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PubMed2001

Reliability of monosynaptic sensory transmission in brain stem neurons in vitro.

The timing of events within the nervous system is a critical feature of signal processing and integration. In neurotransmission, the synaptic latency, the time between stimulus delivery and appearance of the synaptic event, is generally thought to be directly related to the complexity of that pathway. In horizontal brain stem slices, we examined synaptic latency and its shock-to-shock variability (synaptic jitter) in medial nucleus tractus solitarius (NTS) neurons in response to solitary tract (ST) electrical activation. Using a visualized patch recording approach, we activated ST 1-3 mm from the recorded neuron with short trains (50-200 Hz) and measured synaptic currents under voltage clamp. Latencies ranged from 1.5 to 8.6 ms, and jitter values (SD of intraneuronal latency) ranged from 26 to 764 micros (n = 49). Surprisingly, frequency of synaptic failure was not correlated with either latency or jitter (P > 0.147; n = 49). Despite conventional expectations, no clear divisions in latency were found from the earliest arriving excitatory postsynaptic currents (EPSCs) to late pharmacologically polysynaptic responses. Shortest latency EPSCs (<3 ms) were mediated by non-N-methyl-D-aspartate (non-NMDA) glutamate receptors. Longer latency responses were a mix of excitatory and inhibitory currents including non-NMDA EPSCs and GABAa receptor-mediated currents (IPSC). All synaptic responses exhibited prominent frequency-dependent depression. In a subset of neurons, we labeled sensory boutons by the anterograde fluorescent tracer, DiA, from aortic nerve baroreceptors and then recorded from anatomically identified second-order neurons. In identified second-order NTS neurons, ST activation evoked EPSCs with short to moderate latency (1.9-4.8 ms) but uniformly minimal jitter (31 to 61 micros) that were mediated by non-NMDA receptors but had failure rates as high as 39%. These monosynaptic EPSCs in identified second-order neurons were significantly different in latency and jitter than GABAergic IPSCs (latency, 2.95 +/- 0.71 vs. 5.56 +/- 0.74 ms, mean +/- SE, P = 0.027; jitter, 42.3 +/- 6.5 vs. 416.3 +/- 94.4 micros, P = 0.013, n = 4, 6, respectively), but failure rates were similar (27.8 +/- 9.0 vs. 9.7 +/- 4.4%, P = 0.08, respectively). Such results suggest that jitter and not absolute latency or failure rate is the most reliable discriminator of mono- versus polysynaptic pathways. The results suggest that brain stem sensory pathways may differ in their principles of integration compared with cortical models and that this importantly impacts synaptic performance. The unique performance properties of the sensory-NTS pathway may reflect stronger axosomatic synaptic processing in brain stem compared with dendritically weighted models typical in cortical structures and thus may reflect very different strategies of spatio-temporal integration in this NTS region and for autonomic regulation.

Action Potentials↗
PubMed2001

Contribution of the hyperpolarization-activated current (I(h)) to membrane potential and GABA release in hippocampal interneurons.

Intrinsic GABAergic interneurons provide inhibitory input to the principal neurons of the hippocampus. The majority of interneurons located in stratum oriens (s.o.) of the CA1 region express the hyperpolarization-activated cation current known as I(h). In an effort to elucidate the role of this current in regulating the baseline excitability of these neurons and its participation in the regulation of the release of GABA onto CA1 pyramidal neurons, we utilized whole cell electrophysiological recordings from both populations of cells. In voltage-clamp experiments, hyperpolarization of the interneuron membrane initiated a large inward current with an estimated activation threshold of 51.6 +/- 7.6 mV and a half-maximal voltage of -73.0 +/- 7.0 mV. This current was blocked by bath application of the I(h) inhibitors ZD 7288 (50 microM) or cesium (2 mM). Current-clamp experiments at the interneuron resting membrane potential (-61.3 +/- 1.2 mV) revealed a significant hyperpolarization, a decrease in the rate of spontaneous action potential discharge, an increase in the cellular input resistance, and the elimination of rebound afterdepolarizations during blockade of I(h) with ZD 7288 (50 microM). The hyperpolarizing effect of ZD 7288 was also substantially larger in interneurons clamped near -80 mV using current injection through the pipette. In addition to neurons exhibiting I(h), recordings were obtained from a small population of s.o. interneurons that did not exhibit this current. These cells demonstrated resting membrane potentials that were significantly more negative (-73.6 +/- 5.5 mV) than those observed in neurons expressing I(h), suggesting that this current contributes to more depolarized membrane potentials in these cells. Recordings from postsynaptic pyramidal neurons demonstrated that blockade of I(h) with ZD 7288 caused a substantial reduction (approximately 43%) in the frequency of spontaneous action potential-dependent inhibitory postsynaptic currents (IPSCs), without altering their average amplitude. However, miniature action-potential-independent IPSC frequency, amplitude, and decay kinetics were unaltered by ZD 7288. These data suggest that I(h) is active at the resting membrane potential in s.o. interneurons and as a result contributes to the spontaneous activity of these cells and to the tonic inhibition of CA1 pyramidal neurons in the hippocampus.

