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Social behaviors, including reproductive behaviors, often display sexual dimorphism. Lordosis, the measure of female sexual receptivity, is one of the most apparent sexually dimorphic reproductive behaviors. Lordosis is regulated by estrogen and progesterone (P4) acting within a hypothalamic-limbic circuit, consisting of the arcuate, medial preoptic, and ventromedial nuclei of the hypothalamus. Social cues are integrated into the circuit through the amygdala. The posterodorsal part of the medial amygdala (MeApd) is involved in sexually dimorphic social and reproductive behaviors, and sends projections to hypothalamic neuroendocrine regions. GABA from the MeApd appears to facilitate social behaviors, while glutamate may play the opposite role. To test these hypotheses, adult female vesicular GABA transporter (VGAT)-Cre and vesicular glutamate transporter 2 (VGluT2)-Cre mice were transfected with halorhodopsin (eNpHR)-expressing or channelrhodopsin-expressing adeno-associated viruses (AAVs), respectively, in the MeApd. The lordosis quotient (LQ) was measured following either photoinhibition of VGAT or photoexcitation of VGluT2 neurons, and brains were assessed for c-Fos immunohistochemistry (IHC). Photoinhibition of VGAT neurons in the MeApd decreased LQ, and decreased c-Fos expression within VGAT neurons, within the MeApd as a whole, and within the ventrolateral part of the ventromedial nucleus (VMHvl). Photoexcitation of VGluT2 neurons did not affect LQ, but did increase time spent self-grooming, and increased c-Fos expression within VGluT2 neurons in the MeApd. Neither condition altered c-Fos expression in the medial preoptic nucleus (MPN) or the arcuate nucleus (ARH). These data support a role for MeApd GABA in the facilitation of lordosis. Glutamate from the MeApd does not appear to be directly involved in the lordosis circuit, but appears to direct behavior away from social interactions.SIGNIFICANCE STATEMENTLordosis, the measure of female sexual receptivity, is a sexually dimorphic behavior regulated within a hypothalamic-limbic circuit. Social cues are integrated through the amygdala, and the posterodorsal part of the medial amygdala (MeApd) is involved in sexually dimorphic social and reproductive behaviors. Photoinhibition of GABAergic neurons in the MeApd inhibited lordosis, while photoactivation of glutamate neurons had no effect on lordosis, but increased self-grooming. These data support a role for MeApd GABA in the facilitation of social behaviors and MeApd glutamate projections in anti-social interactions.

Cell adhesion molecules (CAMs) are key players in the formation of neural circuits during development. The γ-protocadherins (γ-Pcdhs), a family of 22 CAMs encoded by the Pcdhg gene cluster, are known to play important roles in dendrite arborization, axon targeting, and synapse development. We showed previously that multiple γ-Pcdhs interact physically with the autism-associated CAM neuroligin-1, and inhibit the latter's ability to promote excitatory synapse maturation. Here, we show that γ-Pcdhs can also interact physically with the related neuroligin-2, and inhibit this CAM's ability to promote inhibitory synapse development. In an artificial synapse assay, γ-Pcdhs co-expressed with neuroligin-2 in non-neuronal cells reduce inhibitory presynaptic maturation in contacting hippocampal axons. Mice lacking the γ-Pcdhs from the forebrain (including the cortex, the hippocampus, and portions of the amygdala) exhibit increased inhibitory synapse density and increased co-localization of neuroligin-2 with inhibitory postsynaptic markers in vivo. These Pcdhg mutants also exhibit defective social affiliation and an anxiety-like phenotype in behavioral assays. Together, these results suggest that γ-Pcdhs negatively regulate neuroligins to limit synapse density in a manner that is important for normal behavior.

Accumulating evidence implicates a role for brain structures outside the ascending auditory pathway in tinnitus, the phantom perception of sound. In addition to other factors such as age-dependent hearing loss, high-level sound exposure is a prominent cause of tinnitus. Here, we examined how noise exposure altered the distribution of excitatory and inhibitory synaptic inputs in the guinea pig hippocampus and determined whether these changes were associated with tinnitus. In experiment one, guinea pigs were overexposed to unilateral narrow-band noise (98 dB SPL, 2 h). Two weeks later, the density of excitatory (VGLUT-1/2) and inhibitory (VGAT) synaptic terminals in CA1, CA3, and dentate gyrus hippocampal subregions was assessed by immunohistochemistry. Overall, VGLUT-1 density primarily increased, while VGAT density decreased significantly in many regions. Then, to assess whether the noise-induced alterations were persistent and related to tinnitus, experiment two utilized a noise-exposure paradigm shown to induce tinnitus and assessed tinnitus development which was assessed using gap-prepulse inhibition of the acoustic startle (GPIAS). Twelve weeks after sound overexposure, changes in excitatory synaptic terminal density had largely recovered regardless of tinnitus status, but the recovery of GABAergic terminal density was dramatically different in animals expressing tinnitus relative to animals resistant to tinnitus. In resistant animals, inhibitory synapse density recovered to preexposure levels, but in animals expressing tinnitus, inhibitory synapse density remained chronically diminished. Taken together, our results suggest that noise exposure induces striking changes in the balance of excitatory and inhibitory synaptic inputs throughout the hippocampus and reveal a potential role for rebounding inhibition in the hippocampus as a protective factor leading to tinnitus resilience.

