Action potential firing rhythms in the suprachiasmatic nucleus of the diurnal grass rat, Arvicanthis niloticus.

In mammals, daily physiological activities are regulated by a central circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Recently, an increasing number of studies have used diurnal grass rats to analyze neuronal mechanisms regulating diurnal behavior. However, spontaneous action potential firing rhythms in SCN neurons have not been demonstrated clearly in diurnal grass rats. Therefore, the present study examined extracellular single-unit recordings from SCN neurons in acute hypothalamic slices of Arvicanthis niloticus (Nile grass rats). The results of this study found that circadian firing rhythms with the highest frequency occurred at dusk (6.4 Hz at zeitgeber time (ZT)10-12), while the secondary peak occurred at dawn (5.6 Hz at ZT0-2), and the lowest frequency took place in the middle of the night (3.6 Hz at ZT14-16). Locomotor activity recordings from a separate group of animals demonstrated that the Nile grass rats of the laboratory colony used in this study displayed diurnal behaviors that coincided with large crepuscular peaks under 12:12 h light-dark cycles and bimodal rhythms under constant dim red light. Thus, a positive correlation between SCN firing frequencies and locomotor activity levels was observed in the Nile grass rats. Previously, behavioral coupling of action potential firings in SCN neurons has been suggested by in vivo recordings while the present study demonstrates that the sustenance of bimodal firing rhythms in grass rat SCN neurons can last at least one day in vitro.

A candidate gene of Alzheimer diseases was mutated in senescence-accelerated mouse prone (SAMP) 8 mice.

The senescence-accelerated mouse prone (SAMP) 8 strain exhibits age-related learning and memory deficits (LMD) at 2 months of age. We have found strong association of chromosome 12 locus with learning memory deficit (LMD) phenotype in SAMP8 strain. In the course of searching candidate gene, here we identified solute carrier family 24 sodium/potassium/calcium exchanger member 4 (Slc24a4) in SAMP8 chromosome 12 LMD possessing one single nucleotide polymorphism causing amino acid replacement of Threonine at 413 position with Methionine. Since SLC24A4 has been postulated as a candidate of late onset Alzheimer's diseases (LOAD), we further analyze the functional importance of this polymorphism. By expressing Slc24a4 protein in HEK293 cells, here we showed polymorphic SAMP8 type Slc24a4-T413 M causing significant loss of calcium ion (Ca2+) transporter activity in cells compared with that of wild type mouse (Slc24a4-WT). However, no study yet shows any functional association of human SLC24A4 polymorphism with the onset of LOAD pathogenesis. Thus, our present finding may further help to clarify the importance of this ion exchanger with age related cognitive dysfunction.

Canonical versus non-canonical transsynaptic signaling of neuroligin 3 tunes development of sociality in mice.

Neuroligin 3 (NLGN3) and neurexins (NRXNs) constitute a canonical transsynaptic cell-adhesion pair, which has been implicated in autism. In autism spectrum disorder (ASD) development of sociality can be impaired. However, the molecular mechanism underlying NLGN3-mediated social development is unclear. Here, we identify non-canonical interactions between NLGN3 and protein tyrosine phosphatase δ (PTPδ) splice variants, competing with NRXN binding. NLGN3-PTPδ complex structure revealed a splicing-dependent interaction mode and competition mechanism between PTPδ and NRXNs. Mice carrying a NLGN3 mutation that selectively impairs NLGN3-NRXN interaction show increased sociability, whereas mice where the NLGN3-PTPδ interaction is impaired exhibit impaired social behavior and enhanced motor learning, with imbalance in excitatory/inhibitory synaptic protein expressions, as reported in the Nlgn3 R451C autism model. At neuronal level, the autism-related Nlgn3 R451C mutation causes selective impairment in the non-canonical pathway. Our findings suggest that canonical and non-canonical NLGN3 pathways compete and regulate the development of sociality.

Intracellular interplay between cholecystokinin and leptin signalling for satiety control in rats.