Animals↗
PubMed2001

Electrophysiological evidence for vasopressin V(1) receptors on neonatal motoneurons, premotor and other ventral horn neurons.

Prominent arginine-vasopressin (AVP) binding and AVP V(1) type receptors are expressed early in the developing rat spinal cord. We sought to characterize their influence on neural excitability by using patch-clamp techniques to record AVP-induced responses from a population of motoneurons and interneurons in neonatal (5-18 days) rat spinal cord slices. Data were obtained from 58 thoracolumbar (T(7)-L(5)) motoneurons and 166 local interneurons. A majority (>90%) of neurons responded to bath applied AVP (10 nM to 3 microM) and (Phe(2), Orn(8))-vasotocin, a V(1) receptor agonist, but not V(2) or oxytocin receptor agonists. In voltage-clamp, postsynaptic responses in motoneurons were characterized by slowly rising, prolonged (7-10 min) and tetrodotoxin-resistant inward currents associated with a 25% reduction in a membrane potassium conductance that reversed near -100 mV. In interneurons, net AVP-induced inward currents displayed three patterns: decreasing membrane conductance with reversal near -100 mV, i.e., similar to that in motoneurons (24 cells); increasing conductance with reversal near -40 mV (21 cells); small reduction in conductance with no reversal within the current range tested (41 cells). A presynaptic component recorded in most neurons was evident as an increase in the frequency but not amplitude (in motoneurons) of inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs), in large part due to AVP-induced firing in inhibitory (mainly glycinergic) and excitatory (glutamatergic) neurons synapsing on the recorded cells. An increase in frequency but not amplitude of miniature IPSCs and EPSCs also indicated an AVP enhancement of neurotransmitter release from axon terminals of inhibitory and excitatory interneurons. These observations provide support for a broad presynaptic and postsynaptic distribution of AVP V(1) type receptors and indicate that their activation can enhance the excitability of a majority of neurons in neonatal ventral spinal cord.

2-Amino-5-phosphonovalerate↗
PubMed2001

Kinetic and pharmacological properties of GABA(A) receptors in single thalamic neurons and GABA(A) subunit expression.

Synaptic inhibition in the thalamus plays critical roles in sensory processing and thalamocortical rhythm generation. To determine kinetic, pharmacological, and structural properties of thalamic gamma-aminobutyric acid type A (GABA(A)) receptors, we used patch-clamp techniques and single-cell reverse transcriptase polymerase chain reaction (RT-PCR) in neurons from two principal rat thalamic nuclei-the reticular nucleus (nRt) and the ventrobasal (VB) complex. Single-channel recordings identified GABA(A) channels with densities threefold higher in VB than nRt neurons, and with mean open time fourfold longer for nRt than VB [14.6 +/- 2.5 vs. 3.8 +/- 0.7 (SE) ms, respectively]. GABA(A) receptors in nRt and VB cells were pharmacologically distinct. Zn(2+) (100 microM) reduced GABA(A) channel activity in VB and nRt by 84 and 24%, respectively. Clonazepam (100 nM) increased inhibitory postsynaptic current (IPSC) decay time constants in nRt (from 44.3 to 77.9 ms, P < 0.01) but not in VB. Single-cell RT-PCR revealed subunit heterogeneity between nRt and VB cells. VB neurons expressed alpha1-alpha3, alpha5, beta1-3, gamma2-3, and delta, while nRt cells expressed alpha3, alpha5, gamma2-3, and delta. Both cell types expressed more subunits than needed for a single receptor type, suggesting the possibility of GABA(A) receptor heterogeneity within individual thalamic neurons. beta subunits were not detected in nRt cells, which is consistent with very low levels reported in previous in situ hybridization studies but inconsistent with the expected dependence of functional GABA(A) receptors on beta subunits. Different single-channel open times likely underlie distinct IPSC decay time constants in VB and nRt cells. While we can make no conclusion regarding beta subunits, our findings do support alpha subunits, possibly alpha1 versus alpha3, as structural determinants of channel deactivation kinetics and clonazepam sensitivity. As the gamma2 and delta subunits previously implicated in Zn(2+) sensitivity are both expressed in each cell type, the observed differential Zn(2+) actions at VB versus nRt GABA(A) receptors may involve other subunit differences.

