Excitatory Postsynaptic Membrane Research Articles

CNSC-39. INTEGRATED CLINICAL GENETIC ANALYSIS REVEALS NEUROTRANSMITTER RECEPTOR DYSREGULATION IN MENINGIOMAS CAUSING SEIZURE

Abstract Molecular correlates of seizures in meningioma remain unexplored. Previously, we identified MenG molecular grouping, wherein MenG C meningiomas recur more than MenG A or B. We therefore sought to identify molecular and clinical drivers of seizures in meningioma. We identified 144 primary supratentorial meningiomas, excluding those with pre-existing epilepsy. Whole exome sequencing assessed somatic mutation burden. Bulk RNA-sequencing identified differentially expressed genes and gene ontologies (GO). Immunohistochemistry confirmed protein levels. No differences in canonical gene mutation burden (NF2, TRAF7, AKT1, KLF4, SMO, SMARC) was found between seizure and non-seizure groups. MenG C tumors had higher seizure incidence compared to MenG A or B (p=0.03). Seizure causing meningiomas, though extra-axial, exhibited dysregulated neuronal signaling genes. GABAA receptor subunit genes GABRA3 and GABRG1 were downregulated in tumors causing seizure (Log2FC=-1.67, padj=0.014; Log2FC=-2.24, padj=0.009). Immunohistochemistry confirmed expression of GABRB2 subunit, the first documentation of GABA receptor expression in meningiomas. Neuropeptide Y Receptor Y1 (NPY1R) is downregulated in seizure causing meningiomas (Log2FC=-1.64, padj=0.002). NPY1R loss correlates is observed in mesial temporal lobe epilepsy and post-electroconvulsive shock mouse brains. In GO analysis, the terms “GABA signaling pathway,” “neuropeptide signaling,” “excitatory postsynaptic membrane potential,” and “ionotropic glutamate pathway,” were downregulated in meningiomas with seizure (padj<0.05). We assessed which alterations within MenG C tumors caused seizures by identifying genes associated with both seizure presentation and MenG C classification. Glutamate ionotropic receptor AMPA type subunit 1 (GRIA1) is downregulated in MenG C tumors (Log2FC=-2.29, padj=6.02E-07) and seizure causing tumors (Log2FC=-1.75, padj=0.03). Clinically, patients experiencing pre-operative seizures more frequently had cerebral edema, non-homogenous enhancement, intratumoral calcification, higher MIB-1, atypical grade 1, reduced GCS, and ER presentation; they were less likely to have incidental or headache presentation (p<0.05 for all). MenG classification predicts seizure presentation whereas somatic mutation status does not. Meningiomas causing seizures show pathologic neurotransmitter receptor dysregulation.

Exposure to 1-nitropyrene after weaning induces anxiety-like behavior partially by inhibiting steroid hormone synthesis in prefrontal cortex

1-Nitropyrene (1-NP) is a neurodevelopmental toxicant. This study was to evaluate the impact of exposure to 1-NP after weaning on anxiety-like behavior. Five-week-old mice were administered with 1-NP (0.1 or 1 mg/kg) daily for 4 weeks. Anxiety-like behaviour was measured using elevated-plus maze (EPM) and open field test (OFT). In EPM test, time spending in open arm and times entering open arm were reduced in 1-NP-treated mice. In OFT test, time spent in the center region and times entering the center region were diminished in 1-NP-treated mice. Prefrontal dendritic length and number of dendrite branches were decreased in 1-NP-treated mice. Prefrontal PSD95, an excitatory postsynaptic membrane protein, and gephyrin, an inhibitory postsynaptic membrane protein, were downregulated in 1-NP-treated mice. Further analysis showed that peripheral steroid hormones, including serum testosterone (T) and estradiol (E2), testicular T, and ovarian E2, were decreased in 1-NP-treated mice. Interestingly, T and E2 were diminished in 1-NP-treated prefrontal cortex. Prefrontal T and E2 synthases were diminished in 1-NP-treated mice. Mechanistically, GCN2-eIF2α, a critical pathway that regulates ribosomal protein translation, was activated in 1-NP-treated prefrontal cortex. These results indicate that exposure to 1-NP after weaning induces anxiety-like behaviour partially by inhibiting steroid hormone synthesis in prefrontal cortex.

