Regulation of synaptic input to hypothalamic presympathetic neurons by GABA(B) receptors

The hypothalamic paraventricular (PVN) neurons projecting to the spinal cord and brainstem play an important role in the control of homeostasis and the sympathetic nervous system. Although GABA(B) receptors are present in the PVN, their function in the control of synaptic inputs to PVN presympathetic neurons is not clear. Using retrograde tracing and whole-cell patch-clamp recordings in rat brain slices, we determined the role of presynaptic GABA(B) receptors in regulation of glutamatergic and GABAergic inputs to spinally projecting PVN neurons. The GABA(B) receptor agonist baclofen (1-50 microM) dose-dependently decreased the frequency but not the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (sIPSCs). The effect of baclofen on sEPSCs and sIPSCs was completely blocked by 10 microM CGP52432, a selective GABA(B) receptor antagonist. Baclofen also significantly reduced the frequency of both miniature excitatory and miniature inhibitory postsynaptic currents (mEPSCs and mIPSCs). Furthermore, uncoupling pertussis toxin-sensitive G(i/o) proteins with N-ethylmaleimide abolished baclofen-induced inhibition of mEPSCs and mIPSCs. However, the inhibitory effect of baclofen on the frequency of mIPSCs and mEPSCs persisted in the presence of either Cd2+, a voltage-gated Ca2+ channel blocker, or 4-aminopyridine, a blocker of voltage-gated K+ channels. Our results suggest that activation of presynaptic GABA(B) receptors inhibits synaptic GABA and glutamate release to PVN presympathetic neurons. This presynaptic action of GABA(B) receptors is mediated by the N-ethylmaleimide-sensitive G(i/o) proteins, but independent of voltage-gated Ca2+ and K+ channels.     

VR1 receptor activation induces glutamate release and postsynaptic firing in the paraventricular nucleus

Neurons in the paraventricular nucleus (PVN) are important in regulating autonomic function through projections to the brain stem and spinal cord. Although the vanilloid receptors (VR(1)) are present in the PVN, their physiological function is scarcely known. In this study, we determined the role of VR(1) receptors in the regulation of synaptic inputs and the excitability of spinally projecting PVN neurons. Whole cell patch-clamp recordings were performed on the PVN neurons labeled by a retrograde fluorescence tracer injected into the thoracic spinal cord of rats. Capsaicin significantly increased the frequency of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) without changing the amplitude and decay time constant of mEPSCs. On the other hand, capsaicin had no effect on GABAergic miniature inhibitory postsynaptic currents (mIPSCs). The effect of capsaicin on mEPSCs was abolished by a specific VR(1) antagonist, iodo-resiniferatoxin (iodo-RTX), or ruthenium red. Importantly, iodo-RTX per se significantly reduced the amplitude of evoked EPSCs and the frequency of mEPSCs. Removal of extracellular Ca(2+), but not Cd(2+) treatment, also eliminated the effect of capsaicin on mEPSCs. Furthermore, capsaicin caused a large increase in the firing rate of PVN neurons, and such an effect was abolished in the presence of ionotropic glutamate receptor antagonists. Additionally, the double-immunofluorescence labeling revealed that all of the VR(1) immunoreactivity was colocalized with a presynaptic marker, synaptophysin, in the PVN. Thus this study provides the first evidence that activation of VR(1) receptors excites preautonomic PVN neurons through selective potentiation of glutamatergic synaptic inputs. Presynaptic VR(1) receptors and endogenous capsaicin-like substances in the PVN may represent a previously unidentified mechanism in hypothalamic regulation of the autonomic nervous system.     

Nitric oxide inhibits spinally projecting paraventricular neurons through potentiation of presynaptic GABA release

Nitric oxide (NO) in the paraventricular nucleus (PVN) is involved in the regulation of the excitability of PVN neurons. However, the effect of NO on the inhibitory GABAergic and excitatory glutamatergic inputs to spinally projecting PVN neurons has not been studied specifically. In the present study, we determined the role of the inhibitory GABAergic and excitatory glutamatergic inputs in the inhibitory action of NO on spinally projecting PVN neurons. Spinally projecting PVN neurons were retrogradely labeled by a fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocasbocyane (DiI), injected into the spinal cord of rats. Whole cell voltage- and current-clamp recordings were performed on DiI-labeled PVN neurons in the hypothalamic slice. The spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded in DiI-labeled neurons were abolished by 20 microM bicuculline, whereas the miniature excitatory postsynaptic currents (mEPSCs) were eliminated by 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione. Bath application of an NO donor, 100 microM S-nitroso-N-acetyl-penicillamine (SNAP), or the NO precursor, 100 microM L-arginine, both significantly increased the frequency of mIPSCs of DiI-labeled PVN neurons, without altering the amplitude and the decay time constant of mIPSCs. The effect of SNAP and L-arginine on the frequency of mIPSCs was eliminated by an NO scavenger, 2-(4-carboxypheny)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and an NO synthase inhibitor, 1-(2-trifluoromethylphenyl) imidazole, respectively. Neither SNAP nor L-arginine significantly altered the frequency and the amplitude of mEPSCs. Under current-clamp conditions, 100 microM SNAP or 100 microM L-arginine significantly decreased the discharge rate of the DiI-labeled PVN neurons, without significantly affecting the resting membrane potential. On the other hand, 20 microM bicuculline significantly increased the impulse activity of PVN neurons. In the presence of bicuculline, SNAP or L-arginine both failed to inhibit the firing activity of PVN neurons. This electrophysiological study provides substantial new evidence that NO suppresses the activity of spinally projecting PVN neurons through potentiation of the GABAergic synaptic input.     

