences in the method and timing of inactivation. MCN is inactivated during consolidation. The MCN vPAC pathway is inhibited during fear acquisition, with inhibition possibly extending into early consolidation. MCN inhibition is through muscimol, pathway inhibition through use of and DREADDs. These differences introduce unknown variability into the results.There is no description of measurements to determine how effective the inhibition of the MCN or the vPAC was, but interpretation of results is under the assumption that it was complete or near complete. While a systemic injection of CNO will deliver clozapine to all DREADD receptors , it does not inform about the percentage of projecting axons that express the designer receptor. It would have been reassuring to see some electrophysiological proof of the success and extent of the inhibition.The authors don't seem to have performed necessary control experiments to exclude potential effects of CNO on endogenous clozapine binding sites in the brain when using systemic CNO injections.The authors state that MCN inhibition alters the temporal precision of vPAG offset activity. Timing as a contribution of the cerebellum to fear learning is mentioned several times. However, there is no actual data supporting a change in timing or temporal precision. The authors don't analyze the temporal precision of the offset response, only the area under the curve and peak amplitude.In the last paragraph of the introduction the authors claim that the rate of extinction is affected by MCN inhibition. There are no results to support this. In the same paragraph the authors state that ultrasonic vocalizations (USVs) and rearing behavior during fear acquisition are influenced by MCN inputs to vPAC. However, all results for USV and rearing counts are shown for the extinction phase.Experimental findings with an n = 1, as the author's experiment with one rat exposed to trace fear conditioning, are not acceptable.Supplementary Figure 1 A is supposed to show electrode tracks for rats implanted with two drives for electrodes. The left cerebellar illustration shows what is declared as canular tracks in the other illustration. Was that by mistake or where cannulas inserted here?The manuscript overstates several findings. For example, the presented findings do not imply that the cerebellum 'controls' any of these behaviors or activity patterns but rather that it is part of a larger circuit that does.The claim that the cerebellum control aspects of timing in this task is not supported by results. The timing of the off response, which is specifically mentioned, needs to be analyzed to support or refute this claim.The trace fear conditioning experiment should be repeated with at least two more animals .Manipulations of MCN activity and of the MCN-vPAG pathway should occur during the same period in the fear conditioning protocol and preferentially by using the same method to ensure similar time courses.Inhibition of the MCN and especially be confirmed electrophysiologically, at least for some examples.Please show some example raw data of your electrophysiological recordings.eLife. Your article has been reviewed by 3 peer reviewers, including Roy V Sillitoe as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kate Wassum as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Detlef Heck (Reviewer #3).Thank you for resubmitting your article "Cerebellar modulation of fear behaviour and memory encoding in the PAG." for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.This study describes interactions between the cerebellum and periaqueductal grey during fear conditioning behavior in rats. The authors have used a combination of electrophysiology, behavioral paradigms, and DREADDs to uncover critical circuits that expand the role of the cerebellum beyond motor function. The results have far reaching implications as they add new context for how inter-regional connections drive complex behaviors and they will likely stimulate new ideas on the circuits that cause defects in neuropsychiatric diseases.Essential revisions:Please see below the specific recommendations by the reviewers. In addition to the points of clarity as requested, please pay particular attention to:1) Cases where animal numbers need to be provided.2) References as requested.3) Details regarding methodology.eLife policies for statistical reporting: https://reviewer.elifesciences.org/author-guide/full "Report exact p-values wherever possible alongside the summary statistics (and degrees of freedom) and 95% confidence intervals. These should be reported for all key questions and not only when the p-value is less than 0.05."4) Please ensure your manuscript complies with the Please also include a key resource table.Reviewer #1:The authors have submitted a revised version of their manuscript that tests the role of the cerebellum in fear memory. The manuscript has been given a major overhaul with revised text, revised figures, and additional data. The authors have done an excellent job in addressing the comments provided by the reviewers on the initial submission.Reviewer #3:The authors have adequately addressed all my concerns.One small final request would be that the authors mention the results from their test of DREADD activation and the related findings by Locke et al. 2018 in the manuscript. This could just be