Corticotropin-releasing hormone and oxytocin receptors tonically influence nTS activity in rats subjected to chronic intermittent hypoxia

Chronic intermittent hypoxia (CIH) elevates basal sympathetic nervous system activity, blood pressure, respiration and augments their responses to an acute hypoxic challenge. These physiological effects are due, in part, to the integration of several central nuclei. The nucleus tractus solitarii (nTS) is the initial site of peripheral chemoafferent integration and receives input from the paraventricular nucleus of the hypothalamus (PVN) to modulate nTS activity. PVN-to-nTS projections contain oxytocin (OT) and/or corticotropin-releasing hormone (CRH), yet their contribution to nTS activity after CIH is not well understood. We sought to determine the influence of these neuropeptides on nTS synaptic and neuronal activity, hypothesizing they individually increase nTS activity, which is magnified by their co-application, and such responses are enhanced by CIH. Male Sprague-Dawley rats (~3 wks) were exposed to 10d normoxia (21% O2) or CIH (~30s of 6% O2, 10x/hr, 8 h/day). In nTS slices, spontaneous (s) and TS-evoked excitatory postsynaptic currents (EPSCs), membrane potential and action potential discharge (APD) were recorded (n=7,9). Events were examined during OT and CRH or their receptor block (OTR-x, CRHR2-x). Overall, aCSF vehicle did not alter events over the 40-min recording, confirming their stability. Network-driven sEPSCs were enhanced in normoxic slices by only CRH, whereas CRHR2-x did not alter currents. After CIH, the elevation of sEPSCs by CRH was lost and block of OTR and CRHR2-x reduced sEPSC amplitude, indicating a greater tonic activation of these receptors on the nTS neurocircuitry. Afferent (TS)- EPSCs evoked at 0.5 and 20 Hz were not altered by OT or CRH alone nor their receptor block in normoxia. However, CIH elevated TS-EPSCs in response to CRH and CRH+OT, and this effect was greater than normoxia. Block of OTR and CRHR2 after CIH did not alter TS-EPSC amplitude, indicating their minimal tonic influence on sensory integration. Membrane potential was not altered in either group in response to OT and/or CRH application, or their receptor blockade. APD was induced in both groups by current injection. The co-application of OT and CRH increased the APD in normoxic but not CIH neurons. Within the nTS, CIH decreased the expression of OT and CRH (via immunohistochemistry, n = 3-4) and increased OTR and CRHR2 mRNA (via RNA Scope, n=3-4). In agreement with the increased OTR and CRHR2 mRNA expression, calcium imaging in primary dissociated nTS neurons demonstrated enhanced responses to OT+ CRH following CIH vs normoxia (n= 15, 17). Taken together, these data suggest that following CIH, OT and CRH have modality specific effects on nTS activity. NIH R01 HL098602 & R01 HL128454 (DDK). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pathway-Based Polygenic Risk Scores for Schizophrenia and Associations With Reported Psychotic-like Experiences and Neuroimaging Phenotypes in the UK Biobank