Cholecystokinin (CCK) and leptin are satiety-controlling peptides, yet their interactive roles remain unclear. Here, we addressed this issue using in vitro and in vivo models. In rat C6 glioma cells, leptin pre-treatment enhanced Ca2+ mobilization by a CCK agonist (CCK-8s). This leptin action was reduced by Janus kinase inhibitor (AG490) or PI3-kinase inhibitor (LY294002). Meanwhile, leptin stimulation alone failed to mobilize Ca2+ even in cells overexpressing leptin receptors (C6-ObRb). Leptin increased nuclear immunoreactivity against phosphorylated STAT3 (pSTAT3) whereas CCK-8s reduced leptin-induced nuclear pSTAT3 accumulation in these cells. In the rat ventromedial hypothalamus (VMH), leptin-induced action potential firing was enhanced, whereas nuclear pSTAT3 was reduced by co-stimulation with CCK-8s. To further analyse in vivo signalling interplay, a CCK-1 antagonist (lorglumide) was intraperitoneally injected in rats following 1-h restricted feeding. Food access was increased 3-h after lorglumide injection. At this timepoint, nuclear pSTAT3 was increased whereas c-Fos was decreased in the VMH. Taken together, these results suggest that leptin and CCK receptors may both contribute to short-term satiety, and leptin could positively modulate CCK signalling. Notably, nuclear pSTAT3 levels in this experimental paradigm were negatively correlated with satiety levels, contrary to the generally described transcriptional regulation for long-term satiety via leptin receptors.

Posttetanic enhancement of striato-pallidal synaptic transmission.

The striato (Str)-globus pallidus external segment (GPe) projection plays major roles in the control of neuronal activity in the basal ganglia under both normal and pathological conditions. The present study used rat brain slice preparations to characterize the enhancement of Str-GPe synapses observed after repetitive conditioning stimuli (CS) of Str with the whole cell patch-clamp recording technique. The results show that 1) the Str-GPe synapses have a posttetanic enhancement (PTE) mechanism, which is considered to be a combination of an augmentation and a posttetanic potentiation; 2) the degree of PTE observed in GPe neurons had a wide range and was positively correlated with a wide range of paired-pulse ratios assessed before application of CS; 3) a wide range of CS, from frequencies as low as 2 Hz with as few as 5 pulses to as high as 100 Hz with 100 pulses, could induce PTE; 4) the decay time constant of PTE was dependent on the strength of CS and was prolonged greatly, up to 120 s, when strong CS were applied; and 5) the level of postsynaptic Cl(-) became a limiting factor for the degree of PTE when strong CS were applied. These results imply that Str-GPe synapses transmit inhibitions in a nonlinear activity-weighted manner, which may be suited for scaling timing and force of repeated or sequential body movements. Other possible factors controlling the induction of PTE and functional implications are also discussed.

Serotonin-2C receptor involved serotonin-induced Ca²âº mobilisations in neuronal progenitors and neurons in rat suprachiasmatic nucleus.

The hypothalamic suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals, undergoes serotonergic regulation, but the underlying mechanisms remain obscure. Here, we generated a subclone of an SCN progenitor cell line expressing Ca(2+) sensors (SCN2.2YC) and compared its 5-HT receptor signalling with that of rat SCN neurons in brain slices. SCN2.2YC cells expressed 5-HT1A/2A/2B/2C, but not 5A/7, while all six subtypes were expressed in SCN tissues. High K(+) or 5-HT increased cytosolic Ca(2+) in SCN2.2YC cells. The 5-HT responses were inhibited by ritanserin and SB-221284, but resistant to WAY-100635 and RS-127445, suggesting predominant involvement of 5-HT2C for Ca(2+) mobilisations. Consistently, Ca(2+) imaging and voltage-clamp electrophysiology using rat SCN slices demonstrated post-synaptic 5-HT2C expression. Because 5-HT2C expression was postnatally increased in the SCN and 5-HT-induced Ca(2+) mobilisations were amplified in differentiated SCN2.2YC cells and developed SCN neurons, we suggest that this signalling development occurs in accordance with central clock maturations.

Possible involvement of Hcn1 ion channel in learning and memory dysfunction in SAMP8 mice.

The senescence-accelerated mouse prone 8 (SAMP8) strain exhibits age-related learning and memory deficits (LMD) at 2 months of age. Combined linkage analysis of 264 F2 intercross SAMP8 × JF1 mice and RNA-seq analysis identified Hcn1 gene out of 29 genes in the LMD region on chromosome 13. Hcn1 in SAMP8 strain showed 15 times less polyglutamine repetition compared to Japanese fancy mouse 1 (JF1). Whole cell patch clamp analysis showed that Hcn1 ion conductivity was significantly lower in SAMP8 compared to that of JF1, which may be associated with learning and memory deficiency.

