hypocretins on neuronal activity in the paraventricular nucleus of the thalamus in rats in vitro

Peptides 26 (2005) 471–481

Effects of orexins/hypocretins on neuronal activity in the paraventricular nucleus of the thalamus in rats in vitro Masaru Ishibashia , Shinobu Takanoa , Hiroki Yanagidaa , Masafumi Takatsunaa , Kazuki Nakajimaa , Yutaka Oomurab , Matthew J. Waynerc , Kazuo Sasakia,∗ a

Division of Bio-Information Engineering, Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930-8555, Japan b Department of Integrative Physiology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-0054, Japan c Department of Biology, The University of Texas at San Antonio, San Antonio, TX 78249-0662, USA Received 9 August 2004; received in revised form 11 October 2004; accepted 14 October 2004 Available online 23 November 2004

Abstract Orexin-A (ORX-A) and orexin-B (ORX-B), also called hypocretin-1 and hypocretin-2, respectively, act upon orexin 1 (OX1 R) and orexin 2 (OX2 R) receptors, and are involved in the regulation of sleep-wakefulness and energy homeostasis. Orexin neurons in the lateral hypothalamic perifornical region project heavily to the paraventricular nucleus of the thalamus (PVT), which is deeply involved in the control of motivated behaviors. In the present study, electrophysiological and cytosolic Ca2+ concentration ([Ca2+ ]i ) imaging studies on the effects of ORX-A and ORX-B on neurons in the PVT were carried out in rat brain slice preparations. ORX-A and/or ORX-B were applied extracellularly in the perfusate. Extracellular recordings showed that about 80% of the PVT neurons were excited dose-dependently by both ORX-A and ORX-B at concentrations of 10−8 to 10−6 M, and the increase in firing rate was about three times larger for ORX-B than for ORX-A at 10−7 M. When both ORX-A and ORX-B were applied simultaneously at 10−7 M, the increase in firing rate was almost equal to that of ORX-B at 10−7 M, suggesting that the PVT neurons do not show a high affinity to ORX-A which is expected if they have OX1 R receptors. The excitatory effect of ORX-B was seen in low Ca2+ and high Mg2+ ACSF as well as in normal ACSF, and the increase in firing rate was greater in low Ca2+ and high Mg2+ ACSF than in normal ACSF. [Ca2+ ]i imaging studies demonstrated that [Ca2+ ]i was increased in about 50% of the PVT neurons by both 10−7 M ORX-A and ORX-B with a stronger effect for ORX-B, and the increase in [Ca2+ ]i induced by ORX-B was abolished in Ca2+ -free ACSF, suggesting that ORX-B does not release Ca2+ from intracellular Ca2+ stores. Subsequent whole cell patch clamp recordings revealed that an after hyperpolarization seen following each action potential in normal ACSF disappeared in Ca2+ -free ACSF, and the mean magnitude of the depolarization induced by ORX-B was same in normal, Ca2+ -free and TTX-containing Ca2+ -free ACSFs. Furthermore, ORX-B-induced depolarization was reversed to hyperpolarization when membrane potential was lowered to about −97 mV, and an increase of extracellular K+ concentration from 4.25 to 13.25 mM abolished the ORX-B-induced depolarization, indicating that the ORX-B-induced depolarization is associated with an increase in the membrane resistance resulting from a closure of K+ channels. These results suggest that orexins depolarize and excite post-synaptically PVT neurons via OX2 R receptors, and that orexin-activated PVT neurons play a role in the integration of sleep-wakefulness and energy homeostasis, and in the control of motivated behaviors. © 2004 Elsevier Inc. All rights reserved. Keywords: Paraventricular nucleus of the thalamus; PVT; Orexin-A/hypocretin-1; Orexin-B/hypocretin-2; Calcium; Potassium channel; Patch clamp; [Ca2+ ]i imaging

1. Introduction

∗

Corresponding author. Tel.: +81 76 445 6719; fax: +81 76 445 6723. E-mail address: [email protected] (K. Sasaki).

0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.10.014

It has been reported recently that neurons containing novel neuropeptides, orexin-A (ORX-A) and ORX-B (ORX-B), also called hypocretin-1 and hypocretin-2, respectively, are mainly localized in the perifornical region of the lateral