BackgroundSchizophrenia is a heritable psychiatric disorder with a polygenic architecture. Genome-wide association studies have reported that an increasing number of risk-associated variants and polygenic risk scores (PRSs) explain 17% of the variance in the disorder. Substantial heterogeneity exists in the effect of these variants, and aggregating them based on biologically relevant functions may provide mechanistic insight into the disorder. MethodsUsing the largest schizophrenia genome-wide association study conducted to date, we associated PRSs based on 5 gene sets previously found to contribute to schizophrenia pathophysiology—postsynaptic density of excitatory synapses, postsynaptic membrane, dendritic spine, axon, and histone H3-K4 methylation—along with respective whole-genome PRSs, with neuroimaging (n > 29,000) and reported psychotic-like experiences (n > 119,000) variables in healthy UK Biobank subjects. ResultsSeveral variables were significantly associated with the axon gene-set (psychotic-like communications, parahippocampal gyrus volume, fractional anisotropy thalamic radiations, and fractional anisotropy posterior thalamic radiations (β range −0.016 to 0.0916, false discovery rate–corrected p [pFDR] ≤ .05), postsynaptic density gene-set (psychotic-like experiences distress, global surface area, and cingulate lobe surface area [β range −0.014 to 0.0588, pFDR ≤ .05]), and histone gene set (entorhinal surface area: β = −0.016, pFDR = .035). From these, whole-genome PRSs were significantly associated with psychotic-like communications (β = 0.2218, pFDR = 1.34 × 10−7), distress (β = 0.1943, pFDR = 7.28 × 10−16), and fractional anisotropy thalamic radiations (β = −0.0143, pFDR = .036). Permutation analysis revealed that these associations were not due to chance. ConclusionsOur results indicate that genetic variation in 3 gene sets relevant to schizophrenia may confer risk for the disorder through effects on previously implicated neuroimaging variables. Because associations were stronger overall for whole-genome PRSs, findings here highlight that selection of biologically relevant variants is not yet sufficient to address the heterogeneity of the disorder.

Epigenetic mechanisms underlying the effects of triptolide and tripchlorolide on the expression of neuroligin-1 in the hippocampus of APP/PS1 transgenic mice

Context: Neuroligin-1 (NLGN1) is a cell adhesion protein located on the excitatory postsynaptic membrane. β-Amyloid (Aβ)-induced neuroinflammation decreases NLGN1 expression through epigenetic mechanisms. Triptolide (T10) and tripchlorolide (T4) exert protective effects on synapses in Alzheimer's disease (AD) mice, but the mechanisms remain unclear.Objective: The effects of T10 and T4 on hippocampal NLGN1 expression in AD mice and the epigenetic mechanisms were assessed using chromatin immunoprecipitation and methylated DNA immunoprecipitation.Materials and methods: Sixty APP/PS1 transgenic mice were randomly divided into an AD model group, a T10-treated group and a T4-treated group (n = 20); 20 wild-type littermates served as the control group. APP/PS1 transgenic mice were intraperitoneally injected with T10 (0.1 mg/kg) and T4 (25 μg/kg) once per day for 60 days. NLGN1 expression was examined using western blotting and quantitative PCR.Results: T10 and T4 increased the levels of the NLGN1 protein and mRNA in hippocampus of AD mice. T10 and T4 inhibited the binding of HDAC2 (p< 0.01) and MeCP2 (p< 0.01 and p< 0.05, respectively) to the NLGN1 promoter, and cytosine methylation (1.2305 ± 0.1482/1.2554 ± 0.3570 vs. 1.6578 ± 0.1818, p< 0.01) at the NLGN1 promoter in the hippocampus of AD mice. T10 and T4 increased the level of acetylated histone H3 (0.7733 ± 0.1611/0.8241 ± 0.0964 vs. 0.5587 ± 0.0925, p< 0.01) at the NLGN1 promoter in the hippocampus of AD mice.Conclusions: T10 and T4 may increase hippocampal NLGN1 expression in AD mice through epigenetic mechanisms, providing a new explanation for the mechanism underlying the protective effects of T10 and T4 on synapses.

Direct activation of G-protein-gated inward rectifying K+ channels promotes nonrapid eye movement sleep.

