Activation of neuropeptide Y Y1 receptors inhibits glutamate release through reduction of voltage-dependent Ca2+ entry in the rat cerebral cortex nerve terminals: suppression of this inhibitory effect by the protein kinase C-dependent facilitatory pathway

Neuropeptide Y (NPY) is known to regulate the presynaptic glutamate release and neuronal responses to excitatory neurotransmission. The aim of this study was to investigate the effect of NPY on the release of endogenous glutamate from rat cerebrocortical nerve terminals (synaptosomes). NPY inhibited the Ca2+-dependent glutamate release evoked by 4-aminopyridine, and this inhibitory effect was mediated via NPY Y1 receptors, because it was mimicked by the specific NPY Y1 receptor agonist [Leu31 Pro34] NPY and blocked by the NPY Y1 receptor antagonist GR 231118. The inhibitory action of NPY was not due to it decreasing synaptosomal excitability or directly interfering with the release process at some point subsequent to Ca2+ influx, because NPY did not alter the 4-aminopyridine-evoked depolarization of the synaptosomal plasma membrane potential or ionomycin and hypertonic solution-induced glutamate release. Examination of the effect of NPY on the cytosolic [Ca2+] revealed that the inhibition of glutamate release could be attributed to a reduction in voltage-dependent Ca2+ influx. Consistent with this, the NPY-mediated inhibition of glutamate release was completely abolished in synaptosomes pretreated with N- and P/Q-type Ca2+ channel blocker, omega-conotoxin MVIIC. Moreover, NPY-mediated inhibition of 4-aminopyridine-evoked glutamate release was insensitive to KT 5720 and Ro32-0432 but was suppressed when protein kinase C was stimulated with phorbol ester. Together, these results suggest that NPY acting predominantly on NPY Y1 receptors inhibits glutamate release from rat cerebrocortical synaptosomes, likely by a mechanism involving direct coupling of receptors to N- and P/Q-type Ca2+ channels, and this coupling is subject to regulation by protein kinase C-dependent pathway. This implies that selective ligand for NPY receptors may be of value for treatment of conditions characterized by excessive glutamate release in the cerebral cortex.     

Multiple neuropeptide Y receptors regulate K+ and Ca2+ channels in acutely isolated neurons from the rat arcuate nucleus

We examined the effects of neuropeptide Y (NPY) and related peptides on Ca2+ and K+ currents in acutely isolated neurons from the arcuate nucleus of the rat. NPY analogues that activated all of the known NPY receptors (Y1-Y5), produced voltage-dependent inhibition of Ca2+ currents and activation of inwardly rectifying K+ currents in arcuate neurons. Both of these effects could occur simultaneously in the same cells. In some cells, activation of Y4 NPY receptors also caused oscillations in [Ca2+]i. NPY hyperpolarized arcuate neurons through the activation of a K+ conductance and increased the spike threshold. Molecular biological studies indicated that arcuate neurons possessed all of the previously cloned NPY receptor types (Y1, Y2, Y4, and Y5). Thus activation of multiple types NPY receptors on arcuate neurons can regulate both Ca2+ and K+ conductances leading to a reduction in neuronal excitability and a suppression of neurotransmitter release.     

Postsynaptic target regulates functional responses induced by 5-HT3 serotonin receptors on axonal varicosities of NG108-15 hybrid neuroblastoma cells

Rat brain presynaptic 5-HT3 serotonin receptors, members of the ligand-gated ion channel superfamily, induce changes in nerve terminal [Ca2+]i in a manner distinct from that found for somatic 5-HT3 receptors. Here, we assessed the role of postsynaptic target in regulating the nature of presynaptic receptor-induced responses, using the hybrid neuroblastoma cell line NG108-15 as a model neuronal system that expresses 5-HT3 receptors. Using immunocytochemistry, 5-HT3 receptors were found to be present on the presynaptic-like varicosities of differentiated NG108-15 cells, indicating that these receptors possess an inherent ability to localize to potential presynaptic sites. In the absence of postsynaptic target, 5-HT3 receptors localized to the varicosities induce rapid but transient changes in [Ca2+]i that were initiated by voltage-gated Ca2+ channels, as assessed using Ca2+ channel blockers, these properties being typical of those found for somatic 5-HT3 receptors. In co-cultures containing rat myotubes, with which NG108-15 cells form functional cholinergic synapses, the 5-HT3 receptor-induced changes in [Ca2+]i in the axonal varicosities shifted over time (three to 10 days) to that found for brain nerve endings: sustained responses that were insensitive to blockade by antagonists of voltage-gated Ca2+ channels. The effect of co-culturing myotubes with the NG108-15 cells was mimicked by conditioned media from myotube cultures. These results indicate that regulatory molecules from the target postsynaptic cell dictate the functional responses elicited by presynaptic 5-HT3 receptors. Because the target-induced changes required several days before they were evident, we hypothesize that changes in protein expression, perhaps the consequence of altered gene regulation, underlie the changes in the responses to 5-HT3 receptor activation in the axonal varicosities of this neuronal cell line.     