Development of an image processing support system based on fluorescent dye to prevent elderly people with dementia from wandering.

The wandering of elderly people with dementia is a significant behavioral problem and is a heavy burden on caregivers in residential and nursing homes. Thus, warning systems have been developed to prevent elderly people with dementia from leaving the premises. Some of these systems use radio waves. However, systems based on radio waves present several practical problems. For instance, the transmitter must be carried and may become lost; in addition, the battery of the transmitter must be changed. To solve these problems, we developed a support system that prevents elderly people with dementia from wandering. The system employs image processing technology based on fluorescent dye. The composition of the support system can be described as follows: fluorescent dye is painted in a simple shape on the clothes of an elderly person. The fluorescent color becomes visible by irradiation with a long wavelength of ultraviolet light. In the present paper, the relationship between the color of the dye and the cloth was investigated. A 3D video camera was used to acquire a 3D image and detect the simple shape. As a preliminary experiment, 3 colors (red, green and blue) of fluorescent dye were applied to cloths of 9 different colors. All fluorescent colors were detected on 6 of the cloths, but red and blue dye could not be detected on the other 3 cloths. In contrast, green dye was detectable on all 9 of the cloths. Additionally, we determined whether green dye could be detected in an actual environment. A rectangular shaped patch of green fluorescent dye was painted on the shoulder area of a subject, from the scapula to the clavicle. As a result, the green dye was detected on all 9 different colored cloths.

Short-term plasticity shapes activity pattern-dependent striato-pallidal synaptic transmission.

The cortico-striato (Str)-globus pallidus external segment (GPe) projection plays major roles in the control of neuronal activity in the basal ganglia under both normal and pathological conditions. The present study used rat brain-slice preparations to address our hypothesis that the gain of this disynaptic projection is dynamically controlled by activations of short-term plasticity mechanisms of Str-GPe synapses. The Str-GPe projection neurons fire with very different frequency and firing patterns in vivo depending on the condition of the animal. The results show that the Str-GPe synapses have very strong short-term enhancement mechanisms and that repetitive burst activation of the Str-GPe synapses, which mimic oscillatory burst firing of Str neurons, can sustain enhanced states of synaptic transmission for tens of seconds. The results reveal that the short-term enhancement of Str-GPe synapses contributes to the generation of pauses in the firing of GPe neurons and that signal transfer function in the Str-GPe projection is highly dependent on the firing pattern of Str neurons.

Ghrelin postsynaptically depolarizes dorsal raphe neurons in rats in vitro.

Ghrelin promotes growth hormone (GH) secretion and feeding. Recent studies further showed that ghrelin displayed a defending effect against the depressive-like symptoms and affected sleep in animals and humans. Serotonergic system is considered to be implicated in feeding, depression and other mood disorders, and sleep. The dorsal raphe nucleus (DRN) utilizes serotonin (5-HT) as its major neurotransmitter and expresses GH secretagogue receptors (GHS-Rs). Therefore, the present study was carried out to examine the electrophysiological effect of ghrelin on rat DRN neurons in vitro and determine the ionic mechanism involved. Whole-cell recording revealed that ghrelin depolarized DRN neurons dose-dependently in tetrodotoxin-containing artificial cerebrospinal fluid (TTX ACSF). Pretreatment with [D-Lys(3)]-GHRP-6, a selective antagonist for GHS-Rs, antagonized the ghrelin-induced depolarization. The depolarization was significantly reduced in a low-Na(+) TTX ACSF and in a high-K(+) TTX ACSF and was abolished in the combination of both ACSFs, suggesting that the ghrelin-induced depolarization is mediated by a dual ionic mechanism including an increase in nonselective cationic conductance and a decrease in K(+) conductance. The experiments on the reversal potential also supported an involvement of the dual ionic mechanism in the ghrelin-induced depolarization. On the basis of their electrophysiological and pharmacological properties, approximately 80% of DRN neurons were classified as putative 5-HT-containing neurons and ghrelin depolarized 75% of them. These results suggest that DRN neurons, especially 5-HT-containing neurons, might be involved in the neural mechanisms through which ghrelin participates in the development and/or regulation of feeding behavior, sleep-wake states and depressive-like symptoms.