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hypothalamic area (LHA) [40,51]. The LHA has traditionally been considered the feeding center in the brain. ORX-A and ORX-B are cleaved from common prepro-orexin precursor molecules, 130 residues, and their amino acid residues are 33–66 and 69–96, respectively [51]. The orexins bind to orexin 1 receptors (OX1 R) and orexin 2 receptors (OX2 R) which belong to the G protein-coupled receptor superfamily [51]. OX1 R receptors have a higher affinity for ORX-A than for ORX-B, whereas OX2 R receptors have an equal affinity for both orexins. Nerve terminals of orexin neurons in the perifornical region of the LHA are distributed throughout the brain [12,36,40,47]. In the hypothalamus, orexin neurons project to the arcuate nucleus, ventromedial nucleus and paraventricular nucleus, whereas in brain areas outside the hypothalamus, they distribute to the cerebral cortex, thalamus, limbic system including the hippocampus and amygdala, and brain stem including the locus coeruleus (LC), raphe nuclei and tuberomammillary nucleus (TMN). In accordance with the distribution of nerve terminals, OX1 R and/or OX2 R receptors were found in the hypothalamic and extrahypothalamic brain regions from an early stage of development such as embryonic day 15 and post-natal day 5 [10,19,20,34,35,61,63,66]. Potential roles for ORX-A and ORX-B have been demonstrated in the regulation of arousal and feeding behavior [26,41,49,50]. One of the brain regions with the most dense distribution of orexin-containing nerve terminals is the paraventricular nucleus of the thalamus (PVT); one of the midline and intralaminar thalamic nuclei possibly involved in a non-specific arousal system [12,36,40,47]. Immunoreactivity and mRNA for OX1 R and/or OX2 R receptors were also found in the PVT [10,20,34,35,61]. Anatomical studies using anterograde or retrograde tracers [11,28,30,31,37,43–46,64] show that the PVT receives strong serotonergic, noradrenergic, dopaminergic, histaminergic and cholinergic inputs from the LC, dorsal raphe nucleus (DR), ventral tegmental area, TMN, and laterodorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei, respectively. Most of them are implicated in the control of the sleep-wakefulness states. Activities in the LC, DR and TMN neurons increase during wakefulness [29,58,68], whereas those in the LDT and PPT neurons are activated during REM-sleep [5,59]. The PVT also receives circadian inputs, which are important in the regulation of the sleep-wakefulness cycle, from the suprachiasmatic nucleus of the hypothalamus. Other afferents to the PVT originate from the nucleus of solitary tract (NTS) and parabrachial nucleus that convey visceral signals related to energy homeostasis. For example, some neurons in the NTS are activated by a decrease in glucose level in the systemic or portal circulation, and inhibited by an increase in systemic or portal glucose level and by gastric distention [8,71]. Efferents from the PVT project mainly to the prefrontal cortex such as the cingulate cortex and orbital cortex, amygdala, and nucleus accumbens. These parts of the brain are closely associated with motivation. Based on these anatomical relationships, it seems reasonable to assume that the PVT integrates sleep-

wakefulness and visceral energy homeostatic signals, and controls motivated behaviors [30,37,42,46]. Previous studies show that ORX-A and/or ORX-B depolarize and excite neurons in brain regions in which orexin terminals distribute; and that ionic mechanisms for orexin-induced excitation include the K+ channel [1,4,16,23,70], Ca2+ channel [14,54], non-selective cation channel [32,69,70], and/or electrogenic Na+ /Ca2+ exchanger activated by a surge in cytosolic Ca2+ concentration ([Ca2+ ]i ) released from intracellular stores [14]. However, the effects of orexins on PVT neurons and the ionic mechanisms involved have not been determined. Therefore, the purpose of the present study was to examine the electrophysiological effects and changes in [Ca2+ ]i in PVT neurons of rats in response to extracellular application of ORX-A and/or ORXB. The effects of ORX-A and ORX-B on the PVT neurons, ionic mechanisms related to ionic channels and involvement of Ca2+ released from intracellular Ca2+ stores, were determined by extracellular recordings, whole cell patch clamp recordings, and [Ca2+ ]i imaging.

2. Materials and methods 2.1. Animals Male Wistar rats at 1–3 weeks old were used (Sankyo Lab., Shizuoka, Japan). They were housed with their mother rat in a light-controlled room (light on: 06:00–18:00) at a temperature of 23 ± 2 ◦ C for several days before the experiments. The animals and experimental procedures used were approved by the Institutional Animal Care and Use Committee of the Faculty of Engineering of Toyama University. 2.2. Slice preparation Rats were decapitated after ether anesthesia and the brain was rapidly removed from the skull. The brain was then submerged in ice cold, oxygenated (95% O2 –5% CO2 ) artificial cerebrospinal fluid (ACSF) containing in mM: NaCl 126, KCl 3, CaCl2 2.4, MgSO4 1.3, KH2 PO4 1.25, NaHCO3 26 and glucose 10. Frontal thalamic slices 200–400 ␮m thick were cut by a microslicer (ZERO 1, Dosaka EM, Kyoto, Japan). The PVT was identified on the basis of its anatomical location relative to the dorsal third ventricle and stria medullaris. One or two slices including the PVT were selected from each animal and cut with a scalpel at the level of dorsal third ventricle so that brain tissues such as the cortex and hippocampus dorsal to the PVT were removed. The slices containing the PVT were then pre-incubated in a chamber with oxygenated ACSF for about 1 h at room temperature. 2.3. Extracellular recording After pre-incubation, slices were transferred into a recording chamber, and perfused with oxygenated ACSF containing

