Frequency Of Miniature Inhibitory Postsynaptic Currents Research Articles

Delta opioid receptor agonists activate PI3K-mTORC1 signaling in parvalbumin-positive interneurons in mouse infralimbic prefrontal cortex to exert acute antidepressant-lie effects.

The delta opioid receptor (DOP) is a promising target for novel antidepressants due to its potential for rapid action with minimal adverse effects; however, the functional mechanism underlying acute antidepressant actions remains elusive. We report that subcutaneous injection of the selective DOP agonist KNT-127 reduced immobility in the forced swimming test, and that this antidepressant-like response was reversed by intracerebroventricular injection of the selective mechanistic (or mammalian) target of rapamycin (mTOR) inhibitor rapamycin or the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002. KNT-127 also alleviated social avoidance and reduced sucrose consumption (anhedonia) among chronic vicarious social defeat stress model mice, which were similarly reversed by PI3K and mTOR inhibitors. In addition, KNT-127 increased phosphorylation levels of the mTOR signaling-related proteins Akt and p70S6 kinase in medial prefrontal cortex as revealed by immunoblotting. In the forced swimming test, a microinfusion of KNT-127 and another DOP agonist SNC80 in the infralimbic prefrontal cortex (IL-PFC) attenuated the immobility, which were blocked by rapamycin and LY294002. Perfusion of KNT-127 onto IL-PFC slices increased miniature excitatory postsynaptic current frequency and reduced miniature inhibitory postsynaptic current frequency in pyramidal neurons as measured by whole-cell patch-clamping, and both responses were reversed by rapamycin. Imaging of brain slices from transgenic mice with DOP-promoter-driven green fluorescent protein revealed that most DOPs were expressed in parvalbumin-positive interneurons in the IL-PFC. These findings suggest that DOP agonists exert antidepressant-like actions by suppressing GABA release from parvalbumin-positive interneurons via the PI3K-Akt-mTORC1-p70S6 kinase pathway, thereby enhancing IL-PFC pyramidal neuron excitation.

PCDH19 interplay with GABA(A) receptors: a window to DEE9 pathogenetic mechanisms.

PCDH19 interplay with GABA(A) receptors: a window to DEE9 pathogenetic mechanisms.

Glutamate Delta-1 Receptor Regulates Inhibitory Neurotransmission in the Nucleus Accumbens Core and Anxiety-Like Behaviors.

Glutamate delta-1 receptor (GluD1) is a member of the ionotropic glutamate receptor family expressed at excitatory synapses and functions as a synaptogenic protein by interacting with presynaptic neurexin. We have previously shown that GluD1 plays a role in the maintenance of excitatory synapses in a region-specific manner. Loss of GluD1 leads to reduced excitatory neurotransmission in medium spiny neurons (MSNs) in the dorsal striatum, but not in the ventral striatum (both core and shell of the nucleus accumbens (NAc)). Here, we found that GluD1 loss leads to reduced inhibitory neurotransmission in MSNs of the NAc core as evidenced by a reduction in the miniature inhibitory postsynaptic current frequency and amplitude. Presynaptic effect of GluD1 loss was further supported by an increase in paired pulse ratio of evoked inhibitory responses indicating reduced release probability. Furthermore, analysis of GAD67 puncta indicated a reduction in the number of putative inhibitory terminals. The changes in mIPSC were independent of cannabinoid or dopamine signaling. A role of feed-forward inhibition was tested by selective ablation of GluD1 from PV neurons which produced modest reduction in mIPSCs. Behaviorally, local ablation of GluD1 from NAc led to hypolocomotion and affected anxiety- and depression-like behaviors. When GluD1 was ablated from the dorsal striatum, several behavioral phenotypes were altered in opposite manner compared to GluD1 ablation from NAc. Our findings demonstrate that GluD1 regulates inhibitory neurotransmission in the NAc by a combination of pre- and postsynaptic mechanisms which is critical for motor control and behaviors relevant to neuropsychiatric disorders.

