Neonatal GABAergic transmission primes vestibular gating of output for adult spatial navigation.

GABAergic interneurons are poised with the capacity to shape circuit output via inhibitory gating. How early in the development of medial vestibular nucleus (MVN) are GABAergic neurons recruited for feedforward shaping of outputs to higher centers for spatial navigation? The role of early GABAergic transmission in assembling vestibular circuits for spatial navigation was explored by neonatal perturbation. Immunohistochemistry and confocal imaging were utilized to reveal the expression of parvalbumin (PV)-expressing MVN neurons and their perineuronal nets. Whole-cell patch-clamp recording, coupled with optogenetics, was conducted in vitro to examine the synaptic function of MVN circuitry. Chemogenetic targeting strategy was also employed in vivo to manipulate neuronal activity during navigational tests. We found in rats a neonatal critical period before postnatal day (P) 8 in which competitive antagonization of GABAergic transmission in the MVN retarded maturation of inhibitory neurotransmission, as evidenced by deranged developmental trajectory for excitation/inhibition ratio and an extended period of critical period-like plasticity in GABAergic transmission. Despite increased number of PV-expressing GABAergic interneurons in the MVN, optogenetic-coupled patch-clamp recording indicated null-recruitment of these neurons in tuning outputs along the ascending vestibular pathway. Such perturbation not only offset output dynamics of ascending MVN output neurons, but was further accompanied by impaired vestibular-dependent navigation in adulthood. The same perturbations were however non-consequential when applied after P8. Results highlight neonatal GABAergic transmission as key to establishing feedforward output dynamics to higher brain centers for spatial cognition and navigation.

Impairments of GABAergic transmission in hippocampus mediate increased susceptibility of epilepsy in the early stage of Alzheimer's disease.

BACKGROUND: Patients with Alzheimer's disease (AD) are often co-morbid with unprovoked seizures, making clinical diagnosis and management difficult. Although it has an important role in both AD and epilepsy, abnormal γ-aminobutyric acid (GABA)ergic transmission is recognized only as a compensative change for glutamatergic damage. Neuregulin 1 (NRG1)-ErbB4 signaling can promote GABA release and suppress epileptogenesis, but its effects on cognition in AD are still controversial. METHODS: Four-month-old APPswe/PS1dE9 mice (APP mice) were used as animal models in the early stage of AD in this study. Acute/chronic chemical-kindling epilepsy models were established with pentylenetetrazol. Electroencephalogram and Racine scores were performed to assess seizures. Behavioral tests were used to assess cognition and emotion. Electrophysiology, western blot and immunofluorescence were performed to detect the alterations in synapses, GABAergic system components and NRG1-ErbB4 signaling. Furthermore, NRG1 was administrated intracerebroventricularly into APP mice and then its antiepileptic and cognitive effects were evaluated. RESULTS: APP mice had increased susceptibility to epilepsy and resulting hippocampal synaptic damage and cognitive impairment. Electrophysiological analysis revealed decreased GABAergic transmission in the hippocampus. This abnormal GABAergic transmission involved a reduction in the number of parvalbumin interneurons (PV+ Ins) and decreased levels of GABA synthesis and transport. We also found impaired NRG1-ErbB4 signaling which mediated by PV+ Ins loss. And NRG1 administration could effectively reduce seizures and improve cognition in four-month-old APP mice. CONCLUSION: Our results indicated that abnormal GABAergic transmission mediated hippocampal hyperexcitability, further excitation/inhibition imbalance, and promoted epileptogenesis in the early stage of AD. Appropriate NRG1 administration could down-regulate seizure susceptibility and rescue cognitive function. Our study provided a potential direction for intervening in the co-morbidity of AD and epilepsy.

Bumetanide induces post-traumatic microglia-interneuron contact to promote neurogenesis and recovery.

