Search

A unique population of ventral tegmental area (VTA) neurons co-transmits glutamate and GABA. However, the circuit inputs to VTA VGluT2+VGaT+ neurons are unknown, limiting our understanding of their functional capabilities. By coupling monosynaptic rabies tracing with intersectional genetic targeting in male and female mice, we found that VTA VGluT2+VGaT+ neurons received diverse brainwide inputs. The largest numbers of monosynaptic inputs to VTA VGluT2+VGaT+ neurons were from superior colliculus (SC), lateral hypothalamus (LH), midbrain reticular nucleus, and periaqueductal gray, whereas the densest inputs relative to brain region volume were from the dorsal raphe nucleus, lateral habenula, and VTA. Based on these and prior data, we hypothesized that LH and SC inputs were from glutamatergic neurons. Optical activation of glutamatergic LH neurons activated VTA VGluT2+VGaT+ neurons regardless of stimulation frequency and resulted in flee-like ambulatory behavior. In contrast, optical activation of glutamatergic SC neurons activated VTA VGluT2+VGaT+ neurons for a brief period of time at high frequency and resulted in head rotation and arrested ambulatory behavior (freezing). Stimulation of glutamatergic LH neurons, but not glutamatergic SC neurons, was associated with VTA VGluT2+VGaT+ footshock-induced activity and inhibition of LH glutamatergic neurons disrupted VTA VGluT2+VGaT+ tailshock-induced activity. We interpret these results such that inputs to VTA VGluT2+VGaT+ neurons may integrate diverse signals related to the detection and processing of motivationally salient outcomes., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Retinal ganglion cell (RGC) axons provide direct input into several brain regions, including the dorsal lateral geniculate nucleus (dLGN), which is important for image-forming vision, and the ventrolateral geniculate nucleus (vLGN), which is associated with nonimage-forming vision. Through both activity- and morphogen-dependent mechanisms, retinal inputs play important roles in the development of dLGN, including the refinement of retinal projections, morphological development of thalamocortical relay cells (TRCs), timing of corticogeniculate innervation, and recruitment and distribution of inhibitory interneurons. In contrast, little is known about the role of retinal inputs in the development of vLGN. Grossly, vLGN is divided into two domains, the retinorecipient external vLGN (vLGNe) and nonretinorecipient internal vLGN (vLGNi). Studies previously found that vLGNe consists of transcriptionally distinct GABAergic subtypes distributed into at least four adjacent laminae. At present, it remains unclear whether retinal inputs influence the development of these cell-type-specific neuronal laminae in vLGNe. Here, we elucidated the developmental timeline for these laminae in the mouse vLGNe, and results indicate that these laminae are specified at or before birth. We observed that mutant mice without retinal inputs have a normal laminar distribution of GABAergic cells at birth; however, after the first week of postnatal development, these mutants exhibited a dramatic disruption in the laminar organization of inhibitory neurons and clear boundaries between vLGNe and vLGNi. Overall, our results show that while the formation of cell-type-specific layers in mouse vLGNe does not depend on RGC inputs, retinal signals are critical for their maintenance., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Stebbins et al.)

Adolescent intermittent ethanol (AIE) exposure in rats leads to social deficits. Parvalbumin (PV) expressing fast-spiking interneurons in the prelimbic cortex (PrL) contribute to social behavior, and perineuronal nets (PNNs) within the PrL preferentially encompass and regulate PV interneurons. AIE exposure increases PNNs, but it is unknown if this upregulation contributes to AIE-induced social impairments. The current study was designed to determine the effect of AIE exposure on PNN expression in the PrL and to assess whether PNN dysregulation contributes to social deficits elicited by AIE. cFos-LacZ male and female rats were exposed every other day to tap water or ethanol (4 g/kg, 25% w/v) via intragastric gavage between postnatal day (P) 25-45. We evaluated neuronal activation by β-galactosidase expression and PNN levels either at the end of the exposure regimen on P45 and/or in adulthood on P70. In addition, we used Chondroitinase ABC (ChABC) to deplete PNNs following adolescent exposure (P48) and allowed for PNN restoration before social testing in adulthhod. AIE exposure increased PNN expression in the PrL of adult males, but decreased PNNs immediately following AIE. Vesicular glutamate transporter 2 (vGlut2) and vesicular GABA transporter (vGat) near PNNs were downregulated only in AIE-exposed females. Gene expression of PNN components was largely unaffected by AIE exposure. Removal and reestablishment of PrL PNNs by ChABC led to upregulation of PNNs and social impairments in males, regardless of adolescent exposure. These data suggest that AIE exposure in males upregulates PrL PNNs that likely contribute to social impairments induced by AIE., Competing Interests: Declaration of competing interest All co-authors have read and approved the manuscript for submission and have made substantial contributions to the conception, design, gathering, analysis and/or interpretation of the data as well as contributed to the writing and/or intellectual content of the article. All authors have exercised due care in ensuring the integrity of the work, and all animal procedures were conducted according to NIH guidelines and approved by our institutional animal care and use committee. None of the original material contained in the manuscript, outside of conference abstracts, has been submitted for consideration elsewhere. None of the authors have any financial declarations or conflicts of interest related to the submitted study., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Neurotensin (Nts) is a neuropeptide acting as a neuromodulator in the brain. Pharmacological studies have identified Nts as a potent hypothermic agent. The medial preoptic area, a region that plays an important role in the control of thermoregulation, contains a high density of neurotensinergic neurons and Nts receptors. The conditions in which neurotensinergic neurons play a role in thermoregulation are not known. In this study, optogenetic stimulation of preoptic Nts neurons induced a small hyperthermia. In vitro, optogenetic stimulation of preoptic Nts neurons resulted in synaptic release of GABA and net inhibition of the preoptic pituitary adenylate cyclase-activating polypeptide (Adcyap1) neurons firing activity. GABA-A receptor antagonist or genetic deletion of Slc32a1 (VGAT) in Nts neurons unmasked also an excitatory effect that was blocked by a Nts receptor 1 antagonist. Stimulation of preoptic Nts neurons lacking Slc32a1 resulted in excitation of Adcyap1 neurons and hypothermia. Mice lacking Slc32a1 expression in Nts neurons presented changes in the fever response and in the responses to heat or cold exposure as well as an altered circadian rhythm of body temperature. Chemogenetic activation of all Nts neurons in the brain induced a 4-5°C hypothermia, which could be blocked by Nts receptor antagonists in the preoptic area. Chemogenetic activation of preoptic neurotensinergic projections resulted in robust excitation of preoptic Adcyap1 neurons. Taken together, our data demonstrate that endogenously released Nts can induce potent hypothermia and that excitation of preoptic Adcyap1 neurons is the cellular mechanism that triggers this response., Competing Interests: IT No competing interests declared, (© 2024, Tabarean.)

