Orexin-A directly depolarizes dorsomedial hypothalamic neurons, including those innervating the rostral ventrolateral medulla.

The dorsomedial hypothalamus (DMH) receives dense orexinergic innervation. Intra-DMH application of orexins increases arterial pressure and heart rate in rats. We studied the effects of orexin-A on DMH neurons, including those innervating the medullary cardiovascular center, the rostral ventrolateral medulla (RVLM), by using whole-cell recordings in brain slices. In the presence of tetrodotoxin, orexin-A (30-1000 nM) depolarized 56% of DMH neurons (EC50 82.4 ± 4.4 nM). Under voltage-clamp recording, orexin-A (300 nM) induced three types of responses characterized by different current-voltage relationships, namely unchanged, increased, and decreased slope conductance in 68%, 14%, and 18% of orexin-A-responsive neurons, respectively. The reversal potential of the decreased-conductance response was near the equilibrium potential of K+ and became more positive in a high-K+ solution, suggesting that K+ conductance blockade is the underlying mechanism. In a low-Na+ solution, unchanged-, increased-, and decreased-conductance responses were observed in 56%, 11%, and 33% of orexin-A-responsive neurons, respectively, implying that a non-selective cation current (NSCC) underlies orexin-A-induced responses in a small population of DMH neurons. KBR-7943 (70 µM), an inhibitor of Na+-Ca2+ exchanger (NCX), suppressed orexin-A-induced depolarization in 7 of 10 neurons. In the presence of KBR-7943, the majority of orexin-A-responsive neurons exhibited decreased-conductance responses. These findings suggest that NCX activation may underlie orexin-A-induced depolarization in the majority of orexin-responsive DMH neurons. Of 19 RVLM-projecting DMH neurons identified by retrograde labeling, 17 (90%) were orexin-A responsive. In conclusion, orexin-A directly excited over half of DMH neurons, including those innervating the RVLM, through decreasing K+ conductance, activating NCX, and/or increasing NSCC.

Mechanisms of orexin 2 receptor-mediated depolarization in the rat paraventricular nucleus of the hypothalamus.

The paraventricular nucleus of the hypothalamus (PVN) contains dense orexin 2 (OX2) receptor. We examined the mechanisms of OX2 receptor-mediated excitation on electrophysiologically identified type I (putative magnocellular), low-threshold spikes (LTS)-expressing type II (putative preautonomic), and non-LTS type II (putative parvocellular neuroendocrine) neurons. In the presence of tetrodotoxin, an OX2 receptor agonist, ALOXB (30-1000 nM) depolarized 56% of type I, and 73-75% of type II neurons. In type I neurons, ALOXB-induced inward current displayed increased-conductance current-voltage (I-V) relationship and reversed polarity at -27.5 ± 4.8 mV. A Na+-Ca2+ exchanger (NCX) inhibitor, KBR-7943, attenuated ALOXB responses in the majority of type I neurons, while no attenuation was observed in nearly all type II neurons. Type II neurons exhibited three types of I-V relationships in response to ALOXB, characterized by decreased, increased, and unchanged conductance, respectively. The reversal potential of the decreased-conductance responses was near the equilibrium potential of K+ (Ek+) and became more positive in a high-K+ solution, suggesting that K+ conductance blockade is involved. In a low-Na+ solution, non-reversed I-V curves of increased-conductance responses became decreased-conductance responses and reversed polarity near Ek+, suggesting the involvement of both K+ conductance and non-selective cation conductance (NSCC). Approximately 35% of LTS-expressing type II neurons were vasopressin-immunoreactive and 71% of them responded to ALOXB. In conclusion, orexins may activate OX2 receptor on PVN neurons and cause depolarization by promoting NCX and/or NSCC in magnocellular neurons, and by decreasing K+ conductance and/or increasing NSCC in parvocellular neurons. Furthermore, the majority of vasopressinergic preautonomic neurons are under OX2 receptor regulation.

Role of the orexin 2 receptor in palatable-food consumption-associated cardiovascular reactivity in spontaneously hypertensive rats.

