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Synaptic pruning during adolescence is important for appropriate neurodevelopment and synaptic plasticity. Aberrant synaptic pruning may underlie a variety of brain disorders such as schizophrenia, autism and anxiety. Dopamine D2 receptor (Drd2) is associated with several neuropsychiatric diseases and is the target of some antipsychotic drugs. Here we generate self-reporting Drd2 heterozygous (SR-Drd2 +/- ) rats to simultaneously visualize Drd2-positive neurons and downregulate Drd2 expression. Time course studies on the developing anterior cingulate cortex (ACC) from control and SR-Drd2 +/- rats reveal important roles of Drd2 in regulating synaptic pruning rather than synapse formation. Drd2 also regulates LTD, a form of synaptic plasticity which includes some similar cellular/biochemical processes as synaptic pruning. We further demonstrate that Drd2 regulates synaptic pruning via cell-autonomous mechanisms involving activation of mTOR signaling. Deficits of Drd2-mediated synaptic pruning in the ACC during adolescence lead to hyper-glutamatergic function and anxiety-like behaviors in adulthood. Taken together, our results demonstrate important roles of Drd2 in cortical synaptic pruning., (© 2021. The Author(s).)

Neuregulin 1 (NRG1) and its receptor ERBB4 are schizophrenia (SZ) risk genes that control the development of both excitatory and inhibitory cortical circuits. Most studies focused on the characterization ErbB4 deficient mice. However, ErbB4 deletion concurrently perturbs the signaling of Nrg1 and Neuregulin 3 (Nrg3), another ligand expressed in the cortex. In addition, NRG1 polymorphisms linked to SZ locate mainly in non-coding regions and they may partially reduce Nrg1 expression. Here, to study the relevance of Nrg1 partial loss-of-function in cortical circuits we characterized a recently developed haploinsufficient mouse model of Nrg1 (Nrg1 tm1Lex ). These mice display SZ-like behavioral deficits. The cellular and molecular underpinnings of the behavioral deficits in Nrg1 tm1Lex mice remain to be established. With multiple approaches including Magnetic Resonance Spectroscopy (MRS), electrophysiology, quantitative imaging and molecular analysis we found that Nrg1 haploinsufficiency impairs the inhibitory cortical circuits. We observed changes in the expression of molecules involved in GABAergic neurotransmission, decreased density of Vglut1 excitatory buttons onto Parvalbumin interneurons and decreased frequency of spontaneous inhibitory postsynaptic currents. Moreover, we found a decreased number of Parvalbumin positive interneurons in the cortex and altered expression of Calretinin. Interestingly, we failed to detect other alterations in excitatory neurons that were previously reported in ErbB4 null mice suggesting that the Nrg1 haploinsufficiency does not entirely phenocopies ErbB4 deletions. Altogether, this study suggests that Nrg1 haploinsufficiency primarily affects the cortical inhibitory circuits in the cortex and provides new insights into the structural and molecular synaptic impairment caused by NRG1 hypofunction in a preclinical model of SZ., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In this work, modulation by orexin-A of the release of glutamate and GABA from bipolar and amacrine cells respectively was studied by examining the effects of the neuropeptide on miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) of rat retinal ganglion cells (GCs). Using RNAscope in situ hybridization in combination with immunohistochemistry, we showed positive signals for orexin receptor-1 (OX 1 R) mRNA in the bipolar cell terminals and those for orexin receptor-2 (OX 2 R) mRNA in the amacrine cell terminals. With whole-cell patch-clamp recordings in rat retinal slices, we demonstrated that application of orexin-A reduced the interevent interval of mEPSCs of GCs through OX 1 R. However, it increased the interevent interval of mIPSCs, mediated by GABA A receptors, through OX 2 R. Furthermore, orexin-A-induced reduction of mEPSC interevent interval was abolished by the application of PI-PLC inhibitors or PKC inhibitors. In contrast, orexin-A-induced increase of GABAergic mIPSC interevent interval was mimicked by 8-Br-cAMP or an adenylyl cyclase activator, but was eliminated by PKA antagonists. Finally, application of nimodipine, an L-type Ca 2+ channel blocker, increased both mEPSC and mIPSC interevent interval, and co-application of orexin-A no longer changed the mEPSCs and mIPSCs. We conclude that orexin-A increases presynaptic glutamate release onto GCs by activating L-type Ca 2+ channels in bipolar cells, a process that is mediated by an OX 1 R/PI-PLC/PKC signaling pathway. However, orexin-A decreases presynaptic GABA release onto GCs by inhibiting L-type Ca 2+ channels in amacrine cells, a process that is mediated by an OX 2 R/cAMP-PKA signaling pathway., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: In an early stage of Alzheimer's disease (AD), before the formation of amyloid plaques, neuronal network hyperactivity has been reported in both patients and animal models. This suggests an underlying disturbance of the balance between excitation and inhibition. Several studies have highlighted the role of somatic inhibition in early AD, while less is known about dendritic inhibition., Objective: In this study we investigated how inhibitory synaptic currents are affected by elevated Aβ levels., Methods: We performed whole-cell patch clamp recordings of CA1 pyramidal neurons in organotypic hippocampal slice cultures after treatment with Aβ-oligomers and in hippocampal brain slices from AppNL-F-G mice (APP-KI)., Results: We found a reduction of spontaneous inhibitory postsynaptic currents (sIPSCs) in CA1 pyramidal neurons in organotypic slices after 24 h Aβ treatment. sIPSCs with slow rise times were reduced, suggesting a specific loss of dendritic inhibitory inputs. As miniature IPSCs and synaptic density were unaffected, these results suggest a decrease in activity-dependent transmission after Aβ treatment. We observed a similar, although weaker, reduction in sIPSCs in CA1 pyramidal neurons from APP-KI mice compared to control. When separated by sex, the strongest reduction in sIPSC frequency was found in slices from male APP-KI mice. Consistent with hyperexcitability in pyramidal cells, dendritically targeting interneurons received slightly more excitatory input. GABAergic action potentials had faster kinetics in APP-KI slices., Conclusion: Our results show that Aβ affects dendritic inhibition via impaired action potential driven release, possibly due to altered kinetics of GABAergic action potentials. Reduced dendritic inhibition may contribute to neuronal hyperactivity in early AD.

Inborn errors of CACNA1A-encoded P/Q-type calcium channels impair synaptic transmission, producing early and lifelong neurological deficits, including childhood absence epilepsy, ataxia and dystonia. Whether these impairments owe their pathologies to defective channel function during the critical period for thalamic network stabilization in immature brain remains unclear. Here we show that mice with tamoxifen-induced adult-onset ablation of P/Q channel alpha subunit (iKOp/q) display identical patterns of dysfunction, replicating the inborn loss-of-function phenotypes and, therefore demonstrate that these neurological defects do not rely upon developmental abnormality. Unexpectedly, unlike the inborn model, the adult-onset pattern of excitability changes believed to be pathogenic within the thalamic network is non-canonical. Specifically, adult ablation of P/Q channels does not promote Cacna1g-mediated burst firing or T-type calcium current (IT) in the thalamocortical relay neurons; however, burst firing in thalamocortical relay neurons remains essential as iKOp/q mice generated on a Cacna1g deleted background show substantially diminished seizure generation. Moreover, in thalamic reticular nucleus neurons, burst firing is impaired accompanied by attenuated IT. Interestingly, inborn deletion of thalamic reticular nucleus-enriched, human childhood absence epilepsy-linked gene Cacna1h in iKOp/q mice reduces thalamic reticular nucleus burst firing and promotes rather than reduces seizure, indicating an epileptogenic role for loss-of-function Cacna1h gene variants reported in human childhood absence epilepsy cases. Together, our results demonstrate that P/Q channels remain critical for maintaining normal thalamocortical oscillations and motor control in the adult brain, and suggest that the developmental plasticity of membrane currents regulating pathological rhythmicity is both degenerate and age-dependent., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

FOXG1 syndrome is a severe encephalopathy that exhibit intellectual disability, emotional disorder, and limited social communication. To elucidate the contribution of somatostatin-expressing interneurons (SST-INs) to the cellular basis underlying FOXG1 syndrome, here, by crossing SST-cre with a Foxg1fl/fl line, we selectively ablated Foxg1. Loss of Foxg1 resulted in an obvious reduction in the number of SST-INs, accompanied by an altered ratio of subtypes. Foxg1-deficient SST-INs exhibited decreased membrane excitability and a changed ratio of electrophysiological firing patterns, which subsequently led to an excitatory/inhibitory imbalance. Moreover, cognitive defects, limited social interactions, and depression-like behaviors were detected in Foxg1 cKO mice. Treatment with low-dose of clonazepam effectively alleviated the defects. These results identify a link of SST-IN development to the aberrant emotion, cognition, and social capacities in patients. Our findings identify a novel role of Foxg1 in SST-IN development and put new insights into the cellular basis of FOXG1 syndrome., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

The Federal Pain Research Strategy recommended development of nonopioid analgesics as a top priority in its strategic plan to address the significant public health crisis and individual burden of chronic pain faced by >100 million Americans. Motivated by this challenge, a natural product extracts library was screened and identified a plant extract that targets activity of voltage-gated calcium channels. This profile is of interest as a potential treatment for neuropathic pain. The active extract derived from the desert lavender plant native to southwestern United States, when subjected to bioassay-guided fractionation, afforded 3 compounds identified as pentacyclic triterpenoids, betulinic acid (BA), oleanolic acid, and ursolic acid. Betulinic acid inhibited depolarization-evoked calcium influx in dorsal root ganglion (DRG) neurons predominantly through targeting low-voltage-gated (Cav3 or T-type) and CaV2.2 (N-type) calcium channels. Voltage-clamp electrophysiology experiments revealed a reduction of Ca, but not Na, currents in sensory neurons after BA exposure. Betulinic acid inhibited spontaneous excitatory postsynaptic currents and depolarization-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices. Notably, BA did not engage human mu, delta, or kappa opioid receptors. Intrathecal administration of BA reversed mechanical allodynia in rat models of chemotherapy-induced peripheral neuropathy and HIV-associated peripheral sensory neuropathy as well as a mouse model of partial sciatic nerve ligation without effects on locomotion. The broad-spectrum biological and medicinal properties reported, including anti-HIV and anticancer activities of BA and its derivatives, position this plant-derived small molecule natural product as a potential nonopioid therapy for management of chronic pain.