The cholinergic neurons in the pontomesencephalic tegmentum have been shown to discharge in association with and promote cortical activation during active or attentive waking and paradoxical or rapid eye movement sleep. However, GABA neurons lie intermingled with the cholinergic neurons and may contribute to or oppose this activity and role. Here we investigated in vitro and in vivo the properties, activities, and role of GABA neurons within the laterodorsal tegmental and sublaterodorsal tegmental nuclei (LDT/SubLDT) using male and female transgenic mice expressing channelrhodopsin-(ChR2)-EYFP in vesicular GABA transporter (VGAT)-expressing neurons. Presumed GABA (pGABA) neurons were identified by response to photostimulation and verified by immunohistochemical staining following juxtacellular labeling in vivo pGABA neurons were found to be fast-firing neurons with the capacity to burst when depolarized from a hyperpolarized membrane potential. When stimulated in vivo in urethane-anesthetized or unanesthetized mice, the pGABA neurons fired repetitively at relatively fast rates (∼40 Hz) during a continuous light pulse or phasically in bursts (>100 Hz) when driven by rhythmic light pulses at theta (4 or 8 Hz) frequencies. pNon-GABA, which likely included cholinergic, neurons were inhibited during each light pulse to discharge rhythmically in antiphase to the pGABA neurons. The reciprocal rhythmic bursting by the pGABA and pNon-GABA neurons drove rhythmic theta activity in the EEG. Such phasic bursting by GABA neurons also occurred in WT mice in association with theta activity during attentive waking and paradoxical sleep.SIGNIFICANCE STATEMENT Neurons in the pontomesencephalic tegmentum, particularly cholinergic neurons, play an important role in cortical activation, which occurs during active or attentive waking and paradoxical or rapid eye movement sleep. Yet the cholinergic neurons lie intermingled with GABA neurons, which could play a similar or opposing role. Optogenetic stimulation and recording of these GABA neurons in mice revealed that they can discharge in rhythmic bursts at theta frequencies and drive theta activity in limbic cortex. Such phasic burst firing also occurs during natural attentive waking and paradoxical sleep in association with theta activity and could serve to enhance sensory-motor processing and memory consolidation during these states.

Chronic stress in both humans and rodents induces a robust downregulation of neuroligin-2, a key component of the inhibitory synapse, in the NAc that modifies behavioral coping mechanisms and stress resiliency in mice. Here we extend this observation by examining the role of two other inhibitory synapse constituents, vesicular GABA transporter (vGAT) and gephyrin, in the NAc of male mice that underwent chronic social defeat stress (CSDS) and in patients with major depressive disorder (MDD). We first performed transcriptional profiling of vGAT and gephyrin in postmortem NAc samples from a cohort of healthy controls, medicated, and nonmedicated MDD patients. In parallel, we conducted whole-cell electrophysiology recordings in the NAc of stress-susceptible and stress-resilient male mice following 10 d of CSDS. Finally, we used immunohistochemistry to analyze protein levels of vGAT and gephyrin in the NAc of mice after CSDS. We found that decreased vGAT and gephyrin mRNA in the NAc of nonmedicated MDD patients is paralleled by decreased inhibitory synapse markers and decreased frequency of mini inhibitory postsynaptic currents (mIPSC) in the NAc of susceptible mice, indicating a reduction in the number of NAc inhibitory synapses that is correlated with depression-like behavior. Overall, these findings suggest a common state of reduced inhibitory tone in the NAc in depression and stress susceptibility. SIGNIFICANCE STATEMENT Existing studies focus on excitatory synaptic changes after social stress, although little is known about stress-induced inhibitory synaptic plasticity and its relevance for neuropsychiatric disease. These results extend our previous findings on the critical role of impaired inhibitory tone in the NAc following stress and provide new neuropathological evidence for reduced levels of inhibitory synaptic markers in human NAc from nonmedicated major depressive disorder patients. This finding is corroborated in stress-susceptible male mice that have undergone chronic social defeat stress, a mouse model of depression, at both the level of synaptic function and protein expression. These data support the hypothesis that reduced inhibitory synaptic transmission within the NAc plays a critical role in the stress response.

The nucleus of the solitary tract (NTS) regulates life-sustaining functions ranging from appetite and digestion to heart rate and breathing. It is also the brain’s primary sensory nucleus for visceral sensations relevant to symptoms in medical and psychiatric disorders. To better understand which neurons may exert top-down control over the NTS, here we provide a brain-wide map of all neurons that project axons directly to the caudal, viscerosensory NTS, focusing on a medial subregion with aldosterone-sensitive HSD2 neurons. Injecting an axonal tracer (cholera toxin b) into the NTS produces a similar pattern of retrograde labeling in rats and mice. The paraventricular hypothalamic nucleus (PVH), lateral hypothalamic area, and central nucleus of the amygdala (CeA) contain the densest concentrations of NTS-projecting neurons. PVH afferents are glutamatergic (express Slc17a6/Vglut2) and are distinct from neuroendocrine PVH neurons. CeA afferents are GABAergic (express Slc32a1/Vgat) and are distributed largely in the medial CeA subdivision. Other retrogradely labeled neurons are located in a variety of brain regions, including the cerebral cortex (insular and infralimbic areas), bed nucleus of the stria terminalis, periaqueductal gray, Barrington’s nucleus, Kölliker-Fuse nucleus, hindbrain reticular formation, and rostral NTS. Similar patterns of retrograde labeling result from tracer injections into different NTS subdivisions, with dual retrograde tracing revealing that many afferent neurons project axon collaterals to both the lateral and medial NTS subdivisions. This information provides a roadmap for studying descending axonal projections that may influence visceromotor systems and visceral “mind-body” symptoms.

