mIPSC Frequency Research Articles

1319-P: Gabra5 Plays a Sexually Dimorphic Role in POMC Neuron Activity and Glucose Balance

Pro-opiomelanocortin (POMC) neurons are important for body weight and glucose balance. The inhibitory tone to POMC neurons is mediated primarily by GABA receptor complex. However, the detailed mechanisms and functions of GABA receptors are not well understood. We found that α5 subunit of GABA receptor complex, Gabra5, is highly expressed in POMC neurons. To explore the function of Gabra5 in POMC neurons, we deleted Gabra5 specifically from mature POMC neurons using the CRISPR-Cas9 strategy. Body weight and food intake were not affected in either male mice or female mice by the deletion of Gabra5 from POMC neurons. We found that the deletion of Gabra5 caused a significant increase in the firing frequency and rest membrane potential, and a decrease in the amplitude of mIPSC in male POMC neurons. However, the deletion of Gabra5 just modestly decreased the frequency of mIPSC in female POMC neurons. Consistently, loss of Gabra5 from POMC neurons significantly improved the glucose tolerance in male mice but not in female mice. These results revealed a sexually dimorphic role of Gabra5 in POMC neuron activity and glucose balance, independent of body weight control. Disclosure Z.Pei: None. Y.He: None. Y.He: None. Y.Xu: None. C.Wang: None. Funding National Institutes of Health (KDK119471)

Comprehensive Analysis of lncRNAs, miRNAs and mRNAs in Mouse Hippocampus With Hepatic Encephalopathy.

Hepatic encephalopathy (HE) often presents with varying degrees of cognitive impairment. However, the molecular mechanism of its cognitive impairment has not been fully elucidated. Whole transcriptome analysis of hippocampus between normal and HE mice was performed by using RNA sequencing. 229 lncRNAs, 49 miRNAs and 363 mRNAs were differentially expressed in HE mice. The lncRNA-miRNA-mRNA interaction networks were established, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. Dysregulated RNAs in interaction networks were mainly involved in synaptic plasticity and the regulation of learning and memory. In NH4Cl-treated hippocampal neurons, the dendritic spine density and maturity decreased significantly, the amplitude and frequency of mIPSC increased, while the amplitude and frequency of mEPSC decreased. These manifestations can be reversed by silencing SIX3OS1. Further research on these no-coding RNAs may lead to new therapies for the treatment and management of brain dysfunction caused by HE.

Mice With Monoallelic GNAO1 Loss Exhibit Reduced Inhibitory Synaptic Input to Cerebellar Purkinje Cells

GNAO1 encodes Gαo, a heterotrimeric G protein alpha subunit in the Gi/o family. Gαo, the most abundant membrane protein in the brain, mediates downstream signaling pathways by itself, or via regulating free Gβγ subunits. To determine the role of Gαo protein in regulating synaptic transmission, we use electrophysiology to study cerebellar molecular layer interneuron ‐ Purkinje cell synaptic transmission in a haploinsufficient loss‐of‐function (LOF) Gnao1 mouse model, manifest with movement abnormalities and enhanced seizure susceptibility. We hypothesize that a LOF Gαo protein will increase GABA release through presynaptic Gαo ‐ coupled receptors.Inhibitory post synaptic currents (IPSCs) and excitatory post synaptic currents (EPSCs) were recorded using standard patch clamp techniques. Spontaneous IPSCs (sIPSCs) were isolated with glutamatergic receptor inhibitors CNQX and AP‐5. Spontaneous EPSCs (sEPSCs) were isolated with the GABAergic receptor inhibitor bicuculline. Miniature PSCs were isolated with the additional perfusion of tetrodotoxin. Cerebellar molecular layer inhibitory neurons were labeled with the Kit‐eGFP method and observed under an inverted epifluorescence microscope. Gαo and Gβ protein expression levels from flash‐frozen mouse brain tissues were determined using Western Blot.Instead of increase, we observe a significant reduction in both sIPSC (40%) and mIPSC (75%) frequency but not amplitude in the Gnao1 mutant mouse model. Both frequency and amplitude of sEPSCs and mEPSCs remain unchanged. On Western blots, levels of Gαo were reduced by about half, as expected, while Gβ levels were unchanged from control. To determine whether this reduction is due to the loss of cerebellar inhibitory interneuron populations, we compare cerebellar interneuron number in the cerebellar molecular layer between Gnao1 mutant mice and their wildtype littermate control. There is a modest reduction (13%) in interneuron number of cerebellar molecular layer in the Gnao1 mutant mouse.The observed reduction in IPSC frequency is not related to Gαo ‐ coupled GABAergic receptors. Also, the modest change in cerebellar interneuron counts does not seem to account for the marked reduction in IPSC frequency. These observations, and the reduced Gαo expression in brain with the normal levels of Gβ, strongly support that a model with a haploinsufficient LOF Gnao1 mutant mainly functions through receptor‐independent regulation of free Gβγ subunits to suppress inhibitory neurotransmitter release in Gnao1 mutant cerebellum.