Endocannabinoids regulate cerebellar GABAergic transmission in a synapse type‐dependent manner

Endocannabinoids (eCBs) act as ubiquitous modulators of synaptic transmission via the activation of cannabinoid receptors (CBRs). Cerebellar Purkinje cells (PCs) make strong inhibitory synaptic contacts not only with neurons in the deep cerebellar nuclei (DCN) but also with Lugaro cells and globular cells, whose cell bodies are located underneath the PC layer. However, little is known about the modulatory actions of eCBs on GABA release from PC axon terminals. Here, we examined the effects of eCBs on the GABAergic transmission at PC-globular cell synapses and PC-large DCN neuron synapses electrophysiologically using mouse cerebellar slices. We showed that the types 1 and 2 CBR agonist WIN55212 did not affect either spontaneous or miniature inhibitory postsynaptic currents (IPSCs) in globular cells under control conditions and in a state of enhanced synaptic activity. By contrast, another Gi/o protein-coupled receptor agonist, baclofen, significantly reduced the miniature IPSC frequency in globular cells. WIN55212 had no effects on IPSCs in large DCN neurons. A type 2 CBR agonist, HU308, also had no effects on IPSCs in either globular cells or large DCN neurons. Moreover, the PCs' target neurons did not elicit depolarization-induced suppression of inhibition. These results suggest the lack of a functional role of CBRs at PCs' axon terminals. This is in sharp contrast to the fact that PCs receive abundant excitatory and inhibitory inputs that are under eCB-mediated presynaptic inhibitory modulation. The actions of eCBs are selective to distinct synapses and possibly contribute to information processes and rigorous signal transmission in the cerebellum.

Differential sensitivity of human neurons carrying μ opioid receptor (MOR) N40D variants in response to ethanol.

The acute and chronic effects of alcohol on the brain and behavior are linked to alterations in inhibitory synaptic transmission. Alcohol's most consistent effect at the synaptic level is probably a facilitation of γ-aminobutyric acid (GABA) release, as seen from several rodent studies. The impact of alcohol on GABAergic neurotransmission in human neurons is unknown, due to a lack of a suitable experimental model. Human neurons can also be used to model effects of genetic variants linked with alcohol use disorders (AUDs). The A118G single nucleotide polymorphism (SNP rs1799971) of the OPRM1 gene encoding the N40D (D40 minor allele) mu-opioid receptor (MOR) variant has been linked with individuals who have an AUD. However, while N40D is clearly associated with other drugs of abuse, involvement with AUDs is controversial. In this study, we employed Ascl1-and Dlx2-induced inhibitory neuronal cells (AD-iNs) generated from human iPS cell lines carrying N40D variants, and investigated the impact of ethanol acutely and chronically on GABAergic synaptic transmission. We found that N40 AD-iNs display a stronger facilitation (versus D40) of spontaneous and miniature inhibitory postsynaptic current frequency in response to acute ethanol application. Quantitative immunocytochemistry of Synapsin 1+ synaptic puncta revealed a similar synapse number between N40 and D40 iNs, suggesting an ethanol modulation of presynaptic GABA release without affecting synapse density. Interestingly, D40 iNs exposed to chronic intermittent ethanol application caused a significant increase in mIPSC frequency, with only a modest enhancement observed in N40 iNs. These data suggest that the MOR genotype may confer differential sensitivity to synaptic output, which depends on ethanol exposure time and concentration for AD-iNs and may help explain alcohol dependence in individuals who carry the MOR D40 SNPs. Furthermore, this study supports the use of human neuronal cells carrying risk-associated genetic variants linked to disease, as invitro models to assay the synaptic actions of alcohol on human neuronal cells.

Increased GABAergic transmission in neuropeptide Y-expressing neurons in the dopamine-depleted murine striatum.

As the main input nucleus of the basal ganglia, the striatum plays a central role in planning, control, and execution of movement and motor skill learning. More than 90% of striatal neurons, so-called medium spiny neurons (MSN), are GABAergic projection neurons, innervating primarily the substantia nigra pars reticulata or the globus pallidus internus. The remaining neurons are GABAergic and cholinergic interneurons, synchronizing and controlling striatal output by reciprocal connections with MSN. Besides prominent local cholinergic influence, striatal function is globally regulated by dopamine (DA) from the nigrostriatal pathway. Little is known about whether DA depletion, as occurs in Parkinson's disease, affects the activity of striatal interneurons. Here we focused on neuropeptide Y (NPY)-expressing interneurons, which are among the major subgroups of GABAergic interneurons in the striatum. We investigated the effects of striatal DA depletion on GABAergic transmission in NPY interneurons by electrophysiologically recording GABAergic spontaneous (s) and miniature (m) inhibitory postsynaptic currents (IPSCs) in identified NPY interneurons in slices from 6-hydroxydopamine (6-OHDA)- and vehicle-injected transgenic NPY-humanized Renilla green fluorescent protein (hrGFP) mice with the whole cell patch-clamp technique. We report a significant increase in sIPSC and mIPSC frequency as well as the occurrence of giant synaptic and burst sIPSCs in the 6-OHDA group, suggesting changes in GABAergic circuit activity and synaptic transmission. IPSC kinetics remained unchanged, pointing to mainly presynaptic changes in GABAergic transmission. These results show that chronic DA depletion following 6-OHDA injection causes activity-dependent and -independent increase of synaptic GABAergic inhibition onto striatal NPY interneurons, confirming their involvement in the functional impairments of the DA-depleted striatum.NEW & NOTEWORTHY Neuropeptide Y (NPY) interneurons regulate the function of striatal projection neurons and are upregulated upon dopamine depletion in the striatum. Here we investigated how dopamine depletion affects NPY circuits and show electrophysiologically that it leads to the occurrence of giant synaptic and burst GABAergic spontaneous inhibitory postsynaptic currents (IPSCs) and to an activity-independent increase in GABAergic miniature IPSC frequency in NPY neurons. We suggest that degeneration of dopaminergic terminals in the striatum causes functional changes in striatal GABAergic function.