To investigate the effects of estrogen on the threshold and temperature of orofacial pain and explore the influence on the function of glutamate and GABA neurons in the orofacial pain temperature perception pathway by observing the expression of vesicular glutamate transporter 2 (Vglut2) and vesicular GABA transporter 1 (Vgat1).A total of 24 adult female Sprague-Dawley rats were divided into three groups: sham operation (SHAM), ovariectomized (OVX) and ovariectomized plus estrogen intervention (OVX+E) (n = 8 per group). The threshold of mechanical pain of the orofacial region was assessed with von Frey filaments, and the temperature of the rat orofacial region was monitored by infrared thermography. Changes in the expression of Vglut2 and Vgat1 in glutamatergic and GABAergic neurons in the trigeminal ganglion (TG), spinal trigeminal nucleus (Sp5C), lateral parabrachial nucleus (LPB) and ventral posteromedial nucleus of the thalamus (VPM) were assessed by immunostaining and Western blotting.Under low-estrogen conditions, the mechanical pain threshold of the orofacial region of rats decreased significantly, and the temperature of the orofacial region increased significantly. The expression of Vglut2 and Vgat1 in the TG and Sp5C showed a downward trend, and the decline in Vgat1 was greater than that in Vglut2. Conversely, both proteins were upregulated in the LPB and VPM, and the magnitude of the changes in Vglut2 was greater than that in Vgat1. Estrogen therapy reversed these changes.Under low-estrogen conditions, the proportion of glutamate and GABA neurons in the orofacial pain and temperature sensation pathway changes, which leads to the imbalance of neurotransmission function and the enhancement of excitatory transmission of these two kinds of neurons and finally leads to a decrease in the orofacial pain threshold and an increase in temperature.

While dexterity relies on the constant transmission of sensory information, unchecked feedback can be disruptive to behavior. Yet how somatosensory feedback from the hands is regulated as it first enters the brain, and whether this modulation exerts any influence on movement, remain unclear. Leveraging molecular-genetic access in mice, we find that tactile afferents from the hand recruit neurons in the brainstem cuneate nucleus whose activity is modulated by distinct classes of local inhibitory neurons. Selective manipulation of these inhibitory circuits can suppress or enhance the transmission of tactile information, affecting behaviors that rely on movement of the hands. Investigating whether these local circuits are subject to top-down control, we identify distinct descending cortical pathways that innervate cuneate in a complementary pattern. Somatosensory cortical neurons target the core tactile region of cuneate, while a large rostral cortical population drives feed-forward inhibition of tactile transmission through an inhibitory shell. These findings identify a circuit basis for tactile feedback modulation, enabling the effective execution of dexterous movement.

Objective Melanin-concentrating hormone (MCH) plays a key role in regulating energy balance. MCH acts via its receptor MCHR1, and MCHR1 deletion increases energy expenditure and locomotor activity, which is associated with a hyperdopaminergic state. Since MCHR1 expression is widespread, the neurons supporting the effects of MCH on energy expenditure are not clearly defined. There is a high density of MCHR1 neurons in the striatum, and these neurons are known to be GABAergic. We thus determined if MCH acts via this GABAergic neurocircuit to mediate energy balance. Methods We generated a Mchr1-flox mouse and crossed it with the Vgat-cre mouse to assess if MCHR1 deletion from GABAergic neurons expressing the vesicular GABA transporter (vGAT) in female Vgat-Mchr1-KO mice resulted in lower body weights or increased energy expenditure. Additionally, we determined if MCHR1-expressing neurons within the accumbens form part of the neural circuit underlying MCH-mediated energy balance by delivering an adeno-associated virus expressing Cre recombinase to the accumbens nucleus of Mchr1-flox mice. To evaluate if a dysregulated dopaminergic tone leads to their hyperactivity, we determined if the dopamine reuptake blocker GBR12909 prolonged the drug-induced locomotor activity in Vgat-Mchr1-KO mice. Furthermore, we also performed amperometry recordings to test whether MCHR1 deletion increases dopamine output within the accumbens and whether MCH can suppress dopamine release. Results Vgat-Mchr1-KO mice have lower body weight, increased energy expenditure, and increased locomotor activity. Similarly, restricting MCHR1 deletion to the accumbens nucleus also increased locomotor activity. Vgat-Mchr1-KO mice show increased and prolonged sensitivity to GBR12909-induced locomotor activity, and amperometry recordings revealed that GBR12909 elevated accumbens dopamine levels to twice that of controls, thus MCHR1 deletion may lead to a hyperdopaminergic state that mediates their observed hyperactivity. Consistent with the inhibitory effect of MCH, we found that MCH acutely suppresses dopamine release within the accumbens. Conclusions As with established models of systemic MCH or MCHR1 deletion, we found that MCHR1 deletion from GABAergic neurons, specifically those within the accumbens nucleus, also led to increased locomotor activity. A hyperdopaminergic state underlies this increased locomotor activity, and is consistent with our finding that MCH signaling within the accumbens nucleus suppresses dopamine release. In effect, MCHR1 deletion may disinhibit dopamine release leading to the observed hyperactivity., Highlights • Generation of Mchr1-flox mouse enabled cre-mediated deletion of Mchr1. • Mchr1 deletion at GABAergic neurons decreased body weight. • Mchr1 deletion at GABAergic neurons increased locomotor activity. • Mchr1 deletion increased dopaminergic tone in the mesolimbic accumbens circuitry. • MCH suppressed dopamine release in the accumbens nucleus.

