Loss of mGlu5 receptors in somatostatin-expressing neurons alters negative emotional states

Subtype 5 metabotropic glutamate receptors (mGlu5) are known to play an important role in regulating cognitive, social and valence systems. However, it remains largely unknown at which circuits and neuronal types mGlu5 act to influence these behavioral domains. Altered tissue- or cell-specific expression or function of mGlu5 has been proposed to contribute to the exacerbation of neuropsychiatric disorders. Here, we examined how these receptors regulate the activity of somatostatin-expressing (SST+) neurons, as well as their influence on behavior and brain rhythmic activity. Loss of mGlu5 in SST+ neurons elicited excitatory synaptic dysfunction in a region and sex-specific manner together with a range of emotional imbalances including diminished social novelty preference, reduced anxiety-like behavior and decreased freezing during retrieval of fear memories. In addition, the absence of mGlu5 in SST+ neurons during fear processing impaired theta frequency oscillatory activity in the medial prefrontal cortex and ventral hippocampus. These findings reveal a critical role of mGlu5 in controlling SST+ neurons excitability necessary for regulating negative emotional states.


INTRODUCTION
Affective and anxiety disorders represent together one of the leading causes of disability worldwide [1].Despite their enormous impact on public health, they have remained largely refractory to the identification of their causal risk factors and fundamental neurobiology.These disorders show a high degree of comorbidity [2] and lack clear genetic and biological boundaries [3,4], thus possibly sharing common mechanistic features.They are characterized by shared emotional state abnormalities that may result from a dysfunction in the balance between cortical GABAergic and glutamatergic systems [5,6].Deficits in the expression or function of metabotropic glutamate type 5 receptors (mGlu 5 ) have been proposed as possible mechanisms underlying this imbalance [7][8][9].These receptors contribute to a number of important activitydependent synaptic changes, such as long-term potentiation (LTP) and depression (LTD) at many central synapses [10,11].They also regulate N-Methyl-D-Aspartate (NMDA) glutamate receptor activity and neuronal excitability [12][13][14].The distribution of mGlu 5 is mostly observed in telencephalic areas, with highest expression levels in the hippocampus (HPC), striatum, lateral septum (LS) and prefrontal cortex (PFC) [15][16][17] not only in glutamatergic neurons, but also in GABAergic interneurons (INs) and glial cells [18][19][20][21].
So far, relatively few studies have investigated how mGlu 5 in specific neuronal populations and brain circuits influence behavior and could thus contribute to altered cognitive and emotional processing across psychiatric disorders.Depletion of mGlu 5 in glutamatergic principal neurons throughout all cortical regions in mice did not affect anxiety-like behavior, fear-learning, sensorimotor gating or social interaction, but resulted in increased novelty-induced locomotion and dysfunctional stress coping [22].Conversely, deletion of mGlu 5 in forebrain GABAergic neurons gave rise to a complex phenotype mostly characterized by changes in the regulation of locomotion and habituation responses [23], as well as to reduced anxiety-like behavior as shown by lower novelty-suppressed feeding and better coping to unescapable stress [24].These consequences were only marginally recapitulated by the genetic ablation of mGlu 5 in parvalbuminexpressing (PV+) cells.Interestingly, these animals displayed a broad repertoire of memory deficits, including social and nonsocial memory, but displayed intact anxiety-like behavior [25].
In recent years, somatostatin-expressing (SST+) neurons have gained increased attention with respect to affective and anxiety disorders [26], since several studies reported a crucial role played by these neurons in fear, anxiety and aversive learning in the PFC [27], anterior cingulate cortex [28], LS [29], HPC [30], amygdala [31] and other brain structures.SST+ neurons are the second largest group of INs in the cortex after PV+ INs, where they constitute about 1-3% of the overall neuronal population.They are widely present in the whole rodent brain except for the cerebellum and are highly heterogeneous [32].For instance, SST expression can be found in multipolar, bipolar and fusiform cells, in Martinotti, basket, double-bouquet and bi-tufted neurons [32][33][34] and their electrophysiological properties range from low-threshold to burstaccommodating, burst-irregular spiking and non-accommodating firing responses [32,35].In the cortex (neocortex, basolateral amygdala and HPC), SST+ INs express mGlu 5 [19][20][21], though the specific neuron subclasses remain largely unidentified.Little is also known regarding the expression of this receptor in SST+ cells of subcortical brain regions, such as the central amygdala (CeA) and LS.
We hypothesized that mGlu 5 expression in SST+ neurons regulates their activity and in turn controls emotional behaviors.To test this hypothesis, we selectively deleted mGlu 5 in SST+ neurons in mice and explored behavioral phenotypes related to social, cognitive and emotional domains.Moreover, we evaluated the effects on local neuronal activity and assessed brain oscillatory activity in the medial PFC (mPFC) and ventral hippocampus (vHPC) during aversive emotional processing.