Endogenous casein kinase‐1 modulates NMDA receptor activity of hypothalamic presympathetic neurons and sympathetic outflow in hypertension

Key points

• Increased NMDA receptor activity and excitability of presympathetic neurons in the hypothalamus can increase sympathetic nerve discharges leading to hypertension.
• In this study, we determined how protein kinases and phosphatases are involved in regulating NMDA receptor activity and firing activity of presympathetic neurons in the hypothalamus in normotensive and hypertensive rats.
• We show that casein kinase‐1 inhibition increases NMDA receptor activity and excitability of presympathetic neurons in the hypothalamus and augments sympathetic nerve discharges in normotensive, but not in hypertensive, rats.
• Our data indicate that casein kinase‐1 tonically regulates NMDA receptor activity by interacting with casein kinase‐2 and protein phosphatases in the hypothalamus and that imbalance of NMDA receptor phosphorylation can augment the excitability of hypothalamic presympathetic neurons and sympathetic nerve discharges in hypertension.
• These findings help us understand the neuronal mechanism of hypertension, and reducing the NMDA receptor phosphorylation level may be effective for treating neurogenic hypertension.

Abstract

Increased N‐methyl‐d‐aspartate receptor (NMDAR) activity in the paraventricular nucleus (PVN) of the hypothalamus is involved in elevated sympathetic outflow in hypertension. However, the molecular mechanisms underlying augmented NMDAR activity in hypertension remain unclear. In this study, we determined the role of casein kinase‐1 (CK1) in regulating NMDAR activity in the PVN. NMDAR‐mediated excitatory postsynaptic currents (EPSCs) and puff NMDA‐elicited currents were recorded in spinally projecting PVN neurons in spontaneously hypertensive rats (SHRs) and Wistar–Kyoto (WKY) rats. The basal amplitudes of evoked NMDAR‐EPSCs and puff NMDA currents were significantly higher in SHRs than in WKY rats. The CK1 inhibitor PF4800567 or PF670462 significantly increased the amplitude of NMDAR‐EPSCs and puff NMDA currents in PVN neurons in WKY rats but not in SHRs. PF4800567 caused an NMDAR‐dependent increase in the excitability of PVN neurons only in WKY rats. Also, the CK1ε protein level in the PVN was significantly lower in SHRs than in WKY rats. Furthermore, intracerebroventricular infusion of PF4800567 increased blood pressure and lumbar sympathetic nerve activity in WKY rats, and this effect was eliminated by microinjection of the NMDAR antagonist into the PVN. In addition, PF4800567 failed to increase NMDAR activity in brain slices of WKY rats pretreated with the protein phosphatase 1/2A, calcineurin, or casein kinase‐2 inhibitor. Our findings suggest that CK1 tonically suppresses NMDAR activity in the PVN by reducing the NMDAR phosphorylation level. Diminished CK1 activity may contribute to potentiated glutamatergic synaptic input to PVN presympathetic neurons and elevated sympathetic vasomotor tone in neurogenic hypertension.

