Modeling the motion of disease-associated KIF1A heterodimers.

KIF1A is a member of the kinesin-3 motor protein family that transports synaptic vesicle precursors in axons. Mutations in the Kif1a gene cause neuronal diseases. Most patients are heterozygous and have both mutated and intact KIF1A alleles, suggesting that heterodimers composed of wild-type KIF1A and mutant KIF1A are likely involved in pathogenesis. In this study, we propose mathematical models to describe the motility of KIF1A heterodimers composed of wild-type KIF1A and mutant KIF1A. Our models precisely describe run length, run time, and velocity of KIF1A heterodimers using a few parameters obtained from two homodimers. The first model is a simple hand-over-hand model in which stepping and detachment rates from a microtubule of each head are identical to those in the respective homodimers. Although the velocities of heterodimers expected from this model were in good agreement with the experimental results, this model underestimated the run lengths and run times of some heterodimeric motors. To address this discrepancy, we propose the tethered-head affinity model, in which we hypothesize a tethered head, in addition to a microtubule-binding head, contributes to microtubule binding in a vulnerable one-head-bound state. The run lengths and run times of the KIF1A heterodimers predicted by the tethered-head affinity model matched well with experimental results, suggesting a possibility that the tethered head affects the microtubule binding of KIF1A. Our models provide insights into how each head contributes to the processive movement of KIF1A and can be used to estimate motile parameters of KIF1A heterodimers.

Fast backward steps and detachment of single kinesin molecules measured under a wide range of loads.

To understand force generation under a wide range of loads, the stepping of single kinesin molecules was measured at loads from -20 to 42 pN by optical tweezers with high temporal resolution. The optical trap has been improved to halve positional noise and increase bandwidth by using 200-nm beads. The step size of the forward and backward steps was 8.2 nm even over a wide range of loads. Histograms of the dwell times of backward steps and detachment fit well to two independent exponential equations with fast (~0.4 ms) and slow (>3 ms) time constants, indicating the existence of a fast step in addition to the conventional slow step. The dwell times of the fast steps were almost independent of the load and ATP concentration, while those of the slow backward steps and detachment depended on those. We constructed the kinetic model to explain the fast and slow steps under a wide range of loads.

Number of kinesins engaged in axonal cargo transport: A novel biomarker for neurological disorders.

Kinesin motor proteins play crucial roles in anterograde transport of cargo vesicles in neurons, moving them along axons from the cell body towards the synaptic region. Not only the transport force and velocity of single motor protein, but also the number of kinesin molecules involved in transporting a specific cargo, is pivotal for synapse formation. This collective transport by multiple kinesins ensures stable and efficient cargo transport in neurons. Abnormal increases or decreases in the number of engaged kinesin molecules per cargo could potentially act as biomarkers for neurodegenerative diseases such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis (ALS), spastic paraplegia, polydactyly syndrome, and virus transport disorders. We review here a model constructed using physical measurements to quantify the number of kinesin molecules associated with their cargo, which could shed light on the molecular mechanisms of neurodegenerative diseases related to axonal transport.

6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) from Wasabia japonica alleviates inflammatory bowel disease (IBD) by potential inhibition of glycogen synthase kinase 3 beta (GSK-3ß).