Electrophysiological effects of orexin-B and dopamine on rat nucleus accumbens shell neurons in vitro.

Orexin (ORX) plays a critical role in reward-seeking behavior for natural rewards and drugs of abuse. The mesolimbic dopamine (DA) pathway that projects into the nucleus accumbens (NAc) from the ventral tegmental area is deeply involved in the neural mechanisms underlying reward, drug abuse and motivation. A recent study demonstrated that ORX-immunopositive fibers densely project into the shell of the NAc (NAcSh), suggesting that the NAcSh might be a site of the interaction between the ORXergic and DAergic systems for reward-seeking behavior. Therefore, the electrophysiological effects of ORX-B and DA on NAcSh neurons were examined extracellularly in rat brain slice preparations. ORX-B excited approximately 78% of neurons tested and inhibited 4%, whereas DA excited 50% and inhibited 22% of NAcSh neurons. These excitations and inhibitions persisted during synaptic blockade in a low-Ca(2+)/high-Mg(2+) solution. DA-induced excitation was attenuated by SCH23390 or sulpiride, whereas DA-induced inhibition was suppressed by sulpiride. Of the neurons that were excited by ORX-B, 71% and 18% were excited and inhibited by DA, respectively. In 63% of neurons that were excited by ORX-B, the simultaneous application of ORX-B and DA increased the firing rate to two times greater than ORX-B alone, whereas, the simultaneous application significantly decreased the neuronal firing rate by 73% in the remaining 37% compared to ORX-B. These results suggest that an interaction between the ORXergic and DAergic systems occurs in the NAcSh and that the NAcSh is involved in the neural mechanisms in which ORX participates in the regulation of reward-seeking behavior.

Cytosolic calcium elevation induced by orexin/hypocretin in granule cell domain cells of the rat cochlear nucleus in vitro.

Using rat brain slice preparations, we examined the effect of orexin on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) in the granule cell domain (GCD) cells of the cochlear nucleus that carry non-auditory information to the dorsal cochlear nucleus. Application of orexin concentration-dependently increased [Ca(2+)](i), and in two thirds of GCD cells these increases persisted in the presence of tetrodotoxin. There was no significant difference between the dose-response curve for orexin-A and that for orexin-B. Extracellular Ca(2+) removal abolished the [Ca(2+)](i) elevation induced by orexin-B, whereas depletion of intracellular Ca(2+) stores had no effect. The orexin-B-induced elevation of [Ca(2+)](i) was not blocked by inhibitors of reverse-mode Na(+)/Ca(2+) exchanger (NCX) and nonselective cation channel, whereas it was blocked by lowering the extracellular Na(+) or by applying inhibitors of forward-mode NCX and voltage-gated R- and T-type Ca(2+) channels. The ORX-B-induced increase in [Ca(2+)](i) was also blocked by inhibitors of adenylcyclase (AC) and protein kinase A (PKA), but not by inhibitors of phosphatidylcholine-specific and phosphatidylinositol-specific phospholipase C. In electrophysiological experiments using whole-cell patch clamp recordings, half of GCD cells were depolarized by orexin-B, and the depolarization was abolished by a forward-mode NCX inhibitor. These results suggest that orexin increases [Ca(2+)](i) postsynaptically via orexin 2 receptors, and the increase in [Ca(2+)](i) is induced via the AC-PKA-forward-mode NCX-membrane depolarization-mediated activation of voltage-gated R- and T-type Ca(2+) channels. The results further support the hypothesis that the orexin system participates in integrating neural systems that are involved in arousal, sensory processing, energy homeostasis and autonomic function.

Electrophysiological effect of ghrelin and somatostatin on rat hypothalamic arcuate neurons in vitro.