Animals↗
PubMed2026

The Charcot-Marie-Tooth Neuropathy (CMTX3) Complex Structural Variation Causes Differential SOX3 Spatiotemporal Expression.

Charcot-Marie-Tooth (CMT) neuropathy is a clinically and genetically heterogeneous group of diseases characterized by the length-dependent axonal degeneration of peripheral nerves. We previously mapped a rare form of X-linked CMT, CMTX3, to a 5.7-Mb interval on chromosome Xq26.3-q27.1 and excluded the coding region of all known genes in the linkage interval for mutations. Whole genome sequencing subsequently identified a 78-kb region of chromosome 8q24.3 that had been duplicated and inserted into the CMTX3 locus between the genes HAPSTR2 and SOX3. The 78-kb insertion, which contains a partial transcript of ARHGAP39, fully segregated in families with CMTX3 and was absent in neurologically normal controls. To retain the CMTX3 insertion and investigate its consequences in appropriate neuronal tissue, we generated induced pluripotent stem cells (iPSCs) from CMTX3 fibroblasts. Using bulk RNA sequencing of patient-derived spinal motor neurons, ARHGAP39 was deemed nonpathogenic by excluding both the formation of novel fusion transcripts and dosage effects from the partial duplication. Subsequent NanoString expression analyses of candidate genes within the CMTX3 locus, across different stages of neuronal differentiation, identified spatiotemporal dysregulation of SOX3. NanoString showed reduced SOX3 expression in patient iPSCs. RNA sequencing detected SOX3 downregulation in CMTX3 neuroepithelial progenitor cells, which was further confirmed by quantitative proteomics. Given the early onset and relatively rapid progression of CMTX3, these data prioritise SOX3 as a leading candidate gene, consistent with its role as one of the earliest transcription factors expressed in the developing nervous system and a key regulator of neuronal fate.

Humans↗
PubMed1989

Oxiracetam in the treatment of primary degenerative and multi-infarct dementia: a double-blind, placebo-controlled study.

A multicentre, double-blind, between-patient study was carried out to evaluate the efficacy and tolerability of oxiracetam (800-mg tablets), in comparison with placebo, each given twice daily for 12 weeks to patients suffering from primary degenerative, multi-infarct or mixed forms of dementia. Efficacy was assessed by the Inventory of Psychic and Somatic Complaints in the Elderly (IPSC-E), administered at entry and after 4, 8 and 12 weeks of treatment, and by the Blessed Dementia Scale and the Newcastle Memory, Information and Concentration Scale (NMICS), administered at the beginning and at the end of the study. Three hundred and seven patients were enrolled, 18 of whom were excluded from the analysis because of violation of the protocol. Two hundred and eighty-nine patients were analyzed (145 m, 144 f, mean age 73 years) and 272 completed the study; 3 patients in each treatment group were withdrawn because of poor tolerability, 10 because of poor compliance and 1 patient because of the occurrence of a cerebral stroke. A significantly (p less than 0.01) different effect, in favor of oxiracetam, was observed in the three main efficacy criteria (i.e. IPSC-E, Blessed Dementia Scale and NMIC total scores), and confirmed by descriptive analyses carried out on some subitems of the scales used. Thirty-one patients on oxiracetam and 27 on placebo complained of a total of 35 and 32 minor unwanted effects, respectively. No clinically or statistically significant changes were observed on routine laboratory examinations.