Immunofluorescence experiments
For immunofluorescence staining, mice were deeply anaesthetized with thiopental sodium (150 mg/kg, i.p.) and transcardially perfused with a fixative as previously described [42].Following brain extraction, coronal sections were cut (50 μm) on a Leica VT1000S vibratome (Leica Microsystems, Vienna, Austria) and immunostained against mGlu 5 and other cellular markers based on previously described procedures [43].Extended methods are provided as supplementary material.

Pre-embedding immuno-electron microscopy
For immuno-electron microscopy, 0.05% glutaraldehyde was included in the fixative.Immunocytochemistry for electron microscopy was performed as previously described [42] and using similar conditions as used for the immunofluorescence experiments, but omitting Triton X-100 from the buffers.Extended methods are provided as supplementary material.
Multielectrode Array (MEA) excitability recordings.Brain slices were prepared from 12-17 week old mice as previously described [44].The animals were anesthetized with isoflurane (IsoFlo®, Zoetis) and decapitated.Brains were rapidly removed and immersed in ice-cold aCSF.Coronal slices (300 µm) containing the hippocampal areas were cut in sucrose aCSF.The slices were incubated in oxygenated sucrose aCSF at 32 °C for 30 min and subsequently transferred to oxygenated standard aCSF.
Multi-electrode array (MEA) recordings were performed using a MEA2100 recording system as previously described [45].The slices were transferred to planar MEA chips (120MEA200/30iR-Ti, Multi-Channel Systems, Reutlingen, Germany) and a platinum slice grid spanned with nylon fibers was carefully placed on top of the slices.After a short habituation period, spontaneous action potential firing was recorded with a sampling rate of 5 kHz for 5 min.Some slices were pre-treated with the mGlu 5 NAM MTEP (10 µM) and/or mGlu 1 NAM JNJ16259685 (1 µM) for 10 min.For stimulation the non-selective group I mGlu agonist DHPG (50 µM) was bath-applied for 2 min.All data streams were filtered using a 200 Hz high-pass filter.The recorded traces were analyzed using the MC_Rack software (MultiChannel Systems).Spike threshold was set to −5 standard deviations (SD) from baseline.Recording electrodes were classified as active if the firing frequency exceeded 1/60 Hz (i.e., at least one AP per min).Average firing frequencies were calculated for the different pharmacological treatments.
MEA LTP recordings.For LTP recordings, only male mice were used.Electrical stimuli were applied and the strength of the pulse was adapted to obtain field excitatory postsynaptic potentials (fEPSPs) with 50% maximal slope.An initial input output (IO) curve with increasing amplitudes from 500 mV to 4 V was generated using the MC_Stimulus II software.Schaffer collaterals were stimulated in the CA3 hippocampal region using 2-3 electrodes and evoked fEPSPs were recorded in the CA1 area.Biphasic constant pulses (0.1 ms/phase) were delivered every 60 s until a stable baseline was recorded for 15 min.LTP was induced by a theta burst stimulation (TBS) protocol (3 trains of 10 bursts at 5 Hz, 4 pulses at 100 Hz for each burst).Afterwards, test pulses were constantly delivered every 60 s for 40 min.The recordings were analyzed using the MC_Rack software.In order to quantify LTP, the fEPSP amplitudes after TBS stimulation were normalized to fEPSP amplitudes during baseline recording.

Animal behavioral assays
All behavioral tests were performed by experimenters blinded to the genotype of the animals.
Social preference and social novelty.Social behavior was assessed by means of a modified three-chambered social task apparatus as previously described [43,46].Briefly, the procedure involved two phases: social preference and social novelty, each lasting 10 min.Mice were tested in the dark (infrared light; Lux < 5).After a 10 min habituation period, mice were temporarily contained in the center chamber by closing the side walls, and a novel young unfamiliar mouse (5-8 weeks) was placed into a mesh cylinder (15 cm tall, 7 cm diameter) in the least explored side chamber, whereas an identical empty mesh cylinder was placed in the opposite chamber.The test mouse was then allowed to explore the chambers for 10 min (social preference).On the second phase of the test, mice were again contained in the center chamber while another novel young unfamiliar mouse was placed in the chamber that previously contained the empty mesh cylinder, and test mice were allowed to explore the chambers for 10 min (social novelty).Time spent in interaction with the mesh cylinders (<5 cm) was automatically tracked and scored using the Ethovision XT12 software (Noldus; RRID:SCR_000441).Fear conditioning and extinction.Mice were fear-conditioned in a 25 x 25 x 40 cm chamber (Ugo Basile, Comerio, Italy) with transparent walls and a metal grid floor for delivering a foot-shock.After 120 s acclimation period, an 80 dB 6 kHz noise [conditioned stimulus (CS)] lasting 30 s was paired in the last 2 s with a 0.6 mA scrambled foot-shock [unconditioned stimulus (US)] 5 times (120 s inter-pairing interval).Mice were returned to the home cage after 120 s no-stimulus consolidation period.The chamber was cleaned between subjects with 70% ethanol.Fear extinction training sessions were performed 24 h after the conditioning in a novel context with different visual (squared and striped patterned walls) and olfactory cues (1% acetic acid) and consisted of a 120 s baseline period, after which the CS was presented 20 times with a randomized inter-CS interval ranging from 5-40 s.The first three tone presentations on day 2 were taken as an indicator of fear retrieval.Freezing was automatically measured by ANY-maze software (Stoelting Europe, Dublin, Ireland) using a freezing minimum duration threshold of 1 s, and manually cross-checked by an experienced experimenter.