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and the most common known cause of autism. Loss of fragile X mental retardation protein (FMRP) in mice ( Fmr1 KO) leads to altered synaptic and circuit maturation in the hippocampus that is correlated with alterations in hippocampal-dependent behaviors. Previous studies have demonstrated that loss of FMRP increased the rate of proliferation of progenitor cells and altered their fate specification in adult Fmr1 KO mice. While these studies clearly demonstrate a role for FMRP in adult neurogenesis in the hippocampus, it is not known whether the functional synaptic maturation and integration of adult-born dentate granule cells (abDGCs) into hippocampal circuits is affected in Fmr1 KO mice. Here, we used retroviral labeling to birthdate abDGCs in Fmr1 KO mice which allowed us to perform targeted patch clamp recording to measure the development of synaptic inputs to these neurons at precise time points after differentiation. The frequency and amplitude of spontaneous GABAergic events increased over the first three weeks after differentiation; however, this normal development of GABAergic synapses was not altered in Fmr1 KO mice. Furthermore, the relatively depolarized GABA reversal potential ( E GABA ) in immature abDGCs was unaffected by loss of FMRP as was the development of dendritic arbor of the adult generated neurons. These studies systematically characterized the functional development of abDGCs during the first four weeks after differentiation and demonstrate that the maturation of GABAergic synaptic inputs to these neurons is not grossly affected by the loss of FMRP.

While the canonical assembly of a GABA A receptor contains two α subunits, two β subunits, and a fifth subunit, it is unclear which variants of each subunit are necessary for native receptors. We used CRISPR/Cas9 to dissect the role of the GABA A receptor β subunits in inhibitory transmission onto hippocampal CA1 pyramidal cells and found that deletion of all β subunits 1, 2, and 3 completely eliminated inhibitory responses. In addition, only knockout of β3, alone or in combination with another β subunit, impaired inhibitory synaptic transmission. We found that β3 knockout impairs inhibitory input from PV but not SOM expressing interneurons. Furthermore, expression of β3 alone on the background of the β1-3 subunit knockout was sufficient to restore synaptic and extrasynaptic inhibitory transmission. These findings reveal a crucial role for the β3 subunit in inhibitory transmission and identify a synapse-specific role of the β3 subunit in GABAergic synaptic transmission., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Diffuse axonal injury (DAI) plays a major role in cortical network dysfunction posited to cause excitatory/inhibitory imbalance after mild traumatic brain injury (mTBI). Current thought holds that white matter (WM) is uniquely vulnerable to DAI. However, clinically diagnosed mTBI is not always associated with WM DAI. This suggests an undetected neocortical pathophysiology, implicating GABAergic interneurons. To evaluate this possibility, we used mild central fluid percussion injury to generate DAI in mice with Cre-driven tdTomato labeling of parvalbumin (PV) interneurons. We followed tdTomato+ profiles using confocal and electron microscopy, together with patch-clamp analysis to probe for DAI-mediated neocortical GABAergic interneuron disruption. Within 3 h post-mTBI tdTomato+ perisomatic axonal injury (PSAI) was found across somatosensory layers 2-6. The DAI marker amyloid precursor protein colocalized with GAD67 immunoreactivity within tdTomato+ PSAI, representing the majority of GABAergic interneuron DAI. At 24 h post-mTBI, we used phospho-c-Jun, a surrogate DAI marker, for retrograde assessments of sustaining somas. Via this approach, we estimated DAI occurs in ~9% of total tdTomato+ interneurons, representing ~14% of pan-neuronal DAI. Patch-clamp recordings of tdTomato+ interneurons revealed decreased inhibitory transmission. Overall, these data show that PV interneuron DAI is a consistent and significant feature of experimental mTBI with important implications for cortical network dysfunction.

Higher-order thalamic nuclei, such as the posterior medial nucleus (POm) in the somatosensory system or the pulvinar in the visual system, densely innervate the cortex and can influence perception and plasticity. To systematically evaluate how higher-order thalamic nuclei can drive cortical circuits, we investigated cell-type selective responses to POm stimulation in mouse primary somatosensory (barrel) cortex, using genetically targeted whole-cell recordings in acute brain slices. We find that ChR2-evoked thalamic input selectively targets specific cell types in the neocortex, revealing layer-specific modules for the summation and processing of POm input. Evoked activity in pyramidal neurons from deep layers is fast and synchronized by rapid feedforward inhibition from GABAergic parvalbumin-expressing neurons, and activity in superficial layers is weaker and prolonged, facilitated by slow inhibition from GABAergic neurons expressing the 5HT3a receptor. Somatostatin-expressing GABAergic neurons do not receive direct input in either layer and their spontaneous activity is suppressed during POm stimulation. This novel pattern of weak, delayed, thalamus-evoked inhibition in layer 2 suggests a longer integration window for incoming sensory information and may facilitate stimulus detection and plasticity in superficial pyramidal neurons.

Anatomical studies have shown that the majority of callosal axons are glutamatergic. However, a small proportion of callosal axons are also immunoreactive for glutamic acid decarboxylase, an enzyme required for gamma-aminobutyric acid (GABA) synthesis and a specific marker for GABAergic neurons. Here, we test the hypothesis that corticocortical parvalbumin-expressing (CC-Parv) neurons connect the two hemispheres of multiple cortical areas, project through the corpus callosum, and are a functional part of the local cortical circuit. Our investigation of this hypothesis takes advantage of viral tracing and optogenetics to determine the anatomical and electrophysiological properties of CC-Parv neurons of the mouse auditory, visual, and motor cortices. We found a direct inhibitory pathway made up of parvalbumin-expressing (Parv) neurons which connects corresponding cortical areas (CC-Parv neurons → contralateral cortex). Like other Parv cortical neurons, these neurons provide local inhibition onto nearby pyramidal neurons and receive thalamocortical input. These results demonstrate a previously unknown long-range inhibitory circuit arising from a genetically defined type of GABAergic neuron that is engaged in interhemispheric communication.

Cyclin-dependent kinase 5 (Cdk5) acts as an essential modulator for neural development and neurological disorders. Here we show that Cdk5 plays a pivotal role in modulating GABAergic signaling and the maturation of visual system. In adult mouse primary visual cortex, Cdk5 formed complex with the GABA synthetic enzyme glutamate decarboxylase GAD67, but not with GAD65. In addition to enhancement in the surface level of NR2B-containing NMDA receptors, inhibition of Cdk5 reduced the protein levels of GADs and Otx2, while leaving intact the expression of vesicular GABA transporter and subunits of GABA A or AMPA receptors. Whole-cell patch-clamp recording in layer II/III pyramidal neurons revealed a decrease in the frequency of miniature inhibitory postsynaptic current (mIPSC). Consequently, pharmacological inhibition and genetic knockdown of Cdk5 in adult mice led to a restoration of juvenile-like ocular dominance plasticity in vivo and long-term synaptic potential in layer II/III induced by white matter stimulation in vitro. Interestingly, we did not observe an alteration of perineuronal nets of extracellular matrix, but a reinstatement of the capability to evoke long-term depression at inhibitory synapses (iLTD), which depended on presynaptic endocannabinoid receptors and was a sign of the rejuvenated GABAergic synapses. Enhancement of GABA signaling by diazepam impeded ocular dominance plasticity rescued by Cdk5 inhibition. These results thus suggest that a physiological role of Cdk5 in visual cortex is to consolidate and stabilize neural circuits through controlling GABAergic signaling., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cholinergic regulation of dopaminergic inputs into the striatum is critical for normal basal ganglia (BG) function. This regulation of BG function is thought to be primarily mediated by acetylcholine released from cholinergic interneurons (ChIs) acting locally in the striatum. We now report a combination of pharmacological, electrophysiological, optogenetic, chemogenetic, and functional magnetic resonance imaging studies suggesting extra-striatal cholinergic projections from the pedunculopontine nucleus to the substantia nigra pars reticulata (SNr) act on muscarinic acetylcholine receptor subtype 4 (M 4 ) to oppose cAMP-dependent dopamine receptor subtype 1 (D 1 ) signaling in presynaptic terminals of direct pathway striatal spiny projections neurons. This induces a tonic inhibition of transmission at direct pathway synapses and D 1 -mediated activation of motor activity. These studies provide important new insights into the unique role of M 4 in regulating BG function and challenge the prevailing hypothesis of the centrality of striatal ChIs in opposing dopamine regulation of BG output., (Copyright © 2017 Elsevier Inc. All rights reserved.)