Capsaicin causes robust reduction in glycinergic transmission to rat hypoglossal motor neurons via a TRPV1-independent mechanism.

The effect of capsaicin on glycinergic synaptic transmission to juvenile rat hypoglossal motor neurons in acute brainstem slices was evaluated in the presence of TTX. Capsaicin caused a robust decrease in miniature IPSC frequency, amplitude, and half-width, showing that this effect is independent of action potential generation. In the presence of capsazepine, a classic TRPV1 antagonist, capsaicin was still able to reduce spontaneous inhibitory postsynaptic current (IPSC) amplitude and frequency. We further investigated whether the effect of capsaicin on glycinergic transmission to hypoglossal motor neurons is pre- or postsynaptic in nature by recording pairs of evoked IPSCs. Interestingly, capsaicin also reduced evoked IPSC amplitude without affecting paired-pulse ratio, indicating a postsynaptic mechanism of action. Significant reduction was also observed in evoked IPSC half-width, rise time, and decay tau. We also show that capsaicin does not have any effect on either transient (It) or sustained (Is) potassium currents. Finally, we also show that the hyperpolarization-activated cationic current (Ih) also remains unchanged after capsaicin application. NEW & NOTEWORTHY Capsaicin reduces the amplitude of quantal and evoked glycinergic inhibitory neurotransmission to brainstem motor neurons without altering activity-dependent transmitter release. This effect of capsaicin is not due to activation of TRPV1 receptors, as it is not blocked by capsazepine, a TRPV1 receptor antagonist. Capsaicin does not alter voltage-dependent potassium current or the hyperpolarization-activated cationic current in brainstem motor neurons.

Mitochondrial superoxide increases excitatory synaptic strength in spinal dorsal horn neurons of neuropathic mice

Reactive oxygen species has been suggested as a key player in neuropathic pain, causing central sensitization by changing synaptic strengths in spinal dorsal horn neurons. However, it remains unclear as to what type of reactive oxygen species changes what aspect of synaptic strengths for central sensitization in neuropathic pain conditions. In this study, we investigated whether mitochondrial superoxide affects both excitatory and inhibitory synaptic strengths in spinal dorsal horn neurons after peripheral nerve injury. Upregulation of mitochondrial superoxide level by knockout of superoxide dismutase-2 exacerbated neuropathic mechanical hypersensitivity caused by L5 spinal nerve ligation, whereas downregulation of mitochondrial superoxide level by overexpression of superoxide dismutase-2 alleviated the hypersensitivity. In spinal nerve ligation condition, the frequency of miniature excitatory postsynaptic currents increased, while that of miniature inhibitory postsynaptic currents decreased in spinal dorsal horn neurons. Superoxide dismutase-2-knockout augmented, whereas superoxide dismutase-2-overexpression prevented, the spinal nerve ligation-increased miniature excitatory postsynaptic currents frequency. However, superoxide dismutase-2-knockout had no effect on the spinal nerve ligation-decreased miniature inhibitory postsynaptic current frequency, and superoxide dismutase-2-overexpression unexpectedly decreased miniature inhibitory postsynaptic current frequency in the normal condition. When applied to the spinal cord slice during in vitro recordings, mitoTEMPO, a specific scavenger of mitochondrial superoxide, reduced the spinal nerve ligation-increased miniature excitatory postsynaptic currents frequency but failed to normalize the spinal nerve ligation-decreased miniature inhibitory postsynaptic current frequency. These results suggest that in spinal dorsal horn neurons, high levels of mitochondrial superoxide increase excitatory synaptic strength after peripheral nerve injury and contribute to neuropathic mechanical hypersensitivity. However, mitochondrial superoxide does not seem to be involved in the decreased inhibitory synaptic strength in this neuropathic pain condition.