A major challenge in treating insomnia is to find effective medicines with fewer side effects. Activation of G-protein-gated inward rectifying K+ channels (GIRKs) by GABAB agonists baclofen or γ-hydroxybutyric acid (GHB) promotes nonrapid eye movement (NREM) sleep and consolidates sleep. However, baclofen has poor brain penetration, GHB possesses abuse liability, and in rodents both drugs cause spike-wave discharges (SWDs), an absence seizure activity. We tested the hypothesis that direct GIRK activation promotes sleep without inducing SWD using ML297, a potent and selective GIRK activator. Whole-cell patch-clamp recordings from hypocretin/orexin or hippocampal neurons in mouse brain slices were made to study neuronal excitability and synaptic activity; spontaneous activity, locomotion, contextual and tone-conditioned memory, and novel object recognition were assessed. Electroencephalogram/electromyogram (EEG/EMG) recordings were used to study GIRK modulation of sleep. ML297, like baclofen, caused membrane hyperpolarization, decreased input resistance, and blockade of spontaneous action potentials. Unlike baclofen, ML297 (5-10 µM) did not cause significant depression of postsynaptic excitatory and inhibitory currents (EPSCs-IPSCs), indicating preferential postsynaptic inhibition. ML297 (30 mg/kg, i.p.) inhibited wake activity and locomotion, and preferentially increased NREM sleep without altering EEG delta power, REM sleep, inducing SWDs, or impairing conditioned memory and novel object recognition. This study finds that direct activation of neuronal GIRK channels modulates postsynaptic membrane excitability and prolongs NREM sleep without changing sleep intensity, inducing SWDs, or impairing memory in rodents. These results suggest that direct GIRK activation with a selective compound may present an innovative approach for the treatment of chronic insomnia.

Changes in expression of neuroligin1 in excitatory postsynaptic membrane of spinal dorsal horn in a rat model of incisional pain

Objective To evaluate the changes in the expression of neuroligin1 in excitatory postsynaptic membrane of the spinal dorsal horn in a rat model of incisional pain. Methods Forty-eight pathogen-free healthy adult male Sprague-Dawley rats, aged 6-8 weeks, weighing 280-320 g, were divided into control group(group C, n=12)and incisional pain group(group I, n=36)using a random number table.A 1 cm long incision was made in the plantar surface of the right hindpaw in group I. Cumulative pain score(CPS)was assessed and mechanical paw withdrawal threshold to von Frey stimuli was measured at 3 h and 1 and 3 days after operation(T1, 2, 3). The animals were then sacrificed and their lumbar segments(L3-6)of the spinal cord were removed for detection of the expression of neuroligin1, postsynaptic density-95 protein(PSD-95), glutamate receptor 1(GluR1)and GluR2 in the postsynaptic membrane of spinal dorsal horn(by Western blot)and co-expression of neuroligin1 with PSD-95 in spinal dorsal horn(by co-immuno-precipitation). Results Compared with group C, CPS was significantly increased and mechanical paw withdrawal threshold was decreased at T1-3, and the expression of neuroligin1 and GluR1 in the postsynaptic membrane of spinal dorsal horn at T1, 2 and co-expression of neuroligin1 with PSD-95 at T1 were up-regulated in group I(P<0.01 or 0.05). Conclusion The development and maintenance of incisional pain is related to the signaling pathway regulated by neuroligin1 in excitatory postsynaptic membrane of the spinal dorsal horn of rats. Key words: Cell Adhesion Molecules; Pain; Spinal cord; Receptors, AMPA

Bioinformatics analysis of differently expressed microRNAs in anxiety disorder

OBJECTIVE To identify differentially expressed microRNA (miRNA) in peripheral blood mononuclear cells (PBMCs) of anxiety patients and predict their target genes and function by bioinformatics analysis. METHODS The miRNA expression profiles were determined using an Affymetrix array. To validate the results, real-time quantitative polymerase chain reaction (qRT-PCR) analysis in a larger cohort was employed. The targets of the differentially expressed miRNAs were predicted by Target Scan, miRBD, and DIANA-microT-CDS, and the results were analyzed by gene ontology (GO) and KEGG pathway analysis using FunNet. RESULTS MicroRNA microarray chip analysis has identified 7 miRNAs were detected with significant changes in expression in PBMCs of anxiety patients. qRT-PCR analysis has confirmed that the expression levels of 5 miRNAs (has-miR-4484, has-miR-4505, has-miR-4674, has-miR-501-3p and has-miR-663) were up-regulated. Intersecting the genes by Target Scan, miRBD, and DIANA-microT-CDS has predicted 195 targets. GO analysis showed that biological processes regulated by the predicted target genes have included diverse terms. Some terms, e.g., nervous system development, nerve growth factor receptor signaling pathway, neuron migration, dendrite development, regulation of neuron projection development, midbrain development, regulation of excitatory postsynaptic membrane potential, gliogenesis, dendrite morphogenesis, etc. have direct relationship with the central nervous system and brain functions. Pathway analysis showed that a significant enrichment in several pathways related to neuronal brain functions such as glutamatergic synapse, axon guidance, calcium signaling pathway, MAPK signaling pathway, GnRH signaling pathway, Wnt signaling pathway, gap junction, long-term potentiation and VEGF signaling pathway, etc. Among the five microRNAs, has-miR-4484, has-miR-4505, has-miR-4674 and has-miR-501-3p may have more important regulatory functions. CONCLUSION Five miRNAs (has-miR-4484, has-miR-4505, has-miR-4674, has-miR-501-3p and has-miR-663) are up-regulated in PBMCs of anxiety patients and may be closely involved in the pathogenesis of anxiety disorder.