Abbreviations

ABP

arterial blood pressure

aCSF

artificial cerebrospinal fluid

AMPA

α‐amino‐3‐hydroxy‐4‐isoxazoleproprionic acid

AMPAR

AMPA receptor

CK1

casein kinase‐1

CK2

casein kinase‐2

CNQX

6‐cyano‐7‐nitroquinoxaline‐2,3‐dione

EPSC

excitatory postsynaptic current

GABA

γ‐aminobutyric acid

HR

heart rate

LSNA

lumbar sympathetic nerve activity

NMDA

N‐methyl‐d‐aspartate

NMDAR

NMDA receptor

PVN

paraventricular nucleus

PP1

protein phosphatase 1

PP2A

protein phosphatase 2A

PP2B

protein phosphatase 2B

PKC

protein kinase C

RVLM

rostral ventrolateral medulla

SHR

spontaneously hypertensive rat

WKY

Wistar–Kyoto

     

Chronic ethanol and withdrawal differentially modulate pre- and postsynaptic function at glutamatergic synapses in rat basolateral amygdala

Withdrawal anxiety is a significant factor contributing to continued alcohol abuse in alcoholics. This anxiety is long-lasting, can manifest well after the overt physical symptoms of withdrawal, and is frequently associated with relapse in recovering alcoholics. The neurobiological mechanisms governing these withdrawal-associated increases in anxiety are currently unknown. The basolateral amygdala (BLA) is a major emotional center in the brain and regulates the expression of both learned fear and anxiety. Neurotransmitter system alterations within this brain region may therefore contribute to withdrawal-associated anxiety. Because evidence suggests that glutamate-gated neurotransmitter receptors are sensitive to acute ethanol exposure, we examined the effect of chronic intermittent ethanol (CIE) and withdrawal (WD) on glutamatergic synaptic transmission in the BLA. We found that slices prepared from CIE and WD animals had significantly increased contributions by synaptic NMDA receptors. In addition, CIE increased the amplitude of AMPA-receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs), whereas only WD altered the amplitude and kinetics of tetrodotoxin-resistant spontaneous events (mEPSCs). Similarly, the frequency of sEPSCs was increased in both CIE and WD neurons, although only WD increased the frequency of mEPSCs. These data suggest that CIE and WD differentially alter both pre- and postsynaptic properties of BLA glutamatergic synapses. Finally, we show that microinjection of the AMPA-receptor antagonist, DNQX, can attenuate withdrawal-related anxiety-like behavior. Together, our results suggest that increased glutamatergic function may contribute to anxiety expressed during withdrawal from chronic ethanol.     

Regulation of synaptic inputs to paraventricular-spinal output neurons by alpha2 adrenergic receptors

Neurons in the paraventricular nucleus (PVN) that project to the brain stem and spinal cord are important for autonomic regulation. The excitability of preautonomic PVN neurons is controlled by the noradrenergic input from the brain stem. In this study, we determined the role of alpha(2) adrenergic receptors in the regulation of excitatory and inhibitory synaptic inputs to spinally projecting PVN neurons. Excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were recorded using whole cell voltage-clamp techniques on PVN neurons labeled by a retrograde fluorescence tracer injected into the thoracic spinal cord of rats. Bath application of 5-20 muM clonidine, an alpha(2) receptor agonist, significantly reduced the amplitude of evoked GABAergic IPSCs in a dose-dependent manner. Also, 10 microM clonidine significantly decreased the frequency (from 2.68 +/- 0.41 to 1.22 +/- 0.40 Hz) but not the amplitude of miniature IPSCs (mIPSCs), and this effect was blocked by the alpha(2) receptor antagonist yohimbine. Furthermore, clonidine increased the paired-pulse ratio of evoked IPSCs from 1.25 +/- 0.05 to 1.61 +/- 0.08 (P < 0.05). On the other hand, clonidine had little effect on evoked glutamatergic EPSCs, mEPSCs, and the paired-pulse ratio of evoked EPSCs in most labeled cells examined. Additionally, immunofluorescence labeling revealed that the alpha(2A) receptor and GABA immunoreactivities were co-localized in close apposition to labeled PVN neurons. Collectively, these data suggest that stimulation of alpha(2) adrenergic receptors primarily attenuates GABAergic inputs to PVN output neurons to the spinal cord. The presynaptic alpha(2) receptors function as heteroreceptors to modulate synaptic GABA release and contribute to the hypothalamic regulation of sympathetic outflow.     

TRPV1 receptor mediates glutamatergic synaptic input to dorsolateral periaqueductal gray (dl-PAG) neurons