Although the Na-K-Cl cotransporter (NKCC1) inhibitor bumetanide has prominent positive effects on the pathophysiology of many neurological disorders, the mechanism of action is obscure. Attention paid to elucidating the role of Nkcc1 has mainly been focused on neurons, but recent single cell mRNA sequencing analysis has demonstrated that the major cellular populations expressing NKCC1 in the cortex are non-neuronal. We used a combination of conditional transgenic animals, in vivo electrophysiology, two-photon imaging, cognitive behavioural tests and flow cytometry to investigate the role of Nkcc1 inhibition by bumetanide in a mouse model of controlled cortical impact (CCI). Here, we found that bumetanide rescues parvalbumin-positive interneurons by increasing interneuron-microglia contacts shortly after injury. The longitudinal phenotypic changes in microglia were significantly modified by bumetanide, including an increase in the expression of microglial-derived BDNF. These effects were accompanied by the prevention of CCI-induced decrease in hippocampal neurogenesis. Treatment with bumetanide during the first week post-CCI resulted in significant recovery of working and episodic memory as well as changes in theta band oscillations 1 month later. These results disclose a novel mechanism for the neuroprotective action of bumetanide mediated by an acceleration of microglial activation dynamics that leads to an increase in parvalbumin interneuron survival following CCI, possibly resulting from increased microglial BDNF expression and contact with interneurons. Salvage of interneurons may normalize ambient GABA, resulting in the preservation of adult neurogenesis processes as well as contributing to bumetanide-mediated improvement of cognitive performance.

ErbB4 deficiency exacerbates olfactory dysfunction in an early-stage Alzheimer's disease mouse model.

Olfactory dysfunction is increasingly recognized as an early indicator of Alzheimer's disease (AD). Aberrations in GABAergic function and the excitatory/inhibitory (E/I) balance within the olfactory bulb (OB) have been implicated in olfactory impairment during the initial stages of AD. While the neuregulin 1 (NRG1)/ErbB4 signaling pathway is known to regulate GABAergic transmission in the brain and is associated with various neuropsychiatric disorders, its specific role in early AD-related olfactory impairment remains incompletely understood. This study demonstrated that olfactory dysfunction preceded cognitive decline in young adult APP/PS1 mice and was characterized by reduced levels of NRG1 and ErbB4 in the OB. Further investigation revealed that deletion of ErbB4 in parvalbumin interneurons reduced GABAergic transmission and increased hyperexcitability in mitral and tufted cells (M/Ts) in the OB, thereby accelerating olfactory dysfunction in young adult APP/PS1 mice. Additionally, ErbB4 deficiency was associated with increased accumulation of Aß and BACE1-mediated cleavage of APP, along with enhanced CDK5 signaling in the OB. NRG1 infusion into the OB was found to enhance GABAergic transmission in M/Ts and alleviate olfactory dysfunction in young adult APP/PS1 mice. These findings underscore the critical role of NRG1/ErbB4 signaling in regulating GABAergic transmission and E/I balance within the OB, contributing to olfactory impairment in young adult APP/PS1 mice, and provide novel insights for early intervention strategies in AD. This work has shown that ErbB4 deficiency increased the burden of Aß, impaired GABAergic transmission, and disrupted the E/I balance of mitral and tufted cells (M/Ts) in the OB, ultimately resulting in olfactory dysfunction in young adult APP/PS1 mice. NRG1 could enhance GABAergic transmission, rescue E/I imbalance in M/Ts, and alleviate olfactory dysfunction in young adult APP/PS1 mice. OB: olfactory bulb, E/I: excitation/inhibition, Pr: probability of release, PV: parvalbumin interneurons, Aß: ß-amyloid, GABA: gamma-aminobutyric acid.

Contribution of sex hormones to the sexually dimorphic response of colorectal motility to noxious stimuli in rats.