Chronic Intermittent Hypoxia Alters Chloride Gradients in Median Preoptic Nucleus (MnPO) Neurons of Rats: Comparing ClopHensorN and Perforated Patch Recording

Chronic intermittent hypoxia (CIH) is an animal model that simulates the hypoxemia associated with sleep apnea. Male rats exposed to CIH exhibit increased sympathetic tone and reactivity and persistent elevations in blood pressure. In MnPO neurons, CIH induces reductions in GABAa inhibition that are, in some neurons, exacerbated to the point of GABAa mediated excitation. The genetically encoded ratiometric Cl‐ sensor, ClopHensorN, was used to monitor the Cl‐ flux of PVN projecting MnPO neurons in normoxic (Norm) and CIH treated rats in response to GABAa receptor activation. In some experiments, ClopHensorN imaging was combined with perforated patch recordings.Using isoflurane (2‐3%) anesthesia, male Sprague‐Dawley rats (250‐350g) received bilateral infusions (0.4 µL) of a retrograde AAV9‐Cre in the PVN and a DIO‐ClopHensorN in the MnPO. After recovery, rats were subjected to 7 days of CIH (0800‐1600 hr) or normoxia. CIH consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10x/hr for 8 hours (during the normal inactive/sleep phase) on 7 consecutive days. For recordings, rats were anesthetized with isoflurane (2‐3%) and coronal slices (300 µm) containing the MnPO were cut using standard in vitro slice procedures. All cells were tested in standard artificial cerebrospinal fluid for their responses to 10 s focal application of muscimol (100 uM). ClopHensorN images were captured every 3 sec. Cl‐ flux was determined from the ratio of GFP (ex 488 nm, em 500‐550 nm) to tdTomato (ex 594 nm, em 650‐700 nm) fluorescence. For some cells, voltage‐clamp recordings using gramicidin (100 ug/ml in 140 mM K‐gluconate) perforated patch were also collected (in labeled and unlabeled MnPO neurons).MnPO neurons from CIH treated rats (n = 632), 20.1% showed a decrease in the ratio of GFP to tdTomato fluorescence (n = 127) while 0.3% showed increases in this ratio (n = 2) indicative of Cl‐ efflux. In MnPO neurons from Norm rats (n = 482), 41.9% showed a muscimol dependent decrease in the ratiometric Cl‐ signal (n = 202) with 0 cells showing increases. Muscimol dependent decreases in the Cl‐ signal were reduced in the CIH treated rats suggesting reduced GABAa inhibition. During perforated patch recordings, MnPO neurons from Norm rats exhibited mIPSCs and outward currents in response to muscimol. MnPO neurons from CIH treated rats show variable mIPSC frequencies and heterogeneous responses to muscimol.The results demonstrate that CIH alters the Cl‐ flux of PVN projecting MnPO neurons in response to GABAa stimulation. These changes may contribute to the increased sympathetic tone and reactivity, as well as the persistent hypertension associated with CIH.