Summary: Optogenetic stimulation of inhibitory interneurons has become a commonly used strategy for silencing neuronal activity. This is typically achieved using transgenic mice expressing excitatory opsins in inhibitory interneurons throughout the brain, raising the question of how spatially extensive the resulting inhibition is. Here, we characterize neuronal silencing in VGAT-ChR2 mice, which express channelrhodopsin-2 in inhibitory interneurons, as a function of light intensity and distance from the light source in several cortical and subcortical regions. We show that light stimulation, even at relatively low intensities, causes inhibition not only in brain regions targeted for silencing but also in their subjacent areas. In contrast, virus-mediated expression of an inhibitory opsin enables robust silencing that is restricted to the region of opsin expression. Our results reveal important constraints on using inhibitory interneuron activation to silence neuronal activity and emphasize the necessity of carefully controlling light stimulation parameters when using this silencing strategy. : Optogenetic silencing is often achieved using transgenic mice expressing channelrhodopsin in GABAergic neurons. Babl et al. demonstrate that this strategy causes spatially extensive inhibition that can extend beyond brain regions targeted for silencing, whereas virally mediated expression of an inhibitory opsin enables regionally restricted inhibition. Keywords: optogenetics, neuronal silencing, inhibitory interneurons, channelrhodopsin, VGAT-ChR2, ArchT, spatial specificity

The histaminergic neurons of the tuberomammillary nucleus (TMNHDC) of the posterior hypothalamus have long been implicated in promoting arousal. More recently, a role for GABAergic signaling by the TMNHDCneurons in arousal control has been proposed. Here, we investigated the effects of selective chronic disruption of GABA synthesis (via genetic deletion of the GABA synthesis enzyme, glutamic acid decarboxylase 67) or GABAergic transmission (via genetic deletion of the vesicular GABA transporter (VGAT)) in the TMNHDCneurons on sleep–wake in male mice. We also examined the effects of acute chemogenetic activation and optogenetic inhibition of TMNHDCneurons upon arousal in male mice. Unexpectedly, we found that neither disruption of GABA synthesis nor GABAergic transmission altered hourly sleep–wake quantities, perhaps because very few TMNHDCneurons coexpressed VGAT. Acute chemogenetic activation of TMNHDCneurons did not increase arousal levels above baseline but did enhance vigilance when the mice were exposed to a behavioral cage change challenge. Similarly, acute optogenetic inhibition had little effect upon baseline levels of arousal. In conclusion, we could not identify a role for GABA release by TMNHDCneurons in arousal control. Further, if TMNHDCneurons do release GABA, the mechanism by which they do so remains unclear. Our findings support the view that TMNHDCneurons may be important for enhancing arousal under certain conditions, such as exposure to a novel environment, but play only a minor role in behavioral and EEG arousal under baseline conditions.SIGNIFICANCE STATEMENTThe histaminergic neurons of the tuberomammillary nucleus of the hypothalamus (TMNHDC) have long been thought to promote arousal. Additionally, TMNHDCneurons may counter-regulate the wake-promoting effects of histamine through co-release of the inhibitory neurotransmitter, GABA. Here, we show that impairing GABA signaling from TMNHDCneurons does not impact sleep–wake amounts and that few TMNHDCneurons contain the vesicular GABA transporter, which is presumably required to release GABA. We further show that acute activation or inhibition of TMNHDCneurons has limited effects upon baseline arousal levels and that activation enhances vigilance during a behavioral challenge. Counter to general belief, our findings support the view that TMNHDCneurons are neither necessary nor sufficient for the initiation and maintenance of arousal under baseline conditions.

In mammals, the circadian rhythms are regulated by the central clock located in the hypothalamic suprachiasmatic nucleus (SCN), which is composed of heterogeneous neurons with various neurotransmitters. Among them an inhibitory neurotransmitter, γ-Amino-Butyric-Acid (GABA), is expressed in almost all SCN neurons, however, its role in the circadian physiology is still unclear. Here, we show that the SCN of fetal mice lacking vesicular GABA transporter (VGAT−/−) or GABA synthesizing enzyme, glutamate decarboxylase (GAD65−/−/67−/−), shows burst firings associated with large Ca2+ spikes throughout 24 hours, which spread over the entire SCN slice in synchrony. By contrast, circadian PER2 rhythms in VGAT−/− and GAD65−/−/67−/− SCN remain intact. SCN-specific VGAT deletion in adult mice dampens circadian behavior rhythm. These findings indicate that GABA in the fetal SCN is necessary for refinement of the circadian firing rhythm and, possibly, for stabilizing the output signals, but not for circadian integration of multiple cellular oscillations., Daisuke Ono et al. show that neurons in the suprachiasmatic nucleus from mice lacking VGAT or GAD65/67 exhibit burst firing patterns. These bursts are associated with large calcium spikes and did not affect circadian PER2 rhythms, suggesting an important role for hypothalamic GABA in refinement of circadian output rhythms in mice.

Inhibitory circuits in the auditory brainstem undergo multiple postnatal changes that are both activity-dependent and activity-independent. We tested to see if the shift from GABA- to glycinergic transmission, which occurs in the rat medial nucleus of the trapezoid body (MNTB) around the onset of hearing, depends on sound-evoked neuronal activity. We prevented the activity by bilateral cochlear ablations in early postnatal rats and studied ionotropic GABA and glycine receptors in MNTB neurons after hearing onset. The removal of the cochlea decreased responses of GABAA and glycine receptors to exogenous agonists as well as the amplitudes of inhibitory postsynaptic currents. The reduction was accompanied by a decrease in the number of glycine receptor- or vesicular GABA transporter-immunopositive puncta. Furthermore, the ablations markedly affected the switch in presynaptic GABAA to glycine receptors. The increase in the expression of postsynaptic glycine receptors and the shift in inhibitory transmitters were not prevented. The results suggest that inhibitory transmission in the MNTB is subject to multiple developmental signals and support the idea that auditory experience plays a role in the maturation of the brainstem glycinergic circuits.

