Neurons controlling Aplysia feeding inhibit themselves by continuous NO production

Background

Neural activity can be affected by nitric oxide (NO) produced by spiking neurons. Can neural activity also be affected by NO produced in neurons in the absence of spiking?

Methodology/Principal Findings

Applying an NO scavenger to quiescent Aplysia buccal ganglia initiated fictive feeding, indicating that NO production at rest inhibits feeding. The inhibition is in part via effects on neurons B31/B32, neurons initiating food consumption. Applying NO scavengers or nitric oxide synthase (NOS) blockers to B31/B32 neurons cultured in isolation caused inactive neurons to depolarize and fire, indicating that B31/B32 produce NO tonically without action potentials, and tonic NO production contributes to the B31/B32 resting potentials. Guanylyl cyclase blockers also caused depolarization and firing, indicating that the cGMP second messenger cascade, presumably activated by the tonic presence of NO, contributes to the B31/B32 resting potential. Blocking NO while voltage-clamping revealed an inward leak current, indicating that NO prevents this current from depolarizing the neuron. Blocking nitrergic transmission had no effect on a number of other cultured, isolated neurons. However, treatment with NO blockers did excite cerebral ganglion neuron C-PR, a command-like neuron initiating food-finding behavior, both in situ, and when the neuron was cultured in isolation, indicating that this neuron also inhibits itself by producing NO at rest.

Conclusion/Significance

Self-inhibitory, tonic NO production is a novel mechanism for the modulation of neural activity. Localization of this mechanism to critical neurons in different ganglia controlling different aspects of a behavior provides a mechanism by which a humeral signal affecting background NO production, such as the NO precursor L-arginine, could control multiple aspects of the behavior.     

Culture of major pelvic ganglion neurons from adult rat

Successful culturing of neurons from adult animals has been historically difficult for a relatively long time. In this study, we reported the development of a novel method for the isolation and the culture of major pelvic ganglion (MPG) neurons from adult rat. The cultured cells were identified by neuron morphology and staining with neuronal marker (neurofilament-200, NF-200). The results demonstrate that the new protocol we used was reliable in obtaining a relatively high yield of MPG neurons. Furthermore, it improves the speed and simplicity in neuronal isolation. The viability of neurons can be maintained for about 2 weeks, which should be sufficient for investigating physiological and pathological processes occurring in mature major pelvic ganglia. And this may provide a useful assessment to currently available techniques for the culture of adult neurons.     

Transmitter status in cultured rat sympathetic neurons: plasticity and multiple function

Considerable recent study of the development of transmitter status in sympathetic principal neurons, both in vivo and in culture, has produced several surprising findings. In this paper we review work on cultured immature and adult principal neurons dissociated from the superior cervical ganglia of rats. The main points are; 1) Immature principal neurons that display adrenergic properties during the first postnatal week in culture can be shifted to cholinergic status, including formation of functional cholinergic synapses, by coculture with nonneuronal cells (e.g., dissociated heart cells) or by medium conditioned by such cells. Through the use of microcultures that contain only a single neuron grown on heart cells, it has been possible to demonstrate the transition from adrenergic to cholinergic function directly by serial physiological assays of the same neuron at intervals of days or weeks. 2) During this transition, the cultured neurons display adrenergic/cholinergic dual function. This dual function has also been observed in principal neurons isolated from ganglia of adult rats. 3) Some cultured neurons secrete a third transmitter, probably adenosine or a phosphorylated derivative. This purinergic function is expressed with adrenergic or cholinergic function, or with both (triple function). In some cases, the main effect exerted by a neuron on cocultured cardiac myocytes is purinergic.     