Hypertensive subjects often exhibit exaggerated cardiovascular reactivity. An overactive orexin system underlies the pathophysiology of hypertension. We examined orexin's roles in eating-associated cardiovascular reactivity in spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats. Results showed eating regular chow or palatable food (sucrose agar) was accompanied by elevated arterial pressure and heart rate. In both SHRs and WKY rats, the cardiovascular responses associated with sucrose-agar consumption were greater than that with regular-chow consumption. Additionally, SHRs exhibited greater cardiovascular responses than WKY rats did to regular-chow and palatable food consumption. Central orexin 2 receptor (OX2R) blockade attenuated sucrose-agar consumption-associated cardiovascular response only in SHRs. In both SHRs and WKY rats, OX2R blockade did not affect regular-chow consumption-associated cardiovascular responses. Greater numbers of c-Fos-positive cells in the rostral ventrolateral medulla (RVLM) and of c-Fos-positive orexin neurons in the dorsomedial hypothalamus (DMH) were detected in sucrose agar-treated SHRs, compared to regular chow-treated SHRs and to sucrose agar-treated WKY rats. Central OX2R blockade reduced the number of c-Fos-positive cells in the RVLM only in sucrose agar-treated SHRs. We concluded that in SHRs, orexin neurons in the DMH might be overactive during eating palatable food and may further elicit exaggerated cardiovascular responses via an OX2R-RVLM pathway.

Cardiovascular pressor effects of orexins in the dorsomedial hypothalamus.

Orexins are important regulators of cardiovascular functions in various physiological and pathological conditions. The dorsomedial hypothalamus (DMH), an essential mediator of cardiovascular responses to stress, contains dense orexinergic innervations and receptors. We examined whether orexins can regulate cardiovascular functions through their actions in the DMH in anesthetized rats. An intra-DMH injection of orexin A (30pmol) produced elevation of arterial pressure and heart rate. Orexin A-sensitive sites were located within or immediately adjacent to the DMH and larger responses were induced at the compact part of the dorsomedial hypothalamic nucleus. Orexin A-induced responses were attenuated by intra-DMH pretreatment with an orexin receptor 1 (OX1R) antagonist, SB-334867 (15nmol) (17.7 ± 2.8 vs. 5.2 ± 1.0mmHg; 54.6 ± 10.0 vs. 22.8 ± 7.4 beats/min). Intra-DMH applied [Ala11,D-Leu15]-orexin B (300 pmol), an orexin receptor 2 (OX2R) agonist, elicited cardiovascular responses mimicking the responses of orexin A, except for a smaller pressor response (7.4 ± 1.7 vs. 16.4 ± 1.8mmHg). In a series of experiment, effects of orexin B (100pmol) and then orexin A (30pmol), were examined at a same site. Two patterns of responses were observed in 12 intra-DMH sites: (1) both orexin A and B (9 sites), and (2) only orexin A (3 sites) induced cardiovascular responses, respectively suggesting OX1R/OX2R-mediated and OX1R-predominant mechanisms. In conclusion, orexins regulated cardiovascular functions through OX1R/OX2R- or OX1R-mediated mechanisms at different locations in the DMH.

Spontaneously hypertensive rats have more orexin neurons in the hypothalamus and enhanced orexinergic input and orexin 2 receptor-associated nitric oxide signalling in the rostral ventrolateral medulla.