Norepinephrine Restores Inhibitory Tone of Spinal Lamina X Circuitry, thus Contributing to Analgesia Against Inflammatory Pain

Norepinephrine (NE) acts directly on the inhibitory interneurons of spinal lamina X and may act on spinal lamina X neurons for inhibiting nociceptive synaptic transmission against pain. We investigated this mechanism within inflammatory pain model rats. Using immunohistochemical staining and in vivo extracellular recording, the increased number of phosphorylated extracellular signal-regulated kinase profiles in lamina X (n = 6/group) and increased frequency of spontaneous neuronal firing on putative lamina X (n = 14) under the inflammatory pain were significantly suppressed by the direct application of NE (P < 0.01). Following in vivo observation of enhanced spontaneous neuronal firing, we tested the impact of NE on this discharge using an in vitro spinal slice preparation. Using in vitro patch-clamps recording, the baseline level of miniature inhibitory postsynaptic currents (mIPSCs) frequency on spinal lamina X neurons cord is decreased under inflammatory pain. Direct application of NE to spinal lamina X neurons in inflammatory pain model rats facilitates mIPSCs frequency and induces an outward current (n = 8; P < 0.05), and these responses are inhibited by α1A- and α2-receptor antagonists (n = 8; P > 0.05). Considering these results and those of our previous study (Ohashi et al., 2019), NE might act on inhibitory interneurons of spinal lamina X to facilitate inhibitory transmission and induces neurons located in or around lamina X membrane hyperpolarization. These NE-mediated responses acted through α1A- and α2-receptors. These mechanisms of NE on spinal lamina X might contribute to analgesia against inflammatory pain.

Presynaptic HCN channels constrain GABAergic synaptic transmission in pyramidal cells of the medial prefrontal cortex.

ABSTRACTHyperpolarization-activated cyclic nucleotide-gated (HCN) channels are widely expressed in neurons in the central nervous system. It has been documented that HCN channels regulate the intrinsic excitability of pyramidal cells in the medial prefrontal cortex (mPFC) of rodents. Here, we report that HCN channels limited GABAergic transmission onto pyramidal cells in rat mPFC. The pharmacological blockade of HCN channels resulted in a significant increase in the frequency of both spontaneous and miniature inhibitory postsynaptic currents (IPSCs) in mPFC pyramidal cells, whereas potentiation of HCN channels reversely decreases the frequency of mIPSCs. Furthermore, such facilitation effect on mIPSC frequency required presynaptic Ca2+ influx. Immunofluorescence staining showed that HCN channels expressed in presynaptic GABAergic terminals, as well as in both soma and neurite of parvalbumin-expressing (PV-expressing) basket cells in mPFC. The present results indicate that HCN channels in GABAergic interneurons, most likely PV-expressing basket cells, constrain inhibitory control over layer 5–6 pyramidal cells by restricting presynaptic Ca2+ entry.

GHS-R1a activity suppresses synaptic function of primary cultured hippocampal neurons

Ghrelin is an orexigenic gastric hormone that promotes feeding behaviors and regulates energy homeostasis in both humans and rodents. Studies have reported intriguing yet conflicting roles that ghrelin and its receptor growth hormone secretagogue receptor 1a (GHS-R1a) play in regulating multiple brain functions, such as learning and memory; however, the underlying mechanism is largely unknown. In this study, we investigated the effect of ghrelin incubation and virus-mediated GHS-R1a overexpression on synaptic functions of primary cultured hippocampal neurons. Our results demonstrated that ghrelin pre-treatment for 24 h, with a concentration of either 4 nM or 200 nM, suppressed the frequency of miniature excitatory postsynaptic currents (mEPSCs), the frequency and the amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Similarly, GHS-R1a overexpression inhibited both the frequency and the amplitude of mEPSCs, and mIPSCs frequency. Moreover, our in vitro Ca2+-image study with Rhod-3AM reveals that ghrelin pre-treatment for either 3 h or 24 h suppressed glutamate-induced elevation of cytoplasmic [Ca2+]. Our findings thus suggest that GHS-R1a signaling inhibits synaptic function of hippocampal neurons, which may contribute to the blocking effect of ghrelin on memory formation.

Intrathecally administered pizotifen alleviates neuropathic and inflammatory pain in mice by enhancing GABAergic inhibition