The purpose of this study was to determine the role of transient receptor potential vanilloid type 1 (TRPV1) receptor in modulating neuronal activity of the dorsolateral periaqueductal gray (dl-PAG) through excitatory and inhibitory synaptic inputs. First, whole cell voltage-clamp recording was performed to obtain the spontaneous miniature excitatory postsynaptic currents (mEPSCs) and inhibitory postsynaptic currents (mIPSCs) of the dl-PAG neurons. As 1 microM of capsaicin was applied into the perfusion chamber, the frequency of mEPSCs was increased from 3.21 +/- 0.49 to 5.64 +/- 0.64 Hz (P < 0.05, n = 12) without altering the amplitude and the decay time constant of mEPSCs. In contrast, capsaicin had no distinct effect on mIPSCs. A specific TRPV1 receptor antagonist, iodo-resiniferatoxin (i-RTX, 300 nM), decreased the frequency of mEPSCs from 3.51 +/- 0.29 to 2.01 +/- 0.2 Hz (P < 0.05, n = 8) but did not alter the amplitude and decay time. In addition, i-RTX applied into the chamber abolished the effect of capsaicin on mEPSC of the dl-PAG. In another experiment, spontaneous action potential of the dl-PAG neurons was recorded using whole cell current-clamp methods. Capsaicin significantly elevated the discharge rate of the dl-PAG neurons from 3.03 +/- 0.38 to 5.96 +/- 0.87 Hz (n = 8). The increased firing activity was abolished in the presence of glutamate N-methy-D-aspartate (NMDA) and non-NMDA antagonists, 2-amino-5-phosphonopentanoic acid, and 6-cyano-7-nitroquinoxaline-2,3-dione. The results from this study provide the first evidence indicating that activation of TRPV1 receptors increases the neuronal activity of the dl-PAG through selective potentiation of glutamatergic synaptic inputs.     

Presynaptic actions of opioid receptor agonists in ventromedial hypothalamic neurons in estrogen- and oil-treated female mice

Estrogens act upon ventromedial hypothalamic (VMH) neurons, and their effects on female arousal and sexual behaviors mediated by VMH neurons involve several neurotransmitters and neuromodulators. Among these are opioid peptides which might be predicted to oppose estrogenic action on VMH because they tend to decrease CNS arousal. Spontaneous excitatory postsynaptic currents were recorded from VMH neurons from 17beta-estradiol- (E, 10 mug/0.1 ml) or oil-treated control ovariectomized (OVX) mice using whole-cell patch-clamp techniques. To examine the impact of opioidergic inputs, recordings of neurons from both treatment groups were obtained in the presence of the general opioid receptor agonist methionine enkephalin-Arg-Phe (MERF, 3 muM), or mu-receptor specific agonist [d-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO, 1 muM). Compared with oil, E treatment for 48 h significantly increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) without affecting their amplitude. MERF and DAMGO each abolished this E effect, causing significant reductions in sEPSCs. The effect of MERF was abolished by naltrexone (general opioid receptor antagonist, 3 muM) and the effect of DAMGO by d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) (mu-opioid receptor selective antagonist, 1 muM); in contrast, kappa- and delta-opioid receptor agonists, U69593 (300 nM) and [d-Pen(2),d-Pen(5)]-enkephalin (DPDPE, 1 muM) respectively, had little effect on the sEPSCs compared with DAMGO. To consider presynaptic vs. postsynaptic effects of opioids, miniature excitatory postsynaptic currents (mEPSCs) were investigated in E- and oil-treated VMH neurons and opioid receptor antagonist effects on mEPSCs were observed. Both MERF and DAMGO reduced the frequency of mEPSCs, but had no effect on their amplitude. Our findings indicate that opioids suppress excitatory synaptic transmissions in VMH neurons primarily through mu-receptors and could thereby decrease sexual arousal in mice.     

NMDA receptor activation enhances inhibitory GABAergic transmission onto hippocampal pyramidal neurons via presynaptic and postsynaptic mechanisms

N-methyl-D-aspartate (NMDA) receptors (NMDARs) are implicated in synaptic plasticity and modulation of glutamatergic excitatory transmission. Effect of NMDAR activation on inhibitory GABAergic transmission remains largely unknown. Here, we report that a brief application of NMDA could induce two distinct actions in CA1 pyramidal neurons in mouse hippocampal slices: 1) an inward current attributed to activation of postsynaptic NMDARs; and 2) fast phasic synaptic currents, namely spontaneous inhibitory postsynaptic currents (sIPSCs), mediated by GABA(A) receptors in pyramidal neurons. The mean amplitude of sIPSCs was also increased by NMDA. This profound increase in the sIPSC frequency and amplitude was markedly suppressed by the sodium channel blocker TTX, whereas the frequency and mean amplitude of miniature IPSCs were not significantly affected by NMDA, suggesting that NMDA elicits repetitive firing in GABAergic interneurons, thereby leading to GABA release from multiple synaptic sites of single GABAergic axons. We found that the NMDAR open-channel blocker MK-801 injected into recorded pyramidal neurons suppressed the NMDA-induced increase of sIPSCs, which raises the possibility that the firing of interneurons may not be the sole factor and certain retrograde messengers may also be involved in the NMDA-mediated enhancement of GABAergic transmission. Our results from pharmacological tests suggest that the nitric oxide signaling pathway is mobilized by NMDAR activation in CA1 pyramidal neurons, which in turn retrogradely facilitates GABA release from the presynaptic terminals. Thus NMDARs at glutamatergic synapses on both CA1 pyramidal neurons and interneurons appear to exert feedback and feedforward inhibition for determining the spike timing of the hippocampal microcircuit.     