NEW FINDINGS: What is the central question of this study? Our previous study demonstrates that elevated orexin 2 receptor (OX2R) activity within the rostral ventrolateral medulla (RVLM) contributes to hypertension in spontaneously hypertensive rats (SHRs), and a lower OX2R protein level was detected in their RVLM. The present study aims to explore the mechanisms underlying elevated orexinergic activity in the RVLM of SHRs, compared with their normotensive counterparts, Wistar-Kyoto rats. What is the main finding and its importance? Increased orexinergic input into the RVLM and enhanced OX2R responsiveness in the RVLM, which was mainly mediated by augmented OX2R-neuronal nitric oxide synthase signalling, may underlie the elevated OX2R activity within the RVLM of SHRs. Our previous study showed that elevated orexin 2 receptor (OX2R) activity within the rostral ventrolateral medulla (RVLM) contributes to hypertension in spontaneously hypertensive rats (SHRs). Herein, we investigated the mechanism(s) underlying the elevated OX2R activity. The following results were found. (i) More hypothalamic orexin A-immunoreactive (OXA-IR) cells existed in SHRs than in Wistar-Kyoto (WKY) rats at either 4 (2217 ± 43 versus 1809 ± 69) or 16 weeks of age (1829 ± 59 versus 1230 ± 84). The number of OXA-IR cells that project to the RVLM was higher in 16-week-old SHRs than in WKY rats (91 ± 11 versus 52 ± 11). (ii) Higher numbers of OXA-IR and RVLM-projecting OXA-IR cells were found in the dorsomedial and perifornical hypothalamus of 16-week-old SHRs. (iii) Spontaneously hypertensive rats had higher levels of orexin A and B in the hypothalamus and higher levels of orexin A in the RVLM than did WKY rats. (iv) Unilateral intra-RVLM application of OX2R agonist, orexin A or [Ala(11), d-Leu(15)]-orexin B (50 pmol) induced a larger pressor response in SHRs than in WKY rats. (v) Intra-RVLM pretreatment with a neuronal nitric oxide synthase (NOS) inhibitor, 7-nitro-indazole (2.5 pmol), or a soluble guanylate cyclase inhibitor, methylene blue (250 pmol), reduced the intra-RVLM [Ala(11), d-Leu(15) ]-orexin B-induced pressor response in both WKY rats and SHRs. In contrast, an inducible NOS inhibitor, aminoguanidine (100 pmol), was ineffective. (vi) Neuronal NOS was co-expressed with OX2R in RVLM neurons. In conclusion, increased orexinergic input and enhanced OX2R-neuronal NOS signalling may underlie elevated OX2R activity in the RVLM and contribute to the pathophysiology of hypertension in SHRs.

Blockade of central orexin 2 receptors reduces arterial pressure in spontaneously hypertensive rats.

Orexins can raise arterial pressure and sympathetic activity and are involved in tonic and phasic control of cardiovascular homeostasis. We hypothesized that elevated central orexinergic activity contributes to the maintenance of hypertension in spontaneously hypertensive rats (SHRs). We examined this hypothesis by suppressing central orexinergic activity in SHRs and Wistar-Kyoto rats (WKYs) with specific antagonists or antibodies against orexin 1 (OX1R) and 2 receptors (OX2R). Intracerebroventricular administration of an OX1R antagonist, SB-334867 (30 and 100 nmol), induced no significant change in mean arterial pressure (MAP) and heart rate (HR) in SHRs and WKYs except that at 100 nmol it reduced HR in WKYs. In contrast, an OX2R antagonist, TCS-OX2-29 (3-30 nmol) induced long-lasting reductions of MAP and HR in SHRs (21 ± 3 mmHg and 22 ± 2 beats min(-1) at 30 nmol), but not in WKYs. Intracerebroventricular anti-OX2R IgG, but not anti-OX1R IgG or non-immune goat IgG, significantly lowered MAP and HR in SHRs. None of the three IgGs affected MAP or HR in WKYs. The OX2R protein level in the rostral ventrolateral medulla (RVLM) was lower in SHRs than in WKYs, whereas no differences were found between SHRs and WKYs in the paraventricular hypothalamic nucleus, dorsomedial-perifornical hypothalamic area or caudal nucleus tractus solitarii. The OX1R protein levels in these four regions did not differ between SHRs and WKYs. Injection of TCS-OX2-29 (50 pmol) into the RVLM produced a larger reduction of MAP in SHRs than in WKYs. We conclude that elevated OX2R-mediated activity in the brain, especially in the RVLM, may contribute to hypertension in SHRs.

Sex differences in high-fat diet-induced obesity, metabolic alterations and learning, and synaptic plasticity deficits in mice.