Cellular properties and modulation of the stomatogastric ganglion neurons of a stomatopod,Squilla oratoria

Cellular properties and modulation of the identified neurons of the posterior cardiac plate-pyloric system in the stomatogastric ganglion of a stomatopod, Squilla oratoria, were studied electrophysiologically. Each class of neurons involved in the cyclic bursting activity was able to trigger an endogenous, slow depolarizing potential (termed a driver potential) which sustained bursting. Endogenous oscillatory properties were demonstrated by the phase reset behavior in response to brief stimuli during ongoing rhythm. The driver potential was produced by membrane voltage-dependent activation and terminated by an active repolarization. Striking enhancement of bursting properties of all the cell types was induced by synaptic activation via extrinsic nerves, seen as increases in amplitude or duration of driver potentials, spiking rate during a burst, and bursting rate. The motor pattern produced under the influence of extrinsic modulatory inputs continued for a long time, relative to that in the absence of activation of modulatory inputs. Voltage-dependent conductance mechanisms underlying postinhibitory rebound and driver potential responses were modified by inputs. It is concluded that endogenous cellular properties, as well as synaptic circuitry and extrinsic inputs, contribute to generation of the rhythmic motor pattern, and that a motor system and its component neurons have been highly conserved during evolution between stomatopods and decapods.Abbreviations AB anterior burster neuron - CoG commissural ganglion - CPG central pattern generator - lvn lateral ventricular nerve - OG oesophageal ganglion - pcp posterior cardiac plate - PCP pcp constrictor neuron - PD pyloric dilator neuron - PY pyloric constrictor neuron - son superior oesophageal nerve - STG stomatogastric ganglion - stn stomatogastric nerve     

Nitric Oxide Regulates Neuronal Activity via Calcium-Activated Potassium Channels

Nitric oxide (NO) is an unconventional membrane-permeable messenger molecule that has been shown to play various roles in the nervous system. How NO modulates ion channels to affect neuronal functions is not well understood. In gastropods, NO has been implicated in regulating the feeding motor program. The buccal motoneuron, B19, of the freshwater pond snail Helisoma trivolvis is active during the hyper-retraction phase of the feeding motor program and is located in the vicinity of NO-producing neurons in the buccal ganglion. Here, we asked whether B19 neurons might serve as direct targets of NO signaling. Previous work established NO as a key regulator of growth cone motility and neuronal excitability in another buccal neuron involved in feeding, the B5 neuron. This raised the question whether NO might modulate the electrical activity and neuronal excitability of B19 neurons as well, and if so whether NO acted on the same or a different set of ion channels in both neurons. To study specific responses of NO on B19 neurons and to eliminate indirect effects contributed by other cells, the majority of experiments were performed on single cultured B19 neurons. Addition of NO donors caused a prolonged depolarization of the membrane potential and an increase in neuronal excitability. The effects of NO could mainly be attributed to the inhibition of two types of calcium-activated potassium channels, apamin-sensitive and iberiotoxin-sensitive potassium channels. NO was found to also cause a depolarization in B19 neurons in situ, but only after NO synthase activity in buccal ganglia had been blocked. The results suggest that NO acts as a critical modulator of neuronal excitability in B19 neurons, and that calcium-activated potassium channels may serve as a common target of NO in neurons.     

Predictive features of persistent activity emergence in regular spiking and intrinsic bursting model neurons

Proper functioning of working memory involves the expression of stimulus-selective persistent activity in pyramidal neurons of the prefrontal cortex (PFC), which refers to neural activity that persists for seconds beyond the end of the stimulus. The mechanisms which PFC pyramidal neurons use to discriminate between preferred vs. neutral inputs at the cellular level are largely unknown. Moreover, the presence of pyramidal cell subtypes with different firing patterns, such as regular spiking and intrinsic bursting, raises the question as to what their distinct role might be in persistent firing in the PFC. Here, we use a compartmental modeling approach to search for discriminatory features in the properties of incoming stimuli to a PFC pyramidal neuron and/or its response that signal which of these stimuli will result in persistent activity emergence. Furthermore, we use our modeling approach to study cell-type specific differences in persistent activity properties, via implementing a regular spiking (RS) and an intrinsic bursting (IB) model neuron. We identify synaptic location within the basal dendrites as a feature of stimulus selectivity. Specifically, persistent activity-inducing stimuli consist of activated synapses that are located more distally from the soma compared to non-inducing stimuli, in both model cells. In addition, the action potential (AP) latency and the first few inter-spike-intervals of the neuronal response can be used to reliably detect inducing vs. non-inducing inputs, suggesting a potential mechanism by which downstream neurons can rapidly decode the upcoming emergence of persistent activity. While the two model neurons did not differ in the coding features of persistent activity emergence, the properties of persistent activity, such as the firing pattern and the duration of temporally-restricted persistent activity were distinct. Collectively, our results pinpoint to specific features of the neuronal response to a given stimulus that code for its ability to induce persistent activity and predict differential roles of RS and IB neurons in persistent activity expression.     