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in mM: NaCl 124, KCl 5, CaCl2 2.4, MgSO4 1.3, KH2 PO4 1.25, NaHCO3 26 and glucose 10, at 1 ml/min and at 34 ◦ C. The ACSF was used in extracellular recordings to depolarize a neuron slightly and to make it easy to obtain a spontaneous firing of action potentials. Extracellular neuronal activity was recorded via a glass microelectrode filled with ACSF under a biological microscope (SMZ, Nikon, Tokyo, Japan). Neuronal activity was then fed into an amplifier (IR183, Neuro Data, New York, USA) and the output of the amplifier was monitored on an oscilloscope and recorded on a magnetic tape. The output was also passed through a pulse former and then into a computer. The computer calculated the firing rate of a neuron in 1 s intervals (spikes/s) and displayed them on a computer screen as a function of time. The basal firing rate was then calculated as an average for 3 min immediately before the application of orexins, and this value was subtracted from all subsequent changes in firing rate. The response to orexins was evaluated in the change of mean firing rate averaged for the first 3 min of the response time after the drug application. Two criteria (percent change in mean firing rate relative to the basal firing rate and absolute change in mean firing rate) were used to determine the drug effect because the basal firing rate was ranged from 0 to 5.3 spikes/s. Orexins were considered to be effective if the change in mean firing rate after the application of orexins was greater than ±20% for neurons with the basal firing rate equal to or more than 0.3 spikes/s, whereas if it was lager than ±0.06 spikes/s for neurons with the basal firing rate less than 0.3 spikes/s. In electrophysiological experiments, a recording was made from only one neuron in each slice. 2.4. Whole cell patch clamp recording After pre-incubation, slices were transferred into a whole cell patch clamp recording chamber fixed to the stage of an upright microscope (BX-50WI, Olympus, Tokyo, Japan). The recording chamber was perfused with oxygenated ACSF at 1 ml/min and at 34 ◦ C. PVT neurons were monitored on a television screen through an infrared charge coupled device (CCD) camera (C2741-79, Hamamatsu Photonics, Hamamatsu, Japan) and a real-time digital video microscopy processor (XL-20, Olympus, Tokyo, Japan). Electrodes were filled with an internal solution containing in mM: K-gluconate 120, KCl 20, HEPES 10, MgCl2 2.0, CaCl2 0.5, EGTA 1.0, Na-ATP 4.6, and Na-GTP 0.4, pH adjusted to 7.3 with KOH, and electrode resistances were 5 to 9 M. Membrane potentials recorded via the electrodes were fed into a patch clamp amplifier (Axopatch 200B, Axon Instruments, Union City, USA). Series resistance compensation was performed as much as possible by the amplifier. The output of the amplifier was digitized using an A/D converter board (Digidata 1200, Axon Instruments, Union City, USA) with a sampling rate of 10 kHz, and recorded on a hard disk by data acquisition and analysis software (pCLAMP 8, Axon Instruments, Union City, USA). Membrane potentials were

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low-pass filtered at 10 kHz. Furthermore, membrane potentials less than −60 mV were set to about −60 mV by current injections. 2.5. [Ca2+ ]i imaging After pre-incubation, slices were transferred on a membrane filter, which was placed on a culture dish, and submerged in ACSF containing fura-2 acetoxyl methyl (fura-2 AM, 10 ␮M), a calcium indicator, and pluronic F127 (0.01%). Culture dishes were then placed in a tightly sealed pressure box filled with 95% O2 –5% CO2 at a pressure of 50 kPa at room temperature for 1 h. Thus, PVT neurons were loaded with fura-2. The slice was then transferred into a recording chamber which was attached to the stage of an upright microscope (BX51WI, Olympus, Tokyo, Japan) and perfused with oxygenated ACSF at 1 ml/min and at 34 ◦ C. The fluorescence of fura-2 was excited by means of a xenon lamp (U-LH75XEAPO, Olympus, Tokyo, Japan) through a filter exchanger with filters for 340 and 380 nm (C8214, Hamamatsu Photonics, Hamamatsu, Japan). The fluorescent images were obtained from a high-speed cooled digital CCD camera (C6790, Hamamatsu Photonics, Hamamatsu, Japan) every 10 s, and stored on a hard disk via the data acquisition equipment (C7746, Hamamatsu Photonics, Hamamatsu, Japan). Regions of interest were selected to encompass individual 7–10 cells in the PVT in each slice. Fluorescence data were analyzed on-line or off-line on a computer with data analysis software (Aquacosmos, Hamamatsu Photonics, Hamamatsu, Japan), and displayed on a computer screen. [Ca2+ ]i was expressed as the ratio of fluorescence intensity excited at 340 nm to that excited at 380 nm (fura-2 ratio). 2.6. Drugs ORX-A and ORX-B (Peptide Institute, Osaka, Japan) and tetrodotoxin (TTX, Seikagaku Corporation, Tokyo, Japan) were dissolved in saline at 10−4 M for orexins and at 10−3 M for TTX, and stored at −40 ◦ C in small aliquots. Glutamate (Wako Pure Chemical Industries, Tokyo, Japan) was dissolved in distilled water at 10−3 M and stored at 4 ◦ C for 1 week. They were then diluted with ACSF to desired concentrations when they were used. EGTA (Sigma, Tokyo, Japan) was directly dissolved in ACSF at 10−4 M. Fura-2 AM (Dojindo, Tokyo, Japan) and pluronic F127 (Sigma, Tokyo, Japan) were first dissolved in dimethylsulfoxide (DMSO, Wako Pure Chemical Industries, Tokyo, Japan), and then diluted with ACSF. The final concentration of DMSO was 0.15%. All agents were applied extracellularly. 2.7. Statistics All data were expressed as means ± SEMs. For statistical analysis, one-way ANOVA followed by a post-hoc Fisher’s protected least significant difference (PLSD) test and

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Table 1 Summary of ORX-A and ORX-B effects on PVT neurons tested by ORX-A and/or ORX-B Dose (M)

Excitation

No effect

Total

ORX-A

10−8 10−7 10−6

2 (6.7%) 13 (44.8%) 15 (75.0%)

28 (93.3%) 16 (55.2%) 5 (25.0%)

30 (100%) 29 (100%) 20 (100%)

ORX-B

10−8 10−7 10−6

3 (10.3%) 27 (67.5%) 12 (85.7%)

26 (89.7%) 13 (32.5%) 2 (14.3%)

29 (100%) 40 (100%) 14 (100%)

Student’s t-test were used. P < 0.05 was taken as the level of statistical significance.