in writing without adding a figure. [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]Reviewer #1:In this study, Lawrenson et al. have investigated the contribution of cerebellar projections to the ventral periaqueductal gray (vPAG) to fear-associated behaviors. The manuscript extends a prior, albeit incomplete, understanding of how the cerebellum mediates aversive conditioning via the PAG. In particular, the authors show that vPAG neurons respond both to conditioned stimulus onset and offset, but that there are likely 3 populations of neurons that participate in this response, Type 1, Type 1 onset, and Type 1 offset. They show that blocking cerebellar nuclear signaling with muscimol delivered to the medial cerebellar nucleus during consolidation preferentially effects the only vPAG signaling at offset. The correlate to this in terms of fear behavior was seen to be an increase in rearing during early extinction, with no changes seen in USV or freezing behaviors. The authors then extend their electrophysiology analysis by showing that at the population level, neurons in both the fastigial nucleus and the vPAG show alterations in firing amplitude at CS offset, but not onset, in late vs early extinction, further strengthening the case of involvement of the cerebellum in this process. Finally, the authors use inactivation of the fastigial to vPAG circuit during the acquisition phase using DREADD technology and show effects on freezing and USV behaviors. The manuscript does an elegant job of defining a key cerebellar pathway involved in fear and succeeds in broadening the role of the cerebellum beyond basic motor function. The main weaknesses of the results are related to the presentation of certain control data and in some cases a need for additional clarity in how the data were interpreted and presented.(1.1) Experimental design and paradigm: Please include the timing of both DREADD injection and muscimol injection in the experimental design schematic in Figure 1a.We thank the reviewer for this helpful suggestion. However, given that Figure 1 relates to non- drug (control) experiments we do not think this is the best place to include time of muscimol injections. Instead, we have included the schematic in Figure 2 and Figure 6 to show time of muscimol and DREADD infusion respectively.Alongside this, there is some concern regarding the apparent break required in the timing of muscimol injection and washout in the experimental paradigm. This extends the paradigm from the 3 days described in methods  to 5 days , day 2 ). Please clarify.We included a 48hr delay to be confident there was complete washout of muscimol and no residual effects on motor performance during extinction training. The experimental timeline was therefore as follows: Habituation = day 0; Acquisition = day 1; Extinction training = day 2 (tetrodes only control) or day 3 . As suggested in point 1.3 we have compared day 2 (tetrodes only control) and day 3  to show this does not have an effect on our results and this finding is included in Supplementary Figure 5.(1.2) There are some discrepancies in the figure legends and the methods regarding the number of controls. In the methods it is stated that there are 4 saline injected controls, while in the figure this number is stated as 8.We thank the reviewer for spotting this discrepancy. We have corrected the text and clarified that there are 10 control animals in total: 6 were animals with dual tetrode implants to record from PAG and cerebellum (tetrodes only control), while 4 animals had tetrode implants in the PAG and cannulae implanted in the cerebellum, in which saline was infused .(1.3) As the pooled control electrophysiology data (as stated on page 4 lines 5-8) reflects both the 3 and 5 day paradigm, are similar changes seen in EE vs LE in all three groups ? Adding PETH panels similar to Figures2/3 A & B to supplementary figure 2 would be helpful in this regard.We have added additional PETH plots to Supplementary Figure 5, showing that results for day 2 data (tetrodes only control) and day 3  are not statistically different from each other. However, both control groups are significantly different from the muscimol group in EE .(1.4) vPAG offset responses during trace conditioning: While this is a well-designed control, these data appear too preliminary in their current form. Please increase the sample size.We have increased the sample size of offset responses in trace conditioning experiments to 12 vPAG units recorded in a total of 3 animals. Consistent with our initial findings the data show that 6 type 1 units (those with increased activity at CS+ offset), are temporally related to the offset of the CS+ during retrieval and not to the time of the expected occurrence of the US (1s after CS+ offset). We also recorded 6 units with type 4 responses (those with reduced activity at CS+ offset) and in trace conditioning these were also related to time of CS+ offset. A comparison of time to peak of response for units recorded during control versus trace conditioning showed no statistical difference. Results section has been revised to include these additional results and Supplementary Figure 6 has been updated.