Surgeries and electrophysiological recordings in vivo
Adult male mice were stereotactically implanted under ketamine/xylazine (i.p.) and sevoflurane (Sevorane, AbbVie GmbH, Austria) anesthesia with recording electrodes made of twisted 76,2 µm teflon coated, stainless steel wires (Science Products, Hofheim, Germany) into the mPFC (at 3°angle, AP: +1.8, L: +0.5, D:−1.7 mm-from bregma) and vHPC (AP:−3.2,L: + 3.3, D: −2.8 mm from bregma).A silver wire was used as a ground/reference electrode.All electrodes were connected to a 10-pin PCB connector and cemented to the skull with dental acrylic.Following recovery from surgery, mice were fear conditioned in a 25 × 25 × 40 cm chamber (Ugo Basile) with 5 US-CS pairing blocks consisting of a 15 s CS presentation (white noise) followed by a 1 s US foot-shock (0.5 mA) with 1 min inter-trial interval.Fear retrieval was performed 24 h in the same chamber where mice were subjected to fear conditioning but omitting the US.LFP signals were recorded during the retrieval phase on an EXT-9 recording system using a head-stage-commutator assembly (NPI electronic, Germany), allowing the animal to free movement inside the fear conditioning chamber.The raw signal was amplified, filtered, digitized, and stored on a PC for offline analysis with the use of Spike-2 (ver.8.08) software (CED, Cambridge, UK).
Local field potentials (LFP) recorded from mPFC and vHPC during fear retrieval sessions were digitally filtered and used for further analysis.For spectral analysis of the signal, Fast Fourier Transformation (FFT) was calculated for 9-15 artifact-free 1 s samples taken from each CS presentation and transformed (z-scored).Peak power (Pmax) in the theta frequency band (4-12 Hz) and corresponding dominant frequency was calculated with Sudsa22.2S2script.Pmax values obtained during each CS were afterwards normalized and compared between experimental groups.Theta synchronization between mPFC-vHPC during tone presentations (CS1-CS5) was determined by calculating signal cross-correlation via the Spike-2 software of low-pass (16 Hz) filtered waveforms.The level of the second positive peak in the cross-correlogram, corresponding to the theta frequency peak, was quantified after alignment to the maximal positive peak, averaged across individual animals and statistically assessed.

Statistical analyses
Data were analyzed with the Prism 9 (GraphPad Sowftware Inc.) and R (version 3.3.3)software.Sample size was predetermined on the basis of published studies, experimental pilots and in-house expertize.Data are shown as mean ± SEM and, when informative, with individual values for each animal.Data distribution was tested for normality and successively analyzed with appropriate parametric or non-parametric statistical tests.Where applicable, multiple comparisons, following significant ANOVAs, were further analyzed using Bonferroni post hoc tests.Significance levels were set at p values less than 0.05.

Anatomical and functional profiling of mGlu 5 in SST+ neurons
To evaluate the functional implications of mGlu 5 in SST+ neurons, we studied at first their co-expression in neurons from a number of brain areas that were selected based on previous in situ hybridization and immunocytochemical studies and showed large macroscopic overlap [15,16,[47][48][49][50].Consistent with previous studies, immunoreactivity for mGlu 5 was widespread in the telencephalon, conversely SST+ neurons were observed primarily in the hypothalamus, LS and CeA, whereas they were scattered in the neocortex, HPC, basal amygdala and striatum (Fig. 1a, b).Because of the intense mGlu 5 immunostaining in the neuropil and the SST labeling being mostly restricted to the Golgi apparatus and endoplasmic reticulum in neuronal somata, the study of their co-localization required high resolution confocal and electron microscopy (Fig. 1c-e and Suppl.Fig. 1).In cortical INs, we observed a 55-85% co-localization between mGlu 5 and SST, depending on the area (Fig. 1c and Suppl.Fig. 1e; Suppl.Table 2).Different patterns of mGlu 5 somatic staining were observed ranging from a plasma membrane-restricted immunoreactive signal to a dense cytoplasmic labeling (Fig. 1c, d and Suppl.Fig. 1a, e), suggesting a complex and cell-dependent trafficking [51,52].In the analyzed subcortical brain areas, namely the LS and CeA, we unexpectedly did not detect mGlu 5 labeling in SST+ neurons (Fig. 1e; Suppl.Fig. 1 and Suppl.Table 2).In the CeA, mGlu 5 immunoreactivity appeared to be primarily associated with PKCδ -expressing neurons (Suppl.Fig. 1c, d).