Involvement of peripheral neuropeptide Y receptors in sympathetic modulation of acute cutaneous flare induced by intradermal capsaicin

In a recent study, we have demonstrated that the dorsal root reflex (DRR)-mediated acute cutaneous neurogenic inflammation following intradermal injection of capsaicin (CAP) is sympathetically dependent and subject to modulation by peripheral alpha(1)-adrenoceptors. Postganglionic sympathetic neurons contain not only adrenergic neurotransmitters, but also non-adrenergic substances, including neuropeptide Y (NPY). In this study, we examined if peripheral NPY receptors participate in the flare following CAP injection. Different NPY receptor subtypes were studied by using relatively specific agonists and antagonists for the Y(1) and Y(2) subtypes. Changes in cutaneous blood flow on the plantar surface of the foot were measured using a laser Doppler flowmeter. Following CAP injection, cutaneous flare spread more than 20 mm away from the site of CAP injection. Removal of the postganglionic sympathetic nerves by surgical sympathectomy reduced dramatically the CAP-evoked flare. If the foot of sympathectomized rats was pretreated with either NPY or Y(2) receptor agonists by intra-arterial injection, the spread of flare induced by CAP injection could be restored and prolonged. However, if the spinal cord was pretreated with a GABA(A) receptor antagonist, bicuculline, to prevent DRRs, NPY or an Y(2) receptor agonist no longer restored the CAP-evoked flare. A Y(1) receptor agonist did not affect the CAP-evoked flare in sympathectomized rats. In sympathetically intact rats, blockade of either peripheral NPY or Y(2) receptors with [D-Trp(32)]-NPY or BIIE0246 markedly reduced the flare induced by CAP injection, whereas blockade of peripheral Y(1) receptors by BIBP3226 did not obviously affect the flare. It is suggested that NPY is co-released with NE from the postganglionic sympathetic terminals to activate NPY Y(2) and alpha(1) receptors following CAP injection. Both substances are involved, at least in part, in modulation of the responses of CAP sensitive afferents thereby affecting their ability to evoke the release of inflammatory agents from primary afferents.     

Electrophysiological and immunofluorescence characterization of Ca(2+) channels of acutely isolated rat sphenopalatine ganglion neurons

The sphenopalatine ganglion (SPG) is the main parasympathetic ganglion that is involved in regulating cerebral vascular tone and gland secretion. SPG neurons have been implicated in some types of migraine headaches but their precise role has yet to be determined. In addition, very little information is available regarding ion channel modulation by neurotransmitters that are involved in the parasympathetic drive of SPG neurons. In this study, acute isolation of adult rat SPG neurons was developed in order to begin the electrophysiological characterization of this ganglion. Under our dissociation conditions, the average number of neurons obtained per ganglion was greater than 1200. Immunofluorescence imaging results showed positive labeling with acetylcholinesterase (AChE), confirming the parasympathetic nature of SPG neurons. On the other hand, weak tyrosine hydroxylase immunostaining was observed in these neurons. Whole-cell patch-clamp recordings revealed that most of the Ca(2+) current is carried by N-type (53%) and SNX-482 resistant R-type (30%) Ca(2+) channels. In addition, Ca(2+) currents were inhibited in a voltage-dependent manner following exposure to oxotremorine-M (Oxo-M), norepinephrine and ATP via muscarinic acetylcholine receptor 2 (M(2) AChR) subtype, adrenergic and P2Y purinergic receptors, respectively. The peptides VIP and angiotensin II failed to modulate Ca(2+) currents, suggesting that these receptors are not present on the SPG soma or do not couple to Ca(2+) channels. In summary, our data suggest that the Ca(2+) current inhibition mediated by Oxo-M, NE and ATP in adult rat SPG neurons plays an integral part in maintaining parasympathetic control of cranial functions.     