(1.5) Muscimol effect on rearing behavior: Given the very low number of rearings observed in control animals, saline controls, DREADD, and vehicle animals I am left to wonder whether muscimol itself has an effect on rearing independent of the conditioning paradigm used. Is such data published and could that be cited? If not, what is the explanation for this? Clarification of this point would be useful especially as it is the only behavioral outcome associated with the muscimol paradigm.As indicated above (1.1.2) we administered the muscimol to MCN during the consolidation phase of fear conditioning, 48hrs before extinction training and the evaluation of rearing behaviour. It therefore seems reasonable to rule out any direct action of muscimol on the rearing behaviour we studied . Previous studies of the effects of systemic administration of muscimol on rearing behaviour found this reduced the number of rearings in rats . This is the opposite to our findings, supporting the suggestion that the effect we observed was not a direct consequence of the drug administration. However, and as highlighted by reviewer 2 (point 2.3) our original behavioural analysis was based on a small sample of animals (n = 4). We have doubled the sample  and the effect on number of rearings during extinction training, is no longer statistically significant . Given our proposal that the MCN is involved in different aspects of timing during fear conditioning  we have analyzed the larger behavioural dataset in relation to duration of freezing and movement bouts following CS+ . In muscimol treated animals the duration is significantly longer (on average 65.2% longer duration in EE than control). The latter additional finding is included in a new section in Results (The effect of temporary MCN inactivation during fear consolidation on behaviour (i) Effects on freezing behaviour).(1.6) Please discuss and justify why only male animals were used.We confined our study to males because female rats would introduce an additional variable, namely the effects of the oestrous cycle. This would have 3R implications through greatly increasing our sample size. We have included a short section in Methods  recognising that gender differences are an important issue that may influence our findings , but that this was beyond the scope of the current work.(1.7) Please discuss why there is such a large amount of variability in the control condition as shown in Figure 5B for the inter-CS-US interval graph (bottom right).The emission of USVs is a highly variable behaviour in rodents. For example, several studies have reported that only about half of animals emit 22 kHz USVs during fear conditioning . The factors that underlie this variability are unknown, although they may include the anxiety state of an individual, social experience and status . We have included mention of this inherent variability in Methods pages 35, line 32-3.(1.8) What is the level of inhibition achieved with the DREADD experiments? That is, after CNO, what does inhibition actually look like at the single cell level and/or at the local population level? What is the efficiency of inhibition after drug delivery?This is an interesting question but one that is very difficult to answer experimentally. There is good evidence from the literature that using this technique silences presynaptic terminals by inhibiting vesicle release in the local area . However, inhibition of release at terminals may cause a number of effects in the target region depending on the nature of both pre- and post-synaptic neurons. For example, reducing excitatory drive to glutamatergic neurons will reduce subsequent excitation yet reducing inputs to GABAergic neurons may enhance excitation through disinhibition . The network effects are therefore likely to be complex and are beyond the scope of our current experiments. However, our control and experimental evidence point towards a disruption of physiological signalling in regions of interest when using DREADDs. Given the difficulty of determining if the net effect of the DREADDS is inhibitory or disinhibitory we have removed ‘inhibitory’ from the text and instead refer to ‘modulation’.See also response to reviewer (3.2) where we highlight some additional experiments we have carried out to estimate the population of neurons in the MCN-vPAG pathway that have been manipulated. And also some brain slice experiments we have undertaken showing physiological effects of hM4Di DREADDs consistent with Locke et al. (2018).(1.9) For the supplementary figures , for example supplementary figure 1, please show the actual histology and not just the schematized data. There seems to be some ambiguity in the locations and it is much more informative of the reader has the actual data to examine alongside the functional data.We have revised Supplementary Figure 1 to include photomicrographs of example cases. For the summary diagrams we have revised these to show the start and endpoint of individual electrode tracks where units were recorded.Specific items:1. Line 14: change "rat ventral (vPAG)" to "rat ventral PAG (vPAG)"This edit has been made.2. Please clarify whether the vPAG is a subsection of the longitudinal columns of the PAG – some readers may not have a detailed knowledge of this structure.As in our previous work  we have used the term vPAG to include ventral  sectors of the PAG in which our recordings were made (see histology maps point 1.9). The term ventral is used because of the inherent limitation of using multiple