Neuroprotective gap-junction-mediated bystander transformations in the adult zebrafish spinal cord after injury.

The adult zebrafish spinal cord displays an impressive innate ability to regenerate after traumatic insults, yet the underlying adaptive cellular mechanisms remain elusive. Here, we show that while the cellular and tissue responses after injury are largely conserved among vertebrates, the large-size fast spinal zebrafish motoneurons are remarkably resilient by remaining viable and functional. We also reveal the dynamic changes in motoneuron glutamatergic input, excitability, and calcium signaling, and we underscore the critical role of calretinin (CR) in binding and buffering the intracellular calcium after injury. Importantly, we demonstrate the presence and the dynamics of a neuron-to-neuron bystander neuroprotective biochemical cooperation mediated through gap junction channels. Our findings support a model in which the intimate and dynamic interplay between glutamate signaling, calcium buffering, gap junction channels, and intercellular cooperation upholds cell survival and promotes the initiation of regeneration.

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.

Expression of the M3 Muscarinic Receptor on Orexin Neurons that Project to the Rostral Ventrolateral Medulla.

Activation of central cholinergic receptors causes a pressor response in rats, and the hypothalamus is important for this response. Projections from hypothalamic orexin neurons to the rostral ventrolateral medulla (RVLM) are involved in sympatho-excitation of the cardiovascular system. A small population of orexin neurons is regulated by cholinergic inputs through M3 muscarinic acetylcholine receptor (M3 R). To elucidate whether the M3 R on orexin neurons is involved in cardiosympathetic regulation through the RVLM, we examined the presence of the M3 R on retrograde-labeled RVLM-projecting orexin neurons. The retrograde tracer was unilaterally injected into the RVLM. Within the hypothalamus, retrograde-labeled neurons were located predominantly ipsilateral to the injection side. In the anterior hypothalamus (-1.5 to -2.3 mm to the bregma), retrograde-labeled neurons were densely distributed in the paraventricular nuclei and scattered in the retrochiasmatic area. At -2.3 to -3.5 mm from the bregma, labeled neurons were located in the regions where orexin neurons were situated, that is, the tuberal lateral hypothalamic area, perifornical area, and dorsomedial nuclei. Very few retrograde-labeled neurons were observed in the hypothalamus at -3.5 to -4.5 mm from the bregma. About 19.5% ± 1.6% of RVLM-projecting neurons in the tuberal hypothalamus were orexinergic. The M3 R was present on 18.7% ± 3.0% of RVLM-projecting orexin neurons. Injection of a muscarinic agonist, oxotremorine, in the perifornical area resulted in a pressor response, which was attenuated by a pretreatment of atropine. We conclude that cholinergic inputs to orexin neurons may be involved in cardiosympathetic regulation through the M3 R on the orexin neurons that directly project to the RVLM.

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.

Orexins depolarize rostral ventrolateral medulla neurons and increase arterial pressure and heart rate in rats mainly via orexin 2 receptors.

An injection of orexin A or B into the cisterna magna or the rostral ventrolateral medulla (RVLM), where bulbospinal vasomotor neurons are located, elevated arterial pressure (AP) and heart rate (HR). We examined how orexins affected RVLM neurons to regulate cardiovascular functions by using in vitro recordings of neuronal activity of the RVLM and in vivo measurement of cardiovascular functions in rats. Orexin A and B concentration-dependently depolarized RVLM neurons. At 100 nM, both peptides excited 42% of RVLM neurons. Tetrodotoxin failed to block orexin-induced depolarization. In the presence of N-(2-methyl-6-benzoxazolyl)-N'-1, 5-naphthyridin-4-yl urea (SB-334867), an orexin 1 receptor (OX(1)R) antagonist, orexin A depolarized 42% of RVLM neurons with a smaller, but not significantly different, amplitude (4.9 +/- 0.8 versus 7.2 +/- 1.1 mV). In the presence of (2S)-1- (3,4-dihydro-6,7-dimethoxy-2(1H)-isoquinolinyl)-3,3-dimethyl-2-[(4-pyridinylmethyl)amino]-1-butanone hydrochloride (TCS OX2 29), an orexin 2 receptor (OX(2)R) antagonist, orexin A depolarized 25% of RVLM neurons with a significantly smaller amplitude (1.7 +/- 0.5 mV). Coapplication of both antagonists completely eliminated orexin A-induced depolarization. An OX(2)R agonist, [Ala(11),D-Leu(15)]-orexin B, concentration-dependently depolarized RVLM neurons. Regarding neuronal phenotypes, orexins depolarized 88% of adrenergic, 43% of nonadrenergic, and 36 to 41% of rhythmically firing RVLM neurons. Intracisternal TCS OX2 29 (3 and 10 nmol) suppressed intracisternal orexin A-induced increases of AP and HR, whereas intracisternal SB-334867 (3 and 10 nmol) had no effect on the orexin A-induced increase of HR but suppressed the orexin A-induced pressor response at 10 nmol. We concluded that orexins directly excite RVLM neurons, which include bulbospinal vasomotor neurons, and regulate cardiovascular function mainly via the OX(2)R, with a smaller contribution from the OX(1)R.