Perifornical hypothalamic pathway to the adrenal gland: Role for glutamatergic transmission in the glucose counter-regulatory response.

Adrenaline is an important counter-regulatory hormone that helps restore glucose homeostasis during hypoglycaemia. However, the neurocircuitry that connects the brain glucose sensors and the adrenal sympathetic outflow to the chromaffin cells is poorly understood. We used electrical microstimulation of the perifornical hypothalamus (PeH) and the rostral ventrolateral medulla (RVLM) combined with adrenal sympathetic nerve activity (ASNA) recording to examine the relationship between the RVLM, the PeH and ASNA. In urethane-anaesthetised male Sprague-Dawley rats, intermittent single pulse electrical stimulation of the rostroventrolateral medulla (RVLM) elicited an evoked ASNA response that consisted of early (60±3ms) and late peaks (135±4ms) of preganglionic and postganglionic activity. In contrast, RVLM stimulation evoked responses in lumbar sympathetic nerve activity that were almost entirely postganglionic. PeH stimulation also produced an evoked excitatory response consisting of both preganglionic and postganglionic excitatory peaks in ASNA. Both peaks in ASNA following RVLM stimulation were reduced by intrathecal kynurenic acid (KYN) injection. In addition, the ASNA response to systemic neuroglucoprivation induced by 2-deoxy-d-glucose was abolished by bilateral microinjection of KYN into the RVLM. This suggests that a glutamatergic pathway from the perifornical hypothalamus (PeH) relays in the RVLM to activate the adrenal SPN and so modulate ASNA. The main findings of this study are that (i) adrenal premotor neurons in the RVLM may be, at least in part, glutamatergic and (ii) that the input to these neurons that is activated during neuroglucoprivation is also glutamatergic.

Activation of Medulla-Projecting Perifornical Neurons Modulates the Adrenal Sympathetic Response to Hypoglycemia: Involvement of Orexin Type 2 (OX2-R) Receptors.

Iatrogenic hypoglycemia in response to insulin treatment is commonly experienced by patients with type 1 diabetes and can be life threatening. The body releases epinephrine in an attempt to counterregulate hypoglycemia, but the neural mechanisms underlying this phenomenon remain to be elucidated. Orexin neurons in the perifornical hypothalamus (PeH) project to the rostral ventrolateral medulla (RVLM) and are likely to be involved in epinephrine secretion during hypoglycemia. In anesthetized rats, we report that hypoglycemia increases the sympathetic preganglionic discharge to the adrenal gland by activating PeH orexin neurons that project to the RVLM (PeH-RVLM). Electrophysiological characterization shows that the majority of identified PeH-RVLM neurons, including a subpopulation of orexin neurons, are activated in response to hypoglycemia or glucoprivation. Furthermore, the excitatory input from the PeH is mediated by orexin type 2 receptors in the RVLM. These results suggest that activation of orexin PeH-RVLM neurons and orexin type 2 receptors in the RVLM facilitates epinephrine release by increasing sympathetic drive to adrenal chromaffin cells during hypoglycemia.

Cerebrospinal Fluid Hypernatremia Elevates Sympathetic Nerve Activity and Blood Pressure via the Rostral Ventrolateral Medulla.

Elevated NaCl concentrations of the cerebrospinal fluid increase sympathetic nerve activity (SNA) in salt-sensitive hypertension. Neurons of the rostral ventrolateral medulla (RVLM) play a pivotal role in the regulation of SNA and receive mono- or polysynaptic inputs from several hypothalamic structures responsive to hypernatremia. Therefore, the present study investigated the contribution of RVLM neurons to the SNA and pressor response to cerebrospinal fluid hypernatremia. Lateral ventricle infusion of 0.15 mol/L, 0.6 mol/L, and 1.0 mol/L NaCl (5 µL/10 minutes) produced concentration-dependent increases in lumbar SNA, adrenal SNA, and arterial blood pressure, despite no change in splanchnic SNA and a decrease in renal SNA. Ganglionic blockade with chlorisondamine or acute lesion of the lamina terminalis blocked or significantly attenuated these responses, respectively. RVLM microinjection of the gamma-aminobutyric acid (GABAA) agonist muscimol abolished the sympathoexcitatory response to intracerebroventricular infusion of 1 mol/L NaCl. Furthermore, blockade of ionotropic glutamate, but not angiotensin II type 1, receptors significantly attenuated the increase in lumbar SNA, adrenal SNA, and arterial blood pressure. Finally, single-unit recordings of spinally projecting RVLM neurons revealed 3 distinct populations based on discharge responses to intracerebroventricular infusion of 1 mol/L NaCl: type I excited (46%; 11/24), type II inhibited (37%; 9/24), and type III no change (17%; 4/24). All neurons with slow conduction velocities were type I cells. Collectively, these findings suggest that acute increases in cerebrospinal fluid NaCl concentrations selectively activate a discrete population of RVLM neurons through glutamate receptor activation to increase SNA and arterial blood pressure.