Transmembrane protein (TMEM230) is located in secretory/recycling vesicles, including synaptic vesicles in neurons. However, the functional relationship between TMEM230 and epilepsy is still a mystery. The aims of this study were to investigate the expression of TMEM230 in patients with temporal lobe epilepsy (TLE) and two different mice models of chronic epilepsy, and to determine the probable roles of TMEM230 in epilepsy. Our results showed that TMEM230 expression was increased in the temporal neocortex of epileptic patients and the hippocampus and cortex of epileptic mice compared with that in the control tissues. Moreover, TMEM230 was mainly expressed in the neurons in both humans and mice epileptic brain. TMEM230 co-localized with glutamate vesicular transporter 1 (VGLUT-1), but not with vesicular γ-aminobutyric acid (GABA) transporter (VGAT). Mechanistically, coimmunoprecipitation confirmed that TMEM230 interacted with VGLUT-1, but not with VGAT in the hippocampus of epileptic mice. Lentivirus mediated overexpression of TMEM230 increased mice susceptibility to epilepsy and behavioural phenotypes of epileptic seizures during the kainite (KA)-induced chronic phase of epileptic seizures and the pentylenetetrazole (PTZ) kindling process, whereas lentivirus-mediated TMEM230 downregulation had the opposite effect. These results shed light on the functions of TMEM230 in neurons, suggesting that TMEM230 may play a critical role in the regulation of epileptic activity via influencing excitatory neurotransmission.

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 STATEMENT Lordosis, 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., (Copyright © 2021 Johnson et al.)

Although dexterity relies on the constant transmission of sensory information, unchecked feedback can be disruptive. Yet how somatosensory feedback from the hands is regulated and whether this modulation influences movement remain unclear. We found that mouse tactile afferents recruit neurons in the brainstem cuneate nucleus, whose activity is modulated by distinct classes of local inhibitory neurons. Manipulation of these inhibitory circuits suppresses or enhances the transmission of tactile information, which affects manual behaviors. Top-down cortical pathways innervate cuneate in a complementary pattern, with somatosensory cortical neurons targeting the core tactile region of cuneate and a large rostral cortical population driving feed-forward inhibition of tactile transmission through an inhibitory shell. These findings identify a circuit basis for tactile feedback modulation that enables the effective execution of dexterous movement.

Eye-drop recombinant human nerve growth factor (ed-rhNGF) has proved to recover the retina and optic nerve damage in animal models, including the unilateral optic nerve crush (ONC), and to improve visual acuity in humans. These data, associated with evidence that ed-rhNGF stimulates the brain derived neurotrophic factor (BDNF) in retina and cortex, suggests that NGF might exert retino-fugal effects by affecting BDNF and its receptor TrkB. To address these questions, their expression and relationship with the GABAergic and glutamatergic transmission markers, GAD65 and GAD67, vesicular inhibitory amino acid transporter (VGAT), and vesicular glutamate transporters 1 and 2 (VGLUT-1 and VGLUT-2) were investigated in adult ONC rats contralateral and ipsilateral visual cortex (VCx). Ed-rhNGF recovers the ONC-induced alteration of GABAergic and glutamatergic markers in contralateral VCx, induces an upregulation of TrkB, which is positively correlated with BDNF precursor (proBDNF) decrease in both VCx sides, and strongly enhances TrkB+ cell soma and neuronal endings surrounded by GAD65 immuno-reactive afferents. These findings contribute to enlarging the knowledge on the mechanism of actions and cellular targets of exogenously administrated NGF, and suggest that ed-rhNGF might act by potentiating the activity-dependent TrkB expression in GAD+ cells in VCx following retina damage and/or ONC.

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., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

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 GABA A 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 GABA A receptor in people with schizophrenia compared to controls (p < 0.05). Gene expression of somatostatin (SST) was unchanged (p = 0.485). We confirmed the reduction in GAD at the protein level (34%, p < 0.05). When stratifying by inflammation, only 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 GABA A 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 < 0.001) but not controls (GABRA2; r < - 0.200, GABRA3; r < 0.310, all 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.

Homeotic genes and their genomic organization show remarkable conservation across bilaterians. Consequently, the regulatory mechanisms, which control hox gene expression, are also highly conserved. The crucial presence of conserved GA rich motifs between Hox genes has been previously observed but what factor binds to these is still unknown. Previously we have reported that the vertebrate homologue of Drosophila Trl-GAF preferentially binds to GA rich regions in Evx2-hoxd13 intergenic region of vertebrate HoxD cluster. In this study, we show that the vertebrate-GAF (v-GAF) binds at known cis-regulatory elements in the HoxD complex of zebrafish and mouse. We further used morpholino based knockdown and CRISPR-cas9 knockout technique to deplete the v-GAF in zebrafish. We checked expression of the HoxD genes and found gain of the HoxD4 gene in GAF knockout embryos. Further, we partially rescued the morphological phenotypes in GAF depleted embryos by providing GAF mRNA. Our results show that GAF binds at intergenic regions of the HoxD complex and is important for maintaining the spatial domains of HoxD4 expression during embryonic development., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective: To 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., Methods: We 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., Results: Eight 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)., Conclusion: Missense 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., (© 2021 American Academy of Neurology.)

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 α 1A -adrenaline receptor (α 1A -AR) agonist increase inhibitory synaptic inputs onto SDH interneurons expressing gastrin-releasing peptide receptors, which are essential for itch transmission. However, the contribution of α 1A -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 α 1A -ARs in inhibitory interneurons (Vgat-Cre;Adra1a flox/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 α 1A -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 α 1A -ARs in SDH inhibitory interneurons contribute to the regulation of itch signaling with preference for histamine-independent itch.

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., Competing Interests: QC, RS, XH, WW No competing interests declared, (© 2020, Chen et al.)

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.

Objective: Development of novel therapies for temporal lobe epilepsy is hindered by a lack of models suitable for drug screening. While testing the hypothesis that "inhibiting inhibitory neurons" was sufficient to induce seizures, it was discovered that a mild electrical kindling protocol of VGAT-Cre mice led to spontaneous motor and electrographic seizures. This study characterizes these seizures and investigates the mechanism., Methods: Mice were implanted with electroencephalographic (EEG) headsets that included a stimulating electrode in the hippocampus before being electrically kindled. Seizures were evaluated by review of EEG recordings and behavior. γ-Aminobutyric acidergic (GABAergic) neurotransmission was evaluated by quantitative polymerase chain reaction, immunocytochemistry, Western blot, and electrophysiology., Results: Electrical kindling of VGAT-Cre mice induces spontaneous recurring seizures after a short latency (6 days). Seizures occur 1-2 times per day in both male and female mice, with only minimal neuronal death. These mice express Cre recombinase under the control of the vesicular GABA transporter (VGAT), a gene that is specifically expressed in GABAergic inhibitory neurons. The insertion of Cre disrupts the expression of VGAT mRNA and protein, and impairs GABAergic synaptic transmission in the hippocampus., Significance: Kindled VGAT-Cre mice can be used to study the mechanisms involved in epileptogenesis and may be useful for screening novel therapeutics., (© 2020 International League Against Epilepsy.)

