Juhyon Kim

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 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.

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-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.

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 Barringtons 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 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.

Effects of ghrelin on neuronal activity in the ventromedial nucleus of the hypothalamus in infantile rats: an in vitro study.

Ghrelin is an endogenous ligand for the growth hormone (GH) secretagogue (GHS) receptor (GHS-R) and a potent stimulant for GH secretion even in infantile rats before puberty. Although the ventromedial nucleus of the hypothalamus (VMH) might be a site of action for ghrelin to induce GH release, the electrophysiological effect of ghrelin on VMH neurons in infantile rats remains to be elucidated. Thus, the purpose of the present study was to investigate the effect of ghrelin on VMH neurons using hypothalamic slices of infantile rats. Ghrelin excited a majority of VMH neurons in a concentration-dependent manner. VMH neurons that were excited by GH releasing peptide-6 (GHRP-6), a synthetic GHS, were also excited by ghrelin and vice versa. Repeated application of ghrelin to the same VMH neuron decreased progressively the excitatory responses depending on the number of times it was administered. The excitatory effect of ghrelin on VMH neurons in normal artificial cerebrospinal fluid (ACSF) persisted in low Ca2+-high Mg2+ ACSF. The present results indicate that (1) ghrelin excites a majority of VMH neurons dose-dependently and postsynaptically and (2) the excitatory effects of ghrelin are mimicked by GHRP-6 and desensitized by repeated applications of ghrelin.

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.

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.

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.