GABA released from presynaptic sites induces short-lived phasic inhibition mediated by synaptic GABA A receptors (GABA A Rs) and longer-duration tonic inhibition mediated by extrasynaptic GABA A or GABA B receptors (GABA B Rs). A number of studies have found that contactin-associated protein 2 (Cntnap2) knockout (KO) mice, a well-established mouse model of autism, exhibit reduced interneuron numbers and aberrant phasic inhibition. However, little is known about whether tonic inhibition is disrupted in Cntnap2 KO mice and when the disruption of inhibition begins to occur during postnatal development. We examined tonic and phasic inhibition in layer 2/3 pyramidal cells of primary visual cortex of Cntnap2 KO at two different developmental stages, three to four and six to eight weeks of age. We found that both phasic inhibition and GABA A R but not GABA B R-mediated tonic inhibition was reduced in pyramidal cells from six- to eight-week-old Cntnap2 KO mice, while in three- to four-week-old mice, no significant effects of genotype on tonic or phasic inhibition was observed. We further found that activation of tonic currents mediated by δ-subunit-containing GABA A Rs reduced neural excitability, an effect that was attenuated by loss of Cntnap2. While the relative contribution of tonic versus phasic inhibition to autism-related symptoms remains unclear, our data suggest that reduced tonic inhibition may play an important role, and δ-subunit-containing GABA A Rs may be a useful target for therapeutic intervention in autism.

Objective: Mutations of the KCNQ2 gene, which encodes the K v 7.2 subunit of voltage-gated M-type potassium channels, have been associated with epilepsy in the neonatal period. This developmental stage is unique in that the neurotransmitter gamma aminobutyric acid (GABA), which is inhibitory in adults, triggers excitatory action due to a reversed chloride gradient., Methods: To examine whether KCNQ2-related neuronal hyperexcitability involves neonatally excitatory GABA, we examined 1-week-old knockin mice expressing the K v 7.2 variant p.Tyr284Cys (Y284C)., Results: Brain slice electrophysiology revealed elevated CA1 hippocampal GABAergic interneuron activity with respect to presynaptic firing and postsynaptic current frequency. Blockade with the GABA A receptor antagonist bicuculline decreased ictal-like bursting in brain slices with lowered divalent ion concentration, which is consistent with GABA mediating an excitatory function that contributes to the hyperexcitability observed in mutant animals., Significance: We conclude that excitatory GABA contributes to the phenotype in these animals, which raises the question of whether this special type of neurotransmission has broader importance in neonatal epilepsy than is currently recognized., (Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.)

Objective: The mutant γ-aminobutyric acid type A (GABA A ) receptor γ2(Q390X) subunit (Q351X in the mature peptide) has been associated with the epileptic encephalopathy, Dravet syndrome, and the epilepsy syndrome genetic epilepsy with febrile seizures plus (GEFS+). The mutation generates a premature stop codon that results in translation of a stable truncated and misfolded γ2 subunit that accumulates in neurons, forms intracellular aggregates, disrupts incorporation of γ2 subunits into GABA A receptors, and affects trafficking of partnering α and β subunits. Heterozygous Gabrg2 +/Q390X knock-in (KI) mice had reduced cortical inhibition, spike wave discharges on electroencephalography (EEG), a lower seizure threshold to the convulsant drug pentylenetetrazol (PTZ), and spontaneous generalized tonic-clonic seizures. In this proof-of-principal study, we attempted to rescue these deficits in KI mice using a γ2 subunit gene (GABRG2) replacement therapy., Methods: We introduced the GABRG2 allele by crossing Gabrg2 +/Q390X KI mice with bacterial artificial chromosome (BAC) transgenic mice overexpressing HA (hemagglutinin)-tagged human γ2 HA subunits, and compared GABA A receptor subunit expression by Western blot and immunohistochemical staining, seizure threshold by monitoring mouse behavior after PTZ-injection, and thalamocortical inhibition and network oscillation by slice recording., Results: Compared to KI mice, adult mice carrying both mutant allele and transgene had increased wild-type γ2 and partnering α1 and β2/3 subunits, increased miniature inhibitory postsynaptic current (mIPSC) amplitudes recorded from layer VI cortical neurons, reduced thalamocortical network oscillations, and higher PTZ seizure threshold., Significance: Based on these results we suggest that seizures in a genetic epilepsy syndrome caused by epilepsy mutant γ2(Q390X) subunits with dominant negative effects could be rescued potentially by overexpression of wild-type γ2 subunits., (Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.)

Neuronal inhibition mediated by GABA A receptors constrains nociceptive processing in the spinal cord, and loss of GABAergic inhibition can produce allodynia and hyperalgesia. Extrasynaptic α5 subunit-containing GABA A receptors (α5GABA A Rs) generate a tonic conductance that inhibits neuronal activity and constrains learning and memory; however, it is unclear whether α5GABA A Rs similarly generate a tonic conductance in the spinal cord dorsal horn to constrain nociception. We assessed the distribution of α5GABA A Rs in the spinal cord dorsal horn by immunohistochemical analysis, and the activity and function of α5GABA A Rs in neurons of the superficial dorsal horn using electrophysiological and behavioral approaches in male, null-mutant mice lacking the GABA A R α5 subunit (Gabra5-/-) and wild-type mice (WT). The expression of α5GABA A Rs in the superficial dorsal horn followed a laminar pattern of distribution, with a higher expression in lamina II than lamina I. Similarly, the tonic GABA A current in lamina II neurons had a larger contribution from α5GABA A Rs than in lamina I, with no significant contribution of these receptors to synaptic GABA A current. In behavioural tests, WT and Gabra5-/- mice exhibited similar acute thermal and mechanical nociception, and similar mechanical sensitization immediately following intraplantar capsaicin or Complete Freund's Adjuvant (CFA). However, Gabra5-/- mice showed prolonged recovery from sensitization in these models, and increased responses in the late phase of the formalin test. Overall, our data suggest that tonically-active α5GABA A Rs in the spinal cord dorsal horn accelerate the resolution of hyperalgesia and may therefore serve as a novel therapeutic target to promote recovery from pathological pain. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Methylene blue (MB) is commonly used in diagnostic procedures and is also used to treat various medical conditions. Neurological effects of MB have been reported in clinical observations and experimental studies. Thus the modulation of GABA A receptor function by MB was investigated. Whole-cell GABA-activated currents were recorded from HEK293 cells expressing various GABA A receptor subunit configurations. MB inhibition of GABA currents was apparent at 3 μM, and it had an IC 50 of 31 μM in human α1β2γ2 receptors. The MB action was rapid and reversible. MB inhibition was not mediated via the picrotoxin site, as a mutation (T6'F of the β2 subunit) known to confer resistance to picrotoxin had no effect on MB-induced inhibition. Blockade of GABA A receptors by MB was demonstrated across a range of receptors expressing varying subunits, including those expressed at extrasynaptic sites. The sensitivity of α1β2 receptors to MB was similar to that observed in α1β2γ2 receptors, indicating that MB's action via the benzodiazepine or Zn 2+ site is unlikely. MB-induced inhibition of GABA response was competitive with respect to GABA. Furthermore, mutation of α1 F64 to A and β2 Y205 to F in the extracellular N-terminus, both residues which are known to comprise GABA binding pocket, remarkably diminished MB inhibition of GABA currents. These data suggest that MB inhibits GABA A receptor function by direct or allosteric interaction with the GABA binding site. Finally, in mouse hippocampal CA1 pyramidal neurons, MB inhibited GABA-activated currents as well as GABAergic IPSCs. We demonstrate that MB directly inhibits GABA A receptor function, which may underlie some of the effects of MB on the CNS., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The basal forebrain (BF) is an important regulator of hippocampal and cortical activity. In Alzheimer's disease (AD), there is a significant loss and dysfunction of cholinergic neurons within the BF, and also a hypertrophy of fibers containing the neuropeptide galanin. Understanding how galanin interacts with BF circuitry is critical in determining what role galanin overexpression plays in the progression of AD. Here, we examined the location and function of galanin in the medial septum/diagonal band (MS/DBB) region of the BF. We show that galanin fibers are located throughout the MS/DBB and intermingled with both cholinergic and GABAergic neurons. Whole-cell patch clamp recordings from MS/DBB neurons in acute slices reveal that galanin decreases tetrodotoxin-sensitive spontaneous GABA release and dampens muscarinic receptor-mediated increases in GABA release in the MS/DBB. These effects are not blocked by pre-exposure to β-amyloid peptide (Aβ 1-42 ). Optogenetic activation of cholinergic neurons in the MS/DBB increases GABA release back onto cholinergic neurons, forming a functional circuit within the MS/DBB. Galanin disrupts this cholinergic-GABAergic circuit by blocking the cholinergic-induced increase in GABA release. These data suggest that galanin works in the BF to reduce inhibitory input onto cholinergic neurons and to prevent cholinergic-induced increase in inhibitory tone. This disinhibition of cholinergic neurons could serve as a compensatory mechanism to counteract the loss of cholinergic signaling that occurs during the progression of AD.