We next sought to investigate the intrinsic action of mGlu 5 activation in SST+ INs in the hippocampus and neocortex using whole-cell patch-clamp recording.To identify SST+ INs in acute slices, we took advantage of SST Cre ::Ai14 transgenic mice that express the reporter tdTomato in these neurons.First, hyperpolarizing and depolarizing current steps (1 s) were applied to assess passive and active membrane properties.After recording, biocytin-filled cells were morphologically reconstructed.SST+ INs exhibited diverse axonal arborization patterns, including bistratified and oriens-lacunosum moleculare (O-LM) projections.These INs also showed distinct firing properties from PNs (Fig. 1f, i, l; Suppl.Fig. 2g, i).In the CA1 field of the HPC, most SST+ INs exhibited low-threshold regular spiking properties (Fig. 1l) although we also recorded SST+ INs with fast spiking, adapting and stuttering firing patterns (Fig. 1i; Suppl.Fig. 2g), in line with the known heterogeneity of these INs [32,35].Similar diversity in firing properties was observed for the SST+ INs recorded in the temporal association cortex (Suppl.Fig. 2i).
These findings demonstrate functional expression of mGlu 5 in a large proportion of cortical SST+ INs, belonging to different subtypes.The activation of mGlu 5 in these INs led to membrane depolarization and to an increase in firing frequency.Despite we did not detect a co-localization between mGlu 5 and SST in neurons of the LS and CeA, we cannot rule out, only based on our immunocytochemical experiments, that these neurons, or some of them, may actually contain the receptor, though at levels below our threshold of detection at the soma.Future experiments are warranted to resolve this issue using other methodological approaches.
mGlu 5 in SST+ neurons regulate synaptic plasticity and local network activity Next, we generated a mouse line with a selective deletion of mGlu 5 in SST+ neurons via Cre-mediated recombination (Suppl.Fig. 3a) to explore the functional relevance of mGlu 5 in these neurons.RNAscope, immunofluorescence staining and biochemical assays were used to evaluate cell and tissue-specific removal of mGlu 5 expression.In mice with Grm5 conditional KO (cKO) in SST+ neurons (SST Cre -Grm5 −/− ) no mRNA or protein fluorescent labeling for mGlu 5 was detected in cortical SST+ INs (Fig. 2a; Suppl.Fig. 3b), that was consistent with a small reduction in overall mGlu 5 protein levels as measured by western blot analysis (Suppl.Fig. 3c-f).Furthermore, in hippocampal slices of SST Cre -Grm5 −/− mice, bath application of DHPG (10 µM) failed to increase the membrane potential of SST+ INs (n = 6) in the presence of JNJ16259685 (Suppl.Fig. 4).
Ex vivo recordings of hippocampal network activity yielded subregion-and sex-specific differences in frequency ratios upon stimulation with DHPG (Fig. 2e-j; Suppl.Fig. 5a-g).In male, but not female SST Cre -Grm5 −/− mice, we observed higher frequency ratios compared to baseline in CA3 and lower frequency ratios in CA1, as compared to littermate controls (Fig. 2e, f, h, i).Conversely, in the mPFC both male and female SST Cre -Grm5 −/− mice displayed decreased frequency ratios compared to baseline upon DHPG application (Fig. 2g, j).However, these changes in local network activity were not common for other neocortical structures such as the dorsal anterior cingulate cortex (dACC; Suppl.Fig. 5h, i), suggesting that mGlu 5 in SST+ INs can increase or decrease local network activity in a region-specific manner across different cortical structures.
To explore the relative contribution of mGlu 1 and mGlu 5 , given their complex pattern of co-expression in the HPC [16,57], we performed MEA excitability recordings in slices from control mice, in the presence of the mGlu 1 and mGlu 5 NAMs JNJ16259685 and MTEP, respectively.In the CA1 subfield, MTEP produced a much stronger reduction of DHPG-mediated activity than JNJ16259685 (Suppl.Fig. 5j).On the other hand, in the CA3 DHPG-mediated activity was more effectively lowered in the presence of JNJ16259685 as compared to MTEP (Suppl.Fig. 5k).The combination of JNJ16259685 and MTEP fully suppressed DHPGstimulated activity (Suppl.Fig. 5j, k).These findings suggest that the effects onto local network activity are preferentially mediated by mGlu 5 in the CA1 and by mGlu 1 in the CA3.
Altogether, these results show that the loss of mGlu 5 in SST+ INs facilitates synaptic plasticity in CA1 PNs, most likely through the reduction of dendritic inhibition by these INs, and induces area-and sex-specific changes of local network activity.mGlu 5 in SST+ neurons modulate social and anxiety-like behavior in a sex-specific manner Previous studies have shown that germline Grm5 deletion or systemic application of mGlu 5 antagonists change the expression of several emotional behaviors including social interaction, anxiety-like behavior and fear responses [41,46,58].Selective deletion of Grm5 in forebrain GABAergic neurons, but not in glutamatergic PNs [22], to some extent mimics these changes on emotional states [24].The specific ablation of mGlu 5 in PV+ neurons did not seem to importantly contribute to deficits in anxiety-like behavior, and the influence on sociability was attributed to cognitive deficits [25].