Sevoflurane Exposure during the Critical Period Affects Synaptic Transmission and Mitochondrial Respiration but Not Long-term Behavior in Mice.

Anesthesia during the synaptogenic period induces dendritic spine formation, which may affect neurodevelopment. The authors, therefore, evaluated whether changes in synaptic transmission after dendritic spine formation induced by sevoflurane were associated with long-term behavioral changes. The effects of sevoflurane on mitochondrial function were also assessed to further understand the mechanism behind spinogenesis. Postnatal day 16 to 17 mice were exposed to sevoflurane (2.5% for 2 h), and synaptic transmission was measured in the medial prefrontal cortex 6 h or 5 days later. The expression of postsynaptic proteins and mitochondrial function were measured after anesthesia. Long-term behavioral changes were assessed in adult mice. Sevoflurane increased the expression of excitatory postsynaptic proteins in male and female mice (n = 3 to 5 per group). Sevoflurane exposure in male mice transiently increased miniature excitatory postsynaptic current frequency (control: 8.53 ± 2.87; sevoflurane: 11.09 ± 2.58) but decreased miniature inhibitory postsynaptic current frequency (control: 10.18 ± 4.66; sevoflurane: 6.88 ± 2.15). Unexpectedly, sevoflurane increased miniature inhibitory postsynaptic current frequency (control: 1.81 ± 1.11; sevoflurane: 3.56 ± 1.74) in female mice (neurons, n = 10 to 21 per group). Sevoflurane also increased mitochondrial respiration in male mice (n = 5 to 8 per group). However, such changes from anesthesia during the critical period did not induce long-term behavioral consequences. Values are presented as mean ± SD. Sevoflurane exposure during the critical period induces mitochondrial hyperactivity and transient imbalance of excitatory/inhibitory synaptic transmission, without long-lasting behavioral consequences. Further studies are needed to confirm sexual differences and to define the role of mitochondrial activity during anesthesia-induced spine formation.

Chronic cocaine exposure induces noradrenergic modulation of inhibitory synaptic transmission to cholinergic neurons of the laterodorsal tegmental nucleus.

The laterodorsal tegmental nucleus (LDT), which sends cholinergic efferent connections to dopaminergic (DA) neurons in the ventral tegmental area (VTA), plays a critical role in the development of addictive behavior and the reinstatement of cocaine-seeking behavior. Although repeated cocaine exposure elicits plastic changes in excitatory synaptic transmission and intrinsic membrane excitability in LDT cholinergic neurons, it remains unclear whether inhibitory synaptic transmission is modulated by cocaine exposure. The LDT receives fibers containing noradrenaline (NA), a neurotransmitter whose extracellular levels increase with cocaine exposure. Therefore, it is hypothesized that repeated cocaine exposure induces plastic changes in LDT cholinergic neurons via NA. Ex vivo electrophysiological recordings in LDT cholinergic neurons were obtained from rats repeatedly exposed to cocaine. Bath-application of NA induced similar levels of hyperpolarization in both saline- and cocaine-treated neurons. However, NA attenuated the amplitude of inhibitory postsynaptic currents (IPSCs) in cocaine- but not saline-treated neurons through α2 adrenoceptors. This NA-induced IPSC attenuation was observed in the presence of strychnine, but not gabazine, indicating that NA modulated GABAergic but not glycinergic neurotransmission. NA increased the paired-pulse ratios of evoked IPSCs and decreased the frequencies of miniature IPSCs (mIPSCs) without affecting their amplitudes, suggesting a presynaptic mechanism. These findings suggest that repeated cocaine exposure induces neuroplasticity in GABAergic synaptic transmission onto LDT cholinergic neurons by probably modulating presynaptic α2 adrenoceptors. This potentially increases the activity of LDT cholinergic neurons, which might contribute to the development of addictive behavior by enhancing VTA DA neuronal activity.

Revisiting the Lamotrigine-Mediated Effect on Hippocampal GABAergic Transmission.