Objective: Absence seizures in childhood absence epilepsy are initiated in the thalamocortical (TC) system. We investigated if these seizures result from altered development of the TC system before the appearance of seizures in mice containing a point mutation in γ-aminobutyric acid A (GABA A ) receptor γ2 subunits linked to childhood absence epilepsy (R43Q). Findings from conditional mutant mice indicate that expression of normal γ2 subunits during preseizure ages protect from later seizures. This indicates that altered development in the presence of the R43Q mutation is a key contributor to the R43Q phenotype. We sought to identify the cellular processes affected by the R43Q mutation during these preseizure ages., Methods: We examined landmarks of synaptic development at the end of the critical period for somatosensory TC plasticity using electrophysiologic recordings in TC brain slices from wild-type mice and R43Q mice., Results: We found that the level of TC connectivity to layer 4 (L4) principal cells and the properties of TC synapses were unaltered in R43Q mice. Furthermore, we show that, although TC feedforward inhibition and the total level of GABAergic inhibition were normal, there was a reduction in the local connectivity to cortical interneurons. This reduction leads to altered inhibition during bursts of cortical activity., Significance: This altered inhibition demonstrates that alterations in cortical circuitry precede the onset of seizures by more than a week., (© 2017 The Authors. Epilepsia published by Wiley Periodicals Inc. on behalf of International League Against Epilepsy.)

The gene transfer-mediated expression of inhibitory ion channels in nociceptive neurons holds promise for treating intractable pain. Chemogenetics, which involves expressing constructs activated by biologically inert molecules, is of particular interest as it permits tunable neuromodulation. However, current chloride-permeable chemogenetic constructs are problematic as they mediate a tonic chloride influx which over time would deplete the chloride electrochemical gradient and reduce inhibitory efficacy. Inflammatory pain sensitization can be caused by prostaglandin E2-mediated inhibition of glycinergic inhibitory postsynaptic currents in spinal nociceptive neurons. We developed a highly conducting (100 pS) inhibitory chemogenetic construct based on a human glycine receptor (α1 Y279F,A288G ) with high ivermectin sensitivity. When virally infected into spinal neurons, 10 nM ivermectin increased the magnitude and frequency of glycinergic postsynaptic currents without activating a tonic chloride flux. The construct should thus produce analgesia. Its human origin and the well-established biocompatibility of its ligand suggest it may be suited to human use.

GSG1L is an AMPA receptor (AMPAR) auxiliary subunit that regulates AMPAR trafficking and function in hippocampal CA1 pyramidal neurons. However, its physiological roles in other types of neurons remain to be characterized. Here, we investigated the role of GSG1L in hippocampal dentate granule cells and found that GSG1L is important for the regulation of synaptic strength but is not critical for the modulation of AMPAR deactivation and desensitization kinetics. These data demonstrate a neuronal type-specific role of GSG1L and suggest that physiological function of AMPAR auxiliary subunits may vary in different types of neurons., New & Noteworthy: GSG1L is a newly identified AMPA receptor (AMPAR) auxiliary subunit and plays a unique role in the regulation of AMPAR trafficking and function in hippocampal CA1 pyramidal neurons. However, its role in the regulation of AMPARs in hippocampal dentate granule cells remains to be characterized. The current work reveals that GSG1L regulates strength of AMPAR-mediated synaptic transmission but not the receptor kinetic properties in hippocampal dentate granule neurons.

Unlabelled: Synchronous optogenetic activation of striatal cholinergic interneurons ex vivo produces a disynaptic inhibition of spiny projection neurons composed of biophysically distinct GABAAfast and GABAAslow components. This has been shown to be due, at least in part, to activation of nicotinic receptors on GABAergic NPY-neurogliaform interneurons that monosynaptically inhibit striatal spiny projection neurons. Recently, it has been proposed that a significant proportion of this inhibition is actually mediated by activation of presynaptic nicotinic receptors on nigrostriatal terminals that evoke GABA release from the terminals of the dopaminergic nigrostriatal pathway. To disambiguate these the two mechanisms, we crossed mice in which channelrhodopsin is endogenously expressed in cholinergic neurons with Htr3a-Cre mice, in which Cre is selectively targeted to several populations of striatal GABAergic interneurons, including the striatal NPY-neurogliaform interneuron. Htr3a-Cre mice were then virally transduced to express halorhodopsin to allow activation of channelrhodopsin and halorhodopsin, individually or simultaneously. Thus we were able to optogenetically disconnect the interneuron-spiny projection neuron (SPN) cell circuit on a trial-by-trial basis. As expected, optogenetic activation of cholinergic interneurons produced inhibitory currents in SPNs. During simultaneous inhibition of GABAergic interneurons with halorhodopsin, we observed a large, sometimes near complete reduction in both fast and slow components of the cholinergic-evoked inhibition, and a delay in IPSC latency. This demonstrates that the majority of cholinergic-evoked striatal GABAergic inhibition is derived from GABAergic interneurons. These results also reinforce the notion that a semiautonomous circuit of striatal GABAergic interneurons is responsible for transmitting behaviorally relevant cholinergic signals to spiny projection neurons., Significance Statement: The circuitry between neurons of the striatum has been recently described to be far more complex than originally imagined. One example of this phenomenon is that striatal cholinergic interneurons have been shown to provide intrinsic nicotinic excitation of local GABAergic interneurons, which then inhibit the projection neurons of the striatum. As deficits of cholinergic interneurons are reported in patients with Tourette syndrome, the normal functions of these interneurons are of great interest. Whether this novel route of nicotinic input constitutes a major output of cholinergic interneurons remains unknown. The study addressed this question using excitatory and inhibitory optogenetic technology, so that cholinergic interneurons could be selectively activated and GABAergic interneurons selectively inhibited to determine the causal relationship in this circuit., (Copyright © 2016 the authors 0270-6474/16/369505-07$15.00/0.)

Unlabelled: Elucidating patterns of functional synaptic connectivity and deciphering mechanisms of how plasticity influences such connectivity is essential toward understanding brain function. In the mouse olfactory bulb (OB), principal neurons (mitral/tufted cells) make reciprocal connections with local inhibitory interneurons, including granule cells (GCs) and external plexiform layer (EPL) interneurons. Our current understanding of the functional connectivity between these cell types, as well as their experience-dependent plasticity, remains incomplete. By combining acousto-optic deflector-based scanning microscopy and genetically targeted expression of Channelrhodopsin-2, we mapped connections in a cell-type-specific manner between mitral cells (MCs) and GCs or between MCs and EPL interneurons. We found that EPL interneurons form broad patterns of connectivity with MCs, whereas GCs make more restricted connections with MCs. Using an olfactory associative learning paradigm, we found that these circuits displayed differential features of experience-dependent plasticity. Whereas reciprocal connectivity between MCs and EPL interneurons was nonplastic, the connections between GCs and MCs were dynamic and adaptive. Interestingly, experience-dependent plasticity of GCs occurred only in certain stages of neuronal maturation. We show that different interneuron subtypes form distinct connectivity maps and modes of experience-dependent plasticity in the OB, which may reflect their unique functional roles in information processing., Significance Statement: Deducing how specific interneuron subtypes contribute to normal circuit function requires understanding the dynamics of their connections. In the olfactory bulb (OB), diverse interneuron subtypes vastly outnumber principal excitatory cells. By combining acousto-optic deflector-based scanning microscopy, electrophysiology, and genetically targeted expression of Channelrhodopsin-2, we mapped the functional connectivity between mitral cells (MCs) and OB interneurons in a cell-type-specific manner. We found that, whereas external plexiform layer (EPL) interneurons show broadly distributed patterns of stable connectivity with MCs, adult-born granule cells show dynamic and plastic patterns of synaptic connectivity with task learning. Together, these findings reveal the diverse roles for interneuons within sensory circuits toward information learning and processing., (Copyright © 2016 the authors 0270-6474/16/368856-16$15.00/0.)

Unlabelled: Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease., Significance Statement: Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease., (Copyright © 2016 the authors 0270-6474/16/367442-12$15.00/0.)