SST Cre -Grm5 −/− mice showed no overt phenotypic alterations in development or locomotion (Suppl.Fig. 6a, b).We then examined how the loss of mGlu 5 in SST+ neurons influenced a variety of behaviors in the cognitive, social and negative valence domains (Suppl.Fig. 6c).Male as well as female SST Cre -Grm5 −/− mice showed similar social preference to that of littermate controls in the classical three-chamber social interaction test (Fig. 3a-e).When further tested for social novelty, only male SST Cre -Grm5 −/− mice displayed reduced preference for the novel interactor (Fig. 3f-h).The lack of social novelty differences between genotypes in female mice (Fig. 3i, j), however, might have been affected by a lower sensitivity of the test in this sex under our experimental conditions, as evidenced by a less pronounced preference.The deletion of Grm5 in SST+ neurons did not result in any detectable deficit in novel object exploration and in the marble burying test (Suppl.Fig. 6d, e).When tested for measures of anxiety-like behavior, SST Cre -Grm5 −/− male, but not female, mice spent more time in the open arms of the elevated plus maze test as compared to control animals (Fig. 3l).This apparent anxiolytic activity in male mice did not arise from a noveltyinduced hyper-locomotion (Fig. 3m, n; Suppl.Fig. 6b).
Altogether these data suggest sexual dimorphism in the contribution of mGlu 5 to the regulation of social and anxiety-like behavior by SST+ neurons.mGlu 5 in SST+ neurons modulate fear in an estrous-dependent manner To further explore whether the loss of mGlu 5 in SST+ neurons had additional effects on constructs of negative affect, we next used a Pavlovian fear conditioning paradigm (Fig. 4a).SST Cre -Grm5 −/− mice showed similar fear-learning responses in comparison to their respective littermate controls (Fig. 4b, c).In contrast, both male and female SST Cre -Grm5 −/− mice exhibited reduced freezing when subjected to fear retrieval in a novel context (Fig. 4d, e).The acquisition of fear extinction, which is generally considered a proxy of inhibitory learning [59][60][61][62], was also slightly facilitated by the depletion of mGlu 5 in SST+ neurons.However, similar freezing levels were reached by both SST Cre -Grm5 −/− and control mice at the end of the extinction training (Fig. 4f, g).
In order to identify which traits better discriminate one genotype from another, and to reveal which behavioral domain is mostly affected by the loss of mGlu 5 in SST+ neurons, we performed a random forest (RF) classification analysis with a leaveone-out cross-validation of all behavioral responses measured in SST Cre -Grm5 −/− and control mice (Suppl.Fig. 7a-d).The weight of each response to accurately differentiate genotypes was given by the Gini index, with the most discriminative ones being fear retrieval and anxiety-like behavior (Suppl.Fig. 7a).Interestingly, these two phenotypes, measured as time spent in the open arms of the EPM and freezing during fear retrieval, correlated only in WT male mice (Suppl.Fig. 7e-h).The general RF analysis, including all animals regardless of sex, led to a genotype prediction accuracy of 61%.Conversely, individual RF classifications taking into account the two sexes led to substantial differences in prediction errors.A RF classifier for male mice led to an accuracy close to 80% (24/30 mice correctly predicted), whereas a classifier for female mice remained at chance level (13/30 mice correctly predicted).
We next explored whether the lower prediction power of the behavioral classifiers in female mice would reflect potential estrous-dependent contributions in the negative valence domain (Fig. 4h-j).Upon stratification of the estrous stage following fear extinction, we observed that the reduced fear retrieval and facilitated extinction learning by the depletion of mGlu 5 SST+ neurons was only apparent in SST Cre -Grm5 −/− female mice in estrous-proestrus (Fig. 4i, j).Taken together our findings suggest that the lack of mGlu 5 in SST+ neurons primarily affects the negative valence system.While this is more clearly observed in male mice, in female mice it appears to be estrous stage dependent.

Generation of hippocampal theta rhythm during fear retrieval depends on mGlu 5 in SST+ neurons
Negative emotional valence induced by anxiety or fear enhances the generation of theta frequency in HPC [63] and mPFC [40], as well as their synchronized activity [40].Since SST+ INs facilitate HPC-mPFC synchrony [38], we sought to characterize the local neural oscillatory activity in the and mPFC in mice lacking mGlu 5 in SST+ neurons.SST Cre -Grm5 −/− and littermate control male mice were implanted with LFP recording electrodes in the vHPC and mPFC (Fig. 5a; Suppl.Fig. 8a, b) and tested for the most discriminative behavioral trait between the two genotypes (Suppl.Fig. 7a), namely fear retrieval following a Pavlovian fear conditioning paradigm.However, in this paradigm fear retrieval was carried out in the same context where the conditioning was performed (Fig. 5b) in order to induce stronger theta activity during CS presentations.Consistent with our previous experimental protocol, SST Cre -Grm5 −/− mice froze less than controls during the initial CS presentations (Fig. 5c).This behavioral observation was accompanied with substantial changes in the signal power spectra (FFT) between SST Cre -Grm5 −/− and control mice (Fig. 5d, e).This dissimilarity was particularly evident during the first two CS presentations as a pronounced reduction of spectral content (peak power) in the theta frequency band (4-12 Hz) in SST Cre -Grm5 −/− mice, both in the vHPC and in the mPFC (Fig. 5f, g; Suppl.Fig. 9a, b).During the last 3 CS presentations, theta activity gradually resumed in the Grm5 cKO animals, suggesting that lower theta power during CS presentations did not arise from a general inability of SST Cre -Grm5 −/− mice to generate theta rhythms (Fig. 5f, g; Suppl.Fig. 8c).The dominant frequency in the theta frequency band during the CS presentations was at 5-6 Hz in both WT and SST Cre -Grm5 −/− animals (Suppl.Fig. 9c), consistent with type 2 theta oscillations (4-8 Hz) known to be dominant during periods of fear-induced immobility [63][64][65].The maximal peak power (Pmax) in the mPFC showed a positive correlation with the percent of freezing measured during the 5 CS presentations (Suppl.Fig. 9d).