NO-cGMP mediated galanin expression in NGF-deprived or axotomized sensory neurons

Leukaemia inhibitory factor (LIF) and nerve growth factor (NGF) are well characterized regulators of galanin expression. However, LIF knockout mice containing the rat galanin 5' proximal promoter fragment (- 2546 to + 15 bp) driving luciferase responded to axotomy in the same way as control mice. Also, LIF had no effect on reporter gene expression in vitro, neither in the presence or absence of NGF, suggesting that other factors mediate an axotomy response from the galanin promoter. We then addressed the role of nitric oxide (NO) using NGF-deprived rat dorsal root ganglion (DRG) neuron cultures infected with viral vectors containing the above-mentioned construct, and also studied endogenous galanin expression in axotomized DRG in vivo. Blocking endogenous NO in NGF-deprived DRG cultures suppressed galanin promoter activity. Consistent with this, axotomized/NGF-deprived DRG neurons expressed high levels of neuronal NO synthase (nNOS) and galanin. Further, using pharmacological NOS blockers, or adenoviral vectors expressing dominant-negative either for nNOS or soluble guanylate cyclase in vivo and in vitro, we show that the NO-cGMP pathway induces endogenous galanin in DRG neurons. We propose that both LIF and NO, acting at different promoter regions, are important for the up-regulation of galanin, and for DRG neuron survival and regeneration after axotomy.     

Two high voltage-activated calcium currents are present in isolation in adult rat spinal neurons

In neurons enzymatically isolated from adult rat dorsal root ganglia and used during the following 24 hours, the Ca2+ currents were investigated with the whole-cell patch-clamp technique. In contrast to the neonatal neurons, the salient feature of these adult neurons is the well separated (in the voltage-range) activation and inactivation properties of each recorded current. The low-threshold T-, the high-threshold inactivating N-, and the long-lasting L-currents have a threshold for activation at -60, -45 and -10 mV, and a 50% inactivation at -75, -45 and -5 mV respectively. The N and L currents were poorly affected by 100 microM Ni, a known blocker of T channels and completely blocked by 100 microM Cd2+. Frequently we could find neurons with only one type of current present. We conclude that adult sensory neurons are a better preparation for studying, in isolation, the physiological relevance of the three types of Ca2+ channels.     

Catalpol attenuates nitric oxide increase via ERK signaling pathways induced by rotenone in mesencephalic neurons

Catalpol has been shown to rescue neurons from kinds of damage in vitro and in vivo in previous reports. However, the effect of catalpol on the nitric oxide (NO) system via MAPKs signaling pathway of mesencephalic neurons largely remains to be verified. The current study examined that whether catalpol modulated NO and iNOS increase by rotenone in primary mesencephalic neurons and investigated its underlying signaling pathways. Present results indicated that catalpol inhibited primary mesencephalic neurons from apoptosis by morphological assay, immunocytochemistry and flow cytometric evaluation. Moreover, the ERK signaling pathway plays an important role in NO-mediated degeneration of neuron. The current results suggest that catalpol is a potential agent for the prevention of neurons apoptosis by regulating NO and iNOS increase in ERK-mediated neurodegenerative disorders.     