Chronic pain, such as chronic neuropathic pain and chronic inflammatory pain, is often difficult to manage and bring great trouble to patients. 5-HT plays a key role in the process of pain transmission both in centrally and peripherally. Tricyclic antidepressants (TCA) such as amitriptyline are classical 5-HT reuptake inhibitors, are recommended as the first-line treatment for chronic pain. Pizotifen, a 5-HT2 receptor antagonist, is currently used in the prevention of vascular headaches. However, the antinociceptive effect of pizotifen on non-headache pain especially chronic pain in the spinal level is still unknown. Here we find that intrathecal pizotifen attenuates neuropathic and inflammatory pain mainly due to elevated GABAergic synaptic inhibition. Neuropathic pain is induced by segmental spinal nerve ligation (SNL), and inflammatory pain is induced by intraplantar injection of complete Freund's adjuvant (CFA). Both in SNL and CFA mice, spinally administered pizotifen reduced mechanical and thermal hyperalgesia dose-dependently. Since the levels of GAD65/67 were increased, and the frequency of mIPSCs in the spinal dorsal horn was increased, together with the antinociceptive effect being reversed by both GABAAR and GAD blockade, this antinociceptive effect might be generated from strengthened GABAergic inhibition. Furthermore, high dose of pizotifen (5 μg) weakly affected motor performance and did not influence the locomotor activity in normal animals. In summary, our findings suggest that pizotifen strengthens the inhibitory synaptic transmission and exerts antinociceptive effect on both neuropathic pain and inflammatory pain in the spinal cord, and may serve as a promising remedy for chronic pain.

LiCl treatment leads to long-term restoration of spine maturation and synaptogenesis in adult Tbr1 mutants

BackgroundTbr1 encodes a T-box transcription factor and is considered a high confidence autism spectrum disorder (ASD) gene. Tbr1 is expressed in the postmitotic excitatory neurons of the deep neocortical layers 5 and 6. Postnatally and neonatally, Tbr1 conditional mutants (CKOs) have immature dendritic spines and reduced synaptic density. However, an understanding of Tbr1’s function in the adult mouse brain remains elusive.MethodsWe used conditional mutagenesis to interrogate Tbr1’s function in cortical layers 5 and 6 of the adult mouse cortex.ResultsAdult Tbr1 CKO mutants have dendritic spine and synaptic deficits as well as reduced frequency of mEPSCs and mIPSCs. LiCl, a WNT signaling agonist, robustly rescues the dendritic spine maturation, synaptic defects, and excitatory and inhibitory synaptic transmission deficits.ConclusionsLiCl treatment could be used as a therapeutic approach for some cases of ASD with deficits in synaptic transmission.

Dopamine D2L Receptor Deficiency Alters Neuronal Excitability and Spine Formation in Mouse Striatum.

The striatum contains several types of neurons including medium spiny projection neurons (MSNs), cholinergic interneurons (ChIs), and fast-spiking interneurons (FSIs). Modulating the activity of these neurons by the dopamine D2 receptor (D2R) can greatly impact motor control and movement disorders. D2R exists in two isoforms: D2L and D2S. Here, we assessed whether alterations in the D2L and D2S expression levels affect neuronal excitability and synaptic function in striatal neurons. We observed that quinpirole inhibited the firing rate of all three types of striatal neurons in wild-type (WT) mice. However, in D2L knockout (KO) mice, quinpirole enhanced the excitability of ChIs, lost influence on spike firing of MSNs, and remained inhibitory effect on spike firing of FSIs. Additionally, we showed mIPSC frequency (but not mIPSC amplitude) was reduced in ChIs from D2L KO mice compared with WT mice, suggesting spontaneous GABA release is reduced at GABAergic terminals onto ChIs in D2L KO mice. Furthermore, we found D2L deficiency resulted in reduced dendritic spine density in ChIs, suggesting D2L activation plays a role in the formation/maintenance of dendritic spines of ChIs. These findings suggest new molecular and cellular mechanisms for causing ChIs abnormality seen in Parkinson’s disease or drug-induced dyskinesias.

Presynaptic mechanisms underlying GABAB-receptor-mediated inhibition of spontaneous neurotransmitter release.

SUMMARYInhibition of neurotransmitter release by neurotransmitter substances constitutes a fundamental means of neuromodulation. In contrast to well-delineated mechanisms that underlie inhibition of evoked release via suppression of voltage-gated Ca2+ channels, processes that underlie neuromodulatory inhibition of spontaneous release remain unclear. Here, we interrogated inhibition of spontaneous glutamate and GABA release by presynaptic metabotropic GABAB receptors. Our findings show that this inhibition relies on Gβγ subunit action at the membrane, and it is largely independent of presynaptic Ca2+ signaling for both forms of release. In the case of spontaneous glutamate release, inhibition requires Gβγ interaction with the C terminus of the key fusion machinery component SNAP25, and it is modulated by synaptotagmin-1. Inhibition of spontaneous GABA release, on the other hand, is independent of these pathways and likely requires alternative Gβγ targets at the presynaptic terminal.