Leukotriene enhances NMDA-induced inward currents in dorsal horn neurons of the rat spinal cord after peripheral nerve injury.

BackgroundLTB4 is classified as a leukotriene (LT), a group of lipid mediators that are derived from arachidonic acid. It is recognized that leukotrienes are involved in the pathogenesis of many diseases, including peripheral inflammatory pain. However, little is known about the effects of leukotrienes on the spinal dorsal horn during neuropathic pain. Previously, we reported that there was increased expression of 5-lipoxygenase (5-LO) at spinal microglia, and the leukotriene B4 receptor 1 (BLT1), a high affinity receptor of LTB4, in spinal neurons in spared nerve injury (SNI) model rats. In the present study, we examined the effects of LTB4 on spinal dorsal horn neurons in both naïve and SNI model rats using patch-clamp methods.ResultsBath application of LTB4 did not change AMPA receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs) or membrane potentials. However, we found that LTB4 enhanced the amplitude of NMDA receptor-mediated sEPSCs and significantly increased exogenous NMDA-induced inward currents in SNI model rats. This increase of inward currents could be inhibited by a selective LTB4 antagonist, U75302, as well as a GDP-β-S, a G-protein inhibitor. These results indicate that both increased LTB4 from spinal microglia or increased BLT1 in spinal neurons after peripheral nerve injury can enhance the activity of NMDA receptors through intracellular G-proteins in spinal dorsal horn neurons.ConclusionOur findings showed that LTB4, which may originate from microglia, can activate BLT1 receptors which are expressed on the membrane of spinal dorsal horn neurons during neuropathic pain. This glia-neuron interaction induces the enhancement of NMDA currents through intracellular G-proteins. The enhancement of NMDA receptor sensitivity of dorsal horn neurons may lead to central sensitization, leading to mechanical pain hypersensitivity.

NMDAR-Mediated Hippocampal Neuronal Death is Exacerbated by Activities of ASIC1a.

NMDARs and ASIC1a both exist in central synapses and mediate important physiological and pathological conditions, but the functional relationship between them is unclear. Here we report several novel findings that may shed light on the functional relationship between these two ion channels in the excitatory postsynaptic membrane of mouse hippocampus. Firstly, NMDAR activation induced by either NMDA or OGD led to increased [Ca(2+)](i)and greater apoptotic and necrotic cell deaths in cultured hippocampal neurons; these cell deaths were prevented by application of NMDAR antagonists. Secondly, ASIC1a activation induced by pH 6.0 extracellular solution (ECS) showed similar increases in apoptotic and necrotic cell deaths; these cell deaths were prevented by ASIC1a antagonists, and also by NMDAR antagonists. Since increased [Ca(2+)](i)leads to increased cell deaths and since NMDAR exhibits much greater calcium permeability than ASIC1a, these data suggest that ASIC1a-induced neuronal death is mediated through activation of NMDARs. Thirdly, treatment of hippocampal cultures with both NMDA and acidic ECS induced greater degrees of cell deaths than either NMDA or acidic ECS treatment alone. These results suggest that ASIC1a activation up-regulates NMDAR function. Additional data supporting the functional relationship between ASIC1a and NMDAR are found in our electrophysiology experiments in hippocampal slices, where stimulation of ASIC1a induced a marked increase in NMDAR EPSC amplitude, and inhibition of ASIC1a resulted in a decrease in NMDAR EPSC amplitude. In summary, we present evidence that ASIC1a activity facilitates NMDAR function and exacerbates NMDAR-mediated neuronal death in pathological conditions. These findings are invaluable to the search for novel therapeutic targets in the treatment of brain ischemia.