Transneuronal retrograde viral labeling in the brain stem and hypothalamus is more intense from the left than from the right adrenal gland.

Previous studies using the viral transneuronal tracing technique demonstrated central autonomic circuits involved in the innervation of the adrenal gland. Since increasing number of data indicate laterality in the neuroendocrine system, we aimed to investigate whether the supraspinal innervation of the adrenal gland exhibits asymmetry or not. The central circuitry involved in the innervation of the left and the right adrenal gland was studied in individual rats by dual transneuronal tracing using isogenic recombinant strains (Ba-DupGreen and Ba-Duplac expressing lacZ) of Bartha strain of pseudorabies virus. Viral infection of brain nuclei (dorsal vagal nucleus, nucleus of the solitary tract, caudal raphe nuclei, A5 cell group, hypothalamic paraventricular nucleus) from the left adrenal was more severe than that from the right organ. Dual-infected neurons were present both in the brain stem and in the hypothalamus. The results indicate a predominance in the supraspinal innervation of the left adrenal gland, and that each adrenal gland is innervated both by side-specific neurons and by neurons that project to both organs.

Sympathoadrenal-dependent sexually dimorphic effect of nonhabituating stress on in vivo neutrophil recruitment in the rat.

Since stress both activates the sympathoadrenal axis and profoundly affects inflammation and inflammatory diseases, many of which are sexually dimorphic, we tested whether the effect of stress on neutrophil recruitment, a primary component of the acute inflammatory response, is sexually dimorphic. The effect of intermittent sound (over 4 days), a nonhabituating stress, on lipopolysaccharide (LPS)-induced recruitment of neutrophils was evaluated in vivo in the rat air pouch model. At 24 h following the last stress exposure, LPS-induced neutrophil recruitment was enhanced in male rats, but not in females. When gonadectomized prepubertally and tested as adults, stress significantly inhibited the magnitude of LPS-induced neutrophil recruitment in males, while it still had no effect in gonadectomized females. In males, following adrenal denervation, the increase in LPS-induced neutrophil recruitment produced by stress was prevented. Since these data suggest that the effect of stress is dependent on the sympathoadrenal axis, we tested the hypothesis that catecholamines mediate the stress effects. In male rats, the effect of stress on LPS-induced neutrophil recruitment was significantly attenuated by continuous administration of the beta-adrenergic receptor antagonist, propranolol (4 mg kg(-1) day(-1)), during sound stress exposure, and administration of isoproterenol (10 nmoles, i.v.) significantly increased neutrophil recruitment in males, an effect that was qualitatively and quantitatively similar to the effect of stress. Propranolol significantly increased neutrophil recruitment in nonstressed female rats, but did not significantly affect neutrophil recruitment in stressed females. These findings indicate a marked male sex hormone-dependent sexual dimorphism in the sympathoadrenal-dependent effect of stress on neutrophil migration, a primary component of the inflammatory response, and suggest that the sympathoadrenal axis contributes to this effect via release of epinephrine.

Olfactory stimulation with scent of grapefruit oil affects autonomic nerves, lipolysis and appetite in rats.