Anatomical, molecular, and physiological interactions between astrocytes and neuronal synapses regulate information processing in the brain. The fruit fly Drosophila melanogaster has become a valuable experimental system for genetic manipulation of the nervous system and has enormous potential for elucidating mechanisms that mediate neuron-glia interactions. Here, we show the first electrophysiological recordings from Drosophila astrocytes and characterize their spatial and physiological relationship with particular synapses. Astrocyte intrinsic properties were found to be strongly analogous to those of vertebrate astrocytes, including a passive current-voltage relationship, low membrane resistance, high capacitance, and dye-coupling to local astrocytes. Responses to optogenetic stimulation of glutamatergic premotor neurons were correlated directly with anatomy using serial electron microscopy reconstructions of homologous identified neurons and surrounding astrocytic processes. Robust bidirectional communication was present: neuronal activation triggered astrocytic glutamate transport via excitatory amino acid transporter 1 (Eaat1), and blocking Eaat1 extended glutamatergic interneuron-evoked inhibitory postsynaptic currents in motor neurons. The neuronal synapses were always located within 1 μm of an astrocytic process, but none were ensheathed by those processes. Thus, fly astrocytes can modulate fast synaptic transmission via neurotransmitter transport within these anatomical parameters. J. Comp. Neurol. 524:1979-1998, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Synaptic transmission has been shown to be modulated by glial functions, but the modes of specific glial action may vary in different neural circuits. We have tested the hypothesis, if Bergmann GLIA (BG) are involved in shaping neuronal communication in the mouse cerebellar cortex, using acutely isolated cerebellar slices of wild-type (WT) and of glia-specific receptor knockout mice. Activation of P2Y1 receptors by ADP (100 µM) or glutamatergic receptors by AMPA (0.3 µM) resulted in a robust, reversible and repeatable rise of evoked inhibitory input in Purkinje cells by 80% and 150%, respectively. The ADP-induced response was suppressed by prior application of AMPA, and the AMPA-induced response was suppressed by prior application of ADP. Genetic deletion or pharmacological blockade of either receptor restored the response to the other receptor agonist. Both ADP and AMPA responses were sensitive to Rose Bengal, which blocks vesicular glutamate uptake, and to the NMDA receptor antagonist D-AP5. Our results provide strong evidence that activation of both ADP and AMPA receptors, located on BGs, results in the release of glutamate, which in turn activates inhibitory interneurons via NMDA-type glutamate receptors. This infers that BG cells, by means of metabotropic signaling via their AMPA and P2Y1 receptors, which mutually suppress each other, would interdependently contribute to the fine-tuning of Purkinje cell activity in the cerebellar cortex. GLIA 2016. GLIA 2016;64:1265-1280., (© 2016 Wiley Periodicals, Inc.)

Unlabelled: Although synapsins regulate GABA release, it is unclear which synapsin isoforms are involved. We identified the synapsin isoforms that regulate GABA release via rescue experiments in cultured hippocampal neurons from synapsin I, II, and III triple knock-out (TKO) mice. In situ hybridization indicated that five different synapsin isoforms are expressed in hippocampal interneurons. Evoked IPSC amplitude was reduced in TKO neurons compared with triple wild-type neurons and was rescued by introducing any of the five synapsin isoforms. This contrasts with hippocampal glutamatergic terminals, where only synapsin IIa rescues the TKO phenotype. Deconvolution analysis indicated that the duration of GABA release was prolonged in TKO neurons and this defect in release kinetics was rescued by each synapsin isoform, aside from synapsin IIIa. Because release kinetics remained slow, whereas peak release rate was rescued, there was a 2-fold increase in GABA release in TKO neurons expressing synapsin IIIa. TKO neurons expressing individual synapsin isoforms showed normal depression kinetics aside from more rapid depression in neurons expressing synapsin IIIa. Measurements of the cumulative amount of GABA released during repetitive stimulation revealed that the rate of mobilization of vesicles from the reserve pool to the readily releasable pool and the size of the readily releasable pool of GABAergic vesicles were unaffected by synapsins. Instead, synapsins regulate release of GABA from the readily releasable pool, with all isoforms aside from synapsin IIIa controlling release synchrony. These results indicate that synapsins play fundamentally distinct roles at different types of presynaptic terminals., Significance Statement: Synapsins are a family of proteins that regulate synaptic vesicle (SV) trafficking within nerve terminals. Here, we demonstrate that release of the inhibitory neurotransmitter GABA is supported by many different synapsin types. This contrasts with the release of other neurotransmitters, which typically is supported by only one type of synapsin. We also found that synapsins serve to synchronize the release of GABA in response to presynaptic action potentials, which is different from the synapsin-dependent trafficking of SVs in other nerve terminals. Our results establish that different synapsins play fundamentally different roles at nerve terminals releasing different types of neurotransmitters. This is an important clue to understanding how neurons release their neurotransmitters, a process essential for normal brain function., (Copyright © 2016 the authors 0270-6474/16/366742-16$15.00/0.)

Unlabelled: Neuropathic pain is a common neurobiological disease involving multifaceted maladaptations ranging from gene modulation to synaptic dysfunction, but the interactions between synaptic dysfunction and the genes that are involved in persistent pain remain elusive. In the present study, we found that neuropathic pain induced by the chemotherapeutic drug paclitaxel or L5 ventral root transection significantly impaired the function of GABAergic synapses of spinal dorsal horn neurons via the reduction of the GAD67 expression. We also found that mir-500 expression was significantly increased and involved in the modulation of GAD67 expression via targeting the specific site of Gad1 gene in the dorsal horn. In addition, knock-out of mir-500 or using mir-500 antagomir rescued the GABAergic synapses in the spinal dorsal horn neurons and attenuated the sensitized pain behavior in the rats with neuropathic pain. To our knowledge, this is the first study to investigate the function significance and the underlying molecular mechanisms of mir-500 in the process of neuropathic pain, which sheds light on the development of novel therapeutic options for neuropathic pain., Significance Statement: Neuropathic pain is a common neurobiological disease involving multifaceted maladaptations ranging from gene modulation to synaptic dysfunction, but the underlying molecular mechanisms remain elusive. The present study illustrates for the first time a mir-500-mediated mechanism underlying spinal GABAergic dysfunction and sensitized pain behavior in neuropathic pain induced by the chemotherapeutic drug paclitaxel or L5 ventral root transection, which sheds light on the development of novel therapeutic options for neuropathic pain., (Copyright © 2016 the authors 0270-6474/16/366321-11$15.00/0.)

The dorsolateral striatum mediates habit formation, which is expedited by exposure to alcohol. Across species, alcohol exposure disinhibits the DLS by dampening GABAergic transmission onto this structure's principal medium spiny projection neurons (MSNs), providing a potential mechanistic basis for habitual alcohol drinking. However, the molecular and circuit components underlying this disinhibition remain unknown. To examine this, we used a combination of whole-cell patch-clamp recordings and optogenetics to demonstrate that ethanol potently depresses both MSN- and fast-spiking interneuron (FSI)-MSN GABAergic synaptic transmission in the DLS. Concentrating on the powerfully inhibitory FSI-MSN synapse, we further show that acute exposure of ethanol (50 mM) to striatal slices activates delta opioid receptors that reside on FSI axon terminals and negatively couple to adenylyl cyclase to induce a long-term depression of GABA release onto both direct and indirect pathway MSNs. These findings elucidate a mechanism through which ethanol may globally disinhibit the DLS.

Unlabelled: To investigate excitatory and inhibitory GABA actions in cortical neuronal networks, we present a novel optogenetic approach using a mouse knock-in line with conditional expression of channelrhodopsin-2 (ChR2) in GABAergic interneurons. During whole-cell recordings from hippocampal and neocortical slices from postnatal day (P) 2-P15 mice, photostimulation caused depolarization and excitation of interneurons and evoked barrages of postsynaptic GABAergic currents. Excitatory/inhibitory GABA actions on pyramidal cells were assessed by monitoring the alteration in the frequency of EPSCs during photostimulation of interneurons. We found that in slices from P2-P8 mice, photostimulation evoked an increase in EPSC frequency, whereas in P9-P15 mice the response switched to a reduction in EPSC frequency, indicating a developmental excitatory-to-inhibitory switch in GABA actions on glutamatergic neurons. Using a similar approach in urethane-anesthetized animals in vivo, we found that photostimulation of interneurons reduces EPSC frequency at ages P3-P9. Thus, expression of ChR2 in GABAergic interneurons of mice enables selective photostimulation of interneurons during the early postnatal period, and these mice display a developmental excitatory-to-inhibitory switch in GABA action in cortical slices in vitro, but so far show mainly inhibitory GABA actions on spontaneous EPSCs in the immature hippocampus and neocortex in vivo, Significance Statement: We report a novel optogenetic approach for investigating excitatory and inhibitory GABA actions in mice with conditional expression of channelrhodopsin-2 in GABAergic interneurons. This approach shows a developmental excitatory-to-inhibitory switch in the actions of GABA on glutamatergic neurons in neocortical and hippocampal slices from neonatal mouse pups in vitro, but also reveals inhibitory GABA actions in the neonatal mouse neocortex and hippocampus in vivo., (Copyright © 2016 the authors 0270-6474/16/365961-13$15.00/0.)