Altogether these results suggest that mGlu 5 in SST+ neurons are crucial for the entraining of synchronous theta oscillatory activity in mPFC and vHPC during negative valence processing.

DISCUSSION
Understanding the mechanisms by which cell-and circuit-specific alterations in discrete neurotransmitter receptor function influence the balance between cortical GABAergic and glutamatergic systems is critical for elucidating the fundamental neurobiology underlying many psychiatric disorders.Here we report that the loss of mGlu 5 in SST+ neurons decreased anxiety-like behavior and freezing during the retrieval of fear memory involving a reduction in theta frequency oscillatory activity in the mPFC and vHPC.Our study provides a circuit and functional mechanism contributing to the alleviation of anxiety and affective symptoms exerted by mGlu 5 NAMs in both preclinical and clinical studies [8,[66][67][68][69].In particular, we established that the co-expression of mGlu 5 and SST occurs primarily in cortical INs, though not all of them displayed functional responses to the group I mGlu orthosteric agonist DHPG or labeling for mGlu 5 .The latter ranged from 55-85% according to the cortical area, with the hippocampal CA1 and mPFC apparently having the higher rate of co-localization.We further provided first evidence that ex vivo network activity following stimulation with DHPG was subregion-and sex-specific, most likely reflecting differences in cytoarchitectonics, intrinsic connectivity, diversity of SST+ INs and expression of group I mGlus in distinct neuronal populations.In particular, the known discrete cellular distribution of mGlu 5 and mGlu 1 in the HPC [70] and distinct, or even opposing, effects of these receptors on the excitability of hippocampal CA3 neurons [71] are consistent with the differential DHPG-mediated activity in distinct hippocampal subfields.Future studies should address how cell-and sex-specific changes in mGlu 5 expression and function contribute to these differences.In this respect, it is particularly important for the SST+ INs given their high transcriptomic [72], anatomic and electrophysiologic [32] variability.
In the HPC, the loss of mGlu 5 in SST+ INs facilitated Schaffer collateral to CA1 PN synaptic plasticity.This is fully consistent with a recent finding describing a role for SST+ INs in disinhibition-assisted LTP in the mPFC to amygdala pathway [73].Our data suggest that mGlu 5 in cortical SST+ INs, that mostly form GABAergic synapses onto dendritic spines of PNs [74,75], by facilitating the activation of these INs help to control dendritic spike generation and synaptic plasticity in principal cells [76].
Behaviorally, the genetic-mediated ablation of Grm5 selectively in SST+ neurons induced several alterations in domains of social processing and negative valence that were mostly apparent in males.Our data are consistent with previous work showing that chronic inhibition of cortical SST+ INs, particularly in the mPFC, reduces anxiety-like behavior and overall emotionality under baseline conditions and after chronic stress in adult mice [77,78].Surprisingly, disinhibition of SST+ neurons through inactivation of the γ2 subunit of the GABA-A receptor in these neurons also elicited an anxiolytic-like phenotype [79] apparently at odds with our and other studies [80].On the other hand, the ubiquitous expression of γ2-containing GABA-A receptors in SST+ neurons including the CEA and LS, unlike mGlu 5 , might contribute to explain this discrepancy.In addition, a recent study from Joffe and coworkers showed that restraint stress rapidly potentiates excitatory drive onto SST+ INs in the mPFC through an mGlu 5dependent LTP at BLA inputs, which shunted incoming information from other circuits [81].This mechanism of network processing and gating of information in the mPFC involving mGlu 5 promoted cognitive behavioral adaptations related to acute stress [81].The authors of this study also generated SST Cre -Grm5 −/− mice to confirm that mGlu 5 on SST+ INs mediates restraint stress-induced changes in mPFC physiology, and reported that the loss of mGlu 5 in SST+ neurons prevented stress-induced behavioral adaptations, but did not change baseline anxiety-like behavior [81].However, since in this study behavioral data from both male and female mice were pooled, group differences might have been overlooked.Variations in baseline stress, e.g.introduced by subjecting the mice to repeated trials as in our protocol, may offer an alternative explanation.On the other hand, it is worth mentioning that Joffe and coworkers similarly observed in SST Cre -Grm5 −/− mice a reduction in freezing upon fear recall after cued-fear conditioning [81].