In a previous study, we found that olfactory stimulation with scent of grapefruit oil (SGFO) excites the sympathetic nerve innervating the white adipose tissue in rats. Here we further examined the effects of SGFO in rats and observed that olfactory stimulation with SGFO excited the sympathetic nerves innervating the brown adipose tissue and adrenal gland and inhibited the parasympathetic gastric nerve. Local anesthesia of the nasal mucosa with xylocaine or anosmic treatment using ZnSO4 eliminated the autonomic changes caused by SGFO. Moreover, stimulation with SGFO elevated the plasma glycerol level, and treatment with either ZnSO4 or an intraperitoneal injection of diphenhydramine, a histamine H1 receptor-antagonist, abolished the glycerol elevation by SGFO. Furthermore, a 15-min exposure to SGFO three times a week reduced food intake and body weight. Finally, limonene, a component of grapefruit oil, induced responses similar to those caused by SGFO, and diphenhydramine eliminated the glycerol response to limonene. Thus, the scent of grapefruit oil, and particularly its primary component limonene, affects autonomic nerves, enhances lipolysis through a histaminergic response, and reduces appetite and body weight.

Hypothalamic PI3K and MAPK differentially mediate regional sympathetic activation to insulin.

The action of insulin in the central nervous system produces sympathetic nervous system activation (also called sympathoactivation), although the neuronal intracellular mechanisms that mediate this are unclear. We hypothesized that PI3K and MAPK, the major pathways involved in insulin receptor signaling, mediate sympathetic nerve responses to insulin. Intracerebroventricular administration of insulin in rat increased multifiber sympathetic nerve activity to the hindlimb, brown adipose tissue (BAT), adrenal gland, and kidney. Ex vivo biochemical studies of mediobasal hypothalamic tissue revealed that insulin stimulated the association of insulin receptor substrate-1 with the p85alpha subunit of PI3K and also tyrosine phosphorylation of p42 and p44 subunits of MAPK in the hypothalamus. In order to determine whether PI3K and/or MAPK were involved in insulin-mediated sympathoactivation, we tested the effect of specific inhibitors of PI3K (LY294002 and wortmannin) and MAPK (PD98059 and U0126) on regional sympathetic responses to insulin. Interestingly, regional sympathoactivation to insulin was differentially affected by blockade of PI3K and MAPK. Inhibition of PI3K specifically blocked insulin-induced sympathoactivation to the hindlimb, while inhibition of MAPK specifically blocked insulin-induced sympathoactivation to BAT. Sympathoactivation to corticotrophin-releasing factor, however, was not affected by inhibition of PI3K and MAPK. These data demonstrate that PI3K and MAPK are specific and regionally selective mediators of the action of insulin on the sympathetic nervous system.

Effect of electroacupuncture on blood pressure and adrenal nerve activity in anesthetized rats.

The neural mechanism underlying the effect of electroacupuncture (Ea) on arterial blood pressure (BP) and adrenal nerve activity (ANA) was investigated in anesthetized rats. Tsusanli (St-36) and Hoku (Li-4) were tested with combinations of two different frequencies (3 and 30 Hz) with various stimulation intensities of Ea. At Tsusanli, no effect was found, while at Hoku, an elevation of BP in parallel with ANA was elicited during Ea when the intensity was 5xT or higher. The pattern of the pressor response caused by the low frequency Ea (LFEa, 3 Hz) was a tonic one, while a phasic one was induced by the high frequency Ea (HFEa, 30 Hz). When both Hoku were simultaneously stimulated with the same frequency, the latency to reach the maximal effect was shortened. However, when two different frequencies were used instead, a response characterized by a combination of both phasic and tonic effect was obtained. In bilateral Ea with idential frequency but different onset time, the pressor effect elicited by the latter Ea showed no further increase during the stimulation period, however, when different frequencies were employed, each Ea elicited its own effect independently. The pressor effect elicited by Ea was abolished by regitine but not affected by adrenalectomy. It is concluded that a LFEa and a HFEa at Hoku with appropriate stimulation parameters can increase BP which is mainly due to potentiation of the sympathetic vasoconstrictor tone but via different central mechanisms.

Differential control of renal vs. adrenal sympathetic nerve activity by NTS A2a and P2x purinoceptors.