A novel inositol phosphate selectively inhibits vasoconstriction evoked by the sympathetic co-transmitters neuropeptide Y (NPY) and adenosine triphosphate (ATP).

Postganglionic sympathetic nerves release norepinephrine (NE) as their primary neurotransmitter at vascular and other targets. However, much evidence supports involvement of additional messengers, co-transmitters, which are co-released with NE upon sympathetic nerve stimulation and thereby contribute to their actions, e.g., vasoconstriction. Two such putative co-transmitters, neuropeptide Y (NPY) and adenosine triphosphate (ATP) have been of particular interest since they fulfill several neurotransmitter criteria. Importantly, hitherto it has been difficult to antagonize vasoconstriction evoked by either NPY or ATP with agents that are devoid of intrinsic activity. The present study describes the ability of a novel inositol phosphate, D-myo-inositol 1,2,6-trisphosphate (Ins[1,2,6]P3; PP-56) to in vitro potently block vasoconstrictor responses elicited by NPY and ATP, but not by NE, as studied in guinea-pig isolated basilar artery. The action of Ins[1,2,6]P3 does not seem to occur through antagonism at NPY- or ATP-receptor recognition sites, labeled by 125I-peptide YY and 35S-gamma-ATP, respectively, in membranes of rat cultured vena cava vascular smooth muscle cells. However, it does involve inhibition of the influx of Ca2+ induced by either co-transmitter in these same vena cava cells. It is proposed that Ins[1,2,6]P3 may be a useful functional antagonist of non-adrenergic component(s) of the vasoconstrictor response to sympathetic nerve stimulation.     

Inhibition of vasoconstriction and Ca2+ currents mediated by neuropeptide Y Y2 receptors.

The effects of neuropeptide Y (NPY) and agonists selective for NPY Y1 and Y2 receptors were studied on contraction and Ca2+ currents in arterial smooth muscle. In isolated arterioles from the guinea pig small intestine, small brief constrictions were evoked by depolarising the arteriolar smooth muscle using high K+ solution applied from a micropipette. The constrictions were reduced in amplitude by the Y2-selective agonists PYY(13-36) and N-acetyl[Leu28, Leu31]NPY-(24-36) in concentrations from 20-100 nM. NPY or the Y1 selective agonist [Leu31 Pro34]NPY in concentrations from 50 pM to 100 nM increased the amplitude of the constrictions, with a maximum effect at 10 nM. Smooth muscle cells were isolated from rat small mesenteric arteries, and voltage-activated Ca2+ currents measured by whole cell patch clamping. The peak amplitude of the Ca2+ currents was decreased by N-acetyl[Leu28, Leu31]NPY-(24-36), and by NPY (100 nM). [Leu31, Pro34]NPY either had no effect or slightly increased the Ca2+ currents. We conclude that Y2 receptors on vascular smooth muscle can reduce Ca2+ currents induced by depolarisation, and thus oppose constriction caused by smooth muscle depolarisation.     

Opioid-like actions of neuropeptide Y in rat substantia gelatinosa: Y1 suppression of inhibition and Y2 suppression of excitation

Neuropathic pain that results from injury to the peripheral or CNS responds poorly to opioid analgesics. Y1 and Y2 receptors for neuropeptide Y (NPY) may, however, serve as targets for analgesics that retain their effectiveness in neuropathic pain states. In substantia gelatinosa neurons in spinal cord slices from adult rats, we find that NPY acts via presynaptic Y2 receptors to attenuate excitatory postsynaptic currents (EPSCs) and predominantly on presynaptic Y1 receptors to attenuate glycinergic and GABAergic inhibitory postsynaptic currents (IPSCs). Because NPY attenuates the frequency of TTX-resistant miniature EPSCs and IPSCs, perturbation of the neurotransmitter release process contributes to its actions at both excitatory and inhibitory synapses. These effects, which are reminiscent of those produced by analgesic opioids, provide a cellular basis for previously documented spinal analgesic actions mediated via Y1 and Y2 receptors in neuropathic pain paradigms. They also underline the importance of suppression of inhibition in spinal analgesic mechanisms.     