Using a RF analysis of the individual performances in the behavioral tests, we found that fear retrieval was the most discriminating construct between WT and SST Cre -Grm5 −/− mice and that the lack of mGlu 5 in SST+ neurons could be strongly predicted based on behavioral performances in male, but not in female mice.The estrous cycle in females is known to significantly affect a number of behavioral responses [82][83][84][85].Estrogen receptors (ERs) were reported to interact with group I mGlus [86,87] in female but not in male rodents [88], and this interaction produced sex-dependent responses in conflict-based tests of anxiety-like behavior [89,90].Our results indeed suggest that the loss of mGlu 5 in SST+ neurons affect negative valence specifically during the estrous-proestrous phase, thus supporting a cooperative regulation between ERs and mGlu 5 in SST+ neurons on fear expression.An interesting open question remains as to whether  these interactions might have been responsible for the sexdifferences we observed in both the intrinsic network excitability in vitro and in some of the behavioral tests.
A key finding of our study is that, in addition to altered local network activity, the lack of mGlu 5 in SST+ neurons reduced theta oscillatory activity in the mPFC and vHPC during fear memory retrieval.The marked reduction in theta activity did not arise from a general inability to generate theta oscillations or a shift in the dominant frequency within the theta frequency range, but rather as a result of weakened theta power.Exposure to anxiogenic and threatening contexts triggers the emergence of theta oscillations and their synchronization in the vHPC and mPFC [40,[91][92][93].INs play a well-established role in governing oscillatory states [39,94].SST+ INs are sharply tuned to theta oscillations [91,95] and enable HPC-PFC synchrony [38].Previous studies have shown an important contribution in particular of mPFC SST+ INs in the behavioral expression of fear, conceivably as a result of the disinhibition of mPFC output neurons [27] and consequently the coordination of long range synchrony and information flow between the vHPC and mPFC [38].Consistent with these studies, we show that in SST Cre -Grm5 −/− mice theta synchronization between vHPC and mPFC is markedly decreased in fear retrieval.This suggests that mGlu 5 on SST+ neurons regulate frequencyspecific synchronization between regions and may provide a potential molecular substrate for understanding changes in functional connectivity associated with negative affective states [96].Thus, mGlu 5 could act as gatekeepers of SST + IN inhibitory control onto anxiety network hubs [97], thus indirectly controlling the excitation/inhibition balance underlying the anxiety state of the organism.
Despite the large body of evidence supporting a contribution of mGlu 5 to the development of or susceptibility to affective and anxiety disorders [8,66,67,69,98,99], little was known about the underlying cellular pathophysiological mechanisms.Our study provides a novel mechanistic insight that imbalances in emotional behavior homeostasis, mainly regarding the negative valence domain, can ensue from the long-term loss of mGlu 5 activity in SST+ neurons.While in SST Cre -Grm5 −/− mice region-and sexspecific functional impairments were most likely dependent on specific subclasses of cortical INs, we at present cannot rule out the possible contribution of sparse subcortical neurons coexpressing SST and mGlu 5 or of cortical INs in which coexpression could not be detected.
In conclusion, our study advocates for a distributed but discrete role of mGlu 5 in regulating SST+ neuron excitability and brain oscillatory activity.We envisage that mGlu 5 are crucial determinants in the control that SST+ INs exert over the excitation/inhibition balance across brain regions and neuronal circuits mediating negative emotional states in health and disease.
Elevated plus maze.Mice were allowed to explore an elevated platform (72 cm above the floor) consisting of two opposing open (30 × 5 cm) and two opposing closed arms (30 × 5 cm) for a total of 5 min.Illumination in the open arms was set at 50 Lux.Mice were placed individually in one of the closed arms.The behavior of each mouse was tracked with Ethovision XT12 software.Arm entries were defined as crossing of the center of mass of the animal.Measurements during the test included: time spent in the open arms, entries and distance traveled.The position of the animal within the maze was automatically tracked and scored using Ethovision XT12 software.