Inflammatory bowel disease (IBD) describes a set of disorders involving alterations to gastrointestinal physiology and mucosal immunity. Unravelling its complex pathophysiology is important since many IBD patients are refractory to or suffer adverse side effects from current treatments. Isothiocyanates (ITCs), such as 6-(methylsulfinyl)hexyl ITC (6-MITC) in Wasabia japonica, have potential anti-inflammatory activity. We aimed to elucidate the pathways through which 6-MITC alleviates inflammation by examining its role in the nuclear factor-kappa B (NF-κB) pathway through inhibition of glycogen synthase kinase 3 beta (GSK-3ß) using a chemically induced murine model of IBD, cell-based and in silico techniques. The effects of 6-MITC and two NF-κB inhibitors, sulfasalazine (SS), pyrrolidine dithiolcarbamate (PDTC) were investigated on a dextran sulfate sodium (DSS)-induced murine mouse model of acute and chronic colitis using macroscopic measurements and pro-inflammatory markers. The effect of 6-MITC on NF-κB induction was assessed using a murine macrophage cell line. Complexes of GSK-3ß-6-MITC and GSK-3ß-ATP were generated in silico to elucidate the mechanism of 6-MITC's direct inhibition of GSK-3ß. Changes in pro-inflammatory markers, inducible nitric oxide synthase (iNOS) (increased) and interleukin-6 (IL-6) (decreased) demonstrated that iNOS regulation occurred at the translational level. Intraperitoneal (ip) injection of 6-MITC to the colitis-induced mice ameliorated weight loss whereas oral administration had negligible effect. Fecal blood and colon weight/length ratio parameters improved on treatment with 6-MITC and the other NF-κB inhibitors. Levels of NF-κB decreased upon addition of 6-MITC in vitro while structural studies showed 6-MITC acts competitively to inhibit GSK-3ß at the ATP binding site. In this study we demonstrated that 6-MITC inhibits NF-κB signaling via GSK-3ß inhibition ameliorating fecal blood, colonic alterations and DSS-induced weight loss indirectly indicating reduced intestinal stress. Taken together these results suggest a role for 6-MITC in the treatment of IBD acting to alleviate inflammation through the GSK-3ß/NF-κB pathway. Furthermore, the GSK-3ß-6-MITC model can be utilized as a basis for development of novel therapeutics targeting GSK-3ß for use in other disorders including cancer.

A Unified Walking Model for Dimeric Motor Proteins.

Dimeric motor proteins, kinesin-1, cytoplasmic dynein-1, and myosin-V, move stepwise along microtubules and actin filaments with a regular step size. The motors take backward as well as forward steps. The step ratio r and dwell time τ, which are the ratio of the number of backward steps to the number of forward steps and the time between consecutive steps, respectively, were observed to change with the load. To understand the movement of motor proteins, we constructed a unified and simple mathematical model to explain the load dependencies of r and of τ measured for the above three types of motors quantitatively. Our model consists of three states, and the forward and backward steps are represented by the cycles of transitions visiting different pairs of states among the three, implying that a backward step is not the reversal of a forward step. Each of r and τ is given by a simple expression containing two exponential functions. The experimental data for r and τ for dynein available in the literature are not sufficient for a quantitative analysis, which is in contrast to those for kinesin and myosin-V. We reanalyze the data to obtain r and τ of native dynein to make up the insufficient data to fit them to the model. Our model successfully describes the behavior of r and τ for all of the motors in a wide range of loads from large assisting loads to superstall loads.

Bone regeneration by freeze-dried composite of octacalcium phosphate collagen and teriparatide.

OBJECTIVE: Octacalcium phosphate (OCP) and collagen (col) composite (OCPcol) demonstrated superior bone regeneration properties, and its commercialization appears to be forthcoming. As a practical medical material for new combination products, we developed a freeze-dried composite with OCPcol and teriparatide (TPTD) (OCPcolTPTDf), and investigated its bone regenerative properties. MATERIALS AND METHODS: A disk of OCPcol was made by mixing OCP granules and atelocollagen for medical use. Then, OCPcolTPTDf was prepared by impregnation of the OCPcol disk with 1.0 or 0.1 µg of TPTD solution (OCPcolTPTDf 1.0 and OCPcolTPTDf 0.1, respectively) followed by lyophilization. In vitro release profiles of TPTD from OCPcolTPTDf were determined using an enzyme-linked immunosorbent assay. Implantation of OCPcolTPTDf or OCPcol was carried out for a rat critical-sized calvarial defect. And five defects in each group were collected after 12 weeks of implantation. RESULTS: The retention-release profiles of TPTD from OCPcolTPTDf supported a higher degree of retention of TPTD. Radiographic, histological, and histomorphometric examinations indicated that regenerated bone was filled in most of the defects of the OCPcolTPTDf. Additionally, the OCPcolTPTDf groups showed significantly enhanced bone regeneration compared with the OCPcol group. CONCLUSIONS: These results suggested that this newly developed bone regenerative composite could be a practical medical material.