Self-Administration of Entactogen Psychostimulants Dysregulates Gamma-Aminobutyric Acid (GABA) and Kappa Opioid Receptor Signaling in the Central Nucleus of the Amygdala of Female Wistar Rats.

Male rats escalate intravenous self-administration of entactogen psychostimulants, 3,4-methylenedioxymethcathinone (methylone) and 3,4-methylenedioxymethamphetamine (MDMA) under extended access conditions, as with typical psychostimulants. Here, we investigated whether female rats escalate self-administration of methylone, 3,4-methylenedioxypentedrone (pentylone), and MDMA and then studied consequences of MDMA and pentylone self-administration on GABAA receptor and kappa opioid receptor (KOR) signaling in the central nucleus of the amygdala (CeA), a brain area critically dysregulated by extended access self-administration of alcohol or cocaine. Adult female Wistar rats were trained to self-administer methylone, pentylone, MDMA (0.5 mg/kg/infusion), or saline-vehicle using a fixed-ratio 1 response contingency in 6-h sessions (long-access: LgA) followed by progressive ratio (PR) dose-response testing. The effects of pentylone-LgA, MDMA-LgA and saline on basal GABAergic transmission (miniature post-synaptic inhibitory currents, mIPSCs) and the modulatory role of KOR at CeA GABAergic synapses were determined in acute brain slices using whole-cell patch-clamp. Methylone-LgA and pentylone-LgA rats similarly escalated their drug intake (both obtained more infusions compared to MDMA-LgA rats), however, pentylone-LgA rats reached higher breakpoints in PR tests. At the cellular level, baseline CeA GABA transmission was markedly elevated in pentylone-LgA and MDMA-LgA rats compared to saline-vehicle. Specifically, pentylone-LgA was associated with increased CeA mIPSC frequency (GABA release) and amplitude (post-synaptic GABAA receptor function), while mIPSC amplitudes (but not frequency) was larger in MDMA-LgA rats compared to saline rats. In addition, pentylone-LgA and MDMA-LgA profoundly disrupted CeA KOR signaling such as both KOR agonism (1 mM U50488) and KOR antagonism (200 nM nor-binaltorphimine) decreased mIPSC frequency suggesting recruitment of non-canonical KOR signaling pathways. This study confirms escalated self-administration of entactogen psychostimulants under LgA conditions in female rats which is accompanied by increased CeA GABAergic inhibition and altered KOR signaling. Collectively, our study suggests that CeA GABA and KOR mechanisms play a critical role in entactogen self-administration like those observed with escalation of alcohol or cocaine self-administration.

Developmental changes in GABAergic and glycinergic synaptic transmission to rat motoneurons innervating jaw-closing and jaw-opening muscles

Trigeminal motoneurons (MNs) innervating the jaw-closing and jaw-opening muscles receive numerous inhibitory synaptic inputs from GABAergic and glycinergic neurons, which are essential for oromotor functions, such as the orofacial reflex, suckling, and mastication. The properties of the GABAergic and glycinergic inputs of these MNs undergo developmental alterations during the period in which their feeding behavior proceeds from suckling to mastication; however, the detailed characteristics of the developmental patterns of GABAergic and glycinergic transmission in these neurons remain to be elucidated. This study was conducted to investigate developmental changes in miniature inhibitory postsynaptic currents (mIPSCs) in masseter (jaw-closing) and digastric (jaw-opening) MNs using brainstem slice preparations obtained from Wistar rats on postnatal day (P)2–5, P9–12, and P14–17. The frequency and amplitude of glycinergic mIPSCs substantially increased with age in both the masseter and digastric MNs. The rise time and decay time of glycinergic mIPSCs in both MNs decreased during development. In contrast, the frequency of GABAergic components in masseter MNs was higher at P2–5 than at P14–17, whereas that in the digastric MNs remained unchanged throughout the postnatal period. The proportion of currents mediated by GABA-glycine co-transmission was higher at P2–5, and then it decreased with age in both MNs. These results suggest that characteristics related to the development of inhibitory synaptic inputs differ between jaw-closing and jaw-opening MNs and between GABAergic and glycinergic currents. These distinct developmental characteristics may contribute to the development of feeding behaviors.