Lamotrigine (LTG) is generally considered as a voltage-gated sodium (Nav) channel blocker. However, recent studies suggest that LTG can also serve as a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel enhancer and can increase the excitability of GABAergic interneurons (INs). Perisomatic inhibitory INs, predominantly fast-spiking basket cells (BCs), powerfully inhibit granule cells (GCs) in the hippocampal dentate gyrus. Notably, BCs express abundant Nav channels and HCN channels, both of which are able to support sustained action potential generation. Using whole-cell recording in rat hippocampal slices, we investigated the net LTG effect on BC output. We showed that bath application of LTG significantly decreased the amplitude of evoked compound inhibitory postsynaptic currents (IPSCs) in GCs. In contrast, simultaneous paired recordings from BCs to GCs showed that LTG had no effect on both the amplitude and the paired-pulse ratio of the unitary IPSCs, suggesting that LTG did not affect GABA release, though it suppressed cell excitability. In line with this, LTG decreased spontaneous IPSC (sIPSC) frequency, but not miniature IPSC frequency. When re-examining the LTG effect on GABAergic transmission in the cornus ammonis region 1 (CA1) area, we found that LTG markedly inhibits both the excitability of dendrite-targeting INs in the stratum oriens and the concurrent sIPSCs recorded on their targeting pyramidal cells (PCs) without significant hyperpolarization-activated current (Ih) enhancement. In summary, LTG has no effect on augmenting Ih in GABAergic INs and does not promote GABAergic inhibitory output. The antiepileptic effect of LTG is likely through Nav channel inhibition and the suppression of global neuronal network activity.

Serotonin 5-HT1B receptor-mediated calcium influx-independent presynaptic inhibition of GABA release onto rat basal forebrain cholinergic neurons.

Modulatory roles of serotonin (5-HT) in GABAergic transmission onto basal forebrain cholinergic neurons were investigated, using whole-cell patch-clamp technique in the rat brain slices. GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) were evoked by focal stimulation. Bath application of 5-HT (0.1-300μm) reversibly suppressed the amplitude of evoked IPSCs in a concentration-dependent manner. Application of a 5-HT1B receptor agonist, CP93129, also suppressed the evoked IPSCs, whereas a 5-HT1A receptor agonist, 8-OH-DPAT had little effect on the evoked IPSCs amplitude. In the presence of NAS-181, a 5-HT1B receptor antagonist, 5-HT-induced suppression of evoked IPSCs was antagonised, whereas NAN-190, a 5-HT1A receptor antagonist did not antagonise the 5-HT-induced suppression of evoked IPSCs. Bath application of 5-HT reduced the frequency of spontaneous miniature IPSCs without changing their amplitude distribution. The effect of 5-HT on miniature IPSCs remained unchanged when extracellular Ca(2+) was replaced by Mg(2+) . The paired-pulse ratio was increased by CP93129. In the presence of ω-CgTX, the N-type Ca(2+) channel blocker, ω-Aga-TK, the P/Q-type Ca(2+) channel blocker, or SNX-482, the R-type Ca(2+) channel blocker, 5-HT could still inhibit the evoked IPSCs. 4-AP, a K(+) channel blocker, enhanced the evoked IPSCs, and CP93129 had no longer inhibitory effect in the presence of 4-AP. CP93129 increased the number of action potentials elicited by depolarising current pulses. These results suggest that activation of presynaptic 5-HT1B receptors on the terminals of GABAergic afferents to basal forebrain cholinergic neurons inhibits GABA release in Ca(2+) influx-independent manner by modulation of K(+) channels, leading to enhancement of neuronal activities.

Favouring inhibitory synaptic drive mediated by GABA(A) receptors in the basolateral nucleus of the amygdala efficiently reduces pain symptoms in neuropathic mice.