Our recent studies have shown that noxious stimuli in the colorectum enhance colorectal motility via the brain and spinal defecation centers in male rats. In female rats, however, noxious stimuli have no effect on colorectal motility. The purpose of this study was to determine whether sex hormones are major contributing factors for sex-dependent differences in neural components of the spinal defecation center. Colorectal motility was measured using an in vivo method under ketamine and α-chloralose anesthesia in rats. Capsaicin was administered into the colorectal lumen as noxious stimuli. Orchiectomy in male rats had no effect on the capsaicin-induced response of colorectal motility. However, in ovariectomized female rats, capsaicin administration enhanced colorectal motility, though intact female animals did not show enhanced motility. When estradiol was administered by using a sustained-release preparation in ovariectomized female rats, capsaicin administration did not enhance colorectal motility unless a GABAA receptor antagonist was intrathecally administered to the lumbosacral spinal cord. These findings suggest that estradiol allowed the GABAergic neurons to operate in response to intracolonic administration of capsaicin. The operation of GABAergic inhibition by the action of estradiol could be manifested in male rats only when the effects of male sex hormones were removed by orchiectomy. Taken together, our results indicate that sex hormones contribute to the sexually dimorphic response in colorectal motility enhancement in response to noxious stimuli through modulating GABAergic pathways.NEW & NOTEWORTHY This study demonstrated that estradiol permits inhibitory regulation in the spinal defecation center not only in female rats but also in orchiectomized male rats. GABAergic pathways are likely involved in the effect of estradiol. This is the first report showing that sex hormones affect colorectal motility through the alteration of neural components of the regulatory pathways. Our findings provide a novel insight into pathophysiological mechanisms of defecation disorders related to changes in sex hormones.

Smoothened receptor signaling regulates the developmental shift of GABA polarity in rat somatosensory cortex.

Sonic hedgehog (Shh) and its patched-smoothened receptor complex control a variety of functions in the developing central nervous system, such as neural cell proliferation and differentiation. Recently, Shh signaling components have been found to be expressed at the synaptic level in the postnatal brain, suggesting a potential role in the regulation of synaptic transmission. Using in utero electroporation of constitutively active and negative-phenotype forms of the Shh signal transducer smoothened (Smo), we studied the role of Smo signaling in the development and maturation of GABAergic transmission in the somatosensory cortex. Our results show that enhancing Smo activity during development accelerates the shift from depolarizing to hyperpolarizing GABA in a manner dependent on functional expression of potassium-chloride cotransporter type 2 (KCC2, also known as SLC12A5). On the other hand, blocking Smo activity maintains the GABA response in a depolarizing state in mature cortical neurons, resulting in altered chloride homeostasis and increased seizure susceptibility. This study reveals unexpected functions of Smo signaling in the regulation of chloride homeostasis, through control of KCC2 cell-surface stability, and the timing of the GABA excitatory-to-inhibitory shift in brain maturation.

Thalamocortical bistable switch as a theoretical model of fibromyalgia pathogenesis inferred from a literature survey.

Fibromyalgia (FM) is an unsolved central pain processing disturbance. We aim to provide a unifying model for FM pathogenesis based on a loop network involving thalamocortical regions, i.e., the ventroposterior lateral thalamus (VPL), the somatosensory cortex (SC), and the thalamic reticular nucleus (TRN). The dynamics of the loop have been described by three differential equations having neuron mean firing rates as variables and containing Hill functions to model mutual interactions among the loop elements. A computational analysis conducted with MATLAB has shown a transition from monostability to bistability of the loop behavior for a weakening of GABAergic transmission between TRN and VPL. This involves the appearance of a high-firing-rate steady state, which becomes dominant and is assumed to represent pathogenic pain processing giving rise to chronic pain. Our model is consistent with a bulk of literature evidence, such as neuroimaging and pharmacological data collected on FM patients, and with correlations between FM and immunoendocrine conditions, such as stress, perimenopause, chronic inflammation, obesity, and chronic dizziness. The model suggests that critical targets for FM treatment are to be found among immunoendocrine pathways leading to GABA/glutamate imbalance having an impact on the thalamocortical system.

Restraint Stress and Repeated Corticosterone Administration Differentially Affect Neuronal Excitability, Synaptic Transmission and 5-HT7 Receptor Reactivity in the Dorsal Raphe Nucleus of Young Adult Male Rats.