The effect of desflurane on neuronal communication at a central synapse.

Although general anesthetics are thought to modify critical neuronal functions, their impact on neuronal communication has been poorly examined. We have investigated the effect induced by desflurane, a clinically used general anesthetic, on information transfer at the synapse between mossy fibers and granule cells of cerebellum, where this analysis can be carried out extensively. Mutual information values were assessed by measuring the variability of postsynaptic output in relationship to the variability of a given set of presynaptic inputs. Desflurane synchronized granule cell firing and reduced mutual information in response to physiologically relevant mossy fibers patterns. The decrease in spike variability was due to an increased postsynaptic membrane excitability, which made granule cells more prone to elicit action potentials, and to a strengthened synaptic inhibition, which markedly hampered membrane depolarization. These concomitant actions on granule cells firing indicate that desflurane re-shapes the transfer of information between neurons by providing a less informative neurotransmission rather than completely silencing neuronal activity.

The CNS glucagon-like peptide-2 receptor in the control of energy balance and glucose homeostasis.

The gut-brain axis plays a key role in the control of energy balance and glucose homeostasis. In response to luminal stimulation of macronutrients and microbiota-derived metabolites (secondary bile acids and short chain fatty acids), glucagon-like peptides (GLP-1 and -2) are cosecreted from endocrine L cells in the gut and coreleased from preproglucagonergic neurons in the brain stem. Glucagon-like peptides are proposed as key mediators for bariatric surgery-improved glycemic control and energy balance. Little is known about the GLP-2 receptor (Glp2r)-mediated physiological roles in the control of food intake and glucose homeostasis, yet Glp1r has been studied extensively. This review will highlight the physiological relevance of the central nervous system (CNS) Glp2r in the control of energy balance and glucose homeostasis and focuses on cellular mechanisms underlying the CNS Glp2r-mediated neural circuitry and intracellular PI3K signaling pathway. New evidence (obtained from Glp2r tissue-specific KO mice) indicates that the Glp2r in POMC neurons is essential for suppressing feeding behavior, gastrointestinal motility, and hepatic glucose production. Mice with Glp2r deletion selectively in POMC neurons exhibit hyperphagic behavior, accelerated gastric emptying, glucose intolerance, and hepatic insulin resistance. GLP-2 differentially modulates postsynaptic membrane excitability of hypothalamic POMC neurons in Glp2r- and PI3K-dependent manners. GLP-2 activates the PI3K-Akt-FoxO1 signaling pathway in POMC neurons by Glp2r-p85α interaction. Intracerebroventricular GLP-2 augments glucose tolerance, suppresses glucose production, and enhances insulin sensitivity, which require PI3K (p110α) activation in POMC neurons. Thus, the CNS Glp2r plays a physiological role in the control of food intake and glucose homeostasis. This review will also discuss key questions for future studies.

Glutamate gated spiking Neuron Model.

Biological neuron models mainly analyze the behavior of neural networks. Neurons are described in terms of firing rates viz an analog signal. The Izhikevich neuron model is an efficient, powerful model of spiking neuron. This model is a reduction of Hodgkin-Huxley model to a two variable system and is capable of producing rich firing patterns for many biological neurons. In this paper, the Regular Spiking (RS) neuron firing pattern is used to simulate the spiking of Glutamate gated postsynaptic membrane. Simulation is done in MATLAB environment for excitatory action of synapses. Analogous simulation of spiking of excitatory postsynaptic membrane potential is obtained.