Aged↗
PubMed2005

Propofol suppresses synaptic responsiveness of somatosensory relay neurons to excitatory input by potentiating GABA(A) receptor chloride channels.

Propofol is a widely used intravenous general anesthetic. Propofol-induced unconsciousness in humans is associated with inhibition of thalamic activity evoked by somatosensory stimuli. However, the cellular mechanisms underlying the effects of propofol in thalamic circuits are largely unknown. We investigated the influence of propofol on synaptic responsiveness of thalamocortical relay neurons in the ventrobasal complex (VB) to excitatory input in mouse brain slices, using both current- and voltage-clamp recording techniques. Excitatory responses including EPSP temporal summation and action potential firing were evoked in VB neurons by electrical stimulation of corticothalamic fibers or pharmacological activation of glutamate receptors. Propofol (0.6 - 3 microM) suppressed temporal summation and spike firing in a concentration-dependent manner. The thalamocortical suppression was accompanied by a marked decrease in both EPSP amplitude and input resistance, indicating that a shunting mechanism was involved. The propofol-mediated thalamocortical suppression could be blocked by a GABAA receptor antagonist or chloride channel blocker, suggesting that postsynaptic GABAA receptors in VB neurons were involved in the shunting inhibition. GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) were evoked in VB neurons by electrical stimulation of the reticular thalamic nucleus. Propofol markedly increased amplitude, decay time, and charge transfer of GABAA IPSCs. The results demonstrated that shunting inhibition of thalamic somatosensory relay neurons by propofol at clinically relevant concentrations is primarily mediated through the potentiation of the GABAA receptor chloride channel-mediated conductance, and such inhibition may contribute to the impaired thalamic responses to sensory stimuli seen during propofol-induced anesthesia.

Anesthetics, Intravenous↗
PubMed2026

Tumor-like proliferation of CCM3 knockout endothelial cells: insights from semaxinib treatment and transcriptome profiling of co-cultures.

Cerebral cavernous malformations (CCMs) are vascular lesions associated with severe neurological complications. Increasing evidence suggests that cancer-like mechanisms, like an abnormal expansion of CCM3 knockout (KO) endothelial cells (ECs) in co-culture with wild-type (WT) cells, contribute to lesion formation. Yet, the underlying processes remain poorly understood. Here, we employed a human induced pluripotent stem cell (iPSC)-derived EC co-culture model to screen a cytokine inhibitor library for modulators of this tumor-like behavior. We identified the known VEGFR2 inhibitor semaxinib which selectively suppressed proliferation of WT ECs in co-culture, but not in monoculture. In contrast, CCM3 KO cells maintained their abnormal expansion under semaxinib treatment which was unaffected by modulation of extracellular VEGFA levels. RNA-seq profiling revealed distinct transcriptional responses to semaxinib including extracellular matrix remodeling, stress signaling, and overexpression of growth factors and receptors in CCM3 KO cells, which may contribute to their survival advantage. These findings advance our understanding of the complex interplay between WT and KO cells in CCM pathogenesis and demonstrate that the proliferative advantage of CCM3-deficient cells is not solely driven by CCM3 loss. Finally, our iPSC-based EC co-culture assay provides a scalable platform to study KO/WT interactions and may accelerate the identification of effective therapeutic strategies for CCM disease.

Humans↗
PubMed2006

Rapid inhibition of neurons in the dorsal motor nucleus of the vagus by leptin.