Pain is an emotion and neuropathic pain symptoms are modulated by supraspinal structures such as the amygdala. The central nucleus of the amygdala is often called the 'nociceptive amygdala', but little is known about the role of the basolateral amygdala. Here, we monitored the mechanical nociceptive thresholds in a mouse model of neuropathic pain and infused modulators of the glutamate/GABAergic transmission in the basolateral nucleus of the amygdala (BLA) via chronically-implanted cannulas. We found that an N-methyl-D-aspartate-type glutamate receptor antagonist (MK-801) exerted a potent antiallodynic effect, whereas a transient allodynia was induced after perfusion of bicuculline, a GABA(A) receptor antagonist. Potentiating GABA(A) receptor function using diazepam or etifoxine (a non-benzodiazepine anxiolytic) fully but transiently alleviated mechanical allodynia. Interestingly, the antiallodynic effect of etifoxine disappeared in animals that were incapable of producing 3α-steroids. Diazepam had a similar effect but of shorter duration. As indicated by patch-clamp recordings of BLA neurons, these effects were mediated by a potentiation of GABA(A) receptor-mediated synaptic transmission. Together with a presynaptic elevation of miniature inhibitory postsynaptic current frequency, the duration and amplitude of GABA(A) miniature inhibitory postsynaptic currents were also increased (postsynaptic effect). The analgesic contribution of endogenous neurosteroid seemed to be exclusively postsynaptic. This study highlights the importance of the BLA and the local inhibitory/excitatory neuronal network activity while setting the mechanical nociceptive threshold. Furthermore, it appears that promoting inhibition in this specific nucleus could fully alleviate pain symptoms. Therefore, the BLA could be a novel interesting target for the development of pharmacological or non-pharmacological therapies.

Enhanced GABAergic synaptic transmission at VLPAG neurons and potent modulation by oxycodone in a bone cancer pain model.

Background and PurposeWe demonstrated previously that oxycodone has potent antinociceptive effects at supraspinal sites. In this study, we investigated changes in neuronal function and antinociceptive mechanisms of oxycodone at ventrolateral periaqueductal gray (VLPAG) neurons, which are a major site of opioid action, in a femur bone cancer (FBC) model with bone cancer-related pain.Experimental ApproachWe characterized the supraspinal antinociceptive profiles of oxycodone and morphine on mechanical hypersensitivity in the FBC model. Based on the disinhibition mechanism underlying supraspinal opioid antinociception, the effects of oxycodone and morphine on GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) in VLPAG neurons were evaluated in slices from the FBC model.Key ResultsThe supraspinal antinociceptive effects of oxycodone, but not morphine, were abolished by blocking G protein-gated inwardly rectifying potassium1 (Kir3.1) channels. In slices from the FBC model, GABAergic synaptic transmission at VLPAG neurons was enhanced, as indicated by a leftward shift of the input–output relationship curve of evoked IPSCs, the increased paired-pulse facilitation and the enhancement of miniature IPSC frequency. Following treatment with oxycodone and morphine, IPSCs were reduced in the FBC model, and the inhibition of presynaptic GABA release by oxycodone, but not morphine was enhanced and dependent on Kir3.1 channels.Conclusion and ImplicationsOur results demonstrate that Kir3.1 channels are important for supraspinal antinociception and presynaptic GABA release inhibition by oxycodone in the FBC model. Enhanced GABAergic synaptic transmission at VLPAG neurons in the FBC model is an important site of supraspinal antinociception by oxycodone via Kir3.1 channel activation.

Repeated morphine treatment alters cannabinoid modulation of GABAergic synaptic transmission within the rat periaqueductal grey.

Cannabinoids and opioids produce antinociception by modulating GABAergic synaptic transmission in a descending analgesic pathway from the midbrain periaqueductal grey (PAG). While chronic opioid treatment produces opioid tolerance, it has recently been shown to enhance cannabinoid-induced antinociception within the PAG. This study examined the effect of repeated opioid treatment on opioid and cannabinoid presynaptic modulation of GABAergic synaptic transmission in PAG. Midbrain PAG slices were prepared from untreated rats, and rats that had undergone repeated morphine or saline pretreatment. Whole-cell voltage-clamp recordings were made from neurons within the ventrolateral PAG. In slices from untreated animals, the cannabinoid receptor agonist WIN55212 and the μ receptor agonist DAMGO inhibited electrically evoked GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) IPSCs in PAG neurons, with IC50 s of 30 and 100 nM respectively. The inhibition of evoked IPSCs produced by WIN55212 (30 nM) and DAMGO (100 nM) was similar in PAG neurons from morphine- and saline-treated animals. The cannabinoid CB1 receptor antagonist AM251 increased the frequency of spontaneous miniature IPSCs in PAG neurons from repeated morphine-, but not saline-treated animals. DAMGO inhibition of evoked IPSCs was enhanced in the presence of AM251 in morphine-, but not saline-treated animals. These results indicate that the efficiency of agonist-induced inhibition of GABAergic synaptic transmission is enhanced by morphine treatment, although this is dampened by endocannabinoid-mediated tonic inhibition. Thus, endocannabinoid modulation of synaptic transmission could provide an alternative analgesic approach in a morphine-tolerant state. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

α-MSH exerts direct postsynaptic excitatory effects on NTS neurons and enhances GABAergic signaling in the NTS