Low potency inhibition of Ca channel currents in human neuroblastoma (SH-SY5Y) cells by [Ala]NPY, an l-alanine substituted analogue of neuropeptide Y

Whole-cell Ca²⁺ channel currents were recorded in human neuroblastoma (SH-SY5Y) cells, using the perforated-patch technique with 10 mM Ba²⁺ as charge carrier. Neuropeptide Y (NPY; 10 nM to 1 μM) caused concentration-dependent inhibition of Ca²⁺ channel currents which were associated with a slowing in current activation kinetics. [Ala³¹]NPY, a residue 31 l-alanine substituted analogue of NPY, also inhibited Ca²⁺ channel currents and caused slowing of activation kinetics, but with approximately 6-fold lower potency. In the presence of 100 nM [Ala³¹]NPY (which itself had little or no effect on currents), the actions of NPY were similar in magnitude to its effects in the absence of the analogue. Our results suggest that substitution of isoleucine for alanine at residue 31 results in a NPY analogue which is a full agonist but with lower affinity for Y₂ receptors.     

Effect of high Ba(2+) on norepinephrine-induced inhibition of N-type calcium current in bullfrog sympathetic neurons

The voltage-dependent inhibition of N-type calcium current by neurotransmitters is the best-understood example of neuronal calcium channel inhibition. One of the mechanisms by which this pathway is thought to inhibit the calcium current is by reducing the permeation of divalent cations through the channel. In this study one prediction of this hypothesis was examined, that high concentrations of divalent cations reduce the maximum neurotransmitter-induced inhibition. Norepinephrine (NE)-induced inhibition was compared in external solutions containing either 2 or 100 mM Ba(2+). Initially, NE dose-response curves were generated by averaging data from many neurons, and it was found that the relationship was right shifted in the high-Ba(2+) external solution without an effect on maximum inhibition. The IC(50) was 0.6 and 3 microM in 2 and 100 mM Ba(2+), respectively. This shift was verified by comparing the effect of NE on single neurons exposed to both 2 and 100 mM Ba(2+). The inhibition induced by 1 microM NE was reduced in 100 mM Ba(2+) compared with that in 2 mM Ba(2+). However, the response to 100 microM NE was identical between high and low Ba(2+). Thus, divalent cations appear to act as a competitive inhibitor of NE binding, which likely results from these ions' interacting with negatively charged amino acids that are important for catecholamine binding to adrenergic receptors. Because the maximum inhibition induced by NE was similar in low and high Ba(2+), the effect of inhibition on single N-type calcium channels was not altered by the divalent cation concentration.     

Nerve injury increases an excitatory action of neuropeptide Y and Y2-agonists on dorsal root ganglion neurons

Damage to sensory nerves invokes the expression of neuropeptide Y in the cell bodies of sensory neurons in dorsal root ganglia. We therefore compared the action of this peptide on control dorsal root ganglia neurons with its action on neurons from animals in which the sciatic nerve had been cut. Neuropeptide Y (0.1-1.0 microM) increased the excitability of 24% of control neurons and its effect was stronger and more cells (56%) were affected after axotomy. Increased excitability was mediated via a Y2-receptor and resulted from attenuation of Ca2+-sensitive K+-conductance(s) secondary to suppression of N-type Ca2+ channel current. Y1-agonists potentiated L-type Ca2+ channel current in control neurons without altering excitability. This Y1-effect was attenuated whereas effects mediated via Y2-receptors were enhanced after axotomy. No evidence was found for involvement of Y4- or Y5-receptor subtypes in the actions of neuropeptide Y either on control or on axotomized dorsal root ganglion neurons. It is concluded that neuropeptide Y increases the excitability of sensory neurons by interacting with a Y2-receptor and thereby decreasing N-type Ca2+ channel current and Ca2+-sensitive K+-conductance(s). When peripheral nerves are damaged, dorsal root ganglion neurons start to express neuropeptide Y and its excitatory Y2-excitatory effects are enhanced. The peptide may therefore contribute to the generation of aberrant sensory activity and perhaps to the etiology of injury-induced neuropathic pain.     