Growth hormone (GH) secretion from the pituitary gland is partly regulated by GH releasing hormone (GHRH)-containing neurons located in the hypothalamic arcuate nucleus (ARC). GHRH-containing neurons express the GH secretagogue (GHS) receptor (GHS-R) and the somatostatin (SRIF) receptor. Recently, an endogenous ligand for the GHS-R named ghrelin was found. Therefore, it seems that both ghrelin and SRIF are involved in the hypothalamic regulation of GH release via GHRH-containing neurons in the ARC. In extracellular single unit recordings from in vitro hypothalamic slice preparations from rats, application of 100 nM ghrelin substantially excited ARC neurons (82.5%), whereas 1 microM SRIF substantially inhibited them (81.8%). The ghrelin-induced excitatory and SRIF-induced inhibitory effects on ARC neurons were dose-dependent and persisted during synaptic blockade using low-Ca(2+)/high-Mg(2+) solution. In addition, the effects were antagonized by [D-Lys(3)]-GHRP-6, a GHS-R antagonist, and CYN154806, a SRIF receptor subtype sst2 antagonist, respectively. When ghrelin and SRIF were sequentially applied to ARC neurons, 95.2% were excited by ghrelin and inhibited by SRIF. Similarly, 85.0% of ARC neuroendocrine cells that project to the median eminence were excited by ghrelin and inhibited by SRIF. These results indicate that ARC neuroendocrine cells projecting to the median eminence are dose-dependently, postsynaptically and oppositely regulated by ghrelin through GHS-R and SRIF via the SRIF sst2 receptor subtype. Our results also suggest that most of these ARC neuroendocrine cells are presumably GHRH-containing neurons and are involved in the cellular processes through which ghrelin and SRIF participate in the hypothalamic regulation of GH release.

Microinjection of neuropeptide S into the rat ventral tegmental area induces hyperactivity and increases extracellular levels of dopamine metabolites in the nucleus accumbens shell.

The newly identified neuropeptide S (NPS) is mainly expressed in a group of neurons located between the locus coeruleus and Barrington's nucleus in the brainstem. Central administration of NPS increases motor activity and wakefulness, and it decreases anxiety-like behavior and feeding. The NPS receptor (NPSR) is widely distributed in various brain regions including the ventral tegmental area (VTA). The mesolimbic dopaminergic system originates in the VTA, and activation of the system produces hypermotor activity. Therefore, we hypothesized that NPS-induced hypermotor activity might be mediated by activation of the mesolimbic dopaminergic pathway via the NPSR expressed in the VTA. Intra-VTA injection of NPS significantly and dose-dependently increased horizontal and vertical motor activity in rats, and the hyperactivity was significantly and dose-dependently inhibited by pre-administration of sulpiride, a DA D(2)-like receptor antagonist, into the shell of the nucleus accumbens (NAcSh). Intra-VTA injection of NPS also significantly increased extracellular 3,4-dihydroxy-phenyl acetic acid and homovanillic acid levels in the NAcSh of freely moving rats. These results support the idea that NPS activates the mesolimbic dopaminergic system presumably via the NPSR located in the VTA, thereby stimulating motor activity.

Electrophysiological effects of neuropeptide S on rat ventromedial hypothalamic neurons in vitro.

The newly identified neuropeptide S (NPS) is a ligand for a previously orphan G protein-coupled GPR 154 receptor, now named the NPS receptor (NPSR). Previous studies have shown that NPS induces hyperlocomotion, increases arousal and suppresses anxiety-like behaviors via NPSR. Although NPS also inhibits food intake, nothing is known about the neuronal mechanisms underlying this action. Anatomical studies show that NPSRs are expressed abundantly in the dorsomedial part of the ventromedial hypothalamic nucleus (VMH), a satiety center for food intake. Hence, we examined the electrophysiological effects of NPS on rat VMH neurons in vitro. NPS predominantly depolarized the VMH neurons, and the effects were postsynaptic and dose-dependent. Membrane resistance was significantly decreased during the depolarization, suggesting an opening of some ionic channels. The NPS-induced depolarization was significantly attenuated in Ca(2+)-free, NiCl(2)-containing and mibefradil-containing TTX ACSFs, but it did not disappear. The NPS-induced depolarization was also attenuated in low-Na(+) TTX ACSF, and completely abolished in Ca(2+)-free/low-Na(+) TTX ACSF. Pretreatment with 30 microM KB-R7943, an inhibitor of forward-mode Na(+)/Ca(2+) exchanger, did not have any significant effect on the NPS-induced depolarization in Ca(2+)-free TTX ACSF. These results suggest that NPS depolarizes VMH neurons via activations of R- and T-type Ca(2+) channels and nonselective cation channels, and that VMH neurons might be involved in the cellular process through which NPS participates in the regulation of food intake and energy homeostasis.