The peptide leptin conveys the availability of adipose energy stores to the brain. Increasing evidence implicates a significant role for extrahypothalamic sites of leptin action, including the dorsal vagal complex, a region critical for regulating visceral parasympathetic function. The hypothesis that leptin suppresses cellular activity in the dorsal motor nucleus of the vagus nerve (DMV) was tested using whole-cell patch-clamp recordings in brainstem slices. Leptin caused a rapid membrane hyperpolarization in 50% of rat DMV neurons. Leptin also hyperpolarized a subset of gastric-related neurons (62%), identified after gastric inoculation with a transneuronal retrograde viral tracer. The hyperpolarization was associated with a decrease in input resistance and cellular responsiveness and displayed characteristics consistent with an increased K+ conductance. Perfusion of tolbutamide (200 microM) reversed the leptin-induced hyperpolarization, and tolbutamide or wortmannin (10-100 nM) prevented the hyperpolarization, indicating that leptin activated an ATP-sensitive K+ channel via a phosphoinositide-3-kinase-dependent mechanism. Leptin reduced the frequency of spontaneous and miniature excitatory postsynaptic currents (EPSCs), whereas inhibitory postsynaptic currents (IPSCs) were largely unaffected. Electrical stimulation of the nucleus tractus solitarii (NTS) resulted in constant-latency EPSCs, which were decreased in amplitude by leptin. The paired-pulse ratio was increased, suggesting leptin effects involved activation of receptors presynaptic to the recorded neuron. A leptin-induced suppression of EPSCs, but not IPSCs, evoked by focal photolytic uncaging of glutamate within the NTS was also observed, supportive of leptin effects on the glutamatergic NTS projection to the DMV. Therefore, leptin directly hyperpolarized and indirectly suppressed excitatory synaptic activity to DMV neurons involved in visceral regulation, including gastric-related neurons.

Animals↗
PubMed2004

The effects of isoflurane on desensitized wild-type and alpha 1(S270H) gamma-aminobutyric acid type A receptors.

UNLABELLED: gamma-aminobutyric acid type A receptors (GABA(A)-R) mediate synaptic inhibition and meet many pharmacological criteria required of important general anesthetic targets. During synaptic transmission GABA release is sufficient to saturate, maximally activate, and transiently desensitize postsynaptic GABA(A)-Rs. The resulting inhibitory postsynaptic currents (IPSCs) are prolonged by volatile anesthetics like isoflurane. We investigated the effects of isoflurane on maximally activated and desensitized GABA(A)-R currents expressed in Xenopus oocytes. Wild-type alpha(1)beta(2) and alpha(1)beta(2)gamma(2s) receptors were exposed to 600 microM GABA until currents reached a steady-state desensitized level. At clinical concentrations (0.02-0.3 mM), isoflurane produced a dose-dependent enhancement of steady-state desensitized current in alpha(1)beta(2) receptors, an effect that was less apparent in receptors including a gamma(2s)-subunit. When serine at position 270 is mutated to histidine (alpha(1)(S270H)) in the second transmembrane segment of the alpha(1)-subunit, the currents evoked by sub-saturating concentrations of GABA became less sensitive to isoflurane enhancement. In addition, isoflurane enhancements of desensitized currents were greatly attenuated by this mutation and were undetectable in alpha(1)(S270H)beta(2)gamma(2s) receptors. In conclusion, isoflurane enhancement of GABA(A)-R currents evoked by saturating concentrations of agonist is subunit-dependent. The effects of isoflurane on desensitized receptors may be partly responsible for the prolongation of IPSCs during anesthesia. IMPLICATIONS: Isoflurane enhances desensitized gamma-aminobutyric acid type A receptor (GABA(A)-R) currents, an effect that is subunit-dependent and attenuated by a mutation in an alpha(1)-subunit pore residue of the GABA(A)-R. As GABA release at inhibitory synapses is typically saturating, isoflurane modulation of desensitized receptors may be partly responsible for prolongation of inhibitory postsynaptic currents during anesthesia.

Anesthetics, Inhalation↗
PubMed2004

Brain steroidogenesis mediates ethanol modulation of GABAA receptor activity in rat hippocampus.

