Megumi Tandai-Hiruma

Brain neuronal nitric oxide synthase neuron-mediated sympathoinhibition is enhanced in hypertensive Dahl rats.

Objective To elucidate the role of central neurons containing neuronal nitric oxide synthase (nNOS neurons) in the sympathetic nervous system in hypertensive Dahl salt-sensitive (DS) rats. Design and methods Dahl rats were fed either a regular-salt (0.4% NaCl) or high-salt (8% NaCl) diet for 4 weeks. The effect of intracerebroventricular administration of S-methyl-L-thiocitrulline, a selective nNOS inhibitor, on renal sympathetic nerve activity was examined in chronically instrumented conscious DS rats. The activity and protein amount of brain nNOS was evaluated by enzyme assay and western blot analysis. The distribution and number of nNOS neurons in the brainstem were examined immunohistochemically in hypertensive and normotensive DS rats. Results S-methyl-L-thiocitrulline induced a larger increase in tonic renal sympathetic nerve activity generated before baroreflex-mediated inhibition in hypertensive DS rats than normotensive DS rats. Hypertensive DS rats showed increased nNOS activity in the brainstem, but not in the diencephalon or cerebellum. High nNOS activity was confirmed by an increase in the amount of nNOS protein. nNOS Neurons were localized in several nuclei throughout the brainstem; the dorsolateral periaqueductal gray, pedunculopontine tegmental nucleus, dorsal raphe nucleus, laterodorsal tegmental nucleus, lateral parabrachial nucleus, rostral ventrolateral medulla, nucleus tractus solitarius and raphe magnus. The number of nNOS neurons in these nuclei, except for the two raphes, was significantly greater in hypertensive than in normotensive DS rats. Conclusions These findings suggest that central nNOS-mediated sympathoinhibition may be enhanced in salt-sensitive hypertensive Dahl rats. The upregulated nNOS-mediated inhibition may occur in the central sympathetic control system generated before baroreflex-mediated inhibition.

Neuronal nitric oxide strongly suppresses sympathetic outflow in high-salt Dahl rats.

Objective To investigate the effects of a selective inhibitor of neuronal nitric oxide synthase (nNOS), 7-nitroindazole, on peripheral sympathetic outflow in Dahl rats. Design and methods Dahl salt-sensitive and salt-resistant rats were fed either a regular-salt (0.4% NaCl) or a high-salt (8% NaCl) diet for 4 weeks. In chronically instrumented conscious rats, renal sympathetic nerve activity (RSNA) was measured in both baroreceptor-loaded and baroreceptor-unloaded states. The baroreceptor unload was performed by decreasing arterial pressure with occlusion of the inferior vena cava. Results 7-Nitroindazole (307 μmol/kg intraperitoneally) increased resting RSNA from 24 ± 3% to 38 ± 6% with an increase in mean arterial pressure of 15 ± 3 mmHg, and increased baroreceptor-unloaded RSNA from 100% to 278 ± 16% in salt-sensitive Dahl rats receiving a high-salt diet. However, 7-nitroindazole did not increase resting RSNA, but did increase baroreceptor-unloaded RSNA from 100% to 179 ± 15%, 177 ± 15%, and 133 ± 4% in salt-sensitive Dahl rats receiving a regular-salt diet, salt-resistant Dahl rats receiving a high-salt diet, and salt-resistant Dahl rats receiving a regular-salt diet, respectively. The high-salt diet significantly increased the baroreceptor-unloaded RSNA more than the regular-salt diet did, in both salt-sensitive and salt-resistant rats. After administration of the vehicle for 7-nitroindazole (peanut oil), l-arginine (100 μmol/kg per min for 10 min) decreased both resting and baroreceptor-unloaded RSNA, whereas after pretreatment with 7-nitroindazole, the l-arginine-induced suppression was reversed, in Dahl salt-sensitive rats receiving a high-salt diet. Conclusions Neuronal nitric oxide may suppress the sympathetic discharge generated before baroreflex-mediated inhibition in all rats. This neuronal nitric oxide-mediated suppression was enhanced by the salt load in both salt-resistant and salt-sensitive Dahl rats. Finally, the neuronal nitric oxide-mediated suppression in tonic peripheral sympathetic outflow may be greatly enhanced in salt-sensitive hypertension.

A power-law distribution of inter-spike intervals in renal sympathetic nerve activity in salt-sensitive hypertension-induced chronic heart failure.

To assess sympathetic variability in chronic heart failure (CHF), we evaluated a distribution of inter-spike intervals (ISIs) in renal sympathetic nerve activity (RSNA) in salt-sensitive hypertension-induced CHF (DSSH-CHF) rats. Dahl salt-sensitive rats were fed an 8% NaCl diet for 9 weeks to induce salt-sensitive hypertension-induced CHF. ISIs in RSNA were obtained from chronically instrumented conscious rats, and counts (frequency) and ranks of ISIs in RSNA were plotted with a histogram. We found that ISIs in RSNA followed a power-law distribution in rats, and the power-law distribution of ISIs for RSNA in DSSH-CHF rats was significantly different from that in normal rats. These results indicated that sympathetic variability may be significantly different between salt-sensitive hypertension-induced CHF and healthy individuals, which suggests that sympathetic variability may be used to predict abnormality of the sympathetic regulatory system.