3. Results 3.1. Excitatory effects of ORX-A and ORX-B on PVT neurons Activity of 55 neurons was recorded extracellularly in the PVT. Of these, 35 were tested with ORX-A, and 42 with ORX-B. The doses of ORX-A and ORX-B used were 10−8 , 10−7 , and 10−6 M. Of the 35 neurons tested with ORX-A, 19 were tested with all of the three doses, and the remaining 16 with either one or two of the three doses. Of the 42 neurons tested with ORX-B, 14 were tested with all of the three doses, and the remaining 28 with either one or two of the three doses. The results are summarized in Table 1. When ORX-A and ORX-B were applied, the PVT neurons responded with excitation, and none of the neurons were suppressed by ORX-A and ORX-B. As the dose of ORX-A and ORX-B was increased from 10−8 to 10−6 M, the ratio of the responding neurons was increased. When 10−6 M ORXA was applied to 20 neurons, 75.0% (15/20) were excited, whereas when ORX-B was applied with the same dose to 14 neurons, 85.7% (12/14) were excited. Of the 55 neurons, 17, 19, and 10 were tested by both ORX-A and ORX-B at 10−8 , 10−7 , and 10−6 M, respectively. As shown in Table 2, 80.0% (8/10) of the neurons recorded were excited by both ORX-A and ORX-B at 10−6 M, and the remaining 20.0% (2/10) were unaffected, suggesting that at high doses such as 10−6 M the neuron that was excited by ORX-A was also excited by ORX-B or vice versa. Dose-dependent responses in two typical neurons that were tested with all of the three doses of ORX-A and ORXTable 2 Summary of ORX-A and ORX-B effects on PVT neurons tested by both ORX-A and ORX-B Dose (M) Excitation

10−8 10−7 10−6

No effect

Total

Both ORX-A and ORX-B

ORX-A

ORX-B

1 (5.9%) 8 (42.1%) 8 (80.0%)

0 (0.0%) 1 (5.9%) 15 (88.2%) 17 (100%) 0 (0.0%) 6 (31.6%) 5 (26.3%) 19 (100%) 0 (0.0%) 0 (0.0%) 2 (20.0%) 10 (100%)

Fig. 1. Dose-dependent excitatory effects of ORX-A and ORX-B on PVT neurons. A and B: Firing rate in spikes/s is shown as a function of time in min. Upper ORX-A and lower ORX-B records were continuous, and the application of ORX-A and ORX-B (both applied at 10−8 , 10−7 and 10−6 M for the period indicated by solid bars) increased activity in a dosedependent fashion. C: Increase in mean firing rate to ORX-A and ORX-B at 10−8 M (n = 17), 10−7 M (n = 19), and 10−6 M (n = 10). The mean excitatory response for ORX-B was significantly greater than for ORX-A at 10−7 M. *** P < 0.001; ns: no significance.

B are reproduced in Fig. 1A and B. In Fig. 1A, the PVT neuron did not respond to 10−8 M ORX-A and ORX-B, but did respond with excitation to 10−7 and 10−6 M ORX-A and ORX-B. In Fig. 1B, the neuron showed no effects to 10−8 and 10−7 M ORX-A, but was excited by 10−6 M ORXA; whereas it was excited by ORX-B at 10−7 and 10−6 M, but not at 10−8 M. In these neurons shown in Fig. 1A and B, the response magnitude at 10−7 M was obviously greater for ORX-B than for ORX-A. Therefore, the increase in the mean firing rate was statistically compared between ORX-A and ORX-B in 17, 19 and 10 neurons that were tested by both ORX-A and ORX-B at 10−8 , 10−7 and 10−6 M, respectively. As shown in Fig. 1C, the mean excitatory response at 10−7 M was significantly greater for ORX-B than for ORX-A

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(P < 0.001, Student’s t-test). There were no significant differences between the mean excitatory responses to ORX-A and ORX-B at 10−8 and 10−6 M. 3.2. Effects of simultaneous application of ORX-A plus ORX-B on PVT neurons OX1 R receptors have a 10 times greater affinity for ORXA than for ORX-B, whereas OX2 R receptors have a similar affinity to both ORX-A and ORX-B. Therefore, it might be possible to examine the extent of the involvement of OX1 R and OX2 R receptors in PVT neurons by utilizing the difference in responses to ORX-A and ORX-B at 10−7 M. Six neurons that were excited by both 10−7 M ORX-A and ORX-B (n = 5) and by only 10−7 M ORX-B (n = 1) were further tested by a simultaneous application of 10−7 M ORX-A plus ORXB (ORX-A + B). A sample record is shown in Fig. 2A. The neuron was excited strongly by ORX-B, but the excitatory response to ORX-A was considerably weak. When ORX-A + B was applied, the neuron showed an excitatory response similar to that induced by ORX-B, but not ORX-A. The other 5 neurons also had the similar response relationships to ORXA, ORX-B, and ORX-A + B. Excitatory responses to ORXA, ORX-B, and ORX-A + B expressed by the increase in mean firing rate are shown in Fig. 2B. One-way ANOVA demonstrated a significant difference in the mean excitatory