Obesity is a potential risk factor for cognitive deficits in the elder humans. Using a high-fat diet (HFD)-induced obese mouse model, we investigated the impacts of HFD on obesity, metabolic and stress hormones, learning performance, and hippocampal synaptic plasticity. Both male and female C57BL/6J mice fed with HFD (3 weeks to 9-12 months) gained significantly more weights than the sex-specific control groups. Compared with the obese female mice, the obese males had similar energy intake but developed more weight gains. The obese male mice developed hyperglycemia, hyperinsulinemia, hypercholesterolemia, and hyperleptinemia, but not hypertriglyceridemia. The obese females had less hyperinsulinemia and hypercholesterolemia than the obese males, and no hyperglycemia and hypertriglyceridemia. In the contextual fear conditioning and step-down passive avoidance tasks, the obese male, but not female, mice showed poorer learning performance than their normal counterparts. These learning deficits were not due to sensorimotor impairment as verified by the open-field and hot-plate tests. Although, basal synaptic transmission characteristics (input-output transfer and paired-pulse facilitation (PPF) ratio) were not significantly different between normal and HFD groups, the magnitudes of synaptic plasticity (long-term potentiation (LTP) and long-term depression (LTD)) were lower at the Schaffer collateral-CA1 synapses of the hippocampal slices isolated from the obese male, but not female, mice, as compared with their sex-specific controls. Our results suggest that male mice are more vulnerable than the females to the impacts of HFD on weight gains, metabolic alterations and deficits of learning, and hippocampal synaptic plasticity.

Electrophysiological study on the effects of leptin in rat dorsal motor nucleus of the vagus.

Immunoreactivity of leptin receptor (Ob-R) has been detected in rat dorsal motor nucleus of the vagus (DMNV). Here, we confirmed the presence of Ob-R immunoreactivity on retrograde-labeled parasympathetic preganglionic neurons in the DMNV of neonatal rats. The present study investigated the effects of leptin on DMNV neurons, including parasympathetic preganglionic neurons, by using whole cell patch-clamp recording technique in brain stem slices of neonatal rats. Leptin (30-300 nM) induced membrane depolarization and hyperpolarization, respectively, in 14 and 15 out of 80 DMNV neurons tested. Both leptin-induced inward and outward currents persisted in the presence of TTX, indicating that leptin affected DNMV neurons postsynaptically. The current-voltage (I-V) curve of leptin-induced inward currents is characterized by negative slope conductance and has an average reversal potential of -90 +/- 3 mV. The reversal potential of the leptin-induced inward current was shifted to a more positive potential level in a high-potassium medium. These results indicate that a decrease in potassium conductance is likely the main ionic mechanism underlying the leptin-induced depolarization. On the other hand, the I-V curve of leptin-induced outward currents is characterized by positive slope conductance and has an average reversal potential of -88 +/- 3 mV, suggesting that an increase in potassium conductance may underlie leptin-induced hyperpolarization. Most of the leptin-responsive DMNV neurons were identified as being parasympathetic preganglionic neurons. These results suggest that the DMNV is one of the central target sites of leptin, and leptin can regulate parasympathetic outflow from the DMNV by directly acting on the parasympathetic preganglionic neurons of the DMNV.

Central pressor effects of CART peptides in anesthetized rats.

Interrelationships between energy homeostasis and regulation of cardiovascular functions have been suggested by previous observations [Am. J. Physiol. 278 (2000) R692; Regul. Pept. 104 (2002) 75; Am. J. Physiol. 277 (1999) R1780]. Cocaine- and amphetamine-regulated transcript (CART) was first discovered in the striatum of rats treated with cocaine or amphetamine. The CART peptides were later found in the hypothalamus and functioned as anorectic peptides. We observed that intracisternally (I.C.) administered CART peptide fragments (CART 61-102 and CART 55-102) dose-dependently (1-4 nmol) increased heart rate and blood pressure in urethane-anesthetized adult male Sprague-Dawley rats. Intrathecal (levels T2-T3) and intravenous administrations of these peptides, however, showed little or no effects on the heart rate and blood pressure in the rat. Furthermore, an increase of c-Fos-like immunoreactivity in the rat rostral ventrolateral medulla (RVLM) following an I.C. CART 61-102 was observed. The results suggest that central pressor effects of anorectic CART peptides may involve in activation of the medullary sympathetic systems in the rat. Our observations support the hypothesis that energy homeostasis and cardiovascular regulations are closely related and regulated.