Exogenous corticosterone administration reduces GABAergic transmission and impairs its 5-HT7 receptor-dependent modulation in the rat dorsal raphe nucleus (DRN), but it is largely unknown how neuronal functions of the DRN are affected by repeated physical and psychological stress. This study compared the effects of repeated restraint stress and corticosterone injections on DRN neuronal excitability, spontaneous synaptic transmission, and its 5-HT7 receptor-dependent modulation. Male Wistar rats received corticosterone injections for 7 or 14 days or were restrained for 10 min twice daily for 3 days. Repeated restraint stress and repeated corticosterone administration evoked similar changes in performance in the forced swim test. They increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) recorded from DRN neurons. In contrast to the treatment with corticosterone, restraint stress-induced changes in sEPSC kinetics and decreased intrinsic excitability of DRN neurons did not modify inhibitory transmission. Repeated injections of the 5-HT7 receptor antagonist SB 269970 ameliorated the effects of restraint on excitability and sEPSC frequency but did not restore the altered kinetics of sEPSCs. Thus, repeated restraint stress and repeated corticosterone administration differ in consequences for the intrinsic excitability of DRN projection neurons and their excitatory and inhibitory synaptic inputs. Effects of repeated restraint stress on DRN neurons can be partially abrogated by blocking the 5-HT7 receptor.

Investigating the Role of GABA in Neural Development and Disease Using Mice Lacking GAD67 or VGAT Genes.

Normal development and function of the central nervous system involves a balance between excitatory and inhibitory neurotransmission. Activity of both excitatory and inhibitory neurons is modulated by inhibitory signalling of the GABAergic and glycinergic systems. Mechanisms that regulate formation, maturation, refinement, and maintenance of inhibitory synapses are established in early life. Deviations from ideal excitatory and inhibitory balance, such as down-regulated inhibition, are linked with many neurological diseases, including epilepsy, schizophrenia, anxiety, and autism spectrum disorders. In the mammalian forebrain, GABA is the primary inhibitory neurotransmitter, binding to GABA receptors, opening chloride channels and hyperpolarizing the cell. We review the involvement of down-regulated inhibitory signalling in neurological disorders, possible mechanisms for disease progression, and targets for therapeutic intervention. We conclude that transgenic models of disrupted inhibitory signalling-in GAD67+/- and VGAT-/- mice-are useful for investigating the effects of down-regulated inhibitory signalling in a range of neurological diseases.

A GABAB receptor antagonist rescues functional and structural impairments in the perirhinal cortex of a mouse model of CDKL5 deficiency disorder.

CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD) is a severe neurodevelopmental encephalopathy characterized by early-onset epilepsy and intellectual disability. Studies in mouse models have linked CDKL5 deficiency to defects in neuronal maturation and synaptic plasticity, and disruption of the excitatory/inhibitory balance. Interestingly, increased density of both GABAergic synaptic terminals and parvalbumin inhibitory interneurons was recently observed in the primary visual cortex of Cdkl5 knockout (KO) mice, suggesting that excessive GABAergic transmission might contribute to the visual deficits characteristic of CDD. However, the functional relevance of cortical GABAergic circuits abnormalities in these mutant mice has not been investigated so far. Here we examined GABAergic circuits in the perirhinal cortex (PRC) of Cdkl5 KO mice, where we previously observed impaired long-term potentiation (LTP) associated with deficits in novel object recognition (NOR) memory. We found a higher number of GABAergic (VGAT)-immunopositive terminals in the PRC of Cdkl5 KO compared to wild-type mice, suggesting that increased inhibitory transmission might contribute to LTP impairment. Interestingly, while exposure of PRC slices to the GABAA receptor antagonist picrotoxin had no positive effects on LTP in Cdkl5 KO mice, the selective GABAB receptor antagonist CGP55845 restored LTP magnitude, suggesting that exaggerated GABAB receptor-mediated inhibition contributes to LTP impairment in mutants. Moreover, acute in vivo treatment with CGP55845 increased the number of PSD95 positive puncta as well as density and maturation of dendritic spines in PRC, and restored NOR memory in Cdkl5 KO mice. The present data show the efficacy of limiting excessive GABAB receptor-mediated signaling in improving synaptic plasticity and cognition in CDD mice.

Glutamate transmission rather than cellular pacemaking propels excitatory-inhibitory resonance for ictogenesis in amygdala.