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responses between 3 groups (F2,15 = 8.66, P < 0.01). A posthoc PLSD test further revealed that the magnitude of excitatory responses was significantly greater for ORX-B (P < 0.01) and ORX-A + B (P < 0.01) than for ORX-A; and there was no significant difference between ORX-B and ORX-A + B. These results indicate that in the presence of both ORX-A and ORX-B the PVT neurons do not show the high affinity to ORX-A that is expected when they have OX1 R receptors, and suggest that the PVT neurons express OX2 R receptors rather than OX1 R receptors. 3.3. Effect of ORX-B on PVT neurons in low-Ca2+ and high-Mg2+ ACSF To determine whether the excitatory effect of orexins on PVT neurons was pre-synaptic or post-synaptic, neuronal activity was recorded in low-Ca2+ and high-Mg2+ ACSF. 10−7 M ORX-B was applied because it produced greater excitatory responses than 10−7 M ORX-A. As shown in Fig. 3, this neuron excited by ORX-B in normal ACSF was also excited in low-Ca2+ and high-Mg2+ ACSF, and the firing rate was greater in low-Ca2+ and high-Mg2+ ACSF than in normal ACSF. Similar results were obtained in another 4 neurons tested. 3.4. Effects of ORX-A and ORX-B on [Ca2+ ]i of PVT neurons Extracellular recording experiments demonstrated that at 10−7 M more than half of the PVT neurons were excited by both ORX-A and ORX-B or by ORX-B, and the excitatory effect of ORX-B was greater than that of ORX-A. These findings were further examined in the [Ca2+ ]i imaging study. To identify PVT neurons capable of responding to neurotransmitters, glutamate was first applied. Glutamate, a major excitatory neurotransmitter within the thalamus, acts through a variety of ionotropic and/or metabotropic receptors [33,52,56]. Ninety seven PVT neurons responded to 10−5 M glutamate with an increase of [Ca2+ ]i . Of these, 51 neurons (52.6%) also induced an increase of [Ca2+ ]i when 10−7 M ORX-A and ORX-B were applied. Among the 51 neurons,

Fig. 2. Effects of simultaneous application of ORX-A plus ORX-B on PVT neurons. A: Firing rate in spikes/s is shown as a function of time in min. Application of 10−7 M ORX-B (solid bars) produced a greater excitatory response than that of 10−7 M ORX-A (dotted bars), and a simultaneous application of 10−7 M ORX-A plus ORX-B (ORX-A + B, solid bar plus dotted bar) induced a similar excitatory response to that of ORX-B, but not ORX-A. B: Increases in mean firing rate to ORX-A, ORX-B and ORX-A + B applications at 10−7 M (n = 6). Application of ORX-B and ORX-A + B produced significantly greater responses than ORX-A, but there was no significant difference in mean firing rate between ORX-B and ORX-A + B. ** P < 0.01; ns: no significance.

Fig. 3. Excitatory responses induced by ORX-B in low-Ca2+ and high-Mg2+ ACSF; firing rate in spikes/s is shown as a function of time in min. Excitatory response induced by 10−7 M ORX-B in normal ACSF was again induced in low-Ca2+ and high-Mg2+ ACSF. The firing rate was greater in low-Ca2+ and high-Mg2+ ACSF than in normal ACSF. Solid bars: 10−7 M ORX-B; dotted bar: low-Ca2+ and high-Mg2+ ACSF.

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[Ca2+ ]i of 45 neurons was increased by both ORX-A and ORX-B, and that of 6 neurons by only ORX-B. An example of orexin-evoked increases of [Ca2+ ]i is shown in Fig. 4A. A first application of ORX-B produced a large increase of [Ca2+ ]i but the subsequent application of ORX-A induced only a small increase of [Ca2+ ]i . The second application of ORX-B again increased the [Ca2+ ]i to the same extent as the first response. As shown in Fig. 4B, the mean increase of [Ca2+ ]i in 51 neurons expressed by the fura-2 ratio was significantly greater for ORX-B than for ORX-A (P < 0.001, Student’s t-test). These data confirmed the results obtained in the extracellular recordings showing that the mean excitatory responses were significantly greater for ORX-B than for ORX-A at 10−7 M. In additional 21 ORX-B-responding neurons, the effects of 10−7 M ORX-B on [Ca2+ ]i was further tested in Ca2+ -free ACSF containing 10−4 M EGTA to examine an involvement of Ca2+ released from intracellular Ca2+ stores in the ORX-B-induced depolarization. As shown in Fig. 4C, the ORX-B-induced increase of [Ca2+ ]i was almost completely abolished in Ca2+ -free ACSF containing 10−4 M EGTA. The increase of [Ca2+ ]i by ORX-B was again induced after returning to normal ACSF. The mean magnitudes in the increase of [Ca2+ ]i in normal ACSF and Ca2+ free ACSF containing 10−4 M EGTA were 0.362 ± 0.029 and 0.006 ± 0.013, respectively (Fig. 4D). The difference was statistically significant (P < 0.001, Student’s t-test), suggesting that the increase of [Ca2+ ]i induced by ORX-B is due to an influx of Ca2+ from the extracellular space, but not a release of Ca2+ from intracellular stores.