A biological study establishing the endotoxin limit of biomaterials for bone regeneration in cranial and femoral implantation of rats.

The purpose of this study was to accurately quantify the risk of endotoxin contamination in biomaterials for bone regeneration in order to establish the acceptable endotoxin limit. Collagen sheets containing varying amounts of purified endotoxin from Escherichia coli and dried, heat-killed E. coli or Staphylococcus aureus cells were implanted into cranial or femoral defects in rats. These defects were artificially prepared to a size of 5 × 5 mm or a diameter of 1 mm, respectively. The degree of osteoanagenesis was assessed by soft X-ray radiography and histopathology at 1 and 4 weeks after implantation. The collagen sheet containing the dried E. coli cells showed a dose-dependent delay in cranial and/or femoral osteoanagenesis at endotoxin activities of more than 33.6 EU/mg, at which no inflammatory response was observed. In contrast, no such observation occurred with the collagen sheet containing S. aureus cells. These results suggest that endotoxins may affect the process of osteoanagenesis. Additionally, the no-observed-adverse-effect level was 9.6 EU/mg, corresponding to 255 EU/kg body weight in rats. Interestingly, no delay in osteoanagenesis was induced by the implantation of collagen sheets containing purified endotoxin at any dose tested. This suggested that pure endotoxin implanted into tissues having poor circulation of bodily fluids without bleeding may not be recognized as a foreign substance and may not induce a significant biological response. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1514-1524, 2017.

Giant Acceleration of Diffusion Observed in a Single-Molecule Experiment on F(1)-ATPase.

The giant acceleration (GA) of diffusion is a universal phenomenon predicted by the theoretical analysis given by Reimann et al. [Phys. Rev. Lett. 87, 010602 (2001)]. Here we apply the theory of the GA of diffusion to a single-molecule experiment on a rotary motor protein, F(1), which is a component of F(o)F(1) adenosine triphosphate synthase. We discuss the energetic properties of F(1) and identify a high energy barrier of the rotary potential to be 20k(B)T, with the condition that the adenosine diphosphates are tightly bound to the F(1) catalytic sites. To conclude, the GA of diffusion is useful for measuring energy barriers in nonequilibrium and single-molecule experiments.

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.

Energetics of the single-headed kinesin KIF1A.

KIF1A is a single-headed molecular motor that moves processively and unidirectionally along a microtubule by using the chemical energy released by hydrolyzing adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and inorganic phosphate (P(i)). Although the movement of KIF1A seems to have successfully been explained by a simple Brownian motor model of the flashing ratchet type, this model is not suited to discuss the energetics of KIF1A. We introduce an elaborated model of the ratchet type to investigate how the chemical free energy is converted into mechanical work by taking account of the binding and release of reactant (ATP) and product (ADP and P(i)) molecules to and from the motor. The efficiency of energy transduction, the power output, and other quantities are calculated from the analytically obtained steady-state solution of the Fokker-Planck equations. It turns out that the concentrations of the reactant and product molecules that optimize both the efficiency and the power are close to those in the cell.

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.

Reconstruction of critical-sized bone defect in dog skull by octacalcium phosphate combined with collagen.