Live-cell imaging of receptors around postsynaptic membranes

This protocol describes how to image the trafficking of glutamate receptors around excitatory postsynaptic membrane formed on an adhesion protein-coated glass surface. The protocol was developed to clarify how receptors move during the induction of synaptic plasticity. Dissociated neurons are cultured on a coverslip coated with neurexin, which induces the formation of postsynaptic membrane-like structures on the glass surface. A glutamate receptor tagged with a fluorescent protein is then transfected into neurons, and it is observed with total internal reflection fluorescence microscopy. The whole process takes about 3 weeks. Changes in the amount of cell-surface receptors caused by neuronal activities can be quantified, and individual exocytosis events of receptors can be clearly observed around the pseudo-postsynaptic membrane. This protocol has potential applications for studies of movements of membrane proteins around other specialized regions of the cell membrane, such as the inhibitory postsynaptic membrane, the presynaptic membrane or the immunological synapses.

Hydrogen sulfide augments synaptic neurotransmission in the nucleus of the solitary tract.

Within the brain stem, the nucleus tractus solitarii (NTS) serves as a principal central site for sensory afferent integration from the cardiovascular and respiratory reflexes. Neuronal activity and synaptic transmission in the NTS are highly pliable and subject to neuromodulation. In the central nervous system, hydrogen sulfide (H₂S) is a gasotransmitter generated primarily by the enzyme cystathionine-β-synthase (CBS). We sought to determine the role of H₂S, and its generation by CBS, in NTS excitability. Real-time RT-PCR, immunoblot, and immunohistochemistry analysis identified the presence of CBS in the NTS. Patch-clamp electrophysiology in brain stem slices examined excitatory postsynaptic currents (EPSCs) and membrane properties in monosynaptically driven NTS neurons. Confocal imaging of labeled afferent synaptic terminals in NTS slices monitored intracellular calcium. Exogenous H₂S significantly increased the amplitude of evoked solitary tract (TS)-EPSCs, frequency of miniature (m)EPSCs, and presynaptic terminal calcium fluorescence in the NTS. H₂S did not alter action potential discharge or postsynaptic properties. On the other hand, the CBS inhibitor aminooxyacetate (AOA) significantly reduced the amplitude of TS-EPSCs and presynaptic terminal calcium fluorescence in the NTS without altering postsynaptic properties. Taken together, these data support a presynaptic role for endogenous H₂S in modulation of excitatory neurotransmission in the NTS.

Differentially expressed genes for aggressive pecking behaviour in laying hens

BackgroundAggressive behaviour is an important aspect in the daily lives of animals living in groups. Aggressive animals have advantages, such as better access to food or territories, and they produce more offspring than low ranking animals. The social hierarchy in chickens is measured using the 'pecking order' concept, which counts the number of aggressive pecks given and received. To date, little is known about the underlying genetics of the 'pecking order'.ResultsA total of 60 hens from a high feather pecking selection line were divided into three groups: only receivers (R), only peckers (P) and mixed peckers and receivers (P&R). In comparing the R and P groups, we observed that there were 40 differentially expressed genes [false discovery rate (FDR) P < 0.10]. It was not fully clear how the 40 genes regulated aggressive behaviour; however, gene set analysis detected a number of GO identifiers, which were potentially involved in aggressive behavioural processes. These genes code for synaptosomes (GO:0019797), and proteins involved in the regulation of the excitatory postsynaptic membrane potential (GO:0060079), the regulation of the membrane potential (GO:0042391), and glutamate receptor binding (GO:0035254).ConclusionIn conclusion, our study provides new insights into which genes are involved in aggressive behaviours in chickens. Pecking and receiving hens exhibited different gene expression profiles in their brains. Following confirmation, the identification of differentially expressed genes may elucidate how the pecking order forms in laying hens at a molecular level.

Fentanyl treatment reduces GABAergic inhibition in the CA1 area of the hippocampus 24 h after acute exposure to the drug