Medullary and supramedullary mechanisms regulating sympathetic vasomotor tone

AIM: Neurons in the rostral ventrolateral medulla (RVLM) that project directly to sympathetic preganglionic neurons in the spinal cord play a critical role in maintaining tonic activity in sympathetic vasomotor nerves. Intracellular recordings in vivo from putative RVLM presympathetic neurons have demonstrated that under resting conditions these neurons display an irregular tonic firing rate, and also receive both excitatory and inhibitory synaptic inputs. This paper will briefly review some recent findings on the role of glutamate, GABA and angiotensin II (Ang II) receptors in maintaining the tonic activity of RVLM presympathetic neurons. RESULTS: Based on these findings, the following hypotheses will be discussed: (1) RVLM neurons receive tonic glutamatergic excitatory inputs, which originate from both medullary and supramedullary sources; (2) at least some neurons that project to and tonically inhibit RVLM presympathetic neurons are themselves tonically inhibited by GABAergic inputs originating from neurons in the caudalmost part of the ventrolateral medulla (caudal pressor area); (3) under normal conditions, Ang II receptors in the RVLM do not contribute significantly to the tonic activity of RVLM presympathetic neurons, but may do so in abnormal conditions such as heart failure or neurogenic hypertension; (4) RVLM presympathetic neurons maintain a significant level of tonic resting activity even when glutamate, GABA and Ang II receptors on the neurons are completely blocked. Under these conditions, the tonic activity is a consequence either of the intrinsic membrane properties of the neurons (autoactivity) or of synaptic inputs mediated by receptors other than glutamate, GABA or Ang II receptors. CONCLUSION: The current evidence indicates that the resting activity of RVLM presympathetic neurons is determined by the balance of powerful tonic excitatory and inhibitory synaptic inputs. Ang II receptors also contribute to the raised resting activity of these neurons in some pathological conditions.     

Activity- and BDNF-induced plasticity of miniature synaptic currents in ES cell-derived neurons integrated in a neocortical network

In vitro differentiated embryonic stem (ES) cells have been proposed as potential donor cells for cell replacement therapies of neurodegenerative diseases. The functional synaptic integration of such cells appears conceivable because ES cell-derived neurons are well known to establish excitatory and inhibitory synapses. However, long-term synaptic plasticity, a prerequisite of memory formation, has not yet been demonstrated at these synapses. After in vitro differentiation and purification by immunoisolation, we co-cultured ES cell-derived neurons with neocortical explants, which strongly innervated the ES cell-derived target neurons. ES cell-derived neurons exhibited action potential firing similar to primary cultured neocortical neurons. The formation of glutamatergic synapses was indicated by AMPA receptor-mediated miniature excitatory postsynaptic currents (AMPA mEPSCs). In addition, a N-methyl-D-aspartate receptor-mediated, D-2-amino-5-phosphonopentanoic acid-sensitive mEPSC component was observed. We first studied activity-dependent homeostatic plasticity (synaptic scaling) of mEPSCs at glutamatergic synapses. Chronic blockade of action potential activity by TTX resulted in an increase in the amplitudes of AMPA mEPSCs. This indicates that ES cell-derived neurons are capable of a homeostatic regulation of postsynaptic AMPA receptors. In addition, we investigated neurotrophin-induced synaptic plasticity of mEPSCs at glutamatergic synapses. Chronic addition of brain-derived neurotrophic factor (BDNF; 100 ng/ml) to the culture medium resulted in an increase in both the frequency and the amplitudes of AMPA mEPSCs. These results suggest that BDNF induces the formation and/or the functional maturation of presynaptic release sites in parallel with an upregulation of postsynaptic AMPA receptors. Thus BDNF represents a potential co-factor that could improve functional synaptic integration of ES cell-derived neurons into neocortical networks.     