Lack of Hyperinhibition of Oriens Lacunosum-Moleculare Cells by Vasoactive Intestinal Peptide-Expressing Cells in a Model of Temporal Lobe Epilepsy.

Temporal lobe epilepsy remains a common disorder with no cure and inadequate treatments, potentially because of an incomplete understanding of how seizures start. CA1 pyramidal cells and many inhibitory interneurons increase their firing rate in the seconds-minutes before a spontaneous seizure in epileptic rats. However, some interneurons fail to do so, including those identified as putative interneurons with somata in oriens and axons targeting lacunosum-moleculare (OLM cells). Somatostatin-containing cells, including OLM cells, are the primary target of inhibitory vasoactive intestinal polypeptide and calretinin-expressing (VIP/CR) bipolar interneuron-selective interneurons, type 3 (ISI-3). The objective of this study was to test the hypothesis that in epilepsy inhibition of OLM cells by ISI-3 is abnormally increased, potentially explaining the failure of OLM recruitment when needed most during the ramp up of activity preceding a seizure. Stereological quantification of VIP/CR cells in a model of temporal lobe epilepsy demonstrated that they survive in epileptic mice, despite a reduction in their somatostatin-expressing (Som) cell targets. Paired recordings of unitary IPSCs (uIPSCs) from ISI-3 to OLM cells did not show increased connection probability or increased connection strength, and failure rate was unchanged. When miniature postsynaptic currents in ISI-3 were compared, only mIPSC frequency was increased in epileptic hippocampi. Nevertheless, spontaneous and miniature postsynaptic potentials were unchanged in OLM cells of epileptic mice. These results are not consistent with the hypothesis of hyperinhibition from VIP/CR bipolar cells impeding recruitment of OLM cells in advance of a seizure.

Mice Expressing Regulators of G protein Signaling-insensitive Gαo Define Roles of μ Opioid Receptor Gαo and Gαi Subunit Coupling in Inhibition of Presynaptic GABA Release.

Regulators of G protein signaling (RGS) proteins modulate signaling by G protein-coupled receptors. Using a knock-in transgenic mouse model with a mutation in Gαo that does not bind RGS proteins (RGS-insensitive), we determined the effect of RGS proteins on presynaptic μ opioid receptor (MOR)-mediated inhibition of GABA release in the ventrolateral periaqueductal gray (vlPAG). The MOR agonists [d-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) and met-enkephalin (ME) inhibited evoked inhibitory postsynaptic currents (eIPSCs) in the RGS-insensitive mice compared with wild-type (WT) littermates, respectively. Fentanyl inhibited eIPSCs similarly in both WT and RGS-insensitive mice. There were no differences in opioid agonist inhibition of spontaneous GABA release between the genotypes. To further probe the mechanism underlying these differences between opioid inhibition of evoked and spontaneous GABA release, specific myristoylated Gα peptide inhibitors for Gαo1 and Gαi1-3 that block receptor-G protein interactions were used to test the preference of agonists for MOR-Gα complexes. The Gαo1 inhibitor reduced DAMGO inhibition of eIPSCs, but Gαi1-3 inhibitors had no effect. Both Gαo1 and Gαi1-3 inhibitors separately reduced fentanyl inhibition of eIPSCs but had no effects on ME inhibition. Gαi1-3 inhibitors blocked the inhibitory effects of ME and fentanyl on miniature postsynaptic current (mIPSC) frequency, but both Gαo1 and Gαi1-3 inhibitors were needed to block the effects of DAMGO. Finally, baclofen-mediated inhibition of GABA release is unaffected in the RGS-insensitive mice and in the presence of Gαo1 and Gαi1-3 inhibitor peptides, suggesting that GABAB receptor coupling to G proteins in vlPAG presynaptic terminals is different than MOR coupling. SIGNIFICANCE STATEMENT: Presynaptic μ opioid receptors (MORs) in the ventrolateral periaqueductal gray are critical for opioid analgesia and are negatively regulated by RGS proteins. These data in RGS-insensitive mice provide evidence that MOR agonists differ in preference for Gαo versus Gαi and regulation by RGS proteins in presynaptic terminals, providing a mechanism for functional selectivity between agonists. The results further define important differences in MOR and GABAB receptor coupling to G proteins that could be exploited for new pain therapies.