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 (LH vgat ) show unique activity patterns during feeding that are reactivated during REM, but not non-REM, sleep. REM sleep-specific optogenetic silencing of LH vgat 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., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

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., (Copyright © 2020 the authors.)

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., (Copyright © 2020 the authors.)

We explore here the cis -regulatory logic that dictates gene expression in specific cell types in the nervous system. We focus on a set of eight genes involved in the synthesis, transport, and breakdown of three neurotransmitter systems: acetylcholine ( unc-17 /VAChT , cha-1 /ChAT , cho-1 /ChT , and ace-2 /AChE ), glutamate ( eat-4 /VGluT ), and γ-aminobutyric acid ( unc-25 /GAD , unc-46 /LAMP , and unc-47 /VGAT ). These genes are specifically expressed in defined subsets of cells in the nervous system. Through transgenic reporter gene assays, we find that the cellular specificity of expression of all of these genes is controlled in a modular manner through distinct cis -regulatory elements, corroborating the previously inferred piecemeal nature of specification of neurotransmitter identity. This modularity provides the mechanistic basis for the phenomenon of "phenotypic convergence," in which distinct regulatory pathways can generate similar phenotypic outcomes ( i.e. , the acquisition of a specific neurotransmitter identity) in different neuron classes. We also identify cases of enhancer pleiotropy, in which the same cis -regulatory element is utilized to control gene expression in distinct neuron types. We engineered a cis -regulatory allele of the vesicular acetylcholine transporter, unc-17 /VAChT , to assess the functional contribution of a "shadowed" enhancer. We observed a selective loss of unc-17 /VAChT expression in one cholinergic pharyngeal pacemaker motor neuron class and a behavioral phenotype that matches microsurgical removal of this neuron. Our analysis illustrates the value of understanding cis -regulatory information to manipulate gene expression and control animal behavior., (Copyright © 2020 by the Genetics Society of America.)

Mu-opioid receptors (MORs) are crucial for analgesia by both exogenous and endogenous opioids. However, the distinct mechanisms underlying these two types of opioid analgesia remain largely unknown. Here, we demonstrate that analgesic effects of exogenous and endogenous opioids on inflammatory pain are mediated by MORs expressed in distinct subpopulations of neurons in mice. We found that the exogenous opioid-induced analgesia of inflammatory pain is mediated by MORs in Vglut2 + glutamatergic but not GABAergic neurons. In contrast, analgesia by endogenous opioids is mediated by MORs in GABAergic rather than Vglut2 + glutamatergic neurons. Furthermore, MORs expressed at the spinal level is mainly involved in the analgesic effect of morphine in acute pain, but not in endogenous opioid analgesia during chronic inflammatory pain. Thus, our study revealed distinct mechanisms underlying analgesia by exogenous and endogenous opioids, and laid the foundation for further dissecting the circuit mechanism underlying opioid analgesia., Competing Interests: XZ, YD, LY, QL, YZ, MW, YS No competing interests declared, (© 2020, Zhang et al.)

LRRTM4 is a transsynaptic adhesion protein regulating glutamatergic synapse assembly on dendrites of central neurons. In the mouse retina, we find that LRRTM4 is enriched at GABAergic synapses on axon terminals of rod bipolar cells (RBCs). Knockout of LRRTM4 reduces RBC axonal GABA A and GABA C receptor clustering and disrupts presynaptic inhibition onto RBC terminals. LRRTM4 removal also perturbs the stereotyped output synapse arrangement at RBC terminals. Synaptic ribbons are normally apposed to two distinct postsynaptic "dyad" partners, but in the absence of LRRTM4, "monad" and "triad" arrangements are also formed. RBCs from retinas deficient in GABA release also demonstrate dyad mis-arrangements but maintain LRRTM4 expression, suggesting that defects in dyad organization in the LRRTM4 knockout could originate from reduced GABA receptor function. LRRTM4 is thus a key synapse organizing molecule at RBC terminals, where it regulates function of GABAergic synapses and assembly of RBC synaptic dyads., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Tremor is currently ranked as the most common movement disorder. The brain regions and neural signals that initiate the debilitating shakiness of different body parts remain unclear. Here, we found that genetically silencing cerebellar Purkinje cell output blocked tremor in mice that were given the tremorgenic drug harmaline. We show in awake behaving mice that the onset of tremor is coincident with rhythmic Purkinje cell firing, which alters the activity of their target cerebellar nuclei cells. We mimic the tremorgenic action of the drug with optogenetics and present evidence that highly patterned Purkinje cell activity drives a powerful tremor in otherwise normal mice. Modulating the altered activity with deep brain stimulation directed to the Purkinje cell output in the cerebellar nuclei reduced tremor in freely moving mice. Together, the data implicate Purkinje cell connectivity as a neural substrate for tremor and a gateway for signals that mediate the disease., Competing Interests: AB, JW, Mv, JZ, TL, RS No competing interests declared, (© 2020, Brown et al.)

Models of basal ganglia (BG) function predict that tonic inhibitory output to motor thalamus (MT) suppresses unwanted movements, and that a decrease in such activity leads to action selection. Further, for unilateral activity changes in the BG, a lateralized effect on contralateral movements can be expected due to ipsilateral thalamocortical connectivity. However, a direct test of these outcomes of thalamic inhibition has not been performed. To conduct such a direct test, we utilized rapid optogenetic activation and inactivation of the GABAergic output of the substantia nigra pars reticulata (SNr) to MT in male and female mice that were trained in a sensory cued left/right licking task. Directional licking tasks have previously been shown to depend on a thalamocortical feedback loop between ventromedial MT and antero-lateral premotor cortex. In confirmation of model predictions, we found that unilateral optogenetic inhibition of GABAergic output from the SNr, during ipsilaterally cued trials, biased decision making towards a contralateral lick without affecting motor performance. In contrast, optogenetic excitation of SNr terminals in MT resulted in an opposite bias towards the ipsilateral direction confirming a bidirectional effect of tonic nigral output on directional decision making. However, direct optogenetic excitation of neurons in the SNr resulted in bilateral movement suppression, which is in agreement with previous results that show such suppression for nigral terminals in the superior colliculus (SC), which receives a bilateral projection from SNr., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Cultured rat cortical neurons co-expressing VGLUT1 and VGAT (mixed synapses) co-release Glu and GABA. Here, mixed synapses were studied in cultured mouse cortical neurons to verify whether in mice mixed synapses co-release Glu and GABA, and to gain insight into how they may influence excitation/inhibition balance. Results showed the existence of synapses and autapses that co-release Glu and GABA in cultured mouse cortical neurons, and the ability of both neurotransmitters to evoke postsynaptic responses mediated by ionotropic receptors. We studied the short-term plasticity of glutamatergic, GABAergic, and mixed responses and we found that the kinetics of mixPSC amplitude depression was similar to that observed in EPSCs, but it was different from that of IPSCs. We found similar presynaptic release characteristics in glutamatergic and mixed synapses. Analysis of postsynaptic features, obtained by measuring AMPAR- and NMDAR-mediated currents, showed that AMPAR-mediated currents were significantly higher in pure glutamatergic than in mixed synapses, whereas NMDAR-mediated currents were not significantly different from those measured in mixed synapses. Overall, our findings demonstrate that glutamatergic and mixed synapses share similar electrophysiological properties. However, co-release of GABA and Glu influences postsynaptic ionotropic glutamatergic receptor subtypes, thus selectively influencing AMPAR-mediated currents. These findings strengthen the view that mixed neurons can play a key role in CNS development and in maintaining the excitation-inhibition balance., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The histaminergic neurons of the tuberomammillary nucleus (TMN HDC ) of the posterior hypothalamus have long been implicated in promoting arousal. More recently, a role for GABAergic signaling by the TMN HDC neurons 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 TMN HDC neurons on sleep-wake in male mice. We also examined the effects of acute chemogenetic activation and optogenetic inhibition of TMN HDC neurons 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 TMN HDC neurons coexpressed VGAT. Acute chemogenetic activation of TMN HDC neurons 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 TMN HDC neurons in arousal control. Further, if TMN HDC neurons do release GABA, the mechanism by which they do so remains unclear. Our findings support the view that TMN HDC neurons 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 STATEMENT The histaminergic neurons of the tuberomammillary nucleus of the hypothalamus (TMN HDC ) have long been thought to promote arousal. Additionally, TMN HDC neurons 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 TMN HDC neurons does not impact sleep-wake amounts and that few TMN HDC neurons contain the vesicular GABA transporter, which is presumably required to release GABA. We further show that acute activation or inhibition of TMN HDC neurons 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 TMN HDC neurons are neither necessary nor sufficient for the initiation and maintenance of arousal under baseline conditions., (Copyright © 2019 the authors.)