Fig. 1
Fig.1Anatomical and functional evidence of the co-expression between mGlu 5 and SST. a Sagittal section of a WT mouse brain immunoreactive for mGlu 5(top)  and SST (bottom).Scale bar: 1 mm.b Higher magnification of the hippocampus.Scale bar: 500 μm.c Representative image of mGlu 5 (in magenta) and SST (turquoise) co-expression in an IN in the stratum oriens (Or) of the mouse HPC.Of note it is the high intracytoplasmic immunofluorescence signal for mGlu 5 in comparison to pyramidal neurons (marked with *), that show mostly labeling associated with the plasma membrane.Scale bar: 5 μm.d Electron micrograph of a CA1 oriens/alveus IN characterized by strong SST immunoreactivity (DAB electron-dense deposits indicated by arrowheads) in the Golgi and ER.The inset shows a dendrite of this IN, in which most of the immunometal particles identifying mGlu 5 are intracytoplasmatic rather than associated with the plasma membrane (arrows).Abbreviations: D, dendrite; ER, endoplasmic reticulum; G, Golgi apparatus; N, nucleus.Scale bar: 5 μm; scale bar inset: 1 μm.e Example image of mGlu 5 and SST expression in the mouse LS depicting the absence of co-localization.Scale bar: 5 μm.The thickness of the confocal z-stacks: c 5.02 μm; e 3.6 μm.f Representative example of a biocytin-filled PN (scale bar: 100 μm) with its respective DIC image (scale bar: 10 μm) and membrane responses to current injections.SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.g Example trace displaying PN membrane depolarization upon DHPG application and inhibition by MTEP pre-treatment.h Quantification of DHPG-mediated membrane potential change in PNs in the absence or presence of MTEP (n = 5).One-way RM ANOVA, Treatment F 4,16 = 4.29, *p < 0.05.Bonferroni multiple comparisons test: *p < 0.05, **p < 0.01.Plot represents mean ± SEM. i Example image of a biocytin-filled CA1 stratum oriens SST+ IN with its respective DIC, Ai14-tdTomato expression and membrane responses to current injections.j Example trace displaying the membrane depolarization upon DHPG application and inhibition by MTEP pre-treatment in the same SST+ IN shown in i. k Quantification of DHPGmediated membrane potential change in CA1 SST+ INs in the absence or presence of MTEP (n = 6).One-way RM ANOVA, Treatment F 4,20 = 4.75, **p < 0.01.Bonferroni multiple comparisons test: *p < 0.05, **p < 0.01.Plot represents mean ± SEM. l Example image of a biocytinfilled CA1 SST+ IN with its respective DIC, Ai14-tdTomato expression, and membrane responses to current injections.m Example trace displaying the membrane depolarization upon DHPG infusion of the same SST+ IN shown in l in the presence of the mGlu 1 NAM JNJ16259685.n Quantification of DHPG-mediated membrane potential change in hippocampal SST+ INs in the presence of JNJ16259685 (n = 6).Wilcoxon matched-pairs rank test, W = 21, *p < 0.05.Data are shown as mean ± SEM; individual data points are also provided.*p < 0.05.

Fig. 2
Fig. 2 mGlu 5 in SST+ neurons modulate network activity in a sex-and region-specific manner.a Example images of mGlu 5 (magenta), SST (turquoise), and PV (yellow) immunoreactivity in O-LM INs from sections of a WT (left panels, scale bar 5 μm, thickness of the z-stack 2.21 μm) and a SST Cre -Grm5 −/− mouse brain (right panels, scale bar 5 μm, thickness of the z-stack: 3.45 μm).The absence of mGlu 5 labeling along the plasma membrane (outlined in white in the merged upper panels) of SST Cre -Grm5 −/− O-LM INs confirms the recombination and, in turn deletion of Grm5 from SST+ neurons.b Image depicting the MEA stimulating and recording electrodes position within a hippocampal mouse slice.Red circles represent stimulating electrodes and blue ones recording electrodes.c Upon TBS, slices from SST Cre -Grm5 −/− mice displayed increased LTP (fEPSP amplitude in %) compared to WT mice.Two-way ANOVA, Genotype F 1,73 = 5.317, *p < 0.05.d The increased LTP in SST Cre -Grm5 −/− compared to WT mice was more prominent in the late LTP phase (Early LTP: Mann-Whitney U = 514, p > 0.05; Late LTP: Mann-Whitney U = 558, *p < 0.05).e Fold change frequency ratios upon DHPG stimulation in the CA3 region of the HPC were larger in SST Cre -Grm5 −/− compared to WT male mice.Mann-Whitney U = 20884, *p < 0.05.f Fold change frequency ratios upon DHPG stimulation in the CA1 region of the HPC were reduced in SST Cre -Grm5 −/− compared to WT male mice.Mann-Whitney U = 24475, *p < 0.05.g Fold change frequency ratios upon DHPG stimulation in the mPFC were reduced in SST Cre -Grm5 −/− compared to WT male mice.Mann-Whitney U = 49767, **p < 0.01.h, i Fold change frequency ratios upon DHPG stimulation in the CA3 (Mann-Whitney U = 28391, p > 0.05) (h) and CA1 (Mann-Whitney U = 11080, p > 0.05), i region of the HPC was similar in SST Cre -Grm5 −/− and WT female mice.j Fold change frequency ratios upon DHPG stimulation in the mPFC were reduced in SST Cre -Grm5 −/− compared to WT female mice.(Mann-Whitney U = 56486, *p < 0.05).Data are shown as mean ± SEM. *p < 0.05, **p < 0.01.

Fig. 6
Fig. 6 Ablation of mGlu 5 receptors in SST + INs disrupts network activity synchronization between the mPFC and vHPC.a Theta phase correlation level and b average cross-correlation values for the second positive peak of mPFC/vHPC activity during freezing expression within all CS presentations.Unpaired t test, t 11 = 2.37, *p < 0.05.Data are shown as mean ± SEM and individual values.