During mouse postnatal eye development, the embryonic hyaloid vascular network regresses from the vitreous as an adaption for high-acuity vision. This process occurs with precisely controlled timing. Here, we show that opsin 5 (OPN5; also known as neuropsin)-dependent retinal light responses regulate vascular development in the postnatal eye. In Opn5-null mice, hyaloid vessels regress precociously. We demonstrate that 380-nm light stimulation via OPN5 and VGAT (the vesicular GABA/glycine transporter) in retinal ganglion cells enhances the activity of inner retinal DAT (also known as SLC6A3; a dopamine reuptake transporter) and thus suppresses vitreal dopamine. In turn, dopamine acts directly on hyaloid vascular endothelial cells to suppress the activity of vascular endothelial growth factor receptor 2 (VEGFR2) and promote hyaloid vessel regression. With OPN5 loss of function, the vitreous dopamine level is elevated and results in premature hyaloid regression. These investigations identify violet light as a developmental timing cue that, via an OPN5-dopamine pathway, regulates optic axis clearance in preparation for visual function.

Pain constitutes the major non-motor symptom in Parkinson’s disease (PD). Its mechanism is still poorly understood although an increase in excitation or a decrease in inhibition have been reported in preclinical studies. The aim of this study was to investigate gamma aminobutyric acid (GABA) inhibition in the 6-hydroxydopamine (6-OHDA) PD rat model. Therefore, the expression of three inhibitory markers parvalbumin, glutamate decarboxylase 67 (GAD67) and vesicular GABA transporter (VGAT) was evaluated, besides cold allodynia, in bilateral 6-OHDA lesioned rat. There was a significant increase in the expression of the three markers labeling within the spinal dorsal horn (SDH) of 6-OHDA lesioned rats. In parallel, there was also an increase of the excitatory marker protein kinase C gamma (PKCγ) . PKCγ cells have a crucial role in pain chronicity and are regulated by GABAergic influences. Central dopamine depletion induced an increase in excitation as reveled by an increase in cFOS expression upon acetone stimulus and the presence of cold allodynia. In addition, dopamine depletion induced increased expression in inhibitory markers, which may reflect a disinhibition or a decreased inhibition in 6-OHDA lesioned rats.

The roles GABAergic and glutamatergic inputs in regulating the activity of the gonadotrophin-releasing hormone (GnRH) neurons at the time of the preovulatory surge remain unclear. We used expansion microscopy to compare the density of GABAergic and glutamatergic synapses on the GnRH neuron cell body and proximal dendrite in dioestrous and pro-oestrous female mice. An evaluation of all synapses immunoreactive for synaptophysin revealed that the highest density of inputs to rostral preoptic area GnRH neurons occurred within the first 45 µm of the primary dendrite (approximately 0.19 synapses µm-1 ) with relatively few synapses on the GnRH neuron soma or beyond 45 µm of the dendrite (0.05-0.08 synapses µm-1 ). Triple immunofluorescence labelling demonstrated a predominance of glutamatergic signalling with twice as many vesicular glutamate transporter 2 synapses detected compared to vesicular GABA transporter. Co-labelling with the GABAA receptor scaffold protein gephyrin and the glutamate receptor postsynaptic density marker Homer1 confirmed these observations, as well as the different spatial distribution of GABA and glutamate inputs along the dendrite. Quantitative assessments revealed no differences in synaptophysin, GABA or glutamate synapses at the proximal dendrite and soma of GnRH neurons between dioestrous and pro-oestrous mice. Taken together, these studies demonstrate that the GnRH neuron receives twice as many glutamatergic synapses compared to GABAergic synapses and that these inputs preferentially target the first 45 µm of the GnRH neuron proximal dendrite. These inputs appear to be structurally stable before the onset of pro-oestrous GnRH surge.

Reductions in the GABAergic neurotransmitter system exist across multiple brain regions in schizophrenia and encompass both pre- and postsynaptic components. While reduced midbrain GABAergic inhibitory neurotransmission may contribute to the hyperdopaminergia thought to underpin psychosis in schizophrenia, molecular changes consistent with this have not been reported. We hypothesised that reduced GABA-related molecular markers would be found in the midbrain of people with schizophrenia and that these would correlate with dopaminergic molecular changes. We hypothesised that downregulation of inhibitory neuron markers would be exacerbated in schizophrenia cases with high levels of neuroinflammation. Eight GABAergic-related transcripts were measured with quantitative PCR, and glutamate decarboxylase (GAD) 65/67 and GABAA alpha 3 (α3) (GABRA3) protein were measured with immunoblotting, in post-mortem midbrain (28/28 and 28/26 control/schizophrenia cases for mRNA and protein, respectively), and analysed by both diagnosis and inflammatory subgroups (as previously defined by higher levels of four pro-inflammatory cytokine transcripts). We found reductions (21 – 44%) in mRNA encoding both presynaptic and postsynaptic proteins, vesicular GABA transporter (VGAT), GAD1, and parvalbumin (PV) mRNAs and four alpha subunits (α1, α2, α3, α5) of the GABAA receptor in people with schizophrenia compared to controls (p SST) was unchanged (p = 0.485). We confirmed the reduction in GAD at the protein level (34%, p GABRA3 mRNA exhibited more pronounced changes in high compared to low inflammatory subgroups in schizophrenia. GABRA3 protein was expressed by 98% of tyrosine hydroxylase-positive neurons and was 23% lower in schizophrenia, though this did not reach statistical significance (p > 0.05). Expression of transcripts for GABAA receptor alpha subunits 2 and 3 (GABRA2, GABRA3) were positively correlated with tyrosine hydroxylase (TH) and dopamine transporter (DAT) transcripts in schizophrenia cases (GABRA2; r > 0.630, GABRA3; r > 0.762, all p GABRA2; r GABRA3; r p > 0.05). Taken together, our results support a profound disruption to inhibitory neurotransmission in the substantia nigra regardless of inflammatory status, which provides a potential mechanism for disinhibition of nigrostriatal dopamine neurotransmission.