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., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The dorsomedial striatum (DMS) is critically involved in motor control and reward processing, but the specific neural circuit mediators are poorly understood. Recent evidence highlights the extensive connectivity of low-threshold spiking interneurons (LTSIs) within local striatal circuitry; however, the in vivo function of LTSIs remains largely unexplored. We employed fiber photometry to assess LTSI calcium activity in a range of DMS-mediated behaviors, uncovering specific reward-related activity that is down-modulated during goal-directed learning. Using two mechanistically distinct manipulations, we demonstrated that this down-modulation of LTSI activity is critical for acquisition of novel contingencies, but not for their modification. In contrast, continued LTSI activation slowed instrumental learning. Similar manipulations of fast-spiking interneurons did not reproduce these effects, implying a specific function of LTSIs. Finally, we revealed a role for the γ-aminobutyric acid (GABA)ergic functions of LTSIs in learning. Together, our data provide new insights into this striatal interneuron subclass as important gatekeepers of goal-directed learning., (Copyright © 2019 Elsevier Inc. All rights reserved.)

EPAC2 is a guanine nucleotide exchange factor that regulates GTPase activity of the small GTPase Rap and Ras and is highly enriched at synapses. Activation of EPAC2 has been shown to induce dendritic spine shrinkage and increase spine motility, effects that are necessary for synaptic plasticity. These morphological effects are dysregulated by rare mutations of Epac2 associated with autism spectrum disorders. In addition, EPAC2 destabilizes synapses through the removal of synaptic GluA2/3-containing AMPA receptors. Previous work has shown that Epac2 knockout mice (Epac2 -/- ) display abnormal social interactions, as well as gross disorganization of the frontal cortex and abnormal spine motility in vivo. In this study we sought to further understand the cellular consequences of knocking out Epac2 on the development of neuronal and synaptic structure and organization of cortical neurons. Using primary cortical neurons generated from Epac2 +/+ or Epac2 -/- mice, we confirm that EPAC2 is required for cAMP-dependent spine shrinkage. Neurons from Epac2 -/- mice also displayed increased synaptic expression of GluA2/3-containing AMPA receptors, as well as of the adhesion protein N-cadherin. Intriguingly, analysis of excitatory and inhibitory synaptic proteins revealed that loss of EPAC2 resulted in altered expression of vesicular GABA transporter (VGAT) but not vesicular glutamate transporter 1 (VGluT1), indicating an altered ratio of excitatory and inhibitory synapses onto neurons. Finally, examination of cortical neurons located within the anterior cingulate cortex further revealed subtle deficits in the establishment of dendritic arborization in vivo. These data provide evidence that loss of EPAC2 enhances the stability of excitatory synapses and increases the number of inhibitory inputs., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The basal forebrain (BF) contains at least three distinct populations of neurons (cholinergic, glutamatergic, and GABA-ergic) across its different regions (medial septum, diagonal band, magnocellular preoptic area, and substantia innominata). Much attention has focused on the BF's ascending projections to cortex, but less is known about descending projections to subcortical regions. Given the neurochemical and anatomical heterogeneity of the BF, we used conditional anterograde tracing to map the patterns of subcortical projections from multiple BF regions and neurochemical cell types using mice that express Cre recombinase only in cholinergic, glutamatergic, or GABAergic neurons. We confirmed that different BF regions innervate distinct subcortical targets, with more subcortical projections arising from neurons in the caudal and lateral BF (substantia innominata and magnocellular preoptic area). Additionally, glutamatergic and GABAergic BF neurons have distinct patterns of descending projections, while cholinergic descending projections are sparse. Considering the intensity of glutamatergic and GABAergic descending projections, the BF likely acts through subcortical targets to promote arousal, motivation, and other behaviors.

Vesicular inhibitory amino acid transporter (VIAAT) is a transmembrane transporter which is responsible for the storage of gamma-aminobutyric acid (GABA) or glycine in synaptic vesicles. According to recent studies, GABA is known to be expressed in the kidney. For clear understanding of the intra-renal GABA signaling, the localization of VIAAT was examined in the present study. Intense immunoreactivity was found largely confined to the distal tubule epithelia, especially distinct in the inner medulla, although the immunoreactivity was discerned more or less in all tubules and glomeruli. No distinct immunoreactivity was seen in capillary endothelia or interstitial fibroblasts. In immuno-DAB and immuno-gold electron microscopy, the immunoreaction was found at the basal infoldings of plasma membranes and basal portions of the lateral plasma membranes, but not in any vesicles or vacuoles within the distal tubular cells. The significance of the enigmatic finding, localization of a vesicular molecule on selected portions of the plasma membrane of distal tubular cells, was discussed in view of the possibility of paracrine or autocrine effects of GABA on some other uriniferous tubular cells or interstitial cells., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Purkinje cells receive synaptic input from several classes of interneurons. Here, we address the roles of inhibitory molecular layer interneurons in establishing Purkinje cell function in vivo. Using conditional genetics approaches in mice, we compare how the lack of stellate cell versus basket cell GABAergic neurotransmission sculpts the firing properties of Purkinje cells. We take advantage of an inducible Ascl1 CreER allele to spatially and temporally target the deletion of the vesicular GABA transporter, Vgat, in developing neurons. Selective depletion of basket cell GABAergic neurotransmission increases the frequency of Purkinje cell simple spike firing and decreases the frequency of complex spike firing in adult behaving mice. In contrast, lack of stellate cell communication increases the regularity of Purkinje cell simple spike firing while increasing the frequency of complex spike firing. Our data uncover complementary roles for molecular layer interneurons in shaping the rate and pattern of Purkinje cell activity in vivo.