tetrode implants in an awake animal. This approach increases unit yield at the expense of spatial accuracy of recording site location when reconstructing individual tetrode tracks post mortem. Our recordings and manipulations were centred on the ventrolateral longitudinal column of PAG but we cannot exclude the possibility that some units were located in neighbouring regions. This point has been added in Results .3. Figure 1C: Please make the red bar translucent, it is difficult to tell whether there is a peak being obscured or not.The red stimulus bar in Figure 1C has been made translucent to make clear there were no data obtained at this time of recording because of the stimulus artefact. A comment has also been added to the figure legend to this effect.4. Please refrain from using qualitative language where statistics are not done. Instead refer to the actual numbers  use "75% (n=24/32)" or simply "majority"). Similarly regarding "small proportion (18.5%)…".The manuscript has been revised throughout to remove qualitative language.Reviewer #2:This is an interesting study describing the interactions between the cerebellum and periaqueductal grey (PAG) in fear conditioning behavior in rats. To this end the authors first studied the PAG cellular responses during an auditory cued fear conditioning paradigm. Then, they performed global cerebellar inactivation with muscimol after fear conditioning, during consolidation period and they revealed ataxia in all animals and no difference during extinction training except for rearing behavior. They also performed chemogenetic experiments to inhibit cerebello-PAG pathway during acquisition and early consolidation and show effects on extinction rate of fear related freezing and ultrasonic vocalizations. The main findings are (1) a change in PAG response to the offset of the CS and an increase solely in rearing  during the extinction phase following cerebellar inhibition immediately after the conditioning, (2) a reduction of vocalization and rate of extinction during the extinction phase following the selective inhibition of cerebellar terminals in the PAG. The introduction and discussion are clear and well written, the experiments seem carefully performed; yet the results seem a bit descriptive in the current state of the manuscript and the different elements that compose the manuscript are still a bit too loosely connected. There are notably several weaknesses which need to be addressed:(2.1) In the experiments with muscimol in MCN (page 11), the infusion induced ataxia. This is a severe condition that likely induces intense stress and involves many structures. The interaction with the fear learning could thus result from an interference between the newly-formed memory and a muscimol-induced fear/stress. An effort to control for this effect would be needed to discard this alternate cause .In our lab, muscimol infusions (with similar and larger drug doses) into the interposed nucleus do not cause ataxia ; only infusions into MCN caused ataxia so unfortunately the control experiment proposed by the reviewer would not be an appropriate comparison.However, we have included the following points in the Methods section  to make the case why we think muscimol induced fear/stress is unlikely to fully explain our findings.i) The ataxia induced by the muscimol infusion into MCN during consolidation ranged across animals from moderate  to mild . There was no difference between these two groups in the amount of conditioned freezing displayed during retrieval, suggesting that fear learning was not related to the severity of the ataxia. More generally, we detected no change in conditioned freezing behaviour on retrieval, suggesting that the ataxia during consolidation had little or no effect on fear learning .ii) The effect of muscimol infusion into MCN was specific to PAG encoding of CS tone offset and not CS onset. If our findings were due to a generalized disruption of fear learning because of fear/stress during consolidation, then it might be expected that this would affect both onset and offset responses. It is also noteworthy that stress levels determined by corticosterone are related to tone onset  so it might be expected that this neural response would be sensitive to any changes in stress caused by ataxia.iii) In relation to anxiety, Oskel et al. (2003) found that systemic administration of muscimol in rats causes a general decrease in motility but has no effect on anxiety levels. And in terms of stress, previous studies  have reported a correlation between freezing and corticosterone levels. Given that conditioned freezing behaviour was unaffected in our experiments, this suggests that stress is also unlikely to fully explain our findings.We have consulted with neuroendocrine experts and carefully considered additional experiments to monitor stress levels , but this would require a large number of additional animals and in any case such experiments would not provide a definitive result.(2.2) The results of figures 2 and 3 are presented in parallel, in a way that needlessly complicates the reading. The CS+ onset and CS+ offset neuronal responses are presented together with respectively the fear responses during and between the CS in separate figures but the link between the electrophysiological and behavioral responses is correlative (see point 4 below). A better presentation would be to provide separate figures for the electrophysiology and the behavior as in the text (or change the text).We have revised the figures to show the electrophysiological and behavioural results separately.