3.5. Effects of ORX-B on PVT neurons examined by whole cell patch clamp recordings Membrane potentials of 122 PVT neurons were recorded under current clamp conditions using whole cell patch clamp recording techniques. The mean resting potential and mean input resistance were −54.6 ± 0.8 mV and 353.9 ± 10.5 M, respectively. One hundred and thirteen out of 122 neurons were tested with a bath application of 10−7 M ORX-B. Of 113 neurons, 91 (80.5%) were depolarized by ORX-B as shown in Fig. 5A. The mean depolarization and mean latency were 16.6 ± 0.8 mV and 76.3 ± 4.4 s, respectively. The depolarization returned to a baseline several min after washing. In 65 out of 91 neurons, the depolarization was accompanied by a firing of action potentials (Fig. 5A). In 26 of 91 neurons, the effects of ORX-B were examined in Ca2+ -free ACSF. Bath applications of ORX-B produced depolarizations of 16.3 ± 1.8 mV again in all neurons tested (Fig. 5B). When the depolarization was accompanied by the firing of action potentials in 24 out of 26 neurons, the firing rate seen in Ca2+ -free ACSF (inset “b” in Fig. 5B) was greater than that seen in normal ACSF (inset “a” in Fig. 5A); because the after hyperpolarization (AHP), as can be seen after each action potential in normal ACSF (inset “a” in Fig. 5A), almost disappeared in Ca2+ -free ACSF (inset “b” in Fig. 5B). Of 26 neurons, 16

Fig. 4. Effects of ORX-A and ORX-B on [Ca2+ ]i in PVT neurons. Data in A and C were recorded from two different cells. A: Application of 10−7 M ORX-B (solid bars) produced a strong increase in fura-2 ratio (F340/F380), but that of 10−7 M ORX-A (dotted bar) had only a weak increase. B: Mean increase in fura-2 ratio for ORX-B was significantly greater than that for ORX-A (n = 51). C: The increase in fura-2 ratio induced by 10−7 M ORX-B (solid bars) in normal ACSF almost disappeared in Ca2+ -free ACSF containing 10−4 M EGTA (hatched bar). D: Mean increase in fura-2 ratio obtained for 10−7 M ORX-B in normal ACSF was significantly attenuated in Ca2+ free ACSF containing 10−4 M EGTA (n = 21). *** P < 0.001.

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Fig. 5. Effects of ORX-B on the membrane potential of PVT neurons. Resting membrane potential was −60 mV. ORX-B at 10−7 M was applied three times; in normal ACSF (A), Ca2+ -free ACSF (B), and Ca2+ -free ACSF containing 10−6 M TTX (C). Records in A, B and C are continuous. A: Application of 10−7 M ORX-B in normal ACSF produced a depolarization accompanied by firing of action potentials. Membrane potential at “a” indicated by down arrow was enlarged in the upper right inset, also labeled “a”. Note that each action potential is associated with an after hyperpolarization (AHP). B: Application of 10−7 M ORX-B in Ca2+ -free ACSF produced a depolarization accompanied by firing of action potentials again. Membrane potential at “b” indicated by down arrow was enlarged in the upper right inset, also labeled “b”. Note that the AHP following each action potential seen in normal ACSF has disappeared, and that the firing rate is increased compared with that in normal ACSF. C: The third application of ORX-B induced a depolarization in Ca2+ -free ACSF containing 10−6 M TTX, but firing of action potentials did not occur. Solid bars: 10−7 M ORX-B; hatched bar: Ca2+ -free ACSF; dotted bar: 10−6 M TTX.

with the firing of action potentials were further tested with ORX-B in 10−6 M TTX-containing Ca2+ -free ACSF. Application of ORX-B induced depolarizations of 21.0 ± 2.6 mV in all neurons tested, but abolished the firing of action potentials as shown in Fig. 5C. One-way ANOVA demonstrated that there was no significant difference between the mean magnitudes of the depolarizations obtained in normal, Ca2+ free and TTX-containing Ca2+ -free ACSFs (F2,130 ) = 1.62, P > 0.05). These results confirm that the depolarizing response due to ORX-B is mediated by a postsynaptic action on orexin receptors, and suggest that TTX-sensitive Na+ channels and Ca2+ channels are not involved in the production of the depolarization.

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containing ACSF under different membrane potentials. These were achieved by injections of hyperpolarizing currents. As shown in Fig. 7A, the depolarizing response to ORX-B was reversed to hyperpolarization between −95 and −101 mV, and the estimated reversal potential was −97.4 mV (Fig. 7B). The mean reversal potential was −97.4 ± 5.9 mV (n = 3). A possibility that the depolarization induced by 10−7 M ORXB was dependent on extracellular K+ concentration ([K+ ]o ) was further tested in another 8 of 19 neurons whose membrane potentials were maintained at about −60 mV. As shown in the left record of Fig. 7C, application of 10−7 M ORX-B depolarized a PVT neuron at 4.25 mM of [K+ ]o . When [K+ ]o was increased to 13.25 mM, estimated equilibrium potential for K+ , 62 mV, however the depolarization disappeared in all the neurons tested (middle record of Fig. 7C). A depolarization was again recorded when [K+ ]o was returned to 4.25 mM (right record of Fig. 7C). These results suggest the possibility that the depolarization of PVT neurons induced by ORX-B is due to the closure of K+ channels. 4. Discussion The present results obtained from extracellular recordings demonstrated that PVT neurons were excited