Appropriate synapse formation during development is necessary for normal brain function, and synapse impairment is often associated with brain dysfunction. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are key factors in regulating synaptic development. We previously reported that BDNF/NT-3 secretion was enhanced by calcium-dependent activator protein for secretion 2 (CADPS2). Although BDNF/NT-3 and CADPS2 are co-expressed in various brain regions, the effect of Cadps2-deficiency on brain region-specific BDNF/NT-3 levels and synaptic development remains elusive. Here, we show developmental changes of BDNF/NT-3 levels and we assess disruption of excitatory/inhibitory synapses in multiple brain regions (cerebellum, hypothalamus, striatum, hippocampus, parietal cortex and prefrontal cortex) of Cadps2 knockout (KO) mice compared with wild-type (WT) mice. Compared with WT, BDNF levels in KO mice were reduced in young/adult hippocampus, but increased in young hypothalamus, while NT-3 levels were reduced in adult cerebellum and young hippocampus, but increased in adult parietal cortex. Immunofluorescence of vGluT1, an excitatory synapse marker, and vGAT, an inhibitory synapse marker, in adult KO showed that vGluT1 was higher in the cerebellum and parietal cortex but lower in the hippocampus, whereas vGAT was lower in the hippocampus and parietal cortex compared with WT. Immunolabeling for both vGluT1 and vGAT was increased in the parietal cortex but vGAT was decreased in the cerebellum in adult KO compared with WT. These data suggest that CADPS2-mediated secretion of BDNF/NT-3 may be involved in development and maturation of synapses and in the balance between inhibitory and excitatory synapses., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Local translation in neurites is a phenomenon that enhances the spatial segregation of proteins and their functions away from the cell body, yet it is unclear how local translation varies across neuronal cell types. Further, it is unclear whether differences in local translation across cell types simply reflect differences in transcription or whether there is also a cell type-specific post-transcriptional regulation of the location and translation of specific mRNAs. Most of the mRNAs discovered as being locally translated have been identified from hippocampal neurons because their laminar organization facilitates neurite-specific dissection and microscopy methods. Given the diversity of neurons across the brain, studies have not yet analyzed how locally translated mRNAs differ across cell types. Here, we used the SynapTRAP method to harvest two broad cell types in the mouse forebrain: GABAergic neurons and layer 5 projection neurons. While some transcripts overlap, the majority of the local translatome is not shared across these cell types. In addition to differences driven by baseline expression levels, some transcripts also exhibit cell type-specific post-transcriptional regulation. Finally, we provide evidence that GABAergic neurons specifically localize mRNAs for peptide neurotransmitters, including somatostatin and cortistatin, suggesting localized production of these key signaling molecules in the neurites of GABAergic neurons. Overall, this work suggests that differences in local translation in neurites across neuronal cell types are poised to contribute substantially to the heterogeneity in neuronal phenotypes.

We established the mouse model of fluoride (68 mg F ion/L deionized water) exposure for 90 days, 120 days and 150 days, and applied diverse methods as behavioral models of anxiety and depression, and analyzed the levels of the anxiety- and depression-like related genes like BDNF1, BDNF4, 5-HT1A, VGLUT, GAD67, and VGAT in the mouse hippocampus. In the mice exposed to NaF for 120 days, compared to the control group, chalky opacity was observed on the enamel of teeth; the results of anxiety-like behavior, like elevated zero maze, light/dark exploration test, novel object recognition test and emergence test were significantly altered, however in the mice exposed for 150 days, only the elevated zero maze and emergence test were significantly altered. Also, the results of depression-like behavior were significantly altered in the 120 days treated mice. Exposure to NaF for 120 days significantly decreased the mRNA expression levels of the BDNF4 with a concomitant increase in the 5-HT1A compared to the control mice. Especially the mRNA expression levels of GAD67 and VGAT were significantly decreased in all the three NaF treated groups. However, no significant changes were observed in the mRNA expression levels of the VGLUT compared to the control mice. In summary, we speculated that fluoride exposure had adverse effects on nervous system, inducing an imbalance between excitation and inhibition, which resulted in abnormal behavior and depression., (Copyright © 2018. Published by Elsevier Ltd.)

In mammals, the extended amygdala is a neural hub for social and emotional information processing. In the rat, the extended amygdala receives inhibitory GABAergic projections from the nucleus incertus (NI) in the pontine tegmentum. NI neurons produce the neuropeptide relaxin-3, which acts via the G i/o -protein-coupled receptor, RXFP3. A putative role for RXFP3 signalling in regulating social interaction was investigated by assessing the effect of intracerebroventricular infusion of the RXFP3 agonist, RXFP3-A2, on performance in the 3-chamber social interaction paradigm. Central RXFP3-A2, but not vehicle, infusion, disrupted the capacity to discriminate between a familiar and novel conspecific subject, but did not alter differentiation between a conspecific and an inanimate object. Subsequent studies revealed that agonist-infused rats displayed increased phosphoERK(pERK)-immunoreactivity in specific amygdaloid nuclei at 20 min post-infusion, with levels similar to control again after 90 min. In parallel, we used immunoblotting to profile ERK phosphorylation dynamics in whole amygdala after RXFP3-A2 treatment; and multiplex histochemical labelling techniques to reveal that after RXFP3-A2 infusion and social interaction, pERK-immunopositive neurons in amygdala expressed vesicular GABA-transporter mRNA and displayed differential profiles of RXFP3 and oxytocin receptor mRNA. Overall, these findings demonstrate that central relaxin-3/RXFP3 signalling can modulate social recognition in rats via effects within the amygdala and likely interactions with GABA and oxytocin signalling.