Central Endogenous Vasopressin Induced by Central Salt‐Loading Participates in Body Fluid Homeostasis through Modulatory Effects on Neurones of the Paraventricular Nucleus in Conscious Rats

Peripherally secreted arginine vasopressin (AVP) plays a role in controlling body fluid homeostasis, and central endogenous AVP acts as a neurotransmitter or neuromodulator. The limbic system, which appears to exert an inhibitory effect on the endocrine hypothalamus, is also innervated by fibres that contain AVP. We examined whether central endogenous AVP is also involved in the control of body fluid homeostasis. To explore this possibility, we examined neuronal activity in the paraventricular nucleus of the hypothalamus (PVN), periventricular parts of the PVN and limbic brain areas, as well as AVP mRNA expression in the PVN and the peripheral secretion of AVP after central salt‐loading in rats that had been pretreated i.c.v. with the AVP V1 receptor antagonist OPC‐21268. Neuronal activity in the PVN evaluated in terms of Fos‐like immunoreactivity (FLI), especially in the parvocellular subdivisions, was suppressed. On the other hand, FLI was enhanced in the lateral septum, the bed nucleus of the stria terminalis and the anterior hypothalamic area. Similarly, AVP mRNA expression was enhanced in the magnocellular subnucleus of the PVN, despite the lack of a significant difference in the peripheral AVP level between OPC‐21268‐ and vehicle‐pretreated groups. We recorded renal sympathetic nerve activity (RSNA) as sympathetic nerve outflow during central salt‐loading. The suppression of RSNA was significantly attenuated by i.c.v. pretreatment with OPC‐21268. These results suggest that the suppression of RSNA during central salt‐loading might be the result of a decrease in neuronal activity in the parvocellular subdivisions of the PVN via the inhibitory action of central endogenous AVP. The parvocellular and magnocellular neurones in the PVN might show different responses to central salt‐loading to maintain body fluid homeostasis as a result of the modulatory role of central endogenous AVP.

Paired stimulation between CA3 and CA1 alters excitability of CA3 in the rat hippocampus

It is generally accepted that the extent of plasticity is localized to the region around synapses and post-synaptic intracellular signaling cascades. We investigated the presence of long-range retrograde plasticity associated with excitability at pre-synaptic neurons (CA3) and regulated by the firing of post-synaptic neurons (CA1). We used acute hippocampus slices from transgenic rats expressing channelrhodopsin-2 (ChR2) in both CA1 and CA3 neurons. We employed a parallel photostimulation technique, which enabled robust and independent evocation of action potentials in either CA3 or CA1 neurons. Optically evoked CA3 firings were paired either with CA1 simultaneous firings or with CA1 suppression after the prolonged stimulation. Pre-synaptic excitability was monitored by measuring the optically-evoked firing rate (Opt-FR). We found that the Opt-FR of CA3 neurons was long-term up-regulated as a result of synchronous pre- and post-synaptic pairing stimulation, but down-regulated by the pre-synaptic stimulation during post-synaptic suppression. Both pairing-dependent up-regulation and down-regulation were retarded by NMDA receptor blocking or colchicine preincubation. This finding suggest that CA3 excitability is regulated by CA1 neuron activity at the time of CA3 firing.

Expression and electrophysiological function of actin in chick cerebellar neurons

Among several monoclonal antibodies obtained by immunizing Balb/c mice with cerebellar synaptic membrane fractions from E20 chick embryos, the antibody, named M35, suppressed Ca-spikes in immature cultured chick cerebellar neurons. M35 immunoprecipitated 43kDa protein from a 125I-labeled embryonic crude cerebellar membrane fraction. Immunohistochemically, the M35 antigen was expressed most intensively in Purkinje cells, but its expression was limited to highly motile structures at developmental neuronal remodeling. Electrophysiologically, M35 facilitated current responses to AMPA and inhibited the responses to GABA in cultured cerebellar Purkinje neurons. The several peptides derived from the affinity-purified 43kDa protein were found to have homologous amino acid sequences to non-muscle actins. These results suggest that the antigen recognized by M35 may play an essential role probably as membrane ion channels modulating synaptic functions in not only the development and growth but also the neuronal activity of chick cerebellar Purkinje cells.

High blood pressure enhances brain stem neuronal nitric oxide synthase activity in Dahl salt-sensitive rats

The aims of the present study were to determine the mechanism underlying enhanced neuronal nitric oxide synthase (nNOS) activity in the brain of hypertensive Dahl salt‐sensitive (DSS) rats and the consequences of enhanced nNOS activity. Male DSS rats were fed either a regular (0.4% NaCl) or high‐salt (8% NaCl) diet, with or without 0.25% nifedipine, for 4 weeks. The effects of nifedipine, which lowers blood pressure peripherally, on central nNOS were determined by measuring nNOS activity, as well as the number of nNOS‐positive neurons in the brain stem and diencephalon. The effects of chronic (12 days) infusion of 7 μg (0.5 μL/h, i.c.v.) S‐methyl‐l‐thiocitrulline (SMTC; a stereoselective competitive nNOS inhibitor) on mean arterial pressure were assessed in conscious DSS rats using a radiotelemetry system. In addition, the number of central nNOS‐positive neurons was compared between DSS and salt‐insensitive Sprague‐Dawley rats. Normalization of blood pressure by nifedipine attenuated the increase in nNOS activity in the brain stem of DSS rats. Chronic i.c.v. infusion of SMTC further enhanced hypertension in DSS rats. Feeding of a high‐salt diet increased nNOS‐positive neurons in the lateral parabrachial nucleus, rostral ventrolateral medulla and nucleus tractus solitarius of DSS compared with Sprague‐Dawley rats, whereas nNOS‐positive neurons in the paraventricular nucleus remained downregulated in DSS rats. The results of the present study suggest that hypertension, rather than a high‐salt diet, increases central nNOS activity in hypertensive DSS rats to buffer high blood pressure. However, this compensatory response may be insufficient to relieve salt‐induced hypertension.