(2.3) The behavioral analysis with n=4 rats (muscimol) does not have much sense given the variability of the behavior(s). More animals are needed for the behavior.We have increased the sample size for the behavioural experiments to a total of n = 8 muscimol animals. The relevant text, stats and graphs have been updated .(2.4) The presence of a correspondence between the changes in electrophysiological and behavioral responses in each animal (explored by pairwise correlations) would reinforce the observations: are the animals with the weakest extinction showing weaker changes in the electrophysiological responses? .Front Psychol, 2017) of electrophysiological response size (integrated area) at onset and offset of conditioned tone as a function of % freezing for control and muscimol experiments. Within animals there was a positive relationship, with the largest electrophysiological response size related to the largest proportion of conditioned freezing. These data are included in a revised Figure 5 .We have carried out a repeated measures correlation analysis  The physiological relevance of the neuronal discharge at the CS offset (where a cerebellar contribution is suspected) remains undefined. The experiments for vPAG offset responses during trace conditioning (page 11) is very interesting in light of the characterization of the offset response. Yet, this result is too anecdotal  and not fully analyzed (no quantification for the EE and LE). The manuscript would benefit greatly from a proper demonstration of this response.We have increased the dataset for trace conditioning to a total of 12 vPAG units from 3 animals and quantified the findings. See point 1.4 above.(2.6) The ERP experiment is suffering from two limitations: (1) it is unclear how the LFP relates to the neuronal activity vs afferent activity; it would be much helpful to show using the available PAG recordings how the LFP relates to the multiunit activity.We have included a Supplementary Figure 4 to show an example case that is typical of the available data, showing the relationship between the ERP at offset and multiunit activity in PAG. This shows that the field waveform and duration of spike activity are broadly coincident.(2.6.2) The number of animals is too limited for the analysis ; testing the covariance of MCN and PAG ERPs for each rat could help circumvent the limited number of animals . More animals should be added otherwise.We have increased the number of ERP animals to 6 and the results updated. We have tested the covariance, with a repeated measures correlation, between MCN and PAG ERPs during extinction training . In both cases there was a significant positive correlation. .(2.7) It is difficult to judge from the specificity of the action of CNO; some cannulas  seem off-target : were these experiments included in the analysis? Terminal labeling has been found in the region bordering the vlPAG: how can the authors be sure that the behavioral effects are not due to an action on these terminals outside of the PAG? The diffusion of CNO needs to be documented to interpret these experiments.3, extending from -6.5 to 8.5 anterior-posterior and up to 2.5 laterally. In terms of being off-target we believe the reviewer is referring to one cannula that was centrally located – in this case spread of infusate would include vlPAG. Also infusions were bilateral and the other cannula of the pair in this case was on target.In the majority of our cases the infusion was centred on vlPAG, but in keeping with studies of this type we cannot exclude spread to neighbouring areas. This limitation is noted in Results . Based on other experiments (unpublished data) and previously published reports  we estimate spread to be ~0.45 mm(2.8) If I understand correctly, the extended response to the CS-offset  is interpreted as poor temporal patterning (discussion p22-23); the average trace indicates longer responses to CS+ offset after muscimol during consolidation; are these responses less precisely associated to the end of the CS ? Because the traces are only presented as a grand average, it is not possible to see whether some responses become poorly correlated to the end of the CS.We thank the reviewer for this helpful point. In a new Figure 4 we now include individual data for all available units as violin plots which show for control and muscimol treated animals the number of significant (≥ 2SD from baseline) peaks in neural activity in the 500ms after CS+ onset and CS+ offset for EE and LE. In control animals at CS+ offset during EE there was on average 1 peak compared to 3 peaks in muscimol animals . Also, the proportion of peaks in different 100 ms time bins is significantly different . For control animals over 80% of peaks occurred in the 200 ms time period immediately following CS+ offset, while the corresponding value is 20% for muscimol treated animals .Reviewer #3:The authors investigated the role of projections from the medial cerebellar nucleus (MCN) to the ventral periaqueductal gray (vPAG) in a conditioned fear learning task in rats, measuring vPAG neuronal activity and behavior before and after