Autism spectrum disorder (ASD) is a neurodevelopmental disorder caused by genetic variants, susceptibility alleles, and environmental perturbations. The autism associated geneMETtyrosine kinase has been implicated in many behavioral domains and endophenotypes of autism, including abnormal neural signaling in human sensory cortex. We investigated somatosensory thalamocortical synaptic communication in mice deficient in Met activity in cortical excitatory neurons to gain insights into aberrant somatosensation characteristic of ASD. The ratio of excitation to inhibition is dramatically increased due to decreased postsynaptic GABAAreceptor-mediated inhibition in the trigeminal thalamocortical pathway of mice lacking active Met in the cerebral cortex. Furthermore, in contrast to wild-type mice, insulin failed to increase GABAAreceptor-mediated response in the barrel cortex of mice with compromised Met signaling. Thus, lacking insulin effects may be a risk factor in ASD pathogenesis., Significance Statement: A proposed common cause of neurodevelopmental disorders is an imbalance in excitatory neural transmission, provided by the glutamatergic neurons, and the inhibitory signals from the GABAergic interneurons. Many genes associated with autism spectrum disorders impair synaptic transmission in the expected cell type. Previously, inactivation of the autism-associated Met tyrosine kinase receptor in GABAergic interneurons led to decreased inhibition. In thus report, decreased Met signaling in glutamatergic neurons had no effect on excitation, but decimated inhibition. Further experiments indicate that loss of Met activity downregulates GABAAreceptors on glutamatergic neurons in an insulin independent manner. These data provide a new mechanism for the loss of inhibition and subsequent abnormal excitation/inhibition balance and potential molecular candidates for treatment or prevention., (Copyright © 2016 the authors 0270-6474/16/363691-07$15.00/0.)

Rac GTPases regulate the development of cortical/hippocampal GABAergic interneurons by affecting the early development and migration of GABAergic precursors. We have addressed the function of Rac1 and Rac3 proteins during the late maturation of hippocampal interneurons. We observed specific phenotypic differences between conditional Rac1 and full Rac3 knockout mice. Rac1 deletion caused greater generalized hyperactivity and cognitive impairment compared with Rac3 deletion. This phenotype matched with a more evident functional impairment of the inhibitory circuits in Rac1 mutants, showing higher excitability and reduced spontaneous inhibitory currents in the CA hippocampal pyramidal neurons. Morphological analysis confirmed a differential modification of the inhibitory circuits: deletion of either Rac caused a similar reduction of parvalbumin-positive inhibitory terminals in the pyramidal layer. Intriguingly, cannabinoid receptor-1-positive terminals were strongly increased only in the CA1 of Rac1-depleted mice. This increase may underlie the stronger electrophysiological defects in this mutant. Accordingly, incubation with an antagonist for cannabinoid receptors partially rescued the reduction of spontaneous inhibitory currents in the pyramidal cells of Rac1 mutants. Our results show that Rac1 and Rac3 have independent roles in the formation of GABAergic circuits, as highlighted by the differential effects of their deletion on the late maturation of specific populations of interneurons., (© The Author 2015. Published by Oxford University Press.)

Synaptic dysfunction is a well-documented manifestation in animal models of Alzheimer's disease pathology. In this context, numerous studies have documented reduction in the functionality of synapses in various models. In addition, recent research has shed more light on increased excitability and its link to seizures and seizure-like activities in AD patients as well as in mouse models. These reports of hyperexcitability contradict the observed reduction in synaptic function and have been suggested to be as a result of the interplay between inhibitory and excitatory neuronal mechanism. The present study therefore investigates functional deficiency in the inhibitory system as complementary to the identified alterations in the glutamate excitatory pathway in AD. Since synaptic function deficit in AD is typically linked to progression/pathology of the disease, it is important to determine whether the deficits in the GABAergic system are functional and can be directly linked to the pattern of the disruption documented in the glutamate system. To build on previous research in this field, experiments were designed to determine if previously documented synaptic dysfunction in AD models is concomitantly observed with excitation/inhibition imbalance as suggested by observation of seizure and seizure-like pathology in such models. We report changes in synaptic function in aged APPPS1 mice not observable in the younger cohort. These changes in synaptic function are furthermore accompanied by alteration in the GABAergic neurotransmission. Thus, age-dependent alteration in the inhibitory/excitatory balance might underpin the symptomatic changes observed with the progression of Alzheimer's disease pathology including sleep disturbance and epileptic events.

An increasing number of genetic variants have been implicated in autism spectrum disorders (ASDs), and the functional study of such variants will be critical for the elucidation of autism pathophysiology. Here, we report a de novo balanced translocation disruption of TRPC6, a cation channel, in a non-syndromic autistic individual. Using multiple models, such as dental pulp cells, induced pluripotent stem cell (iPSC)-derived neuronal cells and mouse models, we demonstrate that TRPC6 reduction or haploinsufficiency leads to altered neuronal development, morphology and function. The observed neuronal phenotypes could then be rescued by TRPC6 complementation and by treatment with insulin-like growth factor-1 or hyperforin, a TRPC6-specific agonist, suggesting that ASD individuals with alterations in this pathway may benefit from these drugs. We also demonstrate that methyl CpG binding protein-2 (MeCP2) levels affect TRPC6 expression. Mutations in MeCP2 cause Rett syndrome, revealing common pathways among ASDs. Genetic sequencing of TRPC6 in 1041 ASD individuals and 2872 controls revealed significantly more nonsynonymous mutations in the ASD population, and identified loss-of-function mutations with incomplete penetrance in two patients. Taken together, these findings suggest that TRPC6 is a novel predisposing gene for ASD that may act in a multiple-hit model. This is the first study to use iPSC-derived human neurons to model non-syndromic ASD and illustrate the potential of modeling genetically complex sporadic diseases using such cells.

Interference between similar or overlapping memories formed at different times poses an important challenge on the hippocampal declarative memory system. Difficulties in managing interference are at the core of disabling cognitive deficits in neuropsychiatric disorders. Computational models have suggested that, in the normal brain, the sparse activation of the dentate gyrus granule cells maintained by tonic inhibitory control enables pattern separation, an orthogonalization process that allows distinct representations of memories despite interference. To test this mechanistic hypothesis, we generated mice with significantly reduced expression of the α5-containing GABAA (α5-GABAARs) receptors selectively in the granule cells of the dentate gyrus (α5DGKO mice). α5DGKO mice had reduced tonic inhibition of the granule cells without any change in fast phasic inhibition and showed increased activation in the dentate gyrus when presented with novel stimuli. α5DGKO mice showed impairments in cognitive tasks characterized by high interference, without any deficiencies in low-interference tasks, suggesting specific impairment of pattern separation. Reduction of fast phasic inhibition in the dentate gyrus through granule cell-selective knock-out of α2-GABAARs or the knock-out of the α5-GABAARs in the downstream CA3 area did not detract from pattern separation abilities, which confirms the anatomical and molecular specificity of the findings. In addition to lending empirical support to computational hypotheses, our findings have implications for the treatment of interference-related cognitive symptoms in neuropsychiatric disorders, particularly considering the availability of pharmacological agents selectively targeting α5-GABAARs., Significance Statement: Interference between similar memories poses a significant limitation on the hippocampal declarative memory system, and impaired interference management is a cognitive symptom in many disorders. Thus, understanding mechanisms of successful interference management or processes that can lead to interference-related memory problems has high theoretical and translational importance. This study provides empirical evidence that tonic inhibition in the dentate gyrus (DG), which maintains sparseness of neuronal activation in the DG, is essential for management of interference. The specificity of findings to tonic, but not faster, more transient types of neuronal inhibition and to the DG, but not the neighboring brain areas, is presented through control experiments. Thus, the findings link interference management to a specific mechanism, proposed previously by computational models., (Copyright © 2015 the authors 0270-6474/15/3513699-15$15.00/0.)

Direction selectivity of direction-selective ganglion cells (DSGCs) in the retina results from patterned excitatory and inhibitory inputs onto DSGCs during motion stimuli. The inhibitory inputs onto DSGCs are directionally tuned to the antipreferred (null) direction and therefore potently suppress spiking during motion in the null direction. However, whether direction-selective inhibition is indispensable for direction selectivity is unclear. Here, we selectively eliminated the directional tuning of inhibitory inputs onto DSGCs by disrupting GABA release from the presynaptic interneuron starburst amacrine cell in the mouse retina. We found that, even without directionally tuned inhibition, direction selectivity can still be implemented in a subset of On-Off DSGCs by direction-selective excitation and a temporal offset between excitation and isotropic inhibition. Our results therefore demonstrate the concerted action of multiple synaptic mechanisms for robust direction selectivity in the retina. Significance statement: The direction-selective circuit in the retina has been a classic model to study neural computations by the brain. An important but unresolved question is how direction selectivity is implemented by directionally tuned excitatory and inhibitory mechanisms. Here we specifically removed the direction tuning of inhibition from the circuit. We found that direction tuning of inhibition is important but not indispensable for direction selectivity of DSGCs' spiking activity, and that the residual direction selectivity is implemented by direction-selective excitation and temporal offset between excitation and inhibition. Our results highlight the concerted actions of synaptic excitation and inhibition required for robust direction selectivity in the retina and provide critical insights into how patterned excitation and inhibition collectively implement sensory processing., (Copyright © 2015 the authors 0270-6474/15/3513219-14$15.00/0.)