Activation of adenosine A2a and ATP P2x purinoceptors in the subpostremal nucleus tractus solitarii (NTS) via microinjection of the selective agonists CGS-21680 and alpha,beta-methylene ATP (alpha, beta-MeATP), respectively, elicits large dose-dependent decreases in arterial pressure and heart rate, differential regional vasodilation, and differential inhibition of regional sympathetic outputs. With marked hypotensive hemorrhage, preganglionic adrenal sympathetic nerve activity (pre-ASNA) increases, whereas renal (RSNA) and postganglionic adrenal sympathetic nerve activity (post-ASNA) decrease. In this setting, adenosine levels in the brain stem increase. Therefore, we investigated whether stimulation of specific purinoceptors in the NTS may evoke differential sympathetic responses. RSNA was recorded simultaneously with pre-ASNA or post-ASNA in chloralose-urethan-anesthetized male Sprague-Dawley rats. CGS-21680 (2 and 20 pmol in 50 nl) inhibited RSNA and post-ASNA, whereas pre-ASNA increased markedly. alpha,beta-MeATP (25 and 100 pmol in 50 nl) inhibited all sympathetic outputs. Sinoaortic denervation plus vagotomy markedly prolonged the responses to P2x-purinoceptor stimulation. Glutamate (100 pmol in 50 nl) caused differential inhibition of all sympathetic outputs similar to that evoked by alpha,beta-MeATP. We conclude that NTS A2a-purinoceptor activation evokes differential sympathetic responses similar to those observed during hemorrhage, whereas P2x-purinoceptor and glutamate-receptor activation evokes differential inhibition of sympathetic outputs similar to arterial baroreflex responses.

Relationship of emotional behaviors induced by electrical stimulation of the hypothalamus to changes in EKG, heart, stomach, adrenal glands, and thymus.

The relationships of hypothalamically elicited emotional behaviors to their accompanying pathophysiological effects were examined as a model of how complex "emotional behaviors" may be related to fundamental psychosomatic disorders. Twenty-two unanesthetized adult cats were studied. EKG alterations and histological changes in the heart, stomach, adrenal glands, and thymus were related to the specific stereotypical emotional behaviors that could be elicited by hypothalamic stimulation in tamed subjects. Restlessness, threat, and searching-biting behaviors were evoked by electrical stimulation of the anteromedial, ventromedial, and lateral hypothalamus, respectively. The occurrence of cardiac arrhythmias, ST and/or T (ST-T) changes in the EKG, histological damage to myocardium, gastric erosion, and adrenal hyperplasia were generally observed in the restlessness and threat groups but not in the searching-biting group. The pathophysiological effects were similar in the restlessness and threat groups with no specific EKG change or organ effect attributable to either site of stimulation. Hypothalamically elicited restlessness or threat behaviors in cats are each associated with cardiac, gastric, and adrenal pathophysiologies.

Central command neurons of the sympathetic nervous system: basis of the fight-or-flight response.

During stress, the activity of the sympathetic nervous system is changed in a global fashion, leading to an increase in cardiovascular function and a release of adrenal catecholamines. This response is thought to be regulated by a common set of brain neurons that provide a dual input to the sympathetic preganglionic neurons regulating cardiac and adrenal medullary functions. By using a double-virus transneuronal labeling technique, the existence of such a set of central autonomic neurons in the hypothalamus and brainstem was demonstrated. These neurons innervate both of the sympathetic outflow systems and likely function in circumstances where parallel sympathetic processing occurs, such as in the fight-or-flight response.

Heart adrenoreactivity during acute haemic hypoxia.

The influence of acute haemic hypoxia of mild and moderate degree, produced by subcutaneous administration of sodium nitrite (25-30 or 50 mg/kg) supporting the blood haemoglobin production, on heart adrenoreactivity was studied in experimental rats. An intensified sympathoadrenal activity was detected, that manifested itself by a marked increase in adrenaline content in the hypothalamus, brain stem and heart. In analogous experiments performed in dogs, with increasing severity of hypoxia a decrease in arterial pressure and left ventricular pressure, increase in left ventricular end-diastolic pressure, and an impairment of myocardial contractile function were observed. Administration of adrenergic substances intensified the effector reaction in the heart. According to the authors' conclusions, experimental, hypoxia of the given model calls forth intensification of heart adrenoreactivity in warm-blood animals.

Adrenal sympathetic nerve activity in response to hypothalamic injections of 2-deoxy-D-glucose.