inhibiting MCN activity or the MCN-vPAG pathway. Inactivation of the MCN and the MCN – vPAG projections both affect fear learning related neuronal activity and behavior but in different ways. MCN inactivation results in a reduced neuronal response at the end of the conditioned tone stimulus, and also more frequent rearing and more frequent ultrasonic vocalizations during extinction. Neither manipulation changed the expression of fear as measured by freezing behavior, or the rate of fear extinction. Several statements in the manuscript are not supported by the results, such as the statement that the timing of neuronal activity signaling the end of the conditioned stimulus is altered by inhibition of the MCN or its projections.3.1. The differences between the observed effects of the two manipulations (MCN or pathway inhibition) on fear learning may be due to the differences in the method and timing of inactivation. MCN is inactivated during consolidation. The MCN vPAC pathway is inhibited during fear acquisition, with inhibition possibly extending into early consolidation. MCN inhibition is through muscimol, pathway inhibition through use of and DREADDs. These differences introduce unknown variability into the results. Manipulations of MCN activity and of the MCN-vPAG pathway should occur during the same period in the fear conditioning protocol and preferentially by using the same method to ensure similar time courses.Our experiments included muscimol to abolish all outflow from MCN (which will include both direct and indirect pathways to the PAG) and the DREADD method to specifically inhibit the direct pathway from MCN to PAG. This experimental design therefore allowed a comparison between blocking all MCN pathways and a specific one to PAG. As stated in the Introduction we focused on effects during retrieval and extinction of fear conditioned responses because of the potential clinical relevance to conditions such as PTSD and chronic pain.The reviewer is correct to note that the two types of manipulation were given at different time points (prior to and after acquisition for DREADD activation and muscimol respectively) and this difference is noted in the Methods .We chose muscimol for our initial manipulation during consolidation because the duration of its effects last longer than DREADDs activation. In our case for Muscimol the motor effects were observed for 3-6 hours and other experiments in the literature have found behavioural effects lasting 5+ hours  meaning we effect a longer time period of consolidation. DREADD activation in comparison is for a relatively short amount of time <60mins .We did not deliver muscimol to MCN during acquisition because of the confounding effects of the drug on movement. There are also significant welfare concerns of delivering aversive stimuli to animals with compromised mobility.However, and as noted by the reviewer, the activation of DREADDs by CNO was likely to include both a period during and after acquisition . This means there was likely to be some overlap in time course of action of the two types of manipulation (i.e. effects on early consolidation for both DREADDS and muscimol). The DREADD method has recently been used in mice at different time points during fear conditioning , including the consolidation period after acquisition. Like our muscimol results no effects on fear conditioned freezing behaviour were reported, suggesting that the two methods are comparable and that the effects of the DREADDs are related mainly to an action on associative memory processes during acquisition. Given the comprehensive results on freezing behaviour published by Frontera et al., (2020) we do not think it is justified to repeat their experiments which are considered in the Discussion .3.2. There is no description of measurements to determine how effective the inhibition of the MCN or the vPAC was, but interpretation of results is under the assumption that it was complete or near complete. While a systemic injection of CNO will deliver clozapine to all DREADD receptors , it does not inform about the percentage of projecting axons that express the designer receptor. It would have been reassuring to see some electrophysiological proof of the success and extent of the inhibition. Inhibition of the MCN and especially be confirmed electrophysiologically, at least for some examples.We have carried out additional experiments to provide (i) an estimate of the population of neurons being manipulated and (ii) the physiological effect of the DREADDs. For (i) we have injected retrograde tracer into vlPAG and DREADD into MCN, and counted the proportion of double labelled neurons in MCN. And for (ii) we have recorded from DREADD transfected neurons in cerebellar slices before and during application of CNO.Experiment (i) is now included in Supplementary Figure 10. The anatomical results from 4 rats show that double labelled neurons in MCN represent, on average 70% and 28% when expressed relative to the PAG and DREADD single labelled populations, respectively.Experiment (ii) An inhibitory DREADD virus (AAV-hSyn-hM4D(Gi)) was injected bilaterally into the MCN under general anaesthesia. Approximately 4 weeks later we performed current clamp recordings from MCN neurons, in cerebellar slices, to investigate how CNO altered spontaneous firing rate. CNO caused an increase in