5-HT excites globus pallidus neurons by multiple receptor mechanisms

Anatomical and neurochemical studies indicated that the globus pallidus receives serotonergic innervation from raphe nuclei but the membrane effects of 5-HT on globus pallidus neurons are not entirely clear. We address this question by applying whole-cell patch-clamp recordings on globus pallidus neurons in immature rat brain slices. Under current-clamp recording, 5-HT depolarized globus pallidus neurons and increased their firing rate, an action blocked by both 5-HT(4) and 5-HT(7) receptor antagonists and attributable to an increase in cation conductance(s). Further experiments indicated that 5-HT enhanced the hyperpolarization-activated inward conductance which is blocked by 5-HT(7) receptor antagonist. To determine if 5-HT exerts any presynaptic effects on GABAergic and glutamatergic inputs, the actions of 5-HT on synaptic currents were studied. At 10 microM, 5-HT increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) but had no effect on both the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). However, 5-HT at a higher concentration (50 microM) decreased the frequency but not the amplitude of the mIPSCs, indicating an inhibition of GABA release from the presynaptic terminals. This effect was sensitive to 5-HT(1B) receptor antagonist. In addition to the presynaptic effects on GABAergic neurotransmission, 5-HT at 50 microM had no consistent effects on glutamatergic neurotransmission, significantly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in 4 of 11 neurons and decreased the frequency of mEPSCs in 3 of 11 neurons. In conclusion, we found that 5-HT could modulate the excitability of globus pallidus neurons by both pre- and post-synaptic mechanisms. In view of the extensive innervation by globus pallidus neurons on other basal ganglia nuclei, this action of 5-HT originated from the raphe may have a profound effect on the operation of the entire basal ganglia network.     

Effect of hypertonic saline on rat hypothalamic paraventricular nucleus magnocellular neurons in vitro

The effect of hypertonic saline on rat hypothalamic paraventricular nucleus (PVN) magnocellular neurons was examined using a whole-cell patch-clamp technique. Under a current-clamp, 58/68 of magnocellular neurons were depolarized by hypertonic stimulation. Under a voltage-clamp, hypertonic saline produced an inward current via increased non-selective cationic conductance and shifting of the reversal potential to more positive values. Furthermore, hypertonic saline even without a change in osmolality increased spontaneous excitatory postsynaptic currents (sEPSCs). A bath application of CNQX almost completely blocked EPSCs. Extracellular application of gadolinium blocked the hypertonic saline- and mannitol-induced response. These results suggest that PVN magnocellular neurons are responsive to osmolality and Na+ concentrations. Hypertonic saline excited PVN magnocellular neurons via osmo-reception, Na+ -detection, and excitatory glutamatergic synaptic input.     

Effect of hypertonic saline on rat hypothalamic paraventricular nucleus parvocellular neurons in vitro

The effects of hypertonic saline on hypothalamic paraventricular nucleus (PVN) parvocellular neurons were examined using whole-cell patch-clamp technique. Under current-clamp, 50% (41/82) of parvocellular neurons were depolarized than the predicted values by hypertonic saline, and associated with increasing action potential frequency. Under voltage-clamp, unless hypertonic saline induced a shift of reverse potential to more positive values, neither mannitol nor hypertonic saline obviously increased the conductance in parvocellular neurons. Moreover, spontaneous excitatory postsynaptic currents (sEPSCs) were increased by isotonic increases in [Na⁺]_o in the parvocellular neurons. Bath application AMPA receptor antagonist CNQX or non-selective glutamate antagonist kynurenic acid almost completely blocked the sEPSCs. Extracellular application of gadolinium (Gd³⁺) blocked the hypertonic saline-induced response. These results suggested that subpopulation of PVN parvocellular neurons are selectively sensitive to NaCl. Hypertonic saline excited the PVN parvocellular neurons through Na⁺-detection and the excitatory glutamatergic synaptic input.     

Properties of miniature glutamatergic EPSCs in neurons of the locomotor regions of the developing zebrafish

As a first step in understanding the development of synaptic activation in the locomotor network of the zebrafish, we examined the properties of spontaneous, glutamatergic miniature excitatory postsynaptic currents (mEPSCs). Whole cell patch-clamp recordings were obtained from visually identified hindbrain reticulospinal neurons and spinal motoneurons of curarized zebrafish 1-5 days postfertilization (larvae hatch after the 2nd day of embryogenesis). In the presence of tetrodotoxin (TTX) and blockers of inhibitory receptors (strychnine and picrotoxin), we detected fast glutamatergic mEPSCs that were blocked by the AMPA/kainate receptor-selective antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). At positive voltages or in the absence of Mg(2+), a second, slower component of the mEPSCs was revealed that the N-methyl-D-aspartate (NMDA) receptor-selective antagonist DL-2-amino-5-phosphonovalerate (AP-5) abolished. In the presence of both CNQX and AP-5, all mEPSCs were eliminated. The NMDA component of reticulospinal mEPSCs had a large single-channel conductance estimated to be 48 pS. Larval AMPA/kainate and NMDA components of the mEPSCs decayed with biexponential time courses that changed little during development. At all stages examined, approximately one-half of synapses had only NMDA responses (lacking AMPA/kainate receptors), whereas the remainder of the synapses were composed of a mixture of AMPA/kainate and NMDA receptors. There was an overall increase in the frequency and amplitude of mEPSCs with an NMDA component in reticulospinal (but not motoneurons) during development. These results indicate that glutamate is a prominent excitatory transmitter in the locomotor regions of the developing zebrafish and that it activates either NMDA receptors alone at functionally silent synapses or together with AMPA/kainate receptors.     