Stimulation of 5-HT(2) receptors in prefrontal pyramidal neurons inhibits Ca(v)1.2 L type Ca(2+) currents via a PLCbeta/IP3/calcineurin signaling cascade

There is growing evidence linking alterations in serotonergic signaling in the prefrontal cortex to the etiology of schizophrenia. Prefrontal pyramidal neurons are richly innervated by serotonergic fibers and express high levels of serotonergic 5-HT(2)-class receptors. It is unclear, however, how activation of these receptors modulates cellular activity. To help fill this gap, whole cell voltage-clamp and single-cell RT-PCR studies of acutely isolated layer V-VI prefrontal pyramidal neurons were undertaken. The vast majority (>80%) of these neurons had detectable levels of 5-HT(2A) or 5-HT(2C) receptor mRNA. Bath application of 5-HT(2) agonists inhibited voltage-dependent Ca(2+) channel currents. L-type Ca(2+) channels were a particularly prominent target of this signaling pathway. The L-type channel modulation was blocked by disruption of G(alphaq) signaling or by inhibition of phospholipase Cbeta. Antagonism of intracellular inositol trisphosphate signaling, chelation of intracellular Ca(2+), or depletion of intracellular Ca(2+) stores also blocked this modulation. Inhibition of the Ca(2+)-dependent phosphatase calcineurin prevented receptor-mediated modulation of L-type currents. Last, the 5-HT(2) receptor modulation was robustly expressed in neurons from Ca(v)1.3 knockout mice. These findings argue that 5-HT(2) receptors couple through G(alphaq) proteins to trigger a phospholipase Cbeta/inositol trisphosphate signaling cascade resulting in the mobilization of intracellular Ca(2+), activation of calcineurin, and inhibition of Ca(v)1.2 L-type Ca(2+) currents. This modulation and its blockade by atypical neuroleptics could have wide-ranging effects on synaptic integration and long-term gene expression in deep-layer prefrontal pyramidal neurons.     

Hypothalamic and hindbrain NPY, AGRP and NE increase consummatory feeding responses

Feeding behavior is comprised of both appetitive and consummatory responses to food. Appetitive responses include the motivated acquisition of food. Consummatory responses, including swallowing, are those that move the food from the mouth to the stomach. Intraoral delivery of liquid food bypasses the requirement for appetitive responses and has been used to examine consummatory responses directly in intact rats. In the present study, we administered neuropeptide Y (NPY), agouti-related protein (AGRP) and norepinephrine (NE), into the paraventricular nucleus of the hypothalamus (PVN) or into the fourth cerebral ventricle to examine their effects on the consummatory component of feeding behavior in the rat. To measure consummatory responses, milk (40% lactose free cow's milk diluted with water) was infused intraorally through a chronic cheek fistula (1 ml/min), using an alternating 5 min on − 1 min off schedule, until rejection occurred. We found that both hypothalamic and fourth ventricle injections of NPY, AGRP and NE significantly increased consumption of the intraorally-delivered milk. Our results indicate that the circuitry for modulation of consummatory ingestive responses includes NE, NPY and AGRP receptors operating in both hypothalamic and hindbrain sites.     

Modulation of ion channels by somatostatin and acetylcholine.