ObjectiveTo identify the causative gene in a large unsolved family with genetic epilepsy with febrile seizures plus (GEFS+), we sequenced the genomes of family members, and then determined the contribution of the identified gene to the pathogenicity of epilepsies by examining sequencing data from 2,772 additional patients.MethodsWe performed whole genome sequencing of 3 members of a GEFS+ family. Subsequently, whole exome sequencing data from 1,165 patients with epilepsy from the Epi4K dataset and 1,329 Australian patients with epilepsy from the Epi25 dataset were interrogated. Targeted resequencing was performed on 278 patients with febrile seizures or GEFS+ phenotypes. Variants were validated and familial segregation examined by Sanger sequencing.ResultsEight previously unreported missense variants were identified in SLC32A1, coding for the vesicular inhibitory amino acid cotransporter VGAT. Two variants cosegregated with the phenotype in 2 large GEFS+ families containing 8 and 10 affected individuals, respectively. Six further variants were identified in smaller families with GEFS+ or idiopathic generalized epilepsy (IGE).ConclusionMissense variants in SLC32A1 cause GEFS+ and IGE. These variants are predicted to alter γ-aminobutyric acid (GABA) transport into synaptic vesicles, leading to altered neuronal inhibition. Examination of further epilepsy cohorts will determine the full genotype–phenotype spectrum associated with SLC32A1 variants.

The BOLD fMRI response in the cortex is often assumed to reflect changes in excitatory neural activity. However, the contribution of inhibitory neurons to BOLD fMRI is unclear. Here, the role of inhibitory and excitatory activity was examined using multimodal approaches: electrophysiological recording, 15.2 T fMRI, optical intrinsic signal imaging, and modeling. Inhibitory and excitatory neuronal activity in the somatosensory cortex were selectively modulated by 20-s optogenetic stimulation of VGAT-ChR2 and CaMKII-ChR2 mice, respectively. Somatosensory stimulation and optogenetic stimulation of excitatory neurons induced positive BOLD responses in the somatosensory network, whereas stimulation of inhibitory neurons produced biphasic responses at the stimulation site, initial positive and later negative BOLD signals, and negative BOLD responses at downstream sites. When the stimulation duration was reduced to 5 s, the hemodynamic response of VGAT-ChR2 mice to optogenetic stimulation was only positive. Lastly, modeling performed from neuronal and hemodynamic data shows that the hemodynamic response function (HRF) of excitatory neurons is similar across different conditions, whereas the HRF of inhibitory neurons is highly sensitive to stimulation frequency and peaks earlier than that of excitatory neurons. Our study provides insights into the neurovascular coupling of excitatory and inhibitory neurons and the interpretation of BOLD fMRI signals.

Fumonisin B1 (FB1) is a neurodegenerative mycotoxin synthesized by Fusarium spp., but the potential neurobehavioral toxicity effects in organisms have not been characterized clearly. Caenorhabditis elegans (C. elegans) has emerged as a promising model organism for neurotoxicological studies due to characteristics such as well-functioning nervous system and rich behavioral phenotypes. To investigate whether FB1 has neurobehavioral toxicity effects on C. elegans, the motor behavior, neuronal structure, neurotransmitter content, and gene expression related with neurotransmission of C. elegans were determined after exposed to 20-200 μg/mL FB1 for 24 h and 48 h, respectively. Results showed that FB1 caused behavioral defects, including body bends, head thrashes, crawling distance, mean speed, mean amplitude, mean wavelength, foraging behavior, and chemotaxis learning ability in a dose-, and time-dependent manner. In addition, when C. elegans was exposed to FB1 at a concentration of 200 μg/mL for 24 h and above 100 μg/mL for 48 h, the GABAergic and serotonergic neurons were damaged, but no effect on dopaminergic, glutamatergic, and cholinergic neurons. The relative content of GABA and serotonin decreased significantly. Furthermore, abnormal expression of mRNA levels associated with GABA and serotonin were found in nematodes treated with FB1, such as unc-30, unc-47, unc-49, exp-1, mod-5, cat-1, and tph-1. The neurobehavioral toxicity effect of FB1 may be mediated by abnormal neurotransmission of GABA and serotonin. This study provides useful information for understanding the neurotoxicity of FB1.

The suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals, is a network structure composed of multiple types of γ-aminobutyric acid (GABA)-ergic neurons and glial cells. However, the roles of GABA-mediated signaling in the SCN network remain controversial. Here, we report noticeable impairment of the circadian rhythm in mice with a specific deletion of the vesicular GABA transporter in arginine vasopressin (AVP)-producing neurons. These mice showed disturbed diurnal rhythms of GABA(A) receptor-mediated synaptic transmission in SCN neurons and marked lengthening of the activity time in circadian behavioral rhythms due to the extended interval between morning and evening locomotor activities. Synchrony of molecular circadian oscillations among SCN neurons did not significantly change, whereas the phase relationships between SCN molecular clocks and circadian morning/evening locomotor activities were altered significantly, as revealed by PER2::LUC imaging of SCN explants and in vivo recording of intracellular Ca(2+) in SCN AVP neurons. In contrast, daily neuronal activity in SCN neurons in vivo clearly showed a bimodal pattern that correlated with dissociated morning/evening locomotor activities. Therefore, GABAergic transmission from AVP neurons regulates the timing of SCN neuronal firing to temporally restrict circadian behavior to appropriate time windows in SCN molecular clocks.

Our previous study showed the intrinsic ability of descending noradrenergic neurons projecting from the locus coeruleus to the spinal dorsal horn (SDH) to suppress itch-related behaviors. Noradrenaline and α1A-adrenaline receptor (α1A-AR) agonist increase inhibitory synaptic inputs onto SDH interneurons expressing gastrin-releasing peptide receptors, which are essential for itch transmission. However, the contribution of α1A-ARs expressed in SDH inhibitory interneurons to itch-related behavior remains to be determined. In this study, RNAscope in situ hybridization revealed that Adra1a mRNA is expressed in SDH inhibitory interneurons that are positive for Slc32a1 mRNA (known as vesicular GABA transporter). Mice with conditional knock-out of α1A-ARs in inhibitory interneurons (Vgat-Cre;Adra1aflox/flox mice) exhibited an increase in scratching behavior when induced by an intradermal injection of chloroquine, but not compound 48/80, which are known as models of histamine-independent and dependent itch, respectively. Furthermore, knockout of inhibitory neuronal α1A-ARs in the SDH using the CRISPR–Cas9 system also increased the scratching behavior elicited by chloroquine but not compound 48/80. Our findings demonstrated for the first time that α1A-ARs in SDH inhibitory interneurons contribute to the regulation of itch signaling with preference for histamine-independent itch. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-021-00768-9.

Cannabinoid receptor type 1 (CB1R) is widely distributed in the substantia nigra pars reticulata (SNpr). However, the role of CB1R at the SNpr level in threatening situations is poorly understood. We investigated the role of CB1R in the SNpr on the expression of fear responses in mice confronted with urutu-cruzeiro pit vipers. First, a bidirectional neurotracer was injected into the SNpr; then, immunostaining of the vesicular GABA transporter was conducted at the levels of the striatum (CPu) and deep layers of the superior colliculus (dlSC). In addition, CB1R immunostaining and GABA labelling were performed in the SNpr. Using a prey-versus-snake paradigm, mice were pretreated with the CB1R antagonist AM251 (100 pmol) and treated with the endocannabinoid anandamide (AEA, 5 pmol) in the SNpr, followed by bicuculline (40 ng) in the dlSC, and were then confronted with a snake. Bidirectional neural tract tracers associated with immunofluorescence showed the GABAergic striatonigral disinhibitory and nigrotectal inhibitory pathways. Furthermore, we showed that CB1R labelling was restricted to axonal fibres surrounding SNpr GABAergic cells. We also demonstrated a decrease in the defensive behaviours of mice treated with AEA in the SNpr, but this effect was blocked by pre-treatment with AM251 in this structure. Taken together, our results show that the panicolytic consequences of the AEA enhancement in the SNpr are signalled by CB1R, suggesting that CB1R localised in axon terminals of CPu GABAergic neurons in the SNpr modulates the activity of the nigrotectal GABAergic pathway during the expression of defensive behaviours in threatening situations.

Previously, we found that in the mammalian retina, inhibitory inputs onto starburst amacrine cells (SACs) are required for robust direction selectivity of On-Off direction-selective ganglion cells (On-Off DSGCs) against noisy backgrounds (Chen et al., 2016). However, the source of the inhibitory inputs to SACs and how this inhibition confers noise resilience of DSGCs are unknown. Here, we show that when visual noise is present in the background, the motion-evoked inhibition to an On-Off DSGC is preserved by a disinhibitory motif consisting of a serially connected network of neighboring SACs presynaptic to the DSGC. This preservation of inhibition by a disinhibitory motif arises from the interaction between visually evoked network dynamics and short-term synaptic plasticity at the SAC-DSGC synapse. Although the disinhibitory microcircuit is well studied for its disinhibitory function in brain circuits, our results highlight the algorithmic flexibility of this motif beyond disinhibition due to the mutual influence between network and synaptic plasticity mechanisms.