Epileptic seizures are automatic, excessive, and synchronized neuronal activities originating from many brain regions especially the amygdala, the allocortices and neocortices. This may reflect a shared principle for network organization and signaling in these telencephalic structures. In theory, the automaticity of epileptic discharges may stem from spontaneously active "oscillator" neurons equipped with intrinsic pacemaking conductances, or from a group of synaptically-connected collaborating "resonator" neurons. In the basolateral amygdalar (BLA) network of pyramidal-inhibitory (PN-IN) neuronal resonators, we demonstrated that rhythmogenic currents are provided by glutamatergic rather than the classic intrinsic or cellular pacemaking conductances (namely the h currents). The excitatory output of glutamatergic neurons such as PNs presumably propels a novel network-based "relay burst mode" of discharges especially in INs, which precondition PNs into a state prone to burst discharges and thus further glutamate release. Also, selective activation of unilateral PNs, but never INs, readily drives bilateral BLA networks into reverberating discharges which are fully synchronized with the behavioral manifestations of seizures (e.g. muscle contractions). Seizures originating in BLA and/or the other structures with similar PN-IN networks thus could be viewed as glutamate-triggered erroneous network oscillations that are normally responsible for information relay.

Sodium salicylate enhances neural excitation via reducing GABAergic transmission in the dentate gyrus area of rat hippocampus in vivo.

Sodium salicylate, one of the non-steroidal anti-inflammatory drugs, is widely prescribed in the clinic, but a high dose of usage can cause hyperactivity in the central nervous system, including the hippocampus. At present, the neural mechanism underlying the induced hyperactivity is not fully understood, in particular, in the hippocampus under an in vivo condition. In this study, we found that systemic administration of sodium salicylate increased the field excitatory postsynaptic potential slope and the population spike amplitude in a dose-dependent manner in the hippocampal dentate gyrus area of rats with in vivo field potential extracellular recordings, which indicates that sodium salicylate enhances basal synaptic transmission and neural excitation. In the presence of picrotoxin, a GABA-A receptor antagonist, sodium salicylate failed to increase the initial slope of the field excitatory postsynaptic potential and the amplitude of the population spike in vivo. To further explore how sodium salicylate enhances the neural excitation, we made whole-cell patch-clamp recordings from hippocampal slices. We found that perfusion of the slice with sodium salicylate decreased electrically evoked GABA receptor-mediated currents, increased paired-pulse ratio, and lowered frequency and amplitude of miniature inhibitory postsynaptic currents. Together, these results demonstrate that sodium salicylate enhances the neural excitation through suppressing GABAergic synaptic transmission in presynaptic and postsynaptic mechanisms in the hippocampal dentate gyrus area. Our findings may help understand the side effects caused by sodium salicylate in the central nervous system.

Disinhibition of CA1 pyramidal cells by low-dose ketamine and other antagonists with rapid antidepressant efficacy.

Low-dose ketamine, an open-channel N-methyl d-aspartate receptor (NMDAR) antagonist, mediates rapid antidepressant effects in humans that are mimicked in preclinical rodent models. Disinhibition of pyramidal cells via decreased output of fast-spiking GABAergic interneurons has been proposed as a key mechanism that triggers the antidepressant response. Unfortunately, to date, disinhibition has not been directly demonstrated. Furthermore, whether disinhibition is a common mechanism shared among other antagonists with rapid antidepressant properties in humans has not been investigated. Using in vitro electrophysiology in acute slices of dorsal hippocampus from adult male Sprague-Dawley rats, we examined the immediate effects of a clinically relevant concentration of ketamine to directly test the disinhibition hypothesis. As a mechanistic comparison, we also tested the effects of the glycine site NMDAR partial agonist/antagonist GLYX-13 (rapastinel), the GluN2B subunit-selective NMDAR antagonist Ro 25-6981, and the muscarinic acetylcholine receptor (mAChR) antagonist scopolamine. Low-dose ketamine, GLYX-13, and scopolamine reduced inhibitory input onto pyramidal cells and increased synaptically driven pyramidal cell excitability measured at the single-cell and population levels. Conversely, Ro 25-6981 increased the strength of inhibitory transmission and did not change pyramidal cell excitability. These results show a decrease in the inhibition/excitation balance that supports disinhibition as a common mechanism shared among those antagonists with rapid antidepressant properties. These data suggest that pyramidal cell disinhibition downstream of NMDAR antagonism could serve as a possible biomarker for the efficacy of rapid antidepressant therapy.