PURPOSE: The present study was designed to investigate whether synthetic octacalcium phosphate (OCP) combined with collagen (OCP/collagen) can repair a critical-sized defect in dog skull. OCP/collagen has been shown to biodegrade and to tend to be replaced by newly formed bone if implanted in rat calvaria defects. MATERIALS AND METHODS: An OCP/collagen disk was prepared from pepsin-digested atelocollagen isolated from porcine dermis and synthetic OCP. Two critical-sized defects (20 mm in diameter) were made in a dog skull. Ten disks of OCP/collagen or collagen (control) were implanted in the bone defects and resected with surrounding tissues at 3, 6, or 12 months after the implantation. The specimens were analyzed radiographically, crystallographically, histologically, and histomorphometrically. RESULTS: X-ray diffraction and FTIR analyses showed that OCP tended to convert to a poorly crystallized hydroxyapatite, similar to that of biological apatite, by 3 months. Radiographic and histologic analyses showed that the implantation of OCP/collagen disks initiated new bone formation in the defects at 3 months after implantation. However, there was no promotion of bone formation by control collagen disks even with prolonged implantation up to 12 months. Histomorphometric analysis revealed that the percentage of newly formed bone in the defect implanted with OCP/collagen increased significantly, from 30.91 ± 6.65 at 3 months to 51.22 ± 5.99 at 12 months, although the value tended to reach a plateau at 6 months (44.49 ± 3.34). On the other hand, the percentage of remaining OCP was estimated at approximately 10% at 3 months and remained nearly unchanged thereafter. CONCLUSION: The results suggest that bone regeneration of a critical-sized bone defect of dog calvaria by OCP/collagen can be enhanced for 3 to 6 months and that OCP/collagen holds potential as a bone substitute material.

Detection of respiratory waveforms using non-contact electrodes during bathing.

The aim of this study is to develop a method for measuring the respiratory waveform using non-contact electrodes during bathing. To determine the most appropriate electrode arrangement, we modeled a composite system consisting of a body submerged in bath water. We calculated the frequency dependence of the impedance amplitude using a three-dimensional finite difference method (3D-FDM). The simulation results showed that an increase in chest size due to inspiration caused a decrease in the impedance amplitude in the frequency range of 0.1 Hz to 1 MHz. Next, bioelectric impedance (BEI) was measured in the frequency range of 4 kHz to 4 MHz at the maximum-end-expiration and maximum-end-inspiration stages. BEI results were consistent with those obtained from the model simulations. We found that 1 MHz was the appropriate frequency for measuring the respiratory waveform, and the time dependence of the impedance amplitude was measured at 1 MHz. The impedance amplitude agreed well with the respiratory waveform obtained from rubber strain gauge plethysmography, which was used as a reference.

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.

Theta oscillations in primate prefrontal and anterior cingulate cortices in forewarned reaction time tasks.

Previously, we introduced a monkey model for human frontal midline theta oscillations as a possible neural correlate of attention. It was based on homologous theta oscillations found in the monkey's prefrontal and anterior cingulate cortices (areas 9 and 32) in a self-initiated hand-movement task. However, it has not been confirmed whether theta activity in the monkey model consistently appears in other situations demanding attention. Here, we examined the detailed properties of theta oscillations in four variations of forewarned reaction time tasks with warning (S1) and imperative (S2) stimuli. We characterized the theta oscillations generated exclusively in areas 9 and 32, as follows: 1) in the S1-S2 interval where movement preparation and reward expectation were presumably involved, the theta power was higher than in the pre-S1 period; 2) in the no-go trials of go/no-go tasks instructed by S1, the theta power in the S1-S2 interval was lower than in the pre-S1 period in an asymmetrical reward condition, whereas it was moderately higher in a symmetrical condition; 3) the theta power after reward delivery was higher than in the unrewarded trials; 4) the theta power in the pre-S1 period was higher than in the resting condition; and 5) when the monkey had to guess the S1-S2 duration internally without seeing S2, the theta power in the pre-S1 period was higher than in the original S1-S2 experiment. These findings suggest that attentional loads associated with different causes can induce the same theta activity, thereby supporting the consistency of attention-dependent theta oscillations in our model.

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.