Synaptic inhibition controls a neuron's output via functionally distinct inputs at two subcellular compartments, the cell body and the dendrites. It is unclear whether the assembly of these distinct inhibitory inputs can be regulated independently by neurotransmission. In the mammalian retina, γ-aminobutyric acid (GABA) release from starburst amacrine cells (SACs) onto the dendrites of on-off direction-selective ganglion cells (ooDSGCs) is essential for directionally selective responses. We found that ooDSGCs also receive GABAergic input on their somata from other amacrine cells (ACs), including ACs containing the vasoactive intestinal peptide (VIP). When net GABAergic transmission is reduced, somatic, but not dendritic, GABA A receptor clusters on the ooDSGC increased in number and size. Correlative fluorescence imaging and serial electron microscopy revealed that these enlarged somatic receptor clusters are localized to synapses. By contrast, selectively blocking vesicular GABA release from either SACs or VIP ACs did not alter dendritic or somatic receptor distributions on the ooDSGCs, showing that neither SAC nor VIP AC GABA release alone is required for the development of inhibitory synapses in ooDSGCs. Furthermore, a reduction in net GABAergic transmission, but not a selective reduction from SACs, increased excitatory drive onto ooDSGCs. This increased excitation may drive a homeostatic increase in ooDSGC somatic GABA A receptors. Differential regulation of GABA A receptors on the ooDSGC's soma and dendrites could facilitate homeostatic control of the ooDSGC's output while enabling the assembly of the GABAergic connectivity underlying direction selectivity to be indifferent to altered transmission., Competing Interests: The authors declare no conflict of interest.

Although extensively studied, the exact role of sleep in learning and memory is still not very clear. Sleep deprivation has been most frequently used to explore the effects of sleep on learning and memory, but the results from such studies are inevitably complicated by concurrent stress and distress. Furthermore, it is not clear whether there is a strict time-window between sleep and memory consolidation. In the present study we were able to induce time-locked slow-wave sleep (SWS) in mice by optogenetically stimulating GABAergic neurons in the parafacial zone (PZ), providing a direct approach to analyze the influences of SWS on learning and memory with precise time-windows. We found that SWS induced by light for 30 min immediately or 15 min after the training phase of the object-in-place task significantly prolonged the memory from 30 min to 6 h. However, induction of SWS 30 min after the training phase did not improve memory, suggesting a critical time-window between the induction of a brief episode of SWS and learning for memory consolidation. Application of a gentle touch to the mice during light stimulation to prevent SWS induction also failed to improve memory, indicating the specific role of SWS, but not the activation of PZ GABAergic neurons itself, in memory consolidation. Similar influences of light-induced SWS on memory consolidation also occurred for Y-maze spatial memory and contextual fear memory, but not for cued fear memory. SWS induction immediately before the test phase had no effect on memory performance, indicating that SWS does not affect memory retrieval. Thus, by induction of a brief-episode SWS we have revealed a critical time window for the consolidation of hippocampus-dependent memory.

Glucagon-like peptide 1 receptor (GLP-1R) agonists are U.S. Food and Drug Administration-approved weight loss drugs. Despite their widespread use, the sites of action through which GLP-1R agonists (GLP1RAs) affect appetite and body weight are still not fully understood. We determined whether GLP-1Rs in either GABAergic or glutamatergic neurons are necessary for the short- and long-term effects of the GLP1RA liraglutide on food intake, visceral illness, body weight, and neural network activation. We found that mice lacking GLP-1Rs in vGAT -expressing GABAergic neurons responded identically to controls in all parameters measured, whereas deletion of GLP-1Rs in vGlut2 -expressing glutamatergic neurons eliminated liraglutide-induced weight loss and visceral illness and severely attenuated its effects on feeding. Concomitantly, deletion of GLP-1Rs from glutamatergic neurons completely abolished the neural network activation observed after liraglutide administration. We conclude that liraglutide activates a dispersed but discrete neural network to mediate its physiological effects and that these effects require GLP-1R expression on glutamatergic but not GABAergic neurons., (© 2018 by the American Diabetes Association.)

Background: The ability to appropriately integrate and respond to rewarding and aversive stimuli is essential for survival. The ventral pallidum (VP) plays a critical role in processing both rewarding and aversive stimuli. However, the VP is a heterogeneous structure, and how VP subpopulations integrate into larger reward networks to ultimately modulate these behaviors is not known. We identify a noncanonical population of glutamatergic VP neurons that play a unique role in responding to aversive stimuli and constraining inappropriate reward seeking., Methods: Using neurochemical, genetic, and electrophysiological approaches, we characterized glutamatergic VP neurons (n = 4-8 mice/group). We performed patch clamp and in vivo electrophysiology recordings in the lateral habenula, rostromedial tegmental nucleus, and ventral tegmental area to determine the effect of glutamatergic VP neuron activation in these target regions (n = 6-10 mice/group). Finally, we selectively optogenetically stimulated glutamatergic VP neurons in a real-time place preference task and ablated these neurons using a virally expressed caspase to determine their necessity for reward seeking., Results: Glutamatergic VP neurons exhibit little overlap with cholinergic or gamma-aminobutyric acidergic markers, the canonical VP subtypes, and exhibit distinct membrane properties. Glutamatergic VP neurons innervate and increase firing activity of the lateral habenula, rostromedial tegmental nucleus, and gamma-aminobutyric acidergic ventral tegmental area neurons. While nonselective optogenetic stimulation of the VP induced a robust place preference, selective activation of glutamatergic VP neurons induced a place avoidance. Viral ablation of glutamatergic VP neurons increased reward responding and abolished taste aversion to sucrose., Conclusions: Glutamatergic VP neurons constitute a noncanonical subpopulation of VP neurons. These glutamatergic VP neurons increase activity of the lateral habenula, rostromedial tegmental nucleus, and gamma-aminobutyric acidergic ventral tegmental area neurons and adaptively constrain reward seeking., (Copyright © 2018 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

The GABAergic neurons in the parafacial zone (PZ) play an important role in sleep-wake regulation and have been identified as part of a sleep-promoting center in the brainstem, but the long-range connections mediating this function remain poorly characterized. Here, we performed whole-brain mapping of both the inputs and outputs of the GABAergic neurons in the PZ of the mouse brain. We used the modified rabies virus EnvA-ΔG-DsRed combined with a Cre/loxP gene-expression strategy to map the direct monosynaptic inputs to the GABAergic neurons in the PZ, and found that they receive inputs mainly from the hypothalamic area, zona incerta, and parasubthalamic nucleus in the hypothalamus; the substantia nigra, pars reticulata and deep mesencephalic nucleus in the midbrain; and the intermediate reticular nucleus and medial vestibular nucleus (parvocellular part) in the pons and medulla. We also mapped the axonal projections of the PZ GABAergic neurons with adeno-associated virus, and defined the reciprocal connections of the PZ GABAergic neurons with their input and output nuclei. The newly-found inputs and outputs of the PZ were also listed compared with the literature. This cell-type-specific neuronal whole-brain mapping of the PZ GABAergic neurons may reveal the circuits underlying various functions such as sleep-wake regulation.