The central melanocortin system plays an essential role in the regulation of energy balance. While anorexigenic effects of α-melanocyte-stimulating hormone (α-MSH) acting in the nucleus of the solitary tract (NTS), a critical medullary autonomic control center, have been established, the cellular events underlying these effects are less well characterized. In this study, we used whole-cell patch-clamp electrophysiology to examine firstly whether α-MSH exerts direct postsynaptic effects on the membrane potential of rat NTS neurons in slice preparation, and secondly whether α-MSH influences GABAergic signaling in the NTS. In normal artificial cerebrospinal fluid, perfusion of α-MSH (500nM) resulted in a depolarization in 39% of cells (n=16, mean 6.14±0.54mV), and a hyperpolarization in 22% of cells (n=9, −6.79±1.02mV). Studies using tetrodotoxin to block neuronal communication revealed α-MSH exerts direct depolarizing effects on some NTS neurons, and indirect inhibitory effects on others. A third subset of neurons is simultaneously directly depolarized and indirectly hyperpolarized by α-MSH, resulting in a net lack of effect on membrane potential. The inhibitory inputs influenced by α-MSH were identified as GABAergic, as α-MSH increased the frequency, but not amplitude, of inhibitory postsynaptic currents (IPSCs) in 50% of NTS neurons. α-MSH had no effect on the frequency or amplitude of miniature IPSCs. Furthermore, pharmacological blockade of GABAA and GABAB receptors, and physical removal of all synaptic inputs via cellular dissociation, abolished hyperpolarizations induced by α-MSH. We conclude α-MSH exerts direct, postsynaptic excitatory effects on a subset of NTS neurons. By exciting GABAergic NTS neurons and presynaptically enhancing GABAergic signaling, α-MSH also indirectly inhibits other NTS cells. These findings provide critical insight into the cellular events underlying medullary melanocortin anorexigenic effects, and expand the understanding of the circuitries involved in central melanocortin signaling.

Targeted disruption of layer 4 during development increases GABAAreceptor neurotransmission in the neocortex

Cortical dysplasia (CD) associates with clinical pathologies, including epilepsy and mental retardation. CD results from impaired migration of immature neurons to their cortical targets, leading to clustering of neural cells and changes in cortical properties. We developed a CD model by administering methylazoxymethanol (MAM), an anti-mitotic, to pregnant ferrets on embryonic day 33; this leads to reduction in cortical thickness in addition to redistribution and increased expression of GABAA receptors (GABAAR). We evaluated the impact of MAM treatment on GABAAR-mediated synaptic transmission in postnatal day 0-1 neurons, leaving the ganglionic eminence (GE) and in layer 2/3 pyramidal cells of postnatal day 28-38 ferrets. Embryonic day 33 MAM treatment significantly increases the amplitude and frequency of spontaneous GABAAR-mediated inhibitory postsynaptic currents (IPSCs) in the cells leaving the GE. In older MAM-treated animals, the amplitude and frequency of GABAAR-mediated spontaneous IPSCs in layer 2/3 pyramidal cells is increased, as are the amplitude and frequency of miniature IPSCs. The kinetics of GABAAR opening also altered following treatment with MAM. Western blot analysis shows that the expression of the GABAAα3R and GABAAγ2R subunits amplified in our model animals. We did not observe any significant change in the passive properties of either the layer 2/3 pyramidal cells or cells leaving the GE after MAM treatment. These observations reinforce the idea that synaptic neurotransmission through GABAAR enhances following treatment with MAM and coincides with our finding of increased GABAAαR expression within the upper cortical layers. Overall, we demonstrate that small amounts of toxins delivered during corticogenesis can result in long-lasting changes in ambient expression of GABAAR that influence intrinsic neuronal properties.

Impairment of cortical GABAergic synaptic transmission in an environmental rat model of autism