Kv1.1/1.2 channels are downstream effectors of nitric oxide on synaptic GABA release to preautonomic neurons in the paraventricular nucleus

The paraventricular nucleus (PVN) of the hypothalamus is important for the neural regulation of cardiovascular function. Nitric oxide (NO) increases synaptic GABA release to presympathetic PVN neurons through the cyclic guanosine monophosphate (cGMP)/protein kinase G signaling pathway. However, the downstream signaling mechanisms underlying the effect of NO on synaptic GABA release remain unclear. In this study, whole-cell voltage-clamp recordings were performed on retrograde-labeled spinally projecting PVN neurons in rat brain slices. Bath application of the NO precursor l-arginine or the NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly increased the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in labeled PVN neurons. A specific antagonist of cyclic ADP ribose, 8-bromo-cyclic ADP ribose (8-Br-cADPR), had no significant effect on l-arginine-induced potentiation of mIPSCs. Surprisingly, blocking of voltage-gated potassium channels (Kv) with 4-aminopyridine or alpha-dendrotoxin eliminated the effect of l-arginine on mIPSCs in all labeled PVN neurons tested. The membrane permeable cGMP analog mimicked the effect of l-arginine on mIPSCs, and this effect was blocked by alpha-dendrotoxin. Furthermore, the specific Kv channel blocker for Kv1.1 (dendrotoxin-K) or Kv1.2 (tityustoxin-Kalpha) abolished the effect of l-arginine on mIPSCs in all neurons tested. SNAP failed to inhibit the firing activity of labeled PVN neurons in the presence of dendrotoxin-K, Kalpha. Additionally, the immunoreactivity of Kv1.1 and Kv1.2 subunits was colocalized extensively with synaptophysin in the PVN. These findings suggest that NO increases GABAergic input to PVN presympathetic neurons through a downstream mechanism involving the Kv1.1 and Kv1.2 channels at the nerve terminals.     

Excitatory mechanisms in the suprachiasmatic nucleus: the role of AMPA/KA glutamate receptors

A variety of evidence suggests that the effects of light on the mammalian circadian system are mediated by direct retinal ganglion cell projection to the suprachiasmatic nucleus (SCN). This synaptic connection is glutamatergic and the release of glutamate is detected by both N-methyl-D-asparate (NMDA) and amino-methyl proprionic acid/kainate (AMPA/KA) iontotropic glutamate receptors (GluRs). It is well established that NMDA GluRs play a critical role in mediating the effects of light on the circadian system; however, the role of AMPA/KA GluRs has received less attention. In the present study, we sought to better understand the contribution of AMPA/KA-mediated currents in the circadian system based in the SCN. First, whole cell patch-clamp electrophysiological techniques were utilized to measure spontaneous excitatory postsynaptic currents (sEPSCs) from SCN neurons. These currents were widespread in the SCN and not just restricted to the retino-recipient region. The sEPSC frequency and amplitude did not vary with the daily cycle. Similarly, currents evoked by the exogenous application of AMPA onto SCN neurons were widespread within the SCN and did not exhibit a diurnal rhythm in their magnitude. Fluorometric techniques were utilized to estimate AMPA-induced calcium (Ca(2+)) concentration changes in SCN neurons. The resulting data indicate that AMPA-evoked Ca(2+) transients were widespread in the SCN and that there was a daily rhythm in the magnitude of AMPA-induced Ca(2+) transients that peaked during the night. By itself, blocking AMPA/KA GluRs with a receptor blocker decreased the spontaneous firing of some SCN neurons as well as reduced resting Ca(2+) levels, suggesting tonic glutamatergic excitation. Finally, immunohistochemical techniques were used to describe expression of the AMPA-preferring GluR subunits GluR1 and GluR2/3s within the SCN. Overall, our data suggest that glutamatergic synaptic transmission mediated by AMPA/KA GluRs play an important role throughout the SCN synaptic circuitry.     