An interaction with the GABA type A (GABA(A)) receptor has long been recognized as one of the main neurochemical mechanisms underlying many of the pharmacological actions of ethanol. However, more recent data have suggested that certain behavioral and electrophysiological actions of ethanol are mediated by an increase in brain concentration of neuroactive steroids that results from stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. Neuroactive steroids such as 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) are, in fact, potent and efficacious endogenous positive modulators of GABA(A) receptor function. Because neurosteroids can be synthesized de novo in the brain, we have investigated whether ethanol might affect both neurosteroid synthesis and GABA(A) receptor function in isolated rat hippocampal tissue. Here, we show that ethanol increases the concentration of 3alpha,5alpha-THP as well as the amplitude of GABA(A) receptor-mediated IPSCs recorded from CA1 pyramidal neurons in isolated hippocampal slices. These effects are shared by the neurosteroid precursor progesterone, the peripheral benzodiazepine receptor-selective agonist CB34, and gamma-hydroxybutyrate, all of which are known to increase the formation of neuroactive steroids in plasma and in the brain. The action of ethanol on GABA(A) receptor-mediated IPSC amplitude is biphasic, consisting of a rapid, direct effect on GABA(A) receptor activity and an indirect effect that appears to be mediated by neurosteroid synthesis. Furthermore, ethanol affects GABA(A) receptor activity through a presynaptic action, an effect that is not dependent on neurosteroid formation. These observations suggest that ethanol may modulate GABA(A) receptor function through an increase in de novo neurosteroid synthesis in the brain that is independent of the HPA axis. This novel mechanism may have a crucial role in mediating specific central effects of ethanol.

Animals↗
PubMed2004

Contribution of AMPA, NMDA, and GABA(A) receptors to temporal pattern of postsynaptic responses in the inferior colliculus of the rat.

The central nucleus of the inferior colliculus (ICC) is a major site of synaptic interaction in the central auditory system. To understand how ICC neurons integrate excitatory and inhibitory inputs for processing temporal information, we examined postsynaptic responses of ICC neurons to repetitive stimulation of the lateral lemniscus at 10-100 Hz in rat brain slices. The excitatory synaptic currents mediated by AMPA and NMDA receptors and the inhibitory current mediated by GABA(A) receptors were pharmacologically isolated and recorded by whole-cell patch-clamp techniques. The response kinetics of AMPA receptor-mediated EPSCs and GABA(A) receptor-mediated IPSCs were similar and much faster than those of NMDA receptor-mediated EPSCs. AMPA EPSCs could follow each pulse of stimulation at a rate of 10-100 Hz but showed response depression during the course of repetitive stimulation. GABA(A) IPSCs could also follow stimulus pulses over this frequency range but showed depression at low rates and facilitation at higher rates. NMDA EPSCs showed facilitation and temporal summation in response to repetitive stimulation, which was most pronounced at higher rates of stimulation. GABA(A) inhibition suppressed activation of NMDA receptors and reduced both the degree of AMPA EPSC depression and the extent of temporal summation of NMDA EPSCs. The results indicate that GABA(A) receptor-mediated inhibition plays a crucial role in maintaining the balance of excitation and inhibition and in allowing ICC neurons to process temporal information more precisely.

Animals↗
PubMed2004

Group I metabotropic glutamate receptors inhibit GABA release at interneuron-Purkinje cell synapses through endocannabinoid production.

Actions of endocannabinoids in the cerebellum can be demonstrated following distinct stimulation protocols in Purkinje cells. First, depolarization-induced elevations of intracellular Ca2+ lead to the suppression of neurotransmitter release from both inhibitory and excitatory afferents. In another case, postsynaptic group I metabotropic glutamate receptors (mGluRs) trigger a strong inhibition of the glutamatergic inputs from parallel and climbing fibers. Both pathways involve endocannabinoids retrogradely acting on type 1 cannabinoid receptors (CB1Rs) at presynaptic terminals. Here, we show that group I mGluR activation also depresses GABAergic transmission at the synapses between molecular layer interneurons and Purkinje cells. Using paired recordings, we found that application of the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine reduced the evoked IPSCs in Purkinje cells. This effect was independent of postsynaptic Ca2+ increases and was completely blocked by a CB1R antagonist. Experiments performed with the GTP-analogues GDP-betaS and GTP-gammaS provided evidence that endocannabinoids released after G-protein activation can also inhibit GABAergic inputs onto nearby, unstimulated Purkinje cells. Block of the enzymes DAG lipase or phospholipase C reduced the group I mGluR-dependent inhibition, suggesting that 2-arachidonyl glycerol could act as retrograde messenger. Finally, group I mGluR activation by brief bursts of activity of the parallel fibers induced a short-lived depression of spontaneous IPSCs via presynaptic CB1Rs. Our results reveal a mechanism with potential physiological importance, by which glutamatergic synapses induce an endocannabinoid-mediated inhibition of the GABAergic inputs onto Purkinje cells.