Adrenal sympathetic nerve activity after microinfusion of 2-deoxy-D-glucose (2-DG) into various hypothalamic nuclei was investigated in anesthetized rats. Infusion of 2-DG into the ventrolateral portion of the lateral hypothalamic area (LHA) induced a large and long-lasting increase (greater than 60 min) in adrenal nerve activity. In contrast, infusion into the dorsal or medial portion of the LHA tended to produce a small decrease with a return to baseline within approximately 60 min after the end of the infusion. The direct administration of 2-DG into either the paraventricular nucleus or the dorsomedial hypothalamic nucleus produced a strong inhibition of adrenal nerve activity. Infusions into the ventromedial hypothalamic nucleus induced either a decrease of adrenal nerve activity or were without effect. These findings provide evidence that induction of glucoprivation in the hypothalamus with 2-DG can excite or inhibit adrenal nerve activity, depending on the hypothalamic region. These data also indicate that the ventrolateral portion of the LHA plays an important role in the regulation of adrenal catecholamine secretion in response to glucoprivic conditions in the central nervous system.

Central and peripheral control of sympathoadrenal activity and energy metabolism in rats.

The role of adrenoceptors in the hypothalamus and the peripheral sympathetic nervous system in the regulation of sympathoadrenal activity and glucose and FFA mobilization was investigated in exercising rats. Apparent close relations within the two parts of the sympathoadrenal system and between factors that regulate glucose and FFA mobilization during exercise were completely disrupted by local hypothalamic infusions of adrenoceptor antagonists or anesthetic drugs. The experiments actually identified specific areas in the hypothalamus that integrate the information regarding the substrate levels in the blood with the "central command" from higher centers in the brain. Furthermore, the results of experiments with exercising intact and adrenodemedullated (Adm) rats, with and without administration of selective adrenoceptor agonists and antagonists, suggest that the activity of the sympathetic nervous system is also regulated at the level of the peripheral sympathetic nerve endings. In particular, presynaptic adrenergic regulatory mechanisms can markedly influence the outflow of NE from the sympathetic nerve endings. In conclusion, the data show that an organ-specific organization of sympathetic output during exercise may take place at different levels within the sympathetic nervous system.

Effects of hypothalamic stimulation and lesion on adrenal nerve activity.

Activity changes of efferent adrenal sympathetic nerves in response to bilateral manipulations of the hypothalamus, partly after intra-third cerebroventricular injection of 2-deoxy-D-glucose (2-DG) were investigated in anesthetized rats. Stimulation of the middle part of the lateral hypothalamic area (LHAm) increased adrenal nerve activity, whereas lesion caused rapid and remarkable decrease. Stimulation of the anterior part of the LHA (LHAa) tended to decrease the activity, and lesion produced either rapid decrease or late moderate increase. Stimulation of the ventromedial hypothalamic nucleus (VMH) did not affect the nerve activity, but lesion increased it gradually and then remarkably. Cerebroventricular infusion of 2-DG caused remarkable increase in activity that was suppressed by LHAm lesion. Subsequent infusion of 2-DG during the period of suppressed activity was no longer effective. The increased firing rate after 2-DG was suppressed by stimulation of the VMH, whereas lesion caused no change. These findings indicate that the central regulation of adrenal nerve activity is connected with individual hypothalamic regions and consequently depends on the degree and mode of activation of the sympathoadrenal system.

Neural control of blood glucose level.

All of the experimental results described above can be categorized as follows: the relationship between glucose levels and pancreatic and adrenal nerve activities; innervations of the liver and their role in the regulation of blood glucose level; central integration of blood glucose level; glucose-sensitive afferent nerve fibers in the liver and regulation of blood glucose; oral and intestinal inputs involved in reflex control of blood glucose level. We showed that an increase in blood glucose content produced an increase in the activity of the pancreatic branch of the vagus nerve, whereas it induced a decrease in the activity of the adrenal nerve. It was also shown that a decrease in blood glucose activated the sympatho-adrenal system and suppressed the vago-pancreatic system. It seems rational that these responses are involved in the maintenance of blood glucose level. Studies on the innervation of the liver led us to a conclusion that sympathetic innervation of the liver might play a role in eliciting a prompt hyperglycemic response through liberation of norepinephrine from the nerve terminals, and that the vagal innervation synergically worked with the humoral factor (insulin) for glycogen synthesis in the hyperglycemic condition. The glucose-sensitive afferents from the liver seem to initiate a reflex control of blood glucose level. The gustatory information on EIR response, reported by STEFFENS, is supported by the electrophysiological observations. MEI's reports also indicated the importance of information from the intestinal glucoreceptors in the reflex control of insulin secretion. The role of integrative functions of the hypothalamus and brainstem through neuronal networks on neural control of blood glucose levels is also evident. A schematic diagram of the nervous networks involved in the regulation of the blood glucose levels is shown in Fig. 3.