A complex containing the O-GlcNAc transferase OGT-1 and the ubiquitin ligase EEL-1 regulates GABA neuron function.

Inhibitory GABAergic transmission is required for proper circuit function in the nervous system. However, our understanding of molecular mechanisms that preferentially influence GABAergic transmission, particularly presynaptic mechanisms, remains limited. We previously reported that the ubiquitin ligase EEL-1 preferentially regulates GABAergic presynaptic transmission. To further explore how EEL-1 functions, here we performed affinity purification proteomics using Caenorhabditis elegans and identified the O-GlcNAc transferase OGT-1 as an EEL-1 binding protein. This observation was intriguing, as we know little about how OGT-1 affects neuron function. Using C. elegans biochemistry, we confirmed that the OGT-1/EEL-1 complex forms in neurons in vivo and showed that the human orthologs, OGT and HUWE1, also bind in cell culture. We observed that, like EEL-1, OGT-1 is expressed in GABAergic motor neurons, localizes to GABAergic presynaptic terminals, and functions cell-autonomously to regulate GABA neuron function. Results with catalytically inactive point mutants indicated that OGT-1 glycosyltransferase activity is dispensable for GABA neuron function. Consistent with OGT-1 and EEL-1 forming a complex, genetic results using automated, behavioral pharmacology assays showed that ogt-1 and eel-1 act in parallel to regulate GABA neuron function. These findings demonstrate that OGT-1 and EEL-1 form a conserved signaling complex and function together to affect GABA neuron function.

Sexually differentiated and neuroanatomically specific co-expression of aromatase neurons and GAD67 in the male and female quail brain.

Testosterone aromatization into estrogens in the preoptic area (POA) is critical for the activation of male sexual behavior in many vertebrates. Yet, the cellular mechanisms mediating actions of neuroestrogens on sexual behavior remain largely unknown. We investigated in male and female Japanese quail by dual-label fluorescent in situ hybridization (FISH) whether aromatase-positive (ARO) neurons express glutamic acid decarboxylase 67 (GAD67), the rate-limiting enzyme in GABA biosynthesis. ARO cells and ARO cells double labeled with GAD67 (ARO-GAD67) were counted at standardized locations in the medial preoptic nucleus (POM) and the medial bed nucleus of the stria terminalis (BST) to produce three-dimensional distribution maps. Overall, males had more ARO cells than females in POM and BST. The number of double-labeled ARO-GAD67 cells was also higher in males than in females and greatly varied as a function of the specific position in these nuclei. Significant sex differences were however present only in the most caudal part of POM. Although both ARO and GAD67 were expressed in the VMN, no colocalization between these markers was detected. Together, these data show that a high proportion of estrogen-synthesizing neurons in POM and BST are inhibitory and the colocalization of GAD67 with ARO exhibits a high degree of anatomical specificity as well as localized sex differences. The fact that many preoptic ARO neurons project to the periaqueductal gray in male quail suggests possible mechanisms through which locally produced estrogens could activate male sexual behavior.

Characterization of Carbonic Anhydrase In Vivo Using Magnetic Resonance Spectroscopy.

Carbonic anhydrase is a ubiquitous metalloenzyme that catalyzes the reversible interconversion of CO2/HCO3-. Equilibrium of these species is maintained by the action of carbonic anhydrase. Recent advances in magnetic resonance spectroscopy have allowed, for the first time, in vivo characterization of carbonic anhydrase in the human brain. In this article, we review the theories and techniques of in vivo 13C magnetization (saturation) transfer magnetic resonance spectroscopy as they are applied to measuring the rate of exchange between CO2 and HCO3- catalyzed by carbonic anhydrase. Inhibitors of carbonic anhydrase have a wide range of therapeutic applications. Role of carbonic anhydrases and their inhibitors in many diseases are also reviewed to illustrate future applications of in vivo carbonic anhydrase assessment by magnetic resonance spectroscopy.