Spinal microglial β-endorphin signaling mediates IL-10 and exenatide-induced inhibition of synaptic plasticity in neuropathic pain.

AimThis study aimed to investigate the regulation of pain hypersensitivity induced by the spinal synaptic transmission mechanisms underlying interleukin (IL)‐10 and glucagon‐like peptide 1 receptor (GLP‐1R) agonist exenatide‐induced pain anti‐hypersensitivity in neuropathic rats through spinal nerve ligations.MethodsNeuropathic pain model was established by spinal nerve ligation of L5/L6 and verified by electrophysiological recording and immunofluorescence staining. Microglial expression of β‐endorphin through autocrine IL‐10‐ and exenatide‐induced inhibition of glutamatergic transmission were performed by behavioral tests coupled with whole‐cell recording of miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) through application of endogenous and exogenous IL‐10 and β‐endorphin.ResultsIntrathecal injections of IL‐10, exenatide, and the μ‐opioid receptor (MOR) agonists β‐endorphin and DAMGO inhibited thermal hyperalgesia and mechanical allodynia in neuropathic rats. Whole‐cell recordings of bath application of exenatide, IL‐10, and β‐endorphin showed similarly suppressed enhanced frequency and amplitude of the mEPSCs in the spinal dorsal horn neurons of laminae II, but did not reduce the frequency and amplitude of mIPSCs in neuropathic rats. The inhibitory effects of IL‐10 and exenatide on pain hypersensitive behaviors and spinal synaptic plasticity were totally blocked by pretreatment of IL‐10 antibody, β‐endorphin antiserum, and MOR antagonist CTAP. In addition, the microglial metabolic inhibitor minocycline blocked the inhibitory effects of IL‐10 and exenatide but not β‐endorphin on spinal synaptic plasticity.ConclusionThis suggests that spinal microglial expression of β‐endorphin mediates IL‐10‐ and exenatide‐induced inhibition of glutamatergic transmission and pain hypersensitivity via presynaptic and postsynaptic MORs in spinal dorsal horn.

Propofol modulates inhibitory inputs in paraventricular thalamic nucleus of mice

The mechanisms of general anaesthetics such as propofol have drawn substantial attention. The effects of propofol on inhibitory postsynaptic currents are not exactly the same in different brain nuclei. Recent studies revealed that the paraventricular thalamic nucleus (PVT) is a critical nucleus modulating wakefulness. However, the effects of propofol on PVT neurons and the mechanisms underlying such effects remain unknown. Here, we performed the whole-cell recording of the PVT neurons in acute brain slices and bath application of propofol. We found that propofol hyperpolarized the membrane potentials of the PVT neurons and suppressed the action potentials induced by step-current injection. Propofol did not affect the spontaneous inhibitory postsynaptic currents (sIPSCs) amplitude or frequency, but prolonged the sIPSCs half-width. Besides, propofol increased miniature inhibitory synaptic currents (mIPSCs) frequency and half-width. Furthermore, propofol could induce GABAA receptors-mediated tonic inhibitory currents dose-dependently. Thus, our results demonstrate that propofol hyperpolarizes PVT neurons by modulating inhibitory currents via GABAA receptors in mice.

Estradiol Regulation of the Prelimbic Cortex and the Reinstatement of Cocaine Seeking in Female Rats.

Relapse susceptibility in women with substance use disorders (SUDs) has been linked to the estrogen, 17β-estradiol (E2). Our previous findings in female rats suggest that the influence of E2 on cocaine seeking can be localized to the prelimbic prefrontal cortex (PrL-PFC). Here, we investigated the receptor mechanisms through which E2 regulates the reinstatement of extinguished cocaine seeking. Sexually mature female rats underwent intravenous cocaine self-administration (0.5 mg/inf; 14 × 2 h daily) and extinction, and then were ovariectomized before reinstatement testing. E2 (10 µg/kg, i.p.) alone did not reinstate cocaine seeking, but it potentiated reinstatement when combined with an otherwise subthreshold priming dose of cocaine. A similar effect was observed following intra-PrL-PFC microinfusions of E2 and by systemic or intra-PrL-PFC administration of the estrogen receptor (ER)β agonist, DPN, but not agonists at ERα or the G-protein-coupled ER1 (GPER1). By contrast, E2-potentiated reinstatement was prevented by intra-PrL-PFC microinfusions of the ERβ antagonist, MPP, or the GPER1 antagonist, G15, but not an ERα antagonist. Whole-cell recordings in PrL-PFC layer (L)5/6 pyramidal neurons revealed that E2 decreases the frequency, but not amplitude, of GABAA-dependent miniature IPSCs (mIPSC). As was the case with E2-potentiated reinstatement, E2 reductions in mIPSC frequency were prevented by ERβ and GPER1, but not ERα, antagonists and mimicked by ERβ, but not GPER1, agonists. Altogether, the findings suggest that E2 activates ERβ and GPER1 in the PrL-PFC to attenuate the GABA-mediated constraint of key outputs that mediate cocaine seeking.