Animals↗
PubMed2004

Noradrenaline triggers GABAA inhibition of bed nucleus of the stria terminalis neurons projecting to the ventral tegmental area.

The lateral part of the ventral bed nucleus of the stria terminalis (vlBNST) is a critical site for the antiaversive effects of noradrenergic drugs during opioid withdrawal. The objective of the present study is to identify the cellular action(s) of noradrenaline in the vlBNST after withdrawal from a 5d treatment with morphine. The vlBNST is a heterogeneous cell group with multiple efferent projections. Therefore, neurons projecting to the midbrain were identified by retrograde transport of fluorescent microspheres injected in the ventral tegmental area (VTA). Whole-cell voltage clamp recordings of these neurons and of those sharing physiological properties were done in brain slices. Noradrenaline activated alpha1-adrenergic receptors to increase GABA(A)-IPSC frequency. Noradrenaline produced a similar increase in GABA(A)-IPSCs during acute opioid withdrawal, but this increase resulted from activation of beta-adrenergic receptors, adenylyl cyclase, and protein kinase A, as well as alpha1-adrenergic receptors. Given that neurons in the vlBNST send an excitatory projection to the VTA, noradrenaline may reduce excitatory drive to mesolimbic dopamine cells. This mechanism might contribute to the withdrawal-induced inhibition of dopamine neurons and explain how noradrenergic drugs microinjected into the vlBNST reduce aversive aspects of opioid withdrawal.

Adrenergic alpha-Antagonists↗
PubMed2006

Ambient GABA constrains the strength of GABAergic synapses at Cajal-Retzius cells in the developing visual cortex.

At early stages of brain development, GABA plays a dual role. It fulfills important trophic functions and provides a major excitatory drive for the immature neuronal network. Here, we investigated whether GABA itself can limit the strength of excitatory GABAergic synapses on Cajal-Retzius (CR) cells in sagittal slices from the mouse visual cortex. (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid (CGP55845), a specific GABAB receptor (GABABR) blocker, increased the frequency of spontaneous Ca2+ transients and spontaneous and miniature IPSCs (mIPSCs) but did not affect mIPSC amplitudes or kinetics. CGP55845 significantly increased evoked IPSC (eIPSC) amplitudes and decreased the paired-pulse ratio (PPR). Baclofen, a specific GABABR agonist, produced opposite effects. The size of the readily releasable pool was not affected by these GABABR modulators. The same CGP55845 actions were observed at physiological temperatures, but they were abolished after glutamate decarboxylase block with 3-mercaptopropionic acid (3-MP). These results indicate that presynaptic GABABRs dynamically regulate GABA release probability. SNAP-5114, a specific GABA transporter-2/3 (GAT-2/3) blocker, enhanced mIPSC frequencies, decreased PPR, and increased eIPSC amplitudes without changing eIPSC kinetics. These effects were blocked by CGP55845 and 3-MP. NO-711, a specific GAT-1 blocker, prolonged eIPSC decay and decreased eIPSC/mIPSC amplitudes. These NO-711-mediated effects were not sensitive to CGP55845 and 3-MP. We conclude that the strength of GABAergic inputs to CR cells is constrained by GABABRs that are persistently activated by ambient GABA. The latter is also provided by GAT-2/3 operating in the reversed mode. Presynaptic GAT-1 functions in the uptake mode and possibly provides GABA for presynaptic vesicle filling.

Animals↗
PubMed2004

Action potential-evoked and ryanodine-sensitive spontaneous Ca2+ transients at the presynaptic terminal of a developing CNS inhibitory synapse.

The existence of spontaneous calcium transients (SCaTs) dependent on intracellular store activation has been reported in putative axonal terminals of cerebellar basket interneurons. We used the two-photon imaging technique to optically identify basket terminals in acute cerebellar slices of young rats (11-16 d old) and study the properties of SCaTs unambiguously localized in these regions. The whole-cell recording configuration and preloading technique were alternatively used to load the calcium-dependent dye in the interneuron and compare SCaTs with action potential evoked calcium transients. SCaTs were observed in the basket terminals at frequencies that were significantly increased after bath application of 10 microm ryanodine and did not depend on P/Q- or N-type voltage-dependent calcium channel activation. They originated at specific sites where bursts of events with temporal separation as small as 200 msec could be generated. Their sites of origin were spaced on average 6 microm apart and were preferentially located near axonal endings. SCaTs had amplitudes comparable with those of Ca2+ rises evoked by single action potentials that lead to release of neurotransmitter, as confirmed by parallel recordings of preloaded terminals and evoked IPSCs in the postsynaptic Purkinje cells. These results support the hypothesis that SCaTs at basket terminals underlie the large miniature IPSCs characteristic of Purkinje cells.