Electrophysiological effects of ghrelin on laterodorsal tegmental neurons in rats: an in vitro study.

Ghrelin, a gut and brain peptide, is a potent stimulant for growth hormone (GH) secretion and feeding. Recent studies further show a critical role of ghrelin in the regulation of sleep-wakefulness. Laterodorsal tegmental nucleus (LDT), that regulates waking and rapid eye movement (REM) sleep, expresses GH secretagogue receptors (GHS-Rs). Thus, the present study was carried out to examine electrophysiological effects of ghrelin on LDT neurons using rat brainstem slices, and to determine the ionic mechanism involved. Whole cell recording revealed that ghrelin depolarizes LDT neurons dose-dependently in normal artificial cerebrospinal fluid (ACSF). The depolarization persisted in tetrodotoxin-containing ACSF (TTX ACSF), and is partially blocked by the application of [D-Lys3]-GHRP-6, a selective antagonist for GHS-Rs. Membrane resistance during the ghrelin-induced depolarization increased by about 18% than that before the depolarization. In addition, the ghrelin-induced depolarization was drastically reduced in high-K+ TTX ACSF with a K+ concentration of 13.25 mM. Reversal potentials obtained from I-V curves before and during the depolarization were about -83 mV, close to the equilibrium potential of the K+ channel. Most of the LDT neurons recorded were characterized by an A-current or both the A-current and a low threshold Ca2+ spike, and they were predominantly cholinergic. These results indicate that ghrelin depolarizes LDT neurons postsynaptically and dose-dependently via GHS-Rs, and that the ionic mechanisms underlying the ghrelin-induced depolarization include a decrease of K+ conductance. The results also suggest that LDT neurons are implicated in the cellular processes through which ghrelin participates in the regulation of sleep-wakefulness.

Orexin-A and ghrelin depolarize the same pedunculopontine tegmental neurons in rats: an in vitro study.

Orexin (ORX), also called hypocretin, and ghrelin are newly identified peptides in the brain and/or peripheral organs, and they are involved in the regulation of sleep-wakefulness as well as feeding. In our previous studies we have found that ORX and ghrelin each depolarizes more than half of the cholinergic neurons recorded in the pedunculopontine tegmental nucleus (PPT) via a dual ionic mechanism including a decrease of K(+) conductance and an increase of nonselective cationic conductance. Thus, the present study was carried out to investigate whether ORX-A and ghrelin both depolarize the same PPT neuron. About 60% of PPT neurons examined was depolarized by both ORX-A and ghrelin, 20% by ORX-A alone, and 10% by ghrelin alone. The remaining 10% did not respond to these peptides. In neurons which were responsive to both ORX-A and ghrelin, the depolarizations induced by ORX-A and ghrelin were additive. In addition, the ORX-A- and ghrelin-induced depolarizations were both blocked by D609, a phosphatidylcholine-specific phospholipase C (PLC) inhibitor. These results suggest that same PPT neurons with receptors for ORX and ghrelin are involved in the cellular process through which ORX and ghrelin participate in the regulation of sleep wakefulness, and that the excitatory effects of ORX and ghrelin on PPT neurons are mediated by PLC.

Electrophysiological effects of orexin/hypocretin on nucleus accumbens shell neurons in rats: an in vitro study.

Orexin-A (ORX-A) and orexin-B (ORX-B) play critical roles in the regulation of sleep-wakefulness, energy homeostasis, neuroendocrine system and autonomic functions. Although ORXs are also implicated in the reward process, their electrophysiological effects on neurons in the shell of nucleus accumbems (NAcSh) have not been described thoroughly. Therefore we examined the electrophysiological effects of ORXs on rat NAcSh neurons. Whole cell patch clamp recording in vitro revealed that ORX-A and ORX-B depolarize NAcSh neurons in normal and/or tetrodotoxin (TTX)-containing artificial cerebrospinal fluid (ACSF). The depolarization accompanied by a decrease of membrane resistance was concentration-dependent, and there was no significant difference between the two dose-response curves obtained by ORX-A and ORX-B. The ORX-B-induced depolarization was reduced in low-Na(+), flufenamic acid-containing, and high-K(+) TTX ACSFs, and completely abolished in low-Na(+)/high-K(+) TTX ACSF. An inhibitor of the Na(+)/Ca(2+) exchanger had no effect on the depolarization. The reversal potential obtained from I-V relationships before and during the ORX-B-induced depolarization in low-Na(+) TTX ACSF was about -84mV, and that obtained in TTX ACSF using patch pipettes with Cs(+)-containing internal solution was about -38mV. These results suggest that ORXs directly depolarize NAcSh neurons via OX(2) receptors and via a dual ionic mechanism including an increase of nonselective cationic conductance and a decrease of K(+) conductance, and that NAcSh neurons are involved in the cellular mechanisms through which ORXs participate in the regulation of the reward process as well as feeding and arousal.