The effect of in vivo fentanyl treatment on synaptic transmission was studied in the CA1 area of the rat hippocampus. Animals were treated either with saline or fentanyl (4 × 80 μg/kg, s.c./15 min). Intracellular in vitro recordings were obtained, 24 h after treatment, from CA1 pyramidal neurons. No difference in pyramidal neuron basic membrane properties or postsynaptic membrane excitability was observed between neurons from saline- and fentanyl-treated animals. The peak amplitude of fast (f-) and slow (s-) components of IPSPs elicited in standard ACSF and the peak amplitude and rate of rise of isolated f- and s-IPSPs elicited in the presence of antagonists (CNQX, 10 μM; AP-5, 10 μM; CGP 55845, 1 μM; and bicuculline methochloride, 10 μM), in response to various stimulus intensities, was smaller in fentanyl-treated animals. Conversely, the rising slope of excitatory responses was similar in neurons from saline- and fentanyl-treated animals. Furthermore, in fentanyl-treated animals, lower stimulus strengths were required to elicit subthreshold excitatory responses of the same amplitude suggesting that acute exposure to fentanyl increases susceptibility of pyramidal neurons to presynaptic stimulation. GABA immunohistochemistry revealed lower GABA content in processes and neuronal somata suggesting diminished GABA release onto pyramidal neurons. We conclude that acute in vivo exposure to fentanyl is sufficient to induce long-lasting reduction in GABA-mediated transmission, rather, than enhanced excitatory transmission or modulation of the intrinsic excitability of pyramidal neurons. These findings provide evidence regarding the mechanisms involved in the early stages of tolerance development towards the analgesic effects of opioids.

PKC-dependent autoregulation of membrane kainate receptors

Agonists of kainate receptors (KARs) cause both the opening of the associated ion channels and the activation of signalling pathways driven by G-proteins and PKC. Here we report the existence of an unknown mechanism of KAR autoregulation, involving the interplay of this two signalling mechanisms. Repetitive activation of native KARs evoked the rundown of the ionotropic responses in a manner that was dependent on the activation of PKC. Experiments on recombinant GluR5 expressed in neuroblastoma cells indicated that KARs trigger the activation of PKC and induce the internalization of membrane receptors. This phenomenon depends on the PKC-mediated phosphorylation of serines 879 and 885 of the GluR5-2b subunits, since mutation of these two residues abolished internalization. These results reveal that the non-canonical signalling of KARs is associated with a sensitive mechanism that detects afferent activity. Such a mechanism represents an active way to limit overactivation of the KAR system, by regulating the number of KARs in the cell membrane.

Facilitation of glutamatergic synaptic transmission in hippocampal CA1 area of rats with traumatic brain injury

We investigated the effects of traumatic brain injury (TBI) on the glutamatergic synaptic transmission in the hippocampal CA1 area. A moderate impact (3.8–4.8 atm) was applied onto the left parietal cerebral cortex by a fluid percussion injury (FPI) device. Conventional intracellular recordings were made from hippocampal CA1 pyramidal neurons in vitro. Electrophysiological properties of these neurons were compared between three groups (control, FPI-ipsilateral, and FPI-contralateral). The excitability of postsynaptic membrane of CA1 pyramidal neurons was not altered by the moderate FPI; however, the evoked glutamatergic excitatory synaptic transmission in the pyramidal neurons of post-FPI-CA1 was enhanced. Paired-pulse facilitation (PPF) was significantly suppressed in both the FPI-ipsilateral and FPI-contralateral groups and the frequencies of mEPSPs in neurons from the bilateral FPI groups were greater than the frequency in the control group. These results suggest that the glutamatergic synaptic transmission in the hipppocampal CA1 area is facilitated through presynaptic mechanisms after TBI.

Dissection of synaptic excitability phenotypes by using a dominant-negative Shaker K + channel subunit

During nervous system development, synapses undergo morphological change as a function of electrical activity. In Drosophila, enhanced activity results in the expansion of larval neuromuscular junctions. We have examined whether these structural changes involve the pre- or postsynaptic partner by selectively enhancing electrical excitability with a Shaker dominant-negative (SDN) potassium channel subunit. We find that the SDN enhances neurotransmitter release when expressed in motoneurons, postsynaptic potential broadening when expressed in muscles and neurons, and selectively suppresses fast-inactivating, Shaker-mediated IA currents in muscles. SDN expression also phenocopies the canonical behavioral phenotypes of the Sh mutation. At the neuromuscular junction, we find that activity-dependent changes in arbor size occur only when SDN is expressed presynaptically. This finding indicates that elevated postsynaptic membrane excitability is by itself insufficient to enhance presynaptic arbor growth. Such changes must minimally involve increased neuronal excitability.