Recent studies have identified an especially important role for basal forebrain GABAergic (BF VGAT ) neurons in the regulation of behavioral waking and fast cortical rhythms associated with cognition. However, BF VGAT neurons comprise several neurochemically and anatomically distinct subpopulations, including parvalbumin-containing BF VGAT neurons and somatostatin-containing BF VGAT neurons (BF SOM neurons), and it was recently reported that optogenetic activation of BF SOM neurons increases the probability of a wakefulness to non-rapid-eye movement (NREM) sleep transition when stimulated during the rest period of the animal. This finding was unexpected given that most BF SOM neurons are not NREM sleep active and that central administration of the synthetic somatostatin analog, octreotide, suppresses NREM sleep or increases REM sleep. Here we used a combination of genetically driven chemogenetic and optogenetic activation, chemogenetic inhibition, and ablation approaches to further explore the in vivo role of BF SOM neurons in arousal control. Our findings indicate that acute activation or inhibition of BF SOM neurons is neither wakefulness nor NREM sleep promoting and is without significant effect on the EEG, and that chronic loss of these neurons is without effect on total 24 h sleep amounts, although a small but significant increase in waking was observed in the lesioned mice during the early active period. Our in vitro cell recordings further reveal electrophysiological heterogeneity in BF SOM neurons, specifically suggesting at least two distinct subpopulations. Together, our data support the more nuanced view that BF SOM neurons are electrically heterogeneous and are not NREM sleep or wake promoting per se, but may exert, in particular during the early active period, a modest inhibitory influence on arousal circuitry. SIGNIFICANCE STATEMENT The cellular basal forebrain (BF) is a highly complex area of the brain that is implicated in a wide range of higher-level neurobiological processes, including regulating and maintaining normal levels of electrocortical and behavioral arousal. The respective in vivo roles of BF cell populations and their neurotransmitter systems in the regulation of electrocortical and behavioral arousal remains incompletely understood. Here we seek to define the neurobiological contribution of GABAergic somatostatin-containing BF neurons to arousal control. Understanding the respective contribution of BF cell populations to arousal control may provide critical insight into the pathogenesis of a host of neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, schizophrenia, and the cognitive impairments of normal aging., (Copyright © 2018 the authors 0270-6474/18/385168-14$15.00/0.)

The circuit organization within the mammalian brainstem respiratory network, specifically within and between the pre-Bötzinger (pre-BötC) and Bötzinger (BötC) complexes, and the roles of these circuits in respiratory pattern generation are continuously debated. We address these issues with a combination of optogenetic experiments and modeling studies. We used transgenic mice expressing channelrhodopsin-2 under the VGAT-promoter to investigate perturbations of respiratory circuit activity by site-specific photostimulation of inhibitory neurons within the pre-BötC or BötC. The stimulation effects were dependent on the intensity and phase of the photostimulation. Specifically: (1) Low intensity (≤ 1.0 mW) pulses delivered to the pre-BötC during inspiration did not terminate activity, whereas stronger stimulations (≥ 2.0 mW) terminated inspiration. (2) When the pre-BötC stimulation ended in or was applied during expiration, rebound activation of inspiration occurred after a fixed latency. (3) Relatively weak sustained stimulation (20 Hz, 0.5-2.0 mW) of pre-BötC inhibitory neurons increased respiratory frequency, while a further increase of stimulus intensity (> 3.0 mW) reduced frequency and finally (≥ 5.0 mW) terminated respiratory oscillations. (4) Single pulses (0.2-5.0 s) applied to the BötC inhibited rhythmic activity for the duration of the stimulation. (5) Sustained stimulation (20 Hz, 0.5-3.0 mW) of the BötC reduced respiratory frequency and finally led to apnea. We have revised our computational model of pre-BötC and BötC microcircuits by incorporating an additional population of post-inspiratory inhibitory neurons in the pre-BötC that interacts with other neurons in the network. This model was able to reproduce the above experimental findings as well as previously published results of optogenetic activation of pre-BötC or BötC neurons obtained by other laboratories. The proposed organization of pre-BötC and BötC circuits leads to testable predictions about their specific roles in respiratory pattern generation and provides important insights into key circuit interactions operating within brainstem respiratory networks.

We report that half striatal cholinergic interneurons are dual transmitter cholinergic and GABAergic interneurons (CGINs) expressing ChAT, GAD65, Lhx7, and Lhx6 mRNAs, labeled with GAD and VGAT, generating monosynaptic dual cholinergic/GABAergic currents and an inhibitory pause response. Dopamine deprivation increases CGINs ongoing activity and abolishes GABAergic inhibition including the cortico-striatal pause because of high [Cl - ] i levels. Dopamine deprivation also dramatically increases CGINs dendritic arbors and monosynaptic interconnections probability, suggesting the formation of a dense CGINs network. The NKCC1 chloride importer antagonist bumetanide, which reduces [Cl - ] i levels, restores GABAergic inhibition, the cortico-striatal pause-rebound response, and attenuates motor effects of dopamine deprivation. Therefore, most of the striatal cholinergic excitatory drive is balanced by a concomitant powerful GABAergic inhibition that is impaired by dopamine deprivation. The attenuation by bumetanide of cardinal features of Parkinson's disease paves the way to a novel therapeutic strategy based on a restoration of low [Cl - ] i levels and GABAergic inhibition.

Mice with targeted gene disruption have provided important information about the molecular mechanisms of circadian clock function. A full understanding of the roles of circadian-relevant genes requires manipulation of their expression in a tissue-specific manner, ideally including manipulation with high efficiency within the suprachiasmatic nuclei (SCN). To date, conditional manipulation of genes within the SCN has been difficult. In a previously developed mouse line, Cre recombinase was inserted into the vesicular GABA transporter (Vgat) locus. Since virtually all SCN neurons are GABAergic, this Vgat-Cre line seemed likely to have high efficiency at disrupting conditional alleles in SCN. To test this premise, the efficacy of Vgat-Cre in excising conditional (fl, for flanked by LoxP) alleles in the SCN was examined. Vgat-Cre-mediated excision of conditional alleles of Clock or Bmal1 led to loss of immunostaining for products of the targeted genes in the SCN. Vgat-Cre + ; Clock fl/fl ; Npas2 m/m mice and Vgat-Cre + ; Bmal1 fl/fl mice became arrhythmic immediately upon exposure to constant darkness, as expected based on the phenotype of mice in which these genes are disrupted throughout the body. The phenotype of mice with other combinations of Vgat-Cre + , conditional Clock, and mutant Npas2 alleles also resembled the corresponding whole-body knockout mice. These data indicate that the Vgat-Cre line is useful for Cre-mediated recombination within the SCN, making it useful for Cre-enabled technologies including gene disruption, gene replacement, and opto- and chemogenetic manipulation of the SCN circadian clock.