Dendritic anatomy and electrotonic transfer properties of cat superior colliculus neurons

Detailed morphometrical and corresponding electrotonic characteristics on three classes of cat superior colliculus (SC) neurons have been derived. The sample of cells selected for analysis comprised ascending projection neurons (APNs), inter-layer neurons (ILNs) and tecto-reticulo-spinal neurons (TRSNs) recorded intracellularly and stained with HRP. Superficial SC neurons (APNs, ILNs) could be attached to the allo- and idiodendritic type while deep layer neurons (TRSNs) belong to the isodendritic type. For each neuron, the branching pattern, lengths and diameters of the dendritic trees were determined. These data served as input to the computer program "DENDRIT" from which electrotonic membrane and transfer properties were calculated. Both the morphometrical data and the electronic properties underline the contrasting features of superficial vs deep layer neurons in the SC. Our results support the hypothesis that on the neuron level a close relationship between dendritic pattern and neuron function might exist.     

急性运动轴索型神经病患者血清对培养脊髓运动神经元的影响

为了观察急性运动轴索型神经病(AMAN)病人血清对培养的胚胎大鼠脊髓运动神经元及其轴突的影响,直接、动态观察致病因素对轴突的损害程度。我们分离了胚胎大鼠脊髓腹侧组织,制备成细胞悬液在体外进行原代培养,应用抗非磷酸化神经微丝单克隆抗体SMI-32对培养细胞染色鉴定为运动神经元。培养6天时给予25％浓度AMAN病人血清进行干预,血清中检测有致病型空肠弯曲菌(Cj)PennerO：19型脂多糖抗体存在,正常人血清作为对照组。观察神经元胞体和突起的变化,并经Guillery Shirra及Webster法进行变性纤维染色。结果表明AMAN病人血清干预9h可引起培养运动神经元的轴突变性,嗜银性增加并染为棕黑色;干预12h,胞体开始肿胀,核偏移,胞浆内有银颗粒的沉积,最终培养神经元在16h开始死亡。对照组神经元生长无变化。我们认为AMAN病人血清中含有致病成分,可引起运动神经元轴突变性和继发性胞体改变,最终神经元死亡。推测这种损害在无补体和巨噬细胞参与下,抗PennerO：19型Cj脂多糖抗体起着重要作用。     

Multiple transmitter neurons in Tritonia. II. Control of gut motility

Two large multiple transmitter neurons are located in each buccal ganglion of Tritonia. One of these neurons (B11) contains large quantities of two neuropeptides and acetylcholine (ACh), whereas the other neuron (B12) appears to contain the same two peptides but no ACh. One of the peptides present in these neurons has recently been sequenced and is termed small cardioactive peptide B (SCPB). Both neurons regulate the motility of the gut. Stimulation of B11 produces a posteriorly directed peristalsis after a short latency. This gut movement may normally accompany swallowing. B11 stimulation also produces an increase in the rate of endogenous contractile activity that is similar to that produced by superfusion of the gut with low concentrations (10(-8) M) of SCPB. Stimulation of B12 produces a vigorous longitudinal contraction of the gut, initiated in the posterior part of the gut and not peristaltic in nature. This movement appears incompatible with swallowing behavior and may be involved in regurgitation.     

Nitric oxide and cyclic nucleotides are regulators of neuronal migration in an insect embryo

The dynamic regulation of nitric oxide synthase (NOS) activity and cGMP levels suggests a functional role in the development of nervous systems. We report evidence for a key role of the NO/cGMP signalling cascade on migration of postmitotic neurons in the enteric nervous system of the embryonic grasshopper. During embryonic development, a population of enteric neurons migrates several hundred micrometers on the surface of the midgut. These midgut neurons (MG neurons) exhibit nitric oxide-induced cGMP-immunoreactivity coinciding with the migratory phase. Using a histochemical marker for NOS, we identified potential sources of NO in subsets of the midgut cells below the migrating MG neurons. Pharmacological inhibition of endogenous NOS, soluble guanylyl cyclase (sGC) and protein kinase G (PKG) activity in whole embryo culture significantly blocks MG neuron migration. This pharmacological inhibition can be rescued by supplementing with protoporphyrin IX free acid, an activator of sGC, and membrane-permeant cGMP, indicating that NO/cGMP signalling is essential for MG neuron migration. Conversely, the stimulation of the cAMP/protein kinase A signalling cascade results in an inhibition of cell migration. Activation of either the cGMP or the cAMP cascade influences the cellular distribution of F-actin in neuronal somata in a complementary fashion. The cytochemical stainings and experimental manipulations of cyclic nucleotide levels provide clear evidence that NO/cGMP/PKG signalling is permissive for MG neuron migration, whereas the cAMP/PKA cascade may be a negative regulator. These findings reveal an accessible invertebrate model in which the role of the NO and cyclic nucleotide signalling in neuronal migration can be analyzed in a natural setting.     