3.6. Ionic mechanism of ORX-B-induced depolarization In additional 27 neurons, a hyperpolarizing current was injected through a patch pipette to measure membrane resistance before, during, and after the application of 10−7 M ORX-B in normal ACSF (n = 8), or 10−6 M TTX-containing ACSF (n = 19). As shown in Fig. 6A and B, the depolarization produced by bath application of ORX-B in 10−6 M TTXcontaining ACSF was associated with an increase of 50% in membrane resistance. In addition, low threshold Ca2+ spikes [24,25,57] were seen following the cessation of hyperpolarizing current (upward deflections in Fig. 6A and asterisks in Fig. 6B). The mean increase of membrane resistance in 27 neurons was 45.0 ± 0.05%, and the low threshold Ca2+ spike was seen in 22 (81.5%) of 27 neurons. To test whether the increase in membrane resistance during ORX-B application might be attributable to a closure of K+ channels, we applied 10−7 M ORX-B in 3 of 19 neurons treated with 10−6 M TTX-

Fig. 6. Effects of ORX-B on the membrane resistance of PVT neurons. A: Application of 10−7 M ORX-B (solid bar) induced a depolarization. Downward deflections indicate a decrease in electrotonic potential amplitude due to constant-current hyperpolarizing pulses (500 ms, 50 pA) passed through the patch pipet to measure membrane resistance. Upward deflections, low threshold Ca2+ spikes. B: Membrane potential changes induced by hyperpolarizing currents (500 ms, 50 pA) at “a” and “b” indicated by downward arrows in (A) were enlarged, and then rearranged to compare the membrane resistances before (“a”) and during (“b”) the effect of ORX-B. Membrane resistance increased 50% in the presence of ORX-B. Following the cessation of hyperpolarizing currents, low threshold Ca2+ spikes were induced as indicated by asterisks.

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Fig. 7. Reversal potential and effects of [K+ ]o in ORX-B-induced depolarization. All records were obtained in ACSF containing 10−6 M TTX. A. Responses to 10−7 M ORX-B were recorded at different membrane potentials indicated by numerals at the left of each record. When the membrane potential was hyperpolarized by passing current from −95 to −101 mV, the response was reversed from depolarization to hyperpolarization. Downward deflections in top, middle, and lower records indicate a decrease in electrotonic potential amplitude as described in Fig. 6A. Upward deflections in the top record, low threshold Ca2+ spikes. Solid bars: 10−7 M ORX-B. B: A plot of the values obtained in (A) shows a reversal of the ORX-B-induced depolarization at −97.4 mV. Abscissa, membrane potential. Ordinate, amplitude of depolarization induced by 10−7 M ORX-B. C: Effects of [K+ ]o on ORX-B-induced depolarization. Depolarization induced by 10−7 M ORX-B at 4.25 mM of [K+ ]o (left record) disappeared when [K+ ]o was increased to 13.25 mM (middle record). Depolarization was induced again as [K+ ]o was returned to 4.25 mM (right record). Solid bars, 10−7 M ORX-B.

dose-dependently by both ORX-A and ORX-B. In addition, whole cell patch clamp recording experiments show that the excitation of the PVT neurons by ORX-B is mediated by depolarization of membrane potential. The excitatory effects of orexins were consistent with previous studies showing that orexins activated neurons in the arcuate nucleus [48], paraventricular nucleus of the hypothalamus [54], LHA [65], basal forebrain area [13], centromedial and rhomboid nuclei of the thalamus [1], TMN [2,14], LC [18,20,23,62], DR [4,32], ventral tegmental area [39], LDT [5,59], dorsal motor nucleus of the vagus [16,21], NTS [70], and area postrema [69]. The excitatory response of PVT neurons evoked by ORXB was recorded in low-Ca2+ and high-Mg2+ ACSF, Ca2+ free ACSF, and TTX-containing Ca2+ -free ACSF. These results suggest that the effects of ORX-B are mediated postsynaptically via orexin receptors. The firing rate of action potentials induced by ORX-B in low-Ca2+ and high-Mg2+ ACSF or Ca2+ -free ACSF was greater than that in normal ACSF. Interestingly, the AHP that followed each action potential in normal ACSF almost disappeared in Ca2+ -free ACSF. As well documented, the AHP in thalamic neu-