GABA signaling has been implicated in neural development; however, in vivo genetic evidence is missing because mutant mice lacking GABA activity die prematurely. Here, we studied synapse development by ablating vesicular GABA transporter ( Vgat ) in ErbB4 + interneurons. We show that inhibitory axo-somatic synapses onto pyramidal neurons vary from one cortical layer to another; however, inhibitory synapses on axon initial segments (AISs) were similar across layers. Conversely, parvalbumin-positive (PV + )/ErbB4 + interneurons and PV-only interneurons receive a higher number of inhibitory synapses from PV + ErbB4 + interneurons compared with ErbB4-only interneurons. Vgat deletion from ErbB4 + interneurons reduced axo-somatic or axo-axonic synapses from PV + ErbB4 + interneurons onto excitatory neurons. This effect was associated with corresponding changes in neurotransmission. However, the Vgat mutation seemed to have little effect on inhibitory synapses onto PV + and/or ErbB4 + interneurons. Interestingly, perineuronal nets, extracellular matrix structures implicated in maturation, survival, protection, and plasticity of PV + interneurons, were increased in the cortex of ErbB4-Vgat -/- mice. No apparent difference was observed between males and females. These results demonstrate that Vgat of ErbB4 + interneurons is essential for the development of inhibitory synapses onto excitatory neurons and suggest a role of GABA in circuit assembly. SIGNIFICANCE STATEMENT GABA has been implicated in neural development, but in vivo genetic evidence is missing because mutant mice lacking GABA die prematurely. Here, we ablated Vgat in ErbB4 + interneurons in an inducible manner. We provide evidence that the formation of inhibitory and excitatory synapses onto excitatory neurons requires Vgat in interneurons. In particular, inhibitory axo-somatic and axo-axonic synapses are more vulnerable. Our results suggest a role of GABA in circuit assembly., (Copyright © 2018 the authors 0270-6474/18/382533-18$15.00/0.)

Inhibitory neurons are crucial for shaping and regulating the dynamics of the entire network, and disturbances in these neurons contribute to brain disorders. Despite the recent progress in genetic labeling techniques, the heterogeneity of inhibitory neurons requires the development of highly characterized tools that allow accurate, convenient, and versatile visualization of inhibitory neurons in the mouse brain. Here, we report a novel genetic technique to visualize the vast majority and/or sparse subsets of inhibitory neurons in the mouse brain without using techniques that require advanced skills. We developed several lines of Cre-dependent tdTomato reporter mice based on the vesicular GABA transporter (VGAT)-BAC, named VGAT-stop-tdTomato mice. The most useful line (line #54) was selected for further analysis based on two characteristics: the inhibitory neuron-specificity of tdTomato expression and the transgene integration site, which confers efficient breeding and fewer adverse effects resulting from transgene integration-related genomic disruption. Robust and inhibitory neuron-specific expression of tdTomato was observed in a wide range of developmental and cellular contexts. By breeding the VGAT-stop-tdTomato mouse (line #54) with a novel Cre driver mouse line, Galntl4-CreER, sparse labeling of inhibitory neurons was achieved following tamoxifen administration. Furthermore, another interesting line (line #58) was generated through the unexpected integration of the transgene into the X-chromosome and will be used to map X-chromosome inactivation of inhibitory neurons. Taken together, our studies provide new, well-characterized tools with which multiple aspects of inhibitory neurons can be studied in the mouse., (© 2017 Wiley Periodicals, Inc.)

Despite decades of research, there is a persistent debate regarding the localization of GABA/glycine neurons responsible for hyperpolarizing somatic motoneurons during paradoxical (or REM) sleep (PS), resulting in the loss of muscle tone during this sleep state. Combining complementary neuroanatomical approaches in rats, we first show that these inhibitory neurons are localized within the ventromedial medulla (vmM) rather than within the spinal cord. We then demonstrate their functional role in PS expression through local injections of adeno-associated virus carrying specific short-hairpin RNA in order to chronically impair inhibitory neurotransmission from vmM. After such selective genetic inactivation, rats display PS without atonia associated with abnormal and violent motor activity, concomitant with a small reduction of daily PS quantity. These symptoms closely mimic human REM sleep behavior disorder (RBD), a prodromal parasomnia of synucleinopathies. Our findings demonstrate the crucial role of GABA/glycine inhibitory vmM neurons in muscle atonia during PS and highlight a candidate brain region that can be susceptible to α-synuclein-dependent degeneration in RBD patients.

Mounting evidence supports an important role of chemokines, produced by spinal cord astrocytes, in promoting central sensitization and chronic pain. In particular, CCL2 (C-C motif chemokine ligand 2) has been shown to enhance N-methyl-D-aspartate (NMDA)-induced currents in spinal outer lamina II (IIo) neurons. However, the exact molecular, synaptic, and cellular mechanisms by which CCL2 modulates central sensitization are still unclear. We found that spinal injection of the CCR2 antagonist RS504393 attenuated CCL2- and inflammation-induced hyperalgesia. Single-cell RT-PCR revealed CCR2 expression in excitatory vesicular glutamate transporter subtype 2-positive (VGLUT2 + ) neurons. CCL2 increased NMDA-induced currents in CCR2 + /VGLUT2 + neurons in lamina IIo; it also enhanced the synaptic NMDA currents evoked by dorsal root stimulation; and furthermore, it increased the total and synaptic NMDA currents in somatostatin-expressing excitatory neurons. Finally, intrathecal RS504393 reversed the long-term potentiation evoked in the spinal cord by C-fiber stimulation. Our findings suggest that CCL2 directly modulates synaptic plasticity in CCR2-expressing excitatory neurons in spinal lamina IIo, and this underlies the generation of central sensitization in pathological pain.

The bed nucleus of the stria terminalis (BNST) is a brain region important for regulating anxiety-related behavior in both humans and rodents. Here we used a chemogenetic strategy to investigate how engagement of G protein-coupled receptor (GPCR) signaling cascades in genetically defined GABAergic BNST neurons modulates anxiety-related behavior and downstream circuit function. We saw that stimulation of vesicular γ-aminobutyric acid (GABA) transporter (VGAT)-expressing BNST neurons using hM3Dq, but neither hM4Di nor rM3Ds designer receptors exclusively activated by a designer drug (DREADD), promotes anxiety-like behavior. Further, we identified that activation of hM3Dq receptors in BNST VGAT neurons can induce a long-term depression-like state of glutamatergic synaptic transmission, indicating DREADD-induced changes in synaptic plasticity. Further, we used DREADD-assisted metabolic mapping to profile brain-wide network activity following activation of G q -mediated signaling in BNST VGAT neurons and saw increased activity within ventral midbrain structures, including the ventral tegmental area and hindbrain structures such as the locus coeruleus and parabrachial nucleus. These results highlight that G q -mediated signaling in BNST VGAT neurons can drive downstream network activity that correlates with anxiety-like behavior and points to the importance of identifying endogenous GPCRs within genetically defined cell populations. We next used a microfluidics approach to profile the receptorome of single BNST VGAT neurons. This approach yielded multiple G q -coupled receptors that are associated with anxiety-like behavior and several potential novel candidates for regulation of anxiety-like behavior. From this, we identified that stimulation of the G q -coupled receptor 5-HT 2C R in the BNST is sufficient to elevate anxiety-like behavior in an acoustic startle task. Together, these results provide a novel profile of receptors within genetically defined BNST VGAT neurons that may serve as therapeutic targets for regulating anxiety states and provide a blueprint for examining how G-protein-mediated signaling in a genetically defined cell type can be used to assess behavior and brain-wide circuit function.