Hyperglycemia impairs glucose and insulin regulation of nitric oxide production in glucose-inhibited neurons in the ventromedial hypothalamus

Physiological changes in extracellular glucose, insulin, and leptin regulate glucose-excited (GE) and glucose-inhibited (GI) neurons in the ventromedial hypothalamus (VMH). Nitric oxide (NO) signaling, which is involved in the regulation of food intake and insulin signaling, is altered in obesity and diabetes. We previously showed that glucose and leptin inhibit NO production via the AMP-activated protein kinase (AMPK) pathway, while insulin stimulates NO production via the phosphatidylinositol-3-OH kinase (PI3K) pathway in VMH GI neurons. Hyperglycemia-induced inhibition of AMPK reduces PI3K signaling by activating the mammalian target of rapamycin (mTOR). We hypothesize that hyperglycemia impairs glucose and insulin-regulated NO production in VMH GI neurons. This hypothesis was tested in VMH neurons cultured in hyperglycemic conditions or from streptozotocin-induced type 1 diabetic rats using NO- and membrane potential-sensitive dyes. Neither decreased extracellular glucose from 2.5 to 0.5 mM, nor 5 nM insulin increased NO production in VMH neurons in either experimental condition. Glucose- and insulin-regulated NO production was restored in the presence of the AMPK activator, 5-aminoimidazole-4-carboxamide-1-b-4-ribofuranoside or the mTOR inhibitor rapamycin. Finally, decreased glucose and insulin did not alter membrane potential in VMH neurons cultured in hyperglycemic conditions or from streptozotocin-induced rats. These data suggest that hyperglycemia impairs glucose and insulin regulation of NO production through AMPK inhibition. Furthermore, glucose and insulin signaling pathways interact via the mTOR pathway.     

Induction of cholinergic function in cultured sympathetic neurons by periosteal cells: cellular mechanisms.

Periosteum, the connective tissue surrounding bone, alters the transmitter properties of its sympathetic innervation during development in vivo and after transplantation. Initial noradrenergic properties are downregulated and the innervation acquires cholinergic and peptidergic properties. To elucidate the cellular mechanisms responsible, sympathetic neurons were cultured with primary periosteal cells or osteoblast cell lines. Both primary cells and an immature osteoblast cell line, MC3T3-E1, induced choline acetyltransferase (ChAT) activity. In contrast, lines representing marrow stromal cells or mature osteoblasts did not increase ChAT. Growth of periosteal cells with sympathetic neurons in transwell cultures that prevent direct contact between the neurons and periosteal cells or addition of periosteal cell-conditioned medium to neuron cultures induced ChAT, indicating that periosteal cells release a soluble cholinergic inducing factor. Antibodies against LIFRbeta, a receptor subunit shared by neuropoietic cytokines, prevented ChAT induction in periosteal cell/neuron cocultures, suggesting that a member of this family is responsible. ChAT activity was increased in neurons grown with periosteal cells or conditioned medium from mice lacking either leukemia inhibitory factor (LIF) or LIF and ciliary neurotrophic factor (CNTF). These results provide evidence that periosteal cells influence sympathetic neuron phenotype by releasing a soluble cholinergic factor that is neither LIF nor CNTF but signals via LIFRbeta.     