Perineuronal Nets in the Deep Cerebellar Nuclei Regulate GABAergic Transmission and Delay Eyeblink Conditioning.

Perineuronal nets (PNNs), composed mainly of chondroitin sulfate proteoglycans, are the extracellular matrix that surrounds cell bodies, proximal dendrites, and axon initial segments of adult CNS neurons. PNNs are known to regulate neuronal plasticity, although their physiological roles in cerebellar functions have yet to be elucidated. Here, we investigated the contribution of PNNs to GABAergic transmission from cerebellar Purkinje cells (PCs) to large glutamatergic neurons in the deep cerebellar nuclei (DCN) in male mice by recording IPSCs from cerebellar slices, in which PNNs were depleted with chondroitinase ABC (ChABC). We found that PNN depletion increased the amplitude of evoked IPSCs and enhanced the paired-pulse depression. ChABC treatment also facilitated spontaneous IPSCs and increased the miniature IPSC frequency without changing not only the amplitude but also the density of PC terminals, suggesting that PNN depletion enhances presynaptic GABA release. We also demonstrated that the enhanced GABAergic transmission facilitated rebound firing in large glutamatergic DCN neurons, which is expected to result in the efficient induction of synaptic plasticity at synapses onto DCN neurons. Furthermore, we tested whether PNN depletion affects cerebellar motor learning. Mice having received the enzyme into the interpositus nuclei, which are responsible for delay eyeblink conditioning, exhibited the conditioned response at a significantly higher rate than control mice. Therefore, our results suggest that PNNs of the DCN suppress GABAergic transmission between PCs and large glutamatergic DCN neurons and restrict synaptic plasticity associated with motor learning in the adult cerebellum.SIGNIFICANCE STATEMENT Perineuronal nets (PNNs) are one of the extracellular matrices of adult CNS neurons and implicated in regulating various brain functions. Here we found that enzymatic PNN depletion in the mouse deep cerebellar nuclei (DCN) reduced the paired-pulse ratio of IPSCs and increased the miniature IPSC frequency without changing the amplitude, suggesting that PNN depletion enhances GABA release from the presynaptic Purkinje cell (PC) terminals. Mice having received the enzyme in the interpositus nuclei exhibited a higher conditioned response rate in delay eyeblink conditioning than control mice. These results suggest that PNNs regulate presynaptic functions of PC terminals in the DCN and functional plasticity of synapses on DCN neurons, which influences the flexibility of adult cerebellar functions.

Compensatory Activation of Cannabinoid CB2 Receptor Inhibition of GABA Release in the Rostral Ventromedial Medulla in Inflammatory Pain.

The rostral ventromedial medulla (RVM) is a relay in the descending pain modulatory system and an important site of endocannabinoid modulation of pain. Endocannabinoids inhibit GABA release in the RVM, but it is not known whether this effect persists in chronic pain states. In the present studies, persistent inflammation induced by complete Freund's adjuvant (CFA) increased GABAergic miniature IPSCs (mIPSCs). Endocannabinoid activation of cannabinoid (CB1) receptors known to inhibit presynaptic GABA release was significantly reduced in the RVM of CFA-treated rats compared with naive rats. The reduction in CFA-treated rats correlated with decreased CB1 receptor protein expression and function in the RVM. Paradoxically, the nonselective CB1/CB2 receptor agonist WIN55212 inhibited GABAergic mIPSCs in both naive and CFA-treated rats. However, WIN55212 inhibition was reversed by the CB1 receptor antagonist rimonabant in naive rats but not in CFA-treated rats. WIN55212-mediated inhibition in CFA-treated rats was blocked by the CB2 receptor-selective antagonist SR144528, indicating that CB2 receptor function in the RVM is increased during persistent inflammation. Consistent with these results, CB2 receptor agonists AM1241 and GW405833 inhibited GABAergic mIPSC frequency only in CFA-treated rats, and the inhibition was reversed with SR144528. When administered alone, SR144528 and another CB2 receptor-selective antagonist AM630 increased mIPSC frequency in the RVM of CFA-treated rats, indicating that CB2 receptors are tonically activated by endocannabinoids. Our data provide evidence that CB2 receptor function emerges in the RVM in persistent inflammation and that selective CB2 receptor agonists may be useful for treatment of persistent inflammatory pain. SIGNIFICANCE STATEMENT: These studies demonstrate that endocannabinoid signaling to CB1 and CB2 receptors in adult rostral ventromedial medulla is altered in persistent inflammation. The emergence of CB2 receptor function in the rostral ventromedial medulla provides additional rationale for the development of CB2 receptor-selective agonists as useful therapeutics for chronic inflammatory pain.