Primary afferent stimulation differentially potentiates excitatory and inhibitory inputs to spinal lamina II outer and inner neurons

Spinal lamina II (substantia gelatinosa) neurons play an important role in processing of nociceptive information from primary afferent nerves. Anatomical studies suggest that neurons in the outer (lamina II_o) and inner (lamina II_i) zone of lamina II receive distinct afferent inputs. The functional significance of this preferential afferent termination in lamina II remains unclear. In this study, we examined the differential synaptic inputs to neurons in lamina II_o and II_i in response to primary afferent stimulation. Whole cell voltage-clamp recordings were performed on neurons in lamina II_o and II_i of the rat spinal cord slice under visual guidance. Capsaicin (1 µM) significantly increased the frequency of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in all 27 lamina II_o neurons and significantly increased the amplitude of mEPSCs in 12 of 27 lamina II_o neurons. However, capsaicin only significantly increased the frequency of mEPSCs in 9 of 22 (40.9%) lamina II_i neurons and increased the amplitude of mEPSCs in 6 of these 9 neurons. Furthermore, the peak amplitude of EPSCs, evoked by electrical stimulation of the attached dorsal root, in 40 lamina II_o neurons was significantly greater than that [160.5 ± 16.7 vs. 87.0 ± 10.4 (SE) pA] in 37 lamina II_i neurons. On the other hand, the peak amplitude of evoked inhibitory postsynaptic currents (IPSCs) in 40 lamina II_o neurons was significantly smaller than that (103.1 ± 11.6 vs. 258.4 ± 24.4 pA) in 37 lamina II_i neurons. In addition, the peak amplitudes of both EPSCs and IPSCs, evoked by direct stimulation of lamina II, were similar in lamina II_o and II_i neurons. This study provides new information that stimulation of primary afferents differentially potentiates synaptic inputs to neurons in lamina II_o and II_i. The quantitative difference in excitatory and inhibitory synaptic inputs to lamina II_o and II_i neurons may be important for integration of sensory information from primary afferent nerves.     

Reduced excitatory drive in interneurons in an animal model of cortical dysplasia

Cortical dysplasia (CD) is strongly associated with epilepsy. Enhanced excitability in dysplastic neuronal networks is believed to contribute to epileptogenesis, but the underlying mechanisms for the hyperexcitability are poorly understood. Cortical GABAergic interneurons provide the principal inhibition in the neuronal networks by forming inhibitory synapses on excitatory neurons. The aim of the present study was to determine if the function of interneurons in CD is compromised. In a rat model of CD, in utero irradiation, we studied spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs) in cortical interneurons using whole cell recording techniques. Two types of interneurons, type I and type II, were identified based on their distinctive spike patterns and short-term synaptic plasticity. We found that the frequencies of sEPSCs and mEPSCs were significantly decreased in both types of interneurons in CD. However, the amplitude and kinetics of sEPSCs and mEPSCs were not different. Five-pulse, 20-Hz stimulation produced short-term depression in type I interneurons in both CD and control tissue. Type II interneurons showed a robust short-term facilitation in both CD and control tissue. Morphological analysis of biocytin-filled neurons revealed that dendritic trees of both types of interneurons were not altered in CD. Our results demonstrate that the excitatory drive, namely sEPSCs and mEPSCs, in two main types of interneuron is largely attenuated in CD, probably due to a reduction in the number of excitatory synapses on both types of interneurons in CD.     

Signaling mechanisms of angiotensin II-induced attenuation of GABAergic input to hypothalamic presympathetic neurons

The hypothalamic paraventricular nucleus (PVN) is an important site for the regulation of sympathetic outflow. Angiotensin II (Ang II) can activate AT(1) receptors to stimulate PVN presympathetic neurons through inhibition of GABAergic input. However, little is known about the downstream pathway involved in this presynaptic action of Ang II in the PVN. In this study, using whole cell recording from retrogradely labeled PVN neurons in rat brain slices, we determined the signaling mechanisms responsible for the effect of Ang II on synaptic GABA release to spinally projecting PVN neurons. Bath application of Ang II reproducibly decreased the frequency of GABAergic miniature postsynaptic inhibitory currents (mIPSCs) in fluorescence-labeled PVN neurons. Ang II failed to change the frequency of mIPSCs in labeled PVN neurons treated with pertussis toxin. However, Ang II-induced inhibition of mIPSCs persisted in the presence of either CdCl(2), a voltage-gated Ca(2+) channel blocker, or 4-aminopyridine, a blocker of voltage-gated K(+) channels. Interestingly, inhibition of superoxide with superoxide dismutase or Mn(III) tetrakis (4-benzoic acid) prophyrin completely blocked Ang II-induced decrease in mIPSCs. By contrast, inhibition of hydroxyl radical formation with the ion chelator deferoxamine did not significantly alter the effect of Ang II. These findings suggest that the presynaptic action of Ang II on synaptic GABA release in the PVN is mediated by the pertussis toxin-sensitive G(i/o) proteins but not by voltage-gated Ca(2+) and K(+) channels. Ang II attenuates GABAergic input to PVN presympathetic neurons through reactive oxygen species, especially superoxide anions.