Animal behaviours that are superficially similar can express different intents in different contexts, but how this flexibility is achieved at the level of neural circuits is not understood. For example, males of many species can exhibit mounting behaviour towards same- or opposite-sex conspecifics1, but it is unclear whether the intent and neural encoding of these behaviours are similar or different. Here we show that female- and male-directed mounting in male laboratory mice are distinguishable by the presence or absence of ultrasonic vocalizations (USVs)2–4, respectively. These and additional behavioural data suggest that most male-directed mounting is aggressive, although in rare cases it can be sexual. We investigated whether USV+ and USV− mounting use the same or distinct hypothalamic neural substrates. Micro-endoscopic imaging of neurons positive for oestrogen receptor 1 (ESR1) in either the medial preoptic area (MPOA) or the ventromedial hypothalamus, ventrolateral subdivision (VMHvl) revealed distinct patterns of neuronal activity during USV+ and USV− mounting, and the type of mounting could be decoded from population activity in either region. Intersectional optogenetic stimulation of MPOA neurons that express ESR1 and vesicular GABA transporter (VGAT) (MPOAESR1∩VGAT neurons) robustly promoted USV+ mounting, and converted male-directed attack to mounting with USVs. By contrast, stimulation of VMHvl neurons that express ESR1 (VMHvlESR1 neurons) promoted USV− mounting, and inhibited the USVs evoked by female urine. Terminal stimulation experiments suggest that these complementary inhibitory effects are mediated by reciprocal projections between the MPOA and VMHvl. Together, these data identify a hypothalamic subpopulation that is genetically enriched for neurons that causally induce a male reproductive behavioural state, and indicate that reproductive and aggressive states are represented by distinct population codes distributed between MPOAESR1 and VMHvlESR1 neurons, respectively. Thus, similar behaviours that express different internal states are encoded by distinct hypothalamic neuronal populations. Ultrasonic vocalizations of male mice distinguish aggressive, male-directed mounting from reproductive, female-directed mounting behaviours, which are represented by distinct ESR1-expressing populations of neurons in the ventromedial hypothalamus and medial preoptic area, respectively.

Background Cerebral microinfarcts (MIs) lead to progressive cognitive impairments in the elderly, and there is currently no effective preventative strategy due to uncertainty about the underlying pathogenic mechanisms. One possibility is the dysfunction of GABAergic transmission and ensuing excitotoxicity. Dysfunction of GABAergic transmission induces excitotoxicity, which contributes to stroke pathology, but the mechanism has kept unknown. The secreted leucine-rich repeat (LRR) family protein slit homologue 2 (Slit2) upregulates GABAergic activity and protects against global cerebral ischemia, but the neuroprotective efficacy of Slit2 against MIs has not been examined. Methods Middle-aged Wild type (WT) and Slit2-Tg mice were divided into sham and MI treatment groups. MIs were induced in parietal cortex by laser-evoked arteriole occlusion. Spatial memory was then compared between sham and MI groups using the Morris water maze (MWM) task. In addition, neuronal activity, blood brain barrier (BBB) permeability, and glymphatic clearance in peri-infarct areas were compared using two-photon imaging, while GABAergic transmission, microglial activation, neuronal loss, and altered cortical connectivity were compared by immunofluorescent staining or western blotting. Results Microinfarcts increased the amplitude and frequency of spontaneous intracellular Ca2+ signals, reduced neuronal survival and connectivity within parietal cortex, decreased the number of GABAergic interneurons and expression of vesicular GABA transporter (VGAT), induced neuroinflammation, and impaired both glymphatic clearance and spatial memory. Alternatively, Slit2 overexpression attenuated dysfunctional neuronal Ca2+ signaling, protected against neuronal death in the peri-infarct area as well as loss of parietal cortex connectivity, increased GABAergic interneuron number and VGAT expression, attenuated neuroinflammation, and improved both glymphatic clearance and spatial memory. Conclusion Our results strongly suggest that overexpression of Slit2 protected against the dysfunction in MIs, which is a potential therapeutic target for cognition impairment in the elderly.

Alterations in macronutrient intake can have profound effects on energy intake and whole-body metabolism. For example, reducing protein intake increases energy expenditure, increases insulin sensitivity and decreases body weight in rodents. Fibroblast growth factor 21 (FGF21) signaling in the brain is necessary for the metabolic effects of dietary protein restriction and has more recently been proposed to promote protein preference. However, the neuron populations through which FGF21 elicits these effects are unknown. Here, we demonstrate that deletion of β-klotho in glutamatergic, but not GABAergic, neurons abrogated the effects of dietary protein restriction on reducing body weight, but not on improving insulin sensitivity in both diet-induced obese and lean mice. Specifically, FGF21 signaling in glutamatergic neurons is necessary for protection against body weight gain and induction of UCP1 in adipose tissues associated with dietary protein restriction. However, β-klotho expression in glutamatergic neurons was dispensable for the effects of dietary protein restriction to increase insulin sensitivity. In addition, we report that FGF21 administration does not alter protein preference, but instead promotes the foraging of other macronutrients primarily by suppressing simple sugar consumption. This work provides important new insights into the neural substrates and mechanisms behind the endocrine control of metabolism during dietary protein dilution.

Significance The lateral hypothalamus encompasses neural circuits that are highly active during feeding behavior and rapid eye movement sleep. Yet, it remains unclear whether these mutually exclusive behaviors share common neural populations. Here, we recorded and perturbed the activity of inhibitory neurons of the lateral hypothalamus (LH) across feeding behavior and sleep in freely behaving mice. We found that feeding is reliably encoded by specific patterns of neuron activity and that these patterns are reactivated during rapid eye movement sleep. Disrupting the activity of these inhibitory neurons specifically during rapid eye movement sleep decreased subsequent feeding behavior. These results suggest that rapid eye movement sleep stabilizes the hypothalamic representation of feeding behavior and modulates future food intake., During rapid eye movement (REM) sleep, behavioral unresponsiveness contrasts strongly with intense brain-wide neural network dynamics. Yet, the physiological functions of this cellular activation remain unclear. Using in vivo calcium imaging in freely behaving mice, we found that inhibitory neurons in the lateral hypothalamus (LHvgat) show unique activity patterns during feeding that are reactivated during REM, but not non-REM, sleep. REM sleep-specific optogenetic silencing of LHvgat cells induced a reorganization of these activity patterns during subsequent feeding behaviors accompanied by decreased food intake. Our findings provide evidence for a role for REM sleep in the maintenance of cellular representations of feeding behavior.