Clenbuterol reduces GABAergic transmission in prefrontal cortex layer 5/6 pyramidal neurons of juvenile rat via reducing action potentials firing frequency of GABAergic interneurons.

Beta-adrenoceptors (ß2 -ARs) have beneficial effects on prefrontal cortex (PFC) working memory, however, the cellular and molecular mechanisms are unclear yet. In this study, we probed the effect of ß2 -AR-selective agonist clenbuterol (Clen) on synaptic transmission in layer 5/6 pyramidal neurons of PFC. Bath application of Clen reduced spontaneous IPSC (sIPSC) frequency without effects on sEPSCs. Clen did not alter the frequency and amplitude of miniature IPSCs (mIPSCs), but exerted heterogeneous effects on evoked IPSCs (eIPSCs) recorded from PFC layer 5/6 pyramidal neurons. Clen decreased the firing rate of action potentials of fast-spiking GABAergic interneurons. Clen-induced hyperpolarization of fast-spiking GABAergic interneurons required potentiation of an inward rectifier K+ channels. Clen-induced hyperpolarization of fast-spiking interneurons was dependent on Gs protein rather than cAMP and protein kinase A. Our findings demonstrate that Clen (10 µM) enhances inward rectifier K+ channels via Gs protein to cause membrane hyperpolarization of fast-spiking GABAergic interneurons resulting in reduction of action potentials firing rate to reduce GABAergic transmission.

Chronic inflammatory pain induced GABAergic synaptic plasticity in the adult mouse anterior cingulate cortex.

Background Chronic pain is a persistent unpleasant sensation that produces pathological synaptic plasticity in the central nervous system. Both human imaging study and animal studies consistently demonstrate that the anterior cingulate cortex is a critical cortical area for nociceptive and chronic pain processing. Thus far, the mechanisms of excitatory synaptic transmission and plasticity have been well characterized in the anterior cingulate cortex for various models of chronic pain. By contrast, the potential contribution of inhibitory synaptic transmission in the anterior cingulate cortex, in models of chronic pain, is not fully understood. Methods Chronic inflammation was induced by complete Freund adjuvant into the adult mice left hindpaw. We performed in vitro whole-cell patch-clamp recordings from layer II/III pyramidal neurons in two to three days after the complete Freund adjuvant injection and examined if the model could cause plastic changes, including transient and tonic type A γ-aminobutyric acid (GABAA) receptor-mediated inhibitory synaptic transmission, in the anterior cingulate cortex. We analyzed miniature/spontaneous inhibitory postsynaptic currents, GABAA receptor-mediated tonic currents, and evoked inhibitory postsynaptic currents. Finally, we studied if GABAergic transmission-related proteins in the presynapse and postsynapse of the anterior cingulate cortex were altered. Results The complete Freund adjuvant model reduced the frequency of both miniature and spontaneous inhibitory postsynaptic currents compared with control group. By contrast, the average amplitude of these currents was not changed between two groups. Additionally, the complete Freund adjuvant model did not change GABAA receptor-mediated tonic currents nor the set of evoked inhibitory postsynaptic currents when compared with control group. Importantly, protein expression of vesicular GABA transporter was reduced within the presynpase of the anterior cingulate cortex in complete Freund adjuvant model. In contrast, the complete Freund adjuvant model did not change the protein levels of GABAA receptors subunits such as α1, α5, ß2, γ2, and δ. Conclusion Our results suggest that the induction phase of inflammatory pain involves spontaneous GABAergic plasticity at presynaptic terminals of the anterior cingulate cortex.