the spontaneous firing rate in MCN neurons  demonstrate three different responses in lateral cerebellar nuclei following bath applied CNO: an increased firing rate, a decreased firing rate and no change. It is proposed that neurons that have an increase in firing rate, similar to our results, may be caused by disinhibition from local circuitry .Nevertheless, our brain slice experiments provide evidence that the expression of hM4DiDREADDs in MCN cells has a physiological effect. Additional evidence that the expression of hM4Di-DREADDs is functional is provided by the behavioural deficits we observed when animals were tested on the balance beam following systemic CNO administration.3.3. The authors don't seem to have performed necessary control experiments to exclude potential effects of CNO on endogenous clozapine binding sites in the brain when using systemic CNO injections.We thank the reviewer for highlighting our oversight of not including the additional control for any systemic effects of CNO itself. These results are now included in Supplementary Figure 9F which shows control animal (virus without DREADD) beam performance before (baseline) and after systemic CNO injection . There was no significant difference, consistent with there being no detectable off target effects of the CNO.3.4. The authors state that MCN inhibition alters the temporal precision of vPAG offset activity. Timing as a contribution of the cerebellum to fear learning is mentioned several times. However, there is no actual data supporting a change in timing or temporal precision. The authors don't analyze the temporal precision of the offset response, only the area under the curve and peak amplitude. The claim that the cerebellum control aspects of timing in this task is not supported by results. The timing of the off response, which is specifically mentioned, needs to be analyzed to support or refute this claim.Additional quantitative analysis is now included in a new Figure 4 which shows that there is a statistically significant difference between control and muscimol treated animals in both the number and timing of peaks in vPAG activity at CS offset, consistent with an effect on temporal precision of vPAG encoding. See point 2.8 for further details.3.5. In the last paragraph of the introduction the authors claim that the rate of extinction is affected by MCN inhibition. There are no results to support this. In the same paragraph the authors state that ultrasonic vocalizations (USVs) and rearing behavior during fear acquisition are influenced by MCN inputs to vPAC. However, all results for USV and rearing counts are shown for the extinction phase.We apologise for the error in wording which has been corrected in light of our increased sample size of behavioural data.3.6. Experimental findings with an n = 1, as the author's experiment with one rat exposed to trace fear conditioning, are not acceptable. The trace fear conditioning experiment should be repeated with at least two more animals .We have increased the sample size to 12 units from 3 animals. See 1. 4 for further details.3.7. Supplementary Figure 1 A is supposed to show electrode tracks for rats implanted with two drives for electrodes. The left cerebellar illustration shows what is declared as canular tracks in the other illustration. Was that by mistake or where cannulas inserted here?We thank the reviewer for pointing out our mistake in panel position in this figure. We have amended Supplementary Figure 1 and in the legend, we have also make clear which are cannular tracks and which are electrode tracks. See also 1.9 and 2.11.3.8. The manuscript overstates several findings. For example, the presented findings do not imply that the cerebellum 'controls' any of these behaviors or activity patterns but rather that it is part of a larger circuit that does.We have amended this, and other statements as suggested.3.9. Please show some example raw data of your electrophysiological recordings.We have included example raw tetrode data in Supplementary Figure 2.[Editors’ note: what follows is the authors’ response to the second round of review.]Essential revisions:Please see below the specific recommendations by the reviewers. In addition to the points of clarity as requested, please pay particular attention to:1) Cases where animal numbers need to be provided.Animal numbers have now been included.2) References as requested.The additional references have now been included.3) Details regarding methodology.More details added to the Methods.4) Please ensure your manuscript complies with the eLife policies for statistical reporting: https://reviewer.elifesciences.org/author-guide/full "Report exact p-values wherever possible alongside the summary statistics (and degrees of freedom) and 95% confidence intervals. These should be reported for all key questions and not only when the p-value is less than 0.05."Please also include a key resource table.eLife policies for statistical reportingWe have updated all stats to comply with Resource table added.Reviewer #3:The authors have adequately addressed all my concerns.One small final request would be that the authors mention the results from their test of DREADD activation and the related findings by Locke et al. 2018 in the manuscript. This could just be in writing without adding a figure.New section added to the Methods .