Somatostatin and muscarinic acetylcholine receptors are similar as far as modulation of voltage-gated Ca2+ channels and anomalously rectifying K+ channels are concerned. Activation of either type of receptors induces inhibition of Ca2+ channels and activation of anomalous K+ channels without depending on intracellular cAMP. Somatostatin appears to act on the same receptor subtype for these two actions since somatostatin receptors are homogenous in pituitary cells (Srikant and Patel, 1982; Tran et al., 1985) where the peptide produces these two effects as well as an inhibition of adenylate cyclase. In the case of muscarinic receptors, however, it remains unclear whether the same subtype of receptors is involved in both inhibition of Ca2+ channels and activation of K+ channels. Activation of muscarinic receptors in hippocampal neurones evidently produces a cAMP-independent suppression of Ca2+ channel. In cardiac cells, however, muscarinic stimulation does not cause a cAMP-independent suppression of Ca2+ channels but does activate an anomalous rectifier. These findings do not necessarily mean that the muscarinic receptor involved in the inhibition of Ca2+ channels in hippocampal neurones is not of m2 type which is assumed to mediate the activation of anomalous K+ channels in cardiac cells. There is no evidence that cardiac Ca2+ channels are identical to hippocampal Ca2+ channels susceptible to muscarinic inhibition. In addition, a similar argument could be applied to G proteins coupling muscarinic receptors to Ca2+ channels in neurones and cardiac myocytes. In this regard, it should be noted that activation of GABAB receptors or mu and delta opiate receptors, an event known to inhibit adenylate cyclase activity through a PTX-sensitive Gi protein, also produces both inhibition of Ca2+ channels and activation of anomalous K channels in a cAMP-independent manner. This close correlation between inhibition of adenylate cyclase activity and cAMP-independent modulation of Ca2+ and K+ channels suggests the possible involvement of m2 subtype in the inhibition of Ca2+ channels in hippocampal neurones. Circumstantial evidence indicates that anomalous K+ channels are directly activated by alpha subunits of Gi, but not Go, proteins. The alpha subunit of Go protein seems to mediate inhibition of the Ca2+ channel, probably in a direct manner. The most striking difference between somatostatin and muscarinic receptors would be their opposite actions on the M channel. All the inhibitory receptors on the M channel, including m1 and m3 receptors, are known to stimulate PI hydrolysis via a PTX-insensitive G protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hindbrain catecholamine neurons control multiple glucoregulatory responses

Reduced brain glucose availability evokes an integrated constellation of responses that protect and restore the brain's glucose supply. These include increased food intake, adrenal medullary secretion, corticosterone secretion and suppression of estrous cycles. Our research has focused on mechanisms and neural circuitry underlying these systemic glucoregulatory responses. Using microinjection techniques, we found that localized glucoprivation of hindbrain but not hypothalamic sites, elicited key glucoregulatory responses, indicating that glucoreceptor cells controlling these responses are located in the hindbrain. Selective destruction of hindbrain catecholamine neurons using the retrogradely transported immunotoxin, anti-dopamine beta-hydroxylase conjugated to saporin (DSAP), revealed that spinally-projecting epinephrine (E) or norepinephrine (NE) neurons are required for the adrenal medullary response to glucoprivation, while E/NE neurons with hypothalamic projections are required for feeding, corticosterone and reproductive responses. We also found that E/NE neurons are required for both consummatory and appetitive phases of glucoprivic feeding, suggesting that multilevel collateral projections of these neurons coordinate various components of the behavioral response. Epinephrine or NE neurons co-expressing neuropeptide Y (NPY) may be the neuronal phenotype required for glucoprivic feeding: they increase NPY mRNA expression in response to glucoprivation and are nearly eliminated by DSAP injections that abolish glucoprivic feeding. In contrast, lesion of arcuate nucleus NPY neurons, using the toxin, NPY-saporin, does not impair glucoprivic feeding or hyperglycemic responses. Thus, hindbrain E/NE neurons orchestrate multiple concurrent glucoregulatory responses. Specific catecholamine phenotypes may mediate the individual components of the overall response. Glucoreceptive control of these neurons resides within the hindbrain.     

Purinergic activation of BK channels in clonal kidney cells (Vero cells)