Action Potentials↗
PubMed2006

Vanilloid-mediated heterosynaptic facilitation of inhibitory synaptic input to neurons of the rat dorsal motor nucleus of the vagus.

Vanilloid type-1 receptors (VR1) are abundant in the dorsal vagal complex, where their function is mostly unknown. We examined the role of VR1 in regulating synaptic inputs to neurons of the dorsal motor nucleus of the vagus (DMV). Using patch-clamp recordings from DMV neurons in brainstem slices, capsaicin was found to increase action potential-independent inhibitory input onto DMV neurons. This rapid effect was mimicked by application of the endogenous cannabinoid, anandamide and blocked by VR1 antagonists. The VR1-mediated facilitation of synaptic inhibition was reduced by ionotropic and metabotropic glutamate receptor antagonists, suggesting an indirect, heterosynaptic enhancement of GABA release caused by a VR1-mediated increase in glutamate release from presynaptic terminals of excitatory neurons. Application of L-glutamate also increased GABA release. The paired-pulse ratio was increased for IPSCs evoked after electrical stimulation of the nucleus tractus solitarius, but the effect was slower than for the enhancement of spontaneous and miniature IPSCs. Capsaicin also increased the frequency of glutamatergic postsynaptic currents in a VR1-mediated manner. Results of these studies suggest that VR1-containing glutamatergic terminals contact DMV neurons. Activation of VR1 potently enhances glutamate release onto GABAergic terminals, facilitating GABA release. Endogenous cannabinoids can thereby rapidly enhance inhibitory input to DMV neurons via VR1-mediated presynaptic mechanisms.

Animals↗
PubMed2004

Mu-opioid receptor trafficking on inhibitory synapses in the rat brainstem.

Whole-cell recordings were made from identified gastric-projecting rat dorsal motor nucleus of the vagus (DMV) neurons. The amplitude of evoked IPSCs (eIPSCs) was unaffected by perfusion with met-enkephalin (ME) or by mu-, delta-, or kappa-opioid receptor selective agonists, namely D-Ala2-N-Me-Phe4-Glycol5-enkephalin (DAMGO), cyclic [D-Pen2-D-Pen5]-enkephalin, or trans-3,4-dichloro-N-methyl-N-[2-(1-pyrolytinil)-cyclohexyl]-benzeneacetamide methane sulfonate (U50,488), respectively. Brief incubation with the adenylate cyclase activator forskolin or the nonhydrolysable cAMP analog 8-bromo-cAMP, thyrotropin releasing hormone, or cholecystokinin revealed the ability of ME and DAMGO to inhibit IPSC amplitude; this inhibition was prevented by pretreatment with the mu-opioid receptor (MOR1) selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2. Conversely, incubation with the adenylate cyclase inhibitor dideoxyadenosine, with the protein kinase A (PKA) inhibitor N-[2-(p-Bromocinnamyl-amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89), or with the Golgi-disturbing agent brefeldin A, blocked the ability of forskolin to facilitate the inhibitory actions of ME. Immunocytochemical experiments revealed that under control conditions, MOR1 immunoreactivity (MOR1-IR) was colocalized with glutamic acid decarboxylase (GAD)-IR in profiles apposing DMV neurons only after stimulation of the cAMP-PKA pathway. Pretreatment with H89 or brefeldin A or incubation at 4 degrees C prevented the forskolin-mediated insertion of MOR1 on GAD-IR-positive profiles. These results suggest that the cAMP-PKA pathway regulates trafficking of mu-opioid receptors into the cell surface of GABAergic nerve terminals. By consequence, the inhibitory actions of opioid peptides in the dorsal vagal complex may depend on the state of activation of brainstem vagal circuits.

Animals↗