In many synapses of the CNS, mobile zinc is packaged into glutamatergic vesicles and co-released with glutamate during neurotransmission. Following synaptic release, the mobilized zinc modulates ligand- and voltage-gated channels and receptors, functioning as an inhibitory neuromodulator. However, the origin and role of tonic, as opposed to phasically released, zinc are less well understood. We investigated tonic zinc in the dorsal cochlear nucleus (DCN), a zinc-rich, auditory brainstem nucleus. Our results show that application of a high-affinity, extracellular zinc chelator (ZX1) enhances spontaneous firing in DCN principal neurons (fusiform cells), consistent with inhibition of this neuronal property by tonic zinc. The enhancing effect was prevented by prior application of strychnine, a glycine receptor antagonist, suggesting that ZX1 interferes with zinc-mediated modulation of spontaneous glycinergic inhibition. In particular, ZX1 decreased the amplitude and the frequency of glycinergic miniature inhibitory postsynaptic currents in fusiform cells, from which we conclude that tonic zinc enhances glycinergic inhibitory neurotransmission. The observed zinc-mediated inhibition in spontaneous firing is present in mice lacking the vesicular zinc transporter (ZnT3), indicating that non-vesicular zinc inhibits spontaneous firing. Noise-induced increase in the spontaneous firing of fusiform cells is crucial for the induction of tinnitus. In this context, tonic zinc provides a powerful break of spontaneous firing that may protect against pathological run-up of spontaneous activity in the DCN., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cannabinoids are known to regulate inhibitory synaptic transmission via activation of presynaptic G protein-coupled cannabinoid CB1 receptors (CB1Rs). Additionally, recent studies suggest that cannabinoids can also directly interact with recombinant GABAA receptors (GABAARs), potentiating currents activated by micromolar concentrations of γ-aminobutyric acid (GABA). However, the impact of this direct interaction on GABAergic inhibition in central nervous system is unknown. Here we report that currents mediated by recombinant GABAARs activated by high (synaptic) concentrations of GABA as well as GABAergic inhibitory postsynaptic currents (IPSCs) at neocortical fast spiking (FS) interneuron to pyramidal neuron synapses are suppressed by exogenous and endogenous cannabinoids in a CB1R-independent manner. This IPSC suppression may account for disruption of inhibitory control of pyramidal neurons by FS interneurons. At FS interneuron to pyramidal neuron synapses, endocannabinoids induce synaptic low-pass filtering of GABAAR-mediated currents evoked by high-frequency stimulation. The CB1R-independent suppression of inhibition is synapse specific. It does not occur in CB1R containing hippocampal cholecystokinin-positive interneuron to pyramidal neuron synapses. Furthermore, in contrast to synaptic receptors, the activity of extrasynaptic GABAARs in neocortical pyramidal neurons is enhanced by cannabinoids in a CB1R-independent manner. Thus, cannabinoids directly interact differentially with synaptic and extrasynaptic GABAARs, providing a potent novel context-dependent mechanism for regulation of inhibition., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Persistent CB1 cannabinoid receptor activity limits neurotransmitter release at various synapses throughout the brain. However, it is not fully understood how constitutively active CB1 receptors, tonic endocannabinoid signaling, and its regulation by multiple serine hydrolases contribute to the synapse-specific calibration of neurotransmitter release probability. To address this question at perisomatic and dendritic GABAergic synapses in the mouse hippocampus, we used a combination of paired whole-cell patch-clamp recording, liquid chromatography/tandem mass spectrometry, stochastic optical reconstruction microscopy super-resolution imaging, and immunogold electron microscopy. Unexpectedly, application of the CB1 antagonist and inverse agonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide], but not the neutral antagonist NESS0327 [8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-5,6-dihydro-4H-benzo[2,3]cyclohepta[2,4-b]pyrazole-3-carboxamine], significantly increased synaptic transmission between CB1-positive perisomatic interneurons and CA1 pyramidal neurons. JZL184 (4-nitrophenyl 4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate), a selective inhibitor of monoacylglycerol lipase (MGL), the presynaptic degrading enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), elicited a robust increase in 2-AG levels and concomitantly decreased GABAergic transmission. In contrast, inhibition of fatty acid amide hydrolase (FAAH) by PF3845 (N-pyridin-3-yl-4-[[3-[5-(trifluoromethyl)pyridin-2-yl]oxyphenyl]methyl]piperidine-1-carboxamide) elevated endocannabinoid/endovanilloid anandamide levels but did not change GABAergic synaptic activity. However, FAAH inhibitors attenuated tonic 2-AG increase and also decreased its synaptic effects. This antagonistic interaction required the activation of the transient receptor potential vanilloid receptor TRPV1, which was concentrated on postsynaptic intracellular membrane cisternae at perisomatic GABAergic symmetrical synapses. Interestingly, neither AM251, JZL184, nor PF3845 affected CB1-positive dendritic interneuron synapses. Together, these findings are consistent with the possibility that constitutively active CB1 receptors substantially influence perisomatic GABA release probability and indicate that the synaptic effects of tonic 2-AG release are tightly controlled by presynaptic MGL activity and also by postsynaptic endovanilloid signaling and FAAH activity., Significance Statement: Tonic cannabinoid signaling plays a critical role in the regulation of synaptic transmission. However, the mechanistic details of how persistent CB1 cannabinoid receptor activity inhibits neurotransmitter release have remained elusive. Therefore, electrophysiological recordings, lipid measurements, and super-resolution imaging were combined to elucidate those signaling molecules and mechanisms that underlie tonic cannabinoid signaling. The findings indicate that constitutive CB1 activity has pivotal function in the tonic control of hippocampal GABA release. Moreover, the endocannabinoid 2-arachidonoylglycerol (2-AG) is continuously generated postsynaptically, but its synaptic effect is regulated strictly by presynaptic monoacylglycerol lipase activity. Finally, anandamide signaling antagonizes tonic 2-AG signaling via activation of postsynaptic transient receptor potential vanilloid TRPV1 receptors. This unexpected mechanistic diversity may be necessary to fine-tune GABA release probability under various physiological and pathophysiological conditions., (Copyright © 2015 Lee et al.)

Activity-dependent strengthening of central synapses is a key factor driving neuronal circuit behavior in the vertebrate CNS. At fast inhibitory synapses, strengthening is thought to occur by increasing the number of GABAA receptors (GABARs) of the same subunit composition to preexisting synapses. Here, we show that strengthening of mouse cerebellar granule cell GABAergic synapses occurs by a different mechanism. Specifically, we show that the neuropeptide hormone, insulin, strengthens inhibitory synapses by recruiting α6-containing GABARs rather than accumulating more α1-containing receptors that are resident to the synapse. Because α6-receptors are targeted to functionally distinct postsynaptic sites from α1-receptors, we conclude that only a subset of all inhibitory synapses are strengthened. Together with our recent findings on stellate cells, we propose a general mechanism by which mature inhibitory synapses are strengthened. In this scenario, α1-GABARs resident to inhibitory synapses form the hardwiring of neuronal circuits with receptors of a different composition fulfilling a fundamental, but unappreciated, role in synapse strengthening., (Copyright © 2015 the authors 0270-6474/15/359676-13$15.00/0.)

Neurons coexpressing neuropeptide Y, agouti-related peptide, and GABA (NAG) play an important role in ingestive behavior and are located in the arcuate nucleus of the hypothalamus. NAG neurons receive both GABAergic and glutamatergic synaptic inputs, however, the developmental time course of synaptic input organization of NAG neurons in mice is unknown. In this study, we show that these neurons have low numbers of GABAergic synapses and that GABA is inhibitory to NAG neurons during early postnatal period. In contrast, glutamatergic inputs onto NAG neurons are relatively abundant by P13 and are comparatively similar to the levels observed in the adult. As mice reach adulthood (9-10 weeks), GABAergic tone onto NAG neurons increases. At this age, NAG neurons received similar numbers of inhibitory and EPSCs. To further differentiate age-associated changes in synaptic distribution, 17- to 18-week-old lean and diet-induced obesity (DIO) mice were studied. Surprisingly, NAG neurons from lean adult mice exhibit a reduction in the GABAergic synapses compared with younger adults. Conversely, DIO mice display reductions in the number of GABAergic and glutamatergic inputs onto NAG neurons. Based on these experiments, we propose that synaptic distribution in NAG neurons is continuously restructuring throughout development to accommodate the animals' energy requirements., (Copyright © 2015 the authors 0270-6474/15/358558-12$15.00/0.)

Of the eight metabotropic glutamate (mGlu) receptor subtypes, only mGlu7 is expressed presynaptically at the Schaffer collateral (SC)-CA1 synapse in the hippocampus in adult animals. Coupled with the inhibitory effects of Group III mGlu receptor agonists on transmission at this synapse, mGlu7 is thought to be the predominant autoreceptor responsible for regulating glutamate release at SC terminals. However, the lack of mGlu7-selective pharmacological tools has hampered direct testing of this hypothesis. We used a novel, selective mGlu7-negative allosteric modulator (NAM), ADX71743, and a newly described Group III mGlu receptor agonist, LSP4-2022, to elucidate the role of mGlu7 in modulating transmission in hippocampal area CA1 in adult C57BL/6J male mice. Interestingly, although mGlu7 agonists inhibit SC-CA1 EPSPs, we found no evidence for activation of mGlu7 by stimulation of SC-CA1 afferents. However, LSP4-2022 also reduced evoked monosynaptic IPSCs in CA1 pyramidal cells and, in contrast to its effect on SC-CA1 EPSPs, ADX71743 reversed the ability of high-frequency stimulation of SC afferents to reduce IPSC amplitudes. Furthermore, blockade of mGlu7 prevented induction of LTP at the SC-CA1 synapse and activation of mGlu7 potentiated submaximal LTP. Together, these data suggest that mGlu7 serves as a heteroreceptor at inhibitory synapses in area CA1 and that the predominant effect of activation of mGlu7 by stimulation of glutamatergic afferents is disinhibition, rather than reduced excitatory transmission. Furthermore, this mGlu7-mediated disinhibition is required for induction of LTP at the SC-CA1 synapse, suggesting that mGlu7 could serve as a novel therapeutic target for treatment of cognitive disorders., (Copyright © 2015 the authors 0270-6474/15/357600-16$15.00/0.)