We have studied the activation of a high-conductance channel in clonal kidney cells from African green monkey (Vero cells) using patch-clamp recordings and microfluorometric (fura-2) measurements of cytosolic Ca2+. The single-channel conductance in excised patches is 170 pS in symmetrical 140 mM KCl. The channel is highly selective for K+ and activated by membrane depolarization and application of Ca2+ to the cytoplasmatic side of the patch. The channel is, thus, a large-conductance Ca2+-activated K+ channel (BK channel). Cell-attached recordings revealed that the channel is inactive in unstimulated cells. Extracellular application of less than 0.1 microM ATP transiently increased the cytosolic Ca2+ concentration ([Ca2+]i) to about 550 nM, and induced membrane hyperpolarization caused by Ca2+-activated K+ currents. ATP stimulation also activated BK channels in cell-attached patches at both the normal-resting potential and during membrane hyperpolarization. The increase in [Ca2+]i was owing to Ca2+ release from internal stores, suggesting that Vero cells express G-protein-coupled purinergic receptors (P2Y) mediating IP3-induced release of Ca2+. The P2Y receptors were sensitive to both uracil triphosphate (UTP) and adenosine diphosphate (ADP), and the rank of agonist potency was ATP > UTP >/= ADP. This result indicates the presence of both P2Y1 and P2Y2 receptors or a receptor subtype with untypical agonist sensitivity. It has previously been shown that hypotonic challenge activates BK channels in both normal and clonal kidney cells. The subsequent loss of KCl may be an important factor in cellular volume regulation. Our results support the idea of an autocrine role of ATP in this process. A minute release of ATP induced by hypotonically evoked membrane stretch may activate the P2Y receptors, subsequently increasing [Ca2+]i and thus causing K+ efflux through BK channels.     

GABAergic protection of hippocampal pyramidal neurons against glutamate insult: deficit in young animals compared to adults

Hypoxia-ischemia (HI) injury in neonatal animals leads to selective regional loss of neurons including CA1 and CA3 pyramidal neurons of the hippocampus as well as nonlethal pathologies. Glutamate-receptor over-activation and Ca2+ influx are involved in these neonatal changes. We examined glutamate-evoked Ca2+ responses in neonatal (PN 7-13) and young adult (PN 21-27) CA1 pyramidal neurons in acute slices from rats. In neonates, transient exposure to glutamate produced large Ca2+ increases throughout neurons, including distal dendrites (primary Ca2+ responses). Repeated exposures resulted in sustained Ca2+ increases in apical dendrites (secondary Ca2+ responses) that were independent of continued glutamate exposure. These responses propagated and invaded the soma, resulting in irrecoverably high Ca2+ elevations. In neurons from adults, identical glutamate exposure evoked primary Ca2+ responses only in somata and proximal apical dendrites. Repeated glutamate exposures in the adult neurons also led to secondary Ca2+ responses, but they arose in the peri-somatic region and then spread outward to distal apical dendrites, again without recovery. More stimuli were required to initiate secondary responses in neurons from adult versus neonates. Block of GABAA receptors in adults caused the primary and secondary responses to revert to the spatial pattern seen in the neonates and greatly increased their vulnerability to glutamate. These findings suggest that neurodegenerative secondary Ca2+ events may be important determinants of susceptibility to HI injury in the developing CNS and that immature CA1 neurons may be more susceptible to excitotoxic injury due at least in part to insufficient development of GABAergic inputs to their dendrites.     

The different time courses of reading different levels of Chinese characters: an ERP study

AMP-activated protein kinase (AMPK) is an energy sensor that is activated by the increase of intracellular AMP:ATP ratio. AMPK in the hypothalamic arcuate nucleus (ARC) is activated during fasting and the activation of AMPK stimulates food intake. To clarify the pathway underlying AMPK-induced feeding, we monitored the activity of single ARC neurons by measuring cytosolic Ca(2+) concentration ([Ca(2+)](i)) with fura-2 fluorescence imaging. An AMPK activator, AICA-riboside (AICAR), at 200 μM increased [Ca(2+)](i) in 24% of ARC neurons. AMPK and acetyl CoA carboxylase were phosphorylated in the neurons with [Ca(2+)](i) responses to AICAR. AICAR-induced [Ca(2+)](i) increases were inhibited by Ca(2+)-free condition but not by thapsigargin, suggesting that AICAR increases [Ca(2+)](i) through Ca(2+) influx from extracellular space. Among AICAR-responding ARC neurons, 38% were neuropeptide Y (NPY)-immunoreactive neurons while no proopiomelanocortin (POMC)-immunoreactive neuron was observed. Intracerebroventricular administration of AICAR increased food intake, and the AICAR-induced food intake was abolished by the co-administration of NPY Y1 receptor antagonist, 1229U91. These results indicate that the activation of AMPK leads to the activation of ARC NPY neurons through Ca(2+) influx, thereby causing NPY-dependent food intake. These mechanisms could be implicated in the stimulation of food intake by physiological orexigenic substances.