Electrophysiological effects of ghrelin on pedunculopontine tegmental neurons in rats: An in vitro study.

Ghrelin is a potent stimulant for growth hormone (GH) secretion and feeding. Recent studies further show a critical role of ghrelin in the regulation of sleep-wakefulness. Pedunculopontine tegmental nucleus (PPT), which regulates waking and rapid eye movement (REM) sleep, expresses GH secretagogue receptors (GHS-Rs). Thus, the present study was carried out to examine electrophysiological effects of ghrelin on PPT neurons using rat brainstem slices, and to determine the ionic mechanism involved. Whole cell recording revealed that ghrelin depolarizes PPT neurons dose-dependently in normal artificial cerebrospinal fluid (ACSF). The depolarization persisted in tetrodotoxin-containing ACSF, although action potentials did not occur. Application of [d-Lys(3)]-GHRP-6, a selective antagonist for GHS-Rs, almost blocked the ghrelin-induced depolarization. Furthermore, the ghrelin-induced depolarization was reduced in high K(+) ACSF or low Na(+) ACSF, and abolished in high K(+)-low Na(+) ACSF or in a combination of low Na(+) ACSF and recordings with Cs(+)-containing pipettes. An inhibitor of Na(+)/Ca(2+) exchanger had no effect on the depolarization. Most of the PPT neurons recorded were characterized by an A-current or both the A-current and a low threshold Ca(2+) spike, and they were predominantly cholinergic as revealed by nicotinamide adenine dinucleotide phosphate-diaphorase staining. These results suggest that ghrelin depolarizes PPT neurons postsynaptically and dose-dependently via GHS-Rs, and that the ionic mechanisms underlying the ghrelin-induced depolarization include a decrease of K(+) conductance and an increase of non-selective cationic conductance. The results also support the notion that ghrelin plays a role in the regulation of sleep-wakefulness.

Electrophysiological effects of orexins/hypocretins on pedunculopontine tegmental neurons in rats: an in vitro study.

Orexin-A (ORX-A) and orexin-B (ORX-B) play critical roles in the regulation of sleep-wakefulness and feeding. ORX neurons project to the pedunculopontine tegmental nucleus (PPT), which regulates waking and rapid eye movement (REM) sleep. Thus, we examined electrophysiological effects of ORXs on rat PPT neurons with a soma size of more than 30 microm. Whole cell patch clamp recording in vitro revealed that ORX-A and ORX-B depolarized PPT neurons dose-dependently in normal and/or tetrodotoxin containing artificial cerebrospinal fluids (ACSFs), and the EC(50) values for ORX-A and ORX-B were 66 nM and 536 nM, respectively. SB-334867, a selective inhibitor for ORX 1 (OX(1)) receptors, significantly suppressed the ORX-A-induced depolarization. The ORX-A-induced depolarization was reduced in high K(+) ACSF with extracellular K(+) concentration of 13.25 mM or N-methyl-d-glucamine (NMDG(+))-containing ACSF in which NaCl was replaced with NMDG-Cl, and abolished in high K(+)-NMDG(+) ACSF or in a combination of NMDG(+) ACSF and recordings with Cs(+)-containing pipettes. An inhibitor of Na(+)/Ca(2+) exchanger and chelating intracellular Ca(2+) had no effect on the depolarization. Most of PPT neurons studied were characterized by an A-current or both A-current and a low threshold Ca(2+) spike, and predominantly cholinergic. These results suggest that ORXs directly depolarize PPT neurons via OX(1) receptors and via a dual ionic mechanism including a decrease of K(+) conductances and an increase of non-selective cationic conductances, and support the notion that ORX neurons affect the activity of PPT neurons directly and/or indirectly to control sleep-wakefulness, especially REM sleep.