The biological mechanisms of autism spectrum disorders (ASDs) are largely unknown in spite of extensive research. ASD is characterized by altered function of multiple brain areas including the temporal cortex and by an increased synaptic excitation:inhibition ratio. While numerous studies searched for evidence of increased excitation in ASD, fewer have investigated the possibility of reduced inhibition. We characterized the cortical γ-amino butyric acid (GABA)ergic system in the rat temporal cortex of an ASD model [offspring of mothers prenatally injected with valproic acid (VPA)], by monitoring inhibitory post-synaptic currents (IPSCs) with patch-clamp. We found that numerous features of inhibition were severely altered in VPA animals compared to controls. Among them were the frequency of miniature IPSCs, the rise time and decay time of electrically-evoked IPSCs, the slope and saturation of their input/output curves, as well as their modulation by adrenergic and muscarinic agonists and by the synaptic GABAA receptor allosteric modulator zolpidem (but not by the extra-synaptic modulator gaboxadol). Our data suggest that both pre- and post-synaptic, but not extra-synaptic, inhibitory transmission is impaired in the offspring of VPA-injected mothers. We speculate that impairment in the GABAergic system critically contributes to an increase in the ratio between synaptic excitation and inhibition, which in genetically predisposed individuals may alter cortical circuits responsible for emotional, communication and social impairments at the core of ASD.

α2A adrenoceptor‐mediated presynaptic inhibition of GABAergic transmission in rat tuberomammillary nucleus neurons

Histaminergic neurons within the tuberomammillary nucleus (TMN) play an important role in the regulation of sleep-wakefulness. Here, we report the adrenergic modulation of GABAergic transmission in rat TMN histaminergic neurons using a conventional whole-cell patch clamp technique. Norepinephrine (NE) reversibly decreased the amplitude of action potential-dependent GABAergic inhibitory post-synaptic currents (IPSCs) and increased the paired pulse ratio. The NE-induced inhibition of GABAergic IPSCs was mimicked by clonidine, a selective α2 adrenoceptor agonist. However, cirazoline and isoproterenol, nonselective α1 and β adrenoceptor agonists, respectively, had no effect on GABAergic IPSCs. The NE-induced inhibition of GABAergic IPSCs was significantly blocked by BRL44408, a selective α2A adrenoceptor antagonist, but not imiloxan or JP1302, a selective α2B and α2C adrenoceptor antagonists. The extent of NE-induced inhibition of GABAergic IPSCs was inversely proportional to the extracellular Ca(2+) concentration. Pharmacological agents affecting the activities of adenylyl cyclase or G-protein-coupled inwardly rectifying K(+) channels did not affect the NE-induced inhibition of GABAergic IPSCs. However, NE had no effect on the frequency and amplitude of GABAergic miniature IPSCs. These results suggest that NE acts on presynaptic α2A adrenoceptor to inhibit action potential-dependent GABA release via the inhibition of Ca(2+) influx from the extracellular space to GABAergic nerve terminals, and that this α2A adrenoceptor-mediated modulation of GABAergic transmission may be involved in regulating the excitability of TMN histaminergic neurons.

Heterotrimeric guanosine triphosphate‐binding protein‐coupled modulatory actions of motilin on K+ channels and postsynaptic γ‐aminobutyric acid receptors in mouse medial vestibular nuclear neurons

Some central nervous system neurons express receptors of gastrointestinal hormones, but their pharmacological actions are not well known. Previous anatomical and unit recording studies suggest that a group of cerebellar Purkinje cells express motilin receptors, and motilin depresses the spike discharges of vestibular nuclear neurons that receive direct cerebellar inhibition in rats or rabbits. Here, by the slice-patch recording method, we examined the pharmacological actions of motilin on the mouse medial vestibular nuclear neurons (MVNs), which play an important role in the control of ocular reflexes. A small number of MVNs, as well as cerebellar floccular Purkinje cells, were labeled with an anti-motilin receptor antibody. Bath application of motilin (0.1 μm) decreased the discharge frequency of spontaneous action potentials in a group of MVNs in a dose-dependent manner (K(d) , 0.03 μm). The motilin action on spontaneous action potentials was blocked by apamin (100 nm), a blocker of small-conductance Ca(2+) -activated K(+) channels. Furthermore, motilin enhanced the amplitudes of inhibitory postsynaptic currents (IPSCs) and miniature IPSCs, but did not affect the frequencies of miniature IPSCs. Intracellular application of pertussis toxin (PTx) (0.5 μg/μL) or guanosine triphosphate-γ-S (1 mm) depressed the motilin actions on both action potentials and IPSCs. Only 30% of MVNs examined on slices obtained from wild-type mice, but none of the GABAergic MVNs that were studied on slices obtained from vesicular γ-aminobutyric acid transporter-Venus transgenic mice, showed such a motilin response on action potentials and IPSCs. These findings suggest that motilin could modulate small-conductance Ca(2+) -activated K(+) channels and postsynaptic γ-aminobutyric acid receptors through heterotrimeric guanosine triphosphate-binding protein-coupled receptor in a group of glutamatergic MVNs.