Sympathetic nervous system response to mechanical stress of the spinal column in rats.

The effects of mechanical stimulation of the spine on blood pressure, heart rate and the activity of selected sympathetic nerves (renal and adrenal) were examined in alpha-chloralose/urethane anesthetized rats. Spinal segments from T10 to T13 or from L4 to L7 were isolated from surrounding muscle and the upper and lower segments of the four segment units were fixed by means of spinal clamps. Forces from 0.5 to 3.0 kg were applied to the lateral aspect of the two mobile segments. Stimulations of the thoracic or the lumbar region produced large decreases in blood pressure (-29.8 +/- 3.1 mmHg) along with small decreases in heart rate (6.1 +/- 1.6 beats/min). Additionally, large and immediate decreases were observed in renal nerve activity. While responses attenuated during the course of stimulation, they generally outlasted the stimulus. The adrenal nerve, after an initial decline in activity, showed subsequent increases which were attributed to baroreceptor effects, since bilateral lesion of the vagi and carotid sinus nerves abolished them. After baroreceptor denervations only initial decreases in activity were observed by mechanical stimulation of the spine. The observed responses were found not to be due to spinal cord compression, but rather were ascribed to afferent fiber mediated reflexes. Cutting dorsal (sensory) roots T10 to L2, bilaterally, abolished the response to lower lumbar stimulation. Additionally, in recording from the peripheral end of the severed dorsal roots, large increases in afferent activity were observed by spine stimulation. The present study has demonstrated potent somatovisceral reflexes from mechanical stimulation of the spinal column. These results are discussed in relation to other studies of somatovisceral responses and with regard to various clinical reports.

The rise of plasma ACTH induced by ether is mediated through neural pathways entering the medial basal hypothalamus.

The effect of ether stress on the release of immunoreactive ACTH was studied in rats with an antero-lateral cut around the medial basal hypothalamus. Ether failed to raise the plasma ACTH level of rats in which an antero-lateral hypothalamic cut and adrenalectomy had been performed 7 to 8 days previously. Plasma ACTH was also unchanged in rats exposed to ether 2 h after an antero-lateral cut. These data suggest that intact neural pathways entering the medial basal hypothalamus from the antero-lateral direction are necessary for the ACTH releasing action of ether stress.

The effect of hypothalamic hemi-islands on compensatory adrenal growth.

Unilateral hypothalamic disconnections were made with a Halász knife in young male rats to determine the effects of these lesions on adrenal weight, and the response to unilateral adrenalectomy. Rats were unilaterally adrenalectomized or sham-adrenalectomized 5-7 days after the hypothalamic surgery. No lesion affected adrenal weight in sham-adrenalectomized rats. Compensatory adrenal growth 3 days after unilateral adrenalectomy is prevented by a unilateral hypothalamic hemi-island on the side ipsilateral, not contralateral, to the first removed adrenal (P less than 0.01). Portions of the ipsilateral hemi-island were examined to determine which interrupted the compensatory adrenal growth response. The anterior 90 degrees portion enhanced compensatory adrenal growth (P less than 0.01). The posterior 90 degrees portion only partially inhibited compensatory adrenal growth (P less than 0.05), while the posterolateral portion completely inhibited the response (P less than 0.01). Plasma ACTH and corticosterone levels at the time of sacrifice were unaffected by unilateral adrenalectomy, but were slightly elevated by the hypothalamic cuts. These studies provide further evidence that compensatory adrenal growth is mediated neurally.