In this study, we analyzed the impact that spontaneous seizures might have on the plasma membrane expression, composition and function of GABAA receptors (GABAARs). For this, the tissue of chronically epileptic rats was collected within 3h of seizure occurrence (≤3h group) or at least 24h after seizure occurrence (≥24h group). A retrospective analysis of seizure frequency revealed that selecting animals on the bases of seizure proximity also grouped animals in terms of overall seizure burden with a higher seizure burden observed in the ≤3h group. A biochemical analysis showed that although animals with more frequent/recent seizures (≤3h group) had similar levels of GABAAR at the plasma membrane they showed deficits in inhibitory neurotransmission. By contrast, the tissue obtained from animals experiencing infrequent seizures (≥24h group) had increased plasma membrane levels of GABAAR and showed no deficit in inhibitory function. Together, our findings offer an initial insight into the molecular changes that might help to explain how alterations in GABAAR function can be associated with differential seizure burden. Our findings also suggest that increased plasma membrane levels of GABAAR might act as a compensatory mechanism to more effectively maintain inhibitory function, repress hyperexcitability and reduce seizure burden. This study is an initial step towards a fuller characterization of the molecular events that trigger alterations in GABAergic neurotransmission during chronic epilepsy., (Copyright © 2015. Published by Elsevier Inc.)

Glia plays an active role in neuronal functions and dysfunctions, some of which depend on the expression of astrocyte connexins, the gap junction channel and hemichannel proteins. Under neuroinflammation triggered by the endotoxin lipopolysacharide (LPS), microglia is primary stimulated and releases proinflammatory agents affecting astrocytes and neurons. Here, we investigate the effects of such microglial activation on astrocyte connexin-based channel functions and their consequences on synaptic activity in an ex vivo model. We found that LPS induces astroglial hemichannel opening in acute hippocampal slices while no change is observed in gap junctional communication. Based on pharmacological and genetic approaches we found that the LPS-induced hemichannel opening is mainly due to Cx43 hemichannel activity. This process primarily requires a microglial stimulation resulting in the release of at least two proinflammatory cytokines, IL-1β and TNF-α. Consequences of the hemichannel-mediated increase in membrane permeability are a calcium rise in astrocytes and an enhanced glutamate release associated to a reduction in excitatory synaptic activity of pyramidal neurons in response to Schaffer's collateral stimulation. As a whole our findings point out astroglial hemichannels as key determinants of the impairment of synaptic transmission during neuroinflammation., (© 2015 Wiley Periodicals, Inc.)

The IL-1 receptor antagonist (IL-1ra), encoded by the Il1rn gene, is an endogenous antagonist of the IL-1 receptor. Studies of Il1rn knockout (KO) and wild type (WT) mice identified differences in several ethanol-related behaviors, some of which may be mediated by GABAergic transmission in the central nucleus of the amygdala (CeA). In this study we examined phasic (both evoked and spontaneous) and tonic GABAergic transmission in the CeA of Il1rn KO and WT mice and the ethanol sensitivity of these GABAergic synapses. The mean amplitude of baseline evoked GABAA-inhibitory postsynaptic potentials (IPSPs), and the baseline frequency of spontaneous GABAA-inhibitory postsynaptic currents (sIPSCs), but not the frequency of miniature GABAA-IPSCs (mIPSCs), were significantly increased in KO compared to WT mice, indicating enhanced presynaptic action potential-dependent GABA release in the CeA of KO mice. In KO mice, we also found a cell-type specific switch in the ongoing tonic GABAA receptor conductance such that the tonic conductance in low threshold bursting (LTB) neurons is lost and a tonic conductance in late spiking (LS) neurons appears. Notably, the ethanol-induced facilitation of evoked and spontaneous GABA release was lost in most of the CeA neurons from KO compared to WT mice. Ethanol superfusion increased the sIPSC rise and decay times in both KO and WT mice, suggesting ethanol-induced postsynaptic effects. The pretreatment of CeA slices with exogenous IL-1ra (Kineret; 100ng/ml) returned sIPSC frequency in KO mice to the levels found in WT. Importantly, Kineret also restored ethanol-induced potentiation of the sIPSC frequency in the KO mice. These results show that IL-1ra regulates baseline GABAergic transmission in the CeA and is critical for the ethanol effects at these synapses., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Glycine receptor (GlyR) chloride channels mediate fast inhibitory neurotransmission in the spinal cord and brainstem. Four GlyR subunits (α1-3, β) have been identified in humans, and their differential anatomical distributions underlie a diversity of synaptic isoforms with unique physiological and pharmacological properties. To improve our understanding of these properties, we induced the formation of recombinant synapses between cultured spinal neurons and HEK293 cells expressing GlyR subunits of interest plus the synapse-promoting molecule, neuroligin-2A. In the heterosynapses thus formed, recombinant α1β and α3β GlyRs mediated fast decaying inhibitory postsynaptic currents (IPSCs) whereas α2β GlyRs mediated slow decaying IPSCs. These results are consistent with the fragmentary information available from native synapses and single channel kinetic studies. As β subunit incorporation is considered essential for localizing GlyRs at the synapse, we were surprised that α1-3 homomers supported robust IPSCs with β subunit incorporation accelerating IPSC rise and decay times in α2β and α3β heteromers only. Finally, heterosynapses incorporating α1(D80A)β and α1(A52S)β GlyRs exhibited accelerated IPSC decay rates closely resembling those recorded in native synapses from mutant mice homozygous for these mutations, providing an additional validation of our technique. Glycinergic heterosynapses should prove useful for evaluating the effects of drugs, hereditary disease mutations or other interventions on defined GlyR subunit combinations under realistic synaptic activation conditions.

Orexins are bioactive peptides, which have been shown to play a pivotal role in vigilance state transitions: the loss of orexin-producing neurons (orexin neurons) leads to narcolepsy with cataplexy in the human. However, the effect of the need for sleep (i.e., sleep pressure) on orexin neurons remains largely unknown. Here, we found that immunostaining intensities of the α1 subunit of the GABAA receptor and neuroligin 2, which is involved in inhibitory synapse specialization, on orexin neurons of mouse brain were significantly increased by 6-h sleep deprivation. In contrast, we noted that immunostaining intensities of the α2, γ2, and β2/3 subunits of the GABAA receptor and Huntingtin-associated protein 1, which is involved in GABAAR trafficking, were not changed by 6-h sleep deprivation. Using a slice patch recording, orexin neurons demonstrated increased sensitivity to a GABAA receptor agonist together with synaptic plasticity changes after sleep deprivation when compared with an ad lib sleep condition. In summary, the GABAergic input property of orexin neurons responds rapidly to sleep deprivation. This molecular response of orexin neurons may thus play a role in the changes that accompany the need for sleep following prolonged wakefulness, in particular the decreased probability of a transition to wakefulness once recovery sleep has begun., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Nitric oxide (NO) is a key retrograde messenger that regulates synaptic transmission in the cerebral cortex. However, little is known about NO-induced modulatory effects and their mechanisms relative to inhibitory synaptic transmission. The present study aimed to examine the effects of NO on unitary inhibitory postsynaptic currents (uIPSCs) and to postulate the synaptic location of NO action. We performed multiple whole-cell patch-clamp recordings from rat insular cortex and divided recorded cells into three subtypes: pyramidal cells (Pyr), fast-spiking interneurons (FS), and non-FS GABAergic interneurons. In the connections from FS to Pyr (FS→Pyr), the application of S-nitroso-N-acetyl-dl-penicillamine (SNAP, 100 μM), an NO donor, suppressed uIPSC amplitudes in 31% of the connections, whereas 39% of the connections showed uIPSC facilitation. The remaining FS→Pyr connections showed little effect of SNAP on uIPSCs. An analysis of paired-pulse ratio (PPR) implied the involvement of presynaptic mechanisms in SNAP-induced effects on uIPSCs. Similar effects of SNAP were observed in FS→FS/non-FS connections; 33%, 54%, and 13% of the connections were facilitated, suppressed, and unchanged, respectively. In contrast, non-FS→Pyr or FS/non-FS showed constant uIPSC suppression by SNAP. PPR analysis supports the hypothesis that these SNAP-induced effects are mediated by presynaptic mechanisms in FS→FS/non-FS and non-FS→Pyr/FS/non-FS connections. The NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazolineoxyl-1-oxyl-3-oxide (PTIO), or the inhibitor of guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), abolished the SNAP-induced uIPSC modulation. These results suggest that NO regulation of inhibitory synaptic transmission is dependent on presynaptic cell subtypes and that, at least in part, the effects are mediated by presynaptic mechanisms., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)