rons is generated by an increase of voltage-dependent and Ca2+ -dependent K+ conductances [24,25,57]. Comparing the two conductances, the Ca2+ -dependent K+ conductance contributes most strongly to the interspike interval via the AHP [24,25,57]. Therefore, it seems that a reduction or an elimination of Ca2+ entering into the cell in low-Ca2+ and highMg2+ ACSF or Ca2+ -free ACSF prevents the increase of the Ca2+ -dependent K+ conductances that causes the membrane hyperpolarization, and leads to the increase in the firing rate. The results obtained in [Ca2+ ]i imaging studies showing that Ca2+ is not released from intracellular Ca2+ stores by ORX-B suggest that the ORX-B-induced depolarization is not due to an activation of Na+ /Ca2+ exchangers, because the activation of Na+ /Ca2+ exchangers is secondary to a surge in [Ca2+ ]i released from intracellular stores [14]. In addition, the fact that the mean magnitude of depolarization induced by ORX-B is same in normal, Ca2+ -free and TTX-containing Ca2+ -free ACSFs indicates that an activation of TTX-sensitive Na+ channels and Ca2+ channels does not induce the ORX-B-induced depolarization. The reversal potential of the depolarization, −97.4 ± 5.9 mV, was close to the equilibrium potential of K+ , and far from a reversal potential expected for non-selective cation channels [27,32,69]. Furthermore, the increase of [K+ ]o from 4.25 to 13.25 mM abolished the depolarization. Consequently, it is most likely that a sole ionic mechanism to induce the ORX-B-induced depolarization is due to an increase of membrane resistance resulting from a closure of K+ channels. The present finding was consistent with the previous results showing that in the centromedial nucleus of the thalamus [1], DR [4], LC [23], and dorsal motor nucleus of the vagus [16], the depolarization induced by orexins was accompanied by a closure of K+ channels. However, other ionic mechanisms in the depolarizing action of orexins have been demonstrated in neurons of other brain regions. For example, the depolarization induced by ORX-A and ORX-B in TMN neurons was associated with a small decrease of membrane resistance, and caused by an activation of both the electrogenic Na+ /Ca2+ exchanger and Ca2+ current [14]. In dissociated rat area postrema neurons, the ORX-A-evoked depolarization was produced by an activation of non-selective cation channels [69]. Furthermore, Shirasaka et al. [54] reported that the ORX-B-evoked depolarization was induced in part by an activation of Cd2+ sensitive Ca2+ channels in putative parvocellular neurons; but not in putative magnocellular neurons in the paraventricular nucleus of the hypothalamus. These results suggest that the ionic mechanisms in the orexin-evoked depolarization can depend upon the brain areas and/or the neuronal cell types involved. As Sakurai et al. [51] reported, using a radioligand binding assay in transfected cell lines expressing the human orexin receptors, OX1 R receptors have 10 times the affinity for ORXA than for ORX-B, whereas OX2 R receptors have an equal affinity for ORX-A and ORX-B. In other words, ORX-A has an equal affinity for OX1 R and OX2 R receptors, while

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ORX-B has a 10 times greater affinity for OX2 R receptors than for OX1 R receptors [49,55]. In the present study, a large portion of PVT neurons was excited by both ORXA and ORX-B. At 10−7 M ORX-B was significantly more potent than ORX-A in increasing the firing rate. Elevation of [Ca2+ ]i that appears to be due to an influx from extracellular space through voltage-dependent Ca2+ channels was also significantly greater for ORX-B than for ORX-A at 10−7 M. The stronger effects of ORX-B on the neuronal activity and [Ca2+ ]i might be explained possibly by differences between rat and human orexin receptors. When ORX-A and ORXB were applied simultaneously at 10−7 M, the magnitude of response in the firing rate was similar to that of ORXB, but not ORX-A, indicating that the PVT neurons do not show the high affinity to ORX-A which is expected if they have OX1 R receptors. Taking theses results into consideration, it seems reasonable to assume that the excitatory effects of orexins in PVT neurons are not mediated by the OX1 R receptors but rather by the OX2 R receptors. In support of this assumption, it has been reported that neurons in the basal forebrain area [13], TMN [2], and centromedial and rhomboid nuclei of the thalamus [1], in which ORX-B-induced excitation was equal to or greater than that of ORX-A, have mainly OX2 R receptors. In addition, the present electrophysiological results are consistent with the previous anatomical data demonstrating that PVT neurons mainly express OX2 R mRNA [61]. Prepro-orexin mRNA transcript levels are upregulated 2 to 4-fold in the LHA during fasting and hypoglycemia in rats or mice [7,17]. Orexin neurons in the LHA are actually glucose-sensitive, and excited by hypoglycemia in rats [6,38]. Neuropeptides that stimulate or inhibit feeding also affect glucose-sensitive orexin neurons. Intracerebroventricular administration of ghrelin induces Fos expression in rat orexin neurons, and ghrelin-induced feeding is inhibited in orexin knockout mice [60]. Orexin neurons co-express a longform leptin receptor in rats [22], and leptin inhibits a large portion of glucose-sensitive neurons in the LHA of rats [53]. In addition, ORX-A levels in the cerebrospinal fluid [72] and Fos expression in orexin neurons [15] increase during active dark phase in rats. Intracerebroventricular administration of ORX-A in rats increases wakefulness and decreases REMsleep [18]. Orexin knockout mice exhibit a phenotype strikingly similar to human narcolepsy [9]. Adaptive augmentation of wakefulness and motor activity in response to fasting is not observed in transgenic mice, in which orexin neurons are ablated [67]. Consequently, Beuckmann and Yanagisawa [3] hypothesize that orexin neurons in the LHA function as a critical link to connect between sleep-wakefulness regulation and energy homeostasis. Taking previous findings into consideration, the present results suggest that orexins interact with PVT neurons through the excitatory effect via OX2 R receptors to coordinate sleep-wakefulness and energy homeostatic signals from the visceral organs and hypothalamus, and to elucidate a role of the PVT in the control of motivated behaviors.

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Acknowledgements This work was partly supported by the Japanese Ministry of Education, Culture, Sports, Science and Technology via a Special Coordination Fund for Promoting Science and Technology (K.S.) and by a Grant-in-Aid for Scientific Research (No. 13670056 to K.S.). One of the authors (K.S.) offers special thanks to Mr. Chikamitsu Nakayama for his encouragement throughout this work.

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