ERK1/2 antagonizes glycogen synthase kinase-3beta-induced apoptosis in cortical neurons

Inhibition of glycogen synthase kinase-3beta (GSK3beta) is one of the mechanisms by which phosphatidylinositol 3-kinase (PI3K) activation protects neurons from apoptosis. Here, we report that inhibition of ERK1/2 increased the basal activity of GSK3beta in cortical neurons and that both ERK1/2 and PI3K were required for brain-derived neurotrophic factor (BDNF) suppression of GSK3beta activity. Moreover, cortical neuron apoptosis induced by expression of recombinant GSK3beta was inhibited by coexpression of constitutively active MKK1 or PI3K. Activation of both endogenous ERK1/2 and PI3K signaling pathways was required for BDNF to block apoptosis induced by expression of recombinant GSK3beta. Furthermore, cortical neuron apoptosis induced by LY294002-mediated activation of endogenous GSK3beta was blocked by expression of constitutively active MKK1 or by BDNF via stimulation of the endogenous ERK1/2 pathway. Although both PI3K and ERK1/2 inhibited GSK3beta activity, neither had an effect on GSK3beta phosphorylation at Tyr-216. Interestingly, PI3K (but not ERK1/2) induced the inhibitory phosphorylation of GSK3beta at Ser-9. Significantly, coexpression of constitutively active MKK1 (but not PI3K) still suppressed neuronal apoptosis induced by expression of the GSK3beta(S9A) mutant. These data suggest that activation of the ERK1/2 signaling pathway protects neurons from GSK3beta-induced apoptosis and that inhibition of GSK3beta may be a common target by which ERK1/2 and PI3K protect neurons from apoptosis. Furthermore, ERK1/2 inhibits GSK3beta activity via a novel mechanism that is independent of Ser-9 phosphorylation and likely does not involve Tyr-216 phosphorylation.     

Morphology of two pairs of identified peptidergic neurons in the buccal ganglia of the mollusc Tritonia diomedea

The morphology of two pairs of identified peptidergic neurons (B11 and B12) located in the buccal ganglia of Tritonia diomedea was described. Both pairs of neurons contained a large quantity of a small cardioactive peptide (SCP) in their somata. One of the pairs (B11), the large dorsal white cells, contained ACh in their somata along with SCP. Both pairs of cells appeared white in live preparations under epi-illumination. Each B11 and B12 was a unipolar neuron and sent its major axonal branch through the ipsilateral gastro-esophageal nerve to the gut. In addition, B12 sent a small branch to the contralateral buccal ganglion. A characteristic feature of both neuron pairs was their vesicular content. Three types of vesicles were observed in B11. Vesicles with electron-lucent core (LCV) and electron-dense core (DCV) were found in the somata. The axon hillock and the beginning of axon contained vesicles with variable electron dense core (VDCV) in addition to LCV and DCV. The ratio of DCV: LCV: VDCV changed from 5:95:0 for the perinuclear cytoplasm to 8:55:37 for the beginning of axon. The average maximum diameters were 97 +/- 23 nm for DCV, 103 +/- 32 nm for LCV and 106 +/- 29 nm for VDCV. B12 somata contained DCV (average maximum diameter 100 +/- 26 nm), LCV (109 +/- 23 nm) and elliptical vesicles with eccentric electron-opaque core (115 +/- 20 nm).     

Multiple transmitter neurons in Tritonia. I. Biochemical studies

The buccal ganglia of the marine mollusc Tritonia control a variety of movements associated with feeding, including gut motility. The buccal ganglia and gut contain a class of peptides termed small cardioactive peptides (SCPs). Cobalt backfilling of the nerve which innervates the gut stains several buccal neurons including two pairs of reidentifiable cells, B11 and B12. Both appear white under epiillumination, a characteristic of peptidergic neurons in gastropods. Enzymatic and biochemical analyses of extracts from microdissected B11 cell bodies demonstrate that this neuron contains two species of SCPs. Labeling in organ culture followed by dissection and extraction of cell bodies indicates that these peptides were synthesized in B11. One of these peptides appears to be identical to SCPB, one of two SCPs that have been sequenced. The other SCP present in these neurons is novel. Less extensive analyses of extracts of B12 somata suggest that it also contains the same SCPs. In addition to the peptides, B11 also contains large quantities of acetylcholine (ACh) as determined by a radioenzymatic assay of cell body extracts. B12 does not contain measureable ACh. The concentration of the two peptides and ACh in the B11 cytoplasm is approximately 1 mM. Neuron B11 appears to be an appropriate model system for studying the biochemical and physiological properties of multiple transmitter neurons.     