Insulin differentially modulates GABA signalling in hippocampal neurons and, in an age-dependent manner, normalizes GABA-activated currents in the tg-APPSwe mouse model of Alzheimer's disease.

We examined if tonic γ-aminobutyric acid (GABA)-activated currents in primary hippocampal neurons were modulated by insulin in wild-type and tg-APPSwe mice, an Alzheimer's disease (AD) model. GABA-activated currents were recorded in dentate gyrus (DG) granule cells and CA3 pyramidal neurons in hippocampal brain slices, from 8 to 10weeks old (young) wild-type mice and in dorsal DG granule cells in adult, 5-6 and 10-12 (aged) months old wild-type and tg-APPSwe mice, in the absence or presence of insulin, by whole-cell patch-clamp electrophysiology. In young mice, insulin (1nmol/L) enhanced the total spontaneous inhibitory postsynaptic current (sIPSCT ) density in both dorsal and ventral DG granule cells. The extrasynaptic current density was only increased by insulin in dorsal CA3 pyramidal neurons. In absence of action potentials, insulin enhanced DG granule cells and dorsal CA3 pyramidal neurons miniature IPSC (mIPSC) frequency, consistent with insulin regulation of presynaptic GABA release. sIPSCT densities in DG granule cells were similar in wild-type and tg-APPSwe mice at 5-6months but significantly decreased in aged tg-APPSwe mice where insulin normalized currents to wild-type levels. The extrasynaptic current density was increased in tg-APPSwe mice relative to wild-type littermates but, only in aged tg-APPSwe mice did insulin decrease and normalize the current. Insulin effects on GABA signalling in hippocampal neurons are selective while multifaceted and context-based. Not only is the response to insulin related to cell-type, hippocampal axis-location, age of animals and disease but also to the subtype of neuronal inhibition involved, synaptic or extrasynaptic GABAA receptors-activated currents.

Factors of sex and age dictate the regulation of GABAergic activity by corticotropin-releasing factor receptor 1 in the medial sub-nucleus of the central amygdala

Adolescents are phenotypically characterized with hyper-sensitivity to stress and inappropriate response to stress-inducing events. Despite behavioral distinctions from adults, investigations of developmental shifts in the function of stress peptide corticotropin-releasing factor (CRF) are generally limited. Rodent models have determined that CRF receptor 1 (CRFR1) activation within the central amygdala is associated with a stress response and induces increased GABAergic synaptic neurotransmission within adult males. To investigate age- and sex-specific function of this system, we performed whole-cell patch clamp electrophysiology in brain slices from naive adolescent (postnatal days (P) 40–49) and adult (>P70) male and female Sprague Dawley rats to assess GABAergic activity in the medial central amygdala (CeM). Our results indicate a dynamic influence of age and sex on neuronal excitability within this region, as well as basal spontaneous and miniature (m) inhibitory post-synaptic currents (IPSCs) in the CeM. In addition to replicating prior findings of CRFR1-regulated increases in mIPSC frequency in adult males, we found that the selective CRFR1 agonist, Stressin-1, attenuated mIPSC frequency in adolescent males, at a concentration that did not produce an effect in adult males. Importantly, this age-specific distinction was absent in females, as Stressin-1 attenuated mIPSC frequency in both adolescent and adult females. Finally, an increase in mIPSC frequency in response to the CRF1R antagonist, NBI 35965, was observed only in the CeM of adult males. Together, these data emphasize the robust influence of age and sex on neurophysiological function of a brain region involved in the production of the stress response.