Frequency-dependent and cell-specific effects of electrical activity on growth cone movements of cultured Helisoma neurons

The present experiments address the question of how stimulation parameters, which evoke action potentials in neuronal cell bodies, influence growth cone movements of different identified neurons. The motility of growth cones of Helisoma buccal neurons B19 and B4 was monitored while somata were stimulated simultaneously via an intracellular microelectrode. The findings show that the responses of growth cones of B19 and B4 contain components that are common as well as unique to each neuron. Whereas rates of growth cone advance were suppressed in a graded fashion by stimulus frequencies beyond a threshold of 2 s-1 for both neurons, B4 was more sensitive to electrical stimulation and exhibited a new response, namely, growth rates were enhanced during the poststimulation recovery period after stimulation at specific frequencies. Thus, electrical activity can result in enhancement as well as in inhibition of growth cone movement. Changes in number of filopodia on B19 and B4 were graded also, with B4 again displaying greater sensitivity. The frequency dependence of filopodia compared to growth rate changes was different and suggests a possible dissociation between filopodial activity and growth cone motility. Patterned electrical activity produced effects similar to constant stimulation for B19 growth cones, whereas it decreased the threshold frequency and eliminated the growth enhancement effect for B4. Taken together, these data demonstrate that the quantitative features of electrical activity as well as intrinsic properties of neurons both determine the growth cone response to changes in neuronal activity.     

Elevated endogenous nitric oxide increases Ca2+ flux via L-type Ca2+ channels by S-nitrosylation in rat hippocampal neurons during severe hypoxia and in vitro ischemia

Nitric oxide (NO) mediates pathogenic changes in the brain subsequent to energy deprivation; yet the NO mechanism involved in the early events remains unclear. We examined the acute effects of severe hypoxia and oxygen-glucose deprivation (OGD) on the endogenous NO production and the NO-mediated pathways involved in the intracellular calcium ([Ca(2+)](i)) response in the rat hippocampal neurons. The levels of NO and [Ca(2+)](i) in the CA1 region of the slices rapidly elevated in hypoxia and were more prominent in OGD, measured by the electrochemical method and spectrofluorometry, respectively. The NO and [Ca(2+)](i) responses were enhanced by L-arginine and were reduced by NO synthase inhibitors, suggesting that the endogenous NO increases the [Ca(2+)](i) response to energy deprivation. Nickel and nifedipine significantly decreased the NO and [Ca(2+)](i) responses to hypoxia and OGD, indicating an involvement of L-type Ca(2+) channels in the NO-mediated mechanisms. In addition, the [Ca(2+)](i) responses were attenuated by ODQ or KT5823, inhibitors of the cGMP-PKG pathway, and by acivicin, an inhibitor of gamma-glutamyl transpeptidase for S-nitrosylation, and by the thiol-alkylating agent N-ethylmaleimide (NEM). Moreover, L-type Ca(2+) currents in cultured hippocampal neurons with whole-cell recording were significantly increased by L-arginine and were decreased by L-NAME. Pretreatment with NO synthase inhibitors or NEM but not ODQ abolished the effect of L-arginine on the Ca(2+) currents. Also, vitamin C, which decomposes nitrosothiol but not disulfide by reduction, reversed the change in the Ca(2+) current with L-arginine. Taken together, the results suggest that an elevated endogenous NO production enhances the influx of Ca(2+) via the hippocampal L-type Ca(2+) channel by S-nitrosylation during an initial phase of energy deprivation.