Willian Seiji Korim

Neural pathways that control the glucose counterregulatory response

Glucose is an essential metabolic substrate for all bodily tissues. The brain depends particularly on a constant supply of glucose to satisfy its energy demands. Fortunately, a complex physiological system has evolved to keep blood glucose at a constant level. The consequences of poor glucose homeostasis are well-known: hyperglycemia associated with uncontrolled diabetes can lead to cardiovascular disease, neuropathy and nephropathy, while hypoglycemia can lead to convulsions, loss of consciousness, coma, and even death. The glucose counterregulatory response involves detection of declining plasma glucose levels and secretion of several hormones including glucagon, adrenaline, cortisol, and growth hormone (GH) to orchestrate the recovery from hypoglycemia. Low blood glucose leads to a low brain glucose level that is detected by glucose-sensing neurons located in several brain regions such as the ventromedial hypothalamus, the perifornical region of the lateral hypothalamus, the arcuate nucleus (ARC), and in several hindbrain regions. This review will describe the importance of the glucose counterregulatory system and what is known of the neurocircuitry that underpins it.

Renal sympathoinhibition induced by hypernatremia: involvement of A1 noradrenergic neurons.

Several findings suggest that A1 noradrenergic neurons in the caudal ventrolateral medulla (CVLM) contribute to body fluid homeostasis and cardiovascular regulation. Recently we demonstrated that the renal vasodilation induced by infusion of hypertonic saline (HS) depends on the integrity of the A1 neurons. Here we determined the effect of lesions of these neurons on the inhibition of the renal sympathetic nerve activity (RSNA) induced by HS infusion. All experiments were performed in Wistar rats (280-350 g). A1 neurons were lesioned by microinjections of antidopamine-beta-hydroxylase-saporin (6.3 ng in 60 nl) into the CVLM (n=5), whereas sham rats received microinjections of free saporin (1.3 ng in 60 nl, n=10). Two weeks later, rats were anesthetized (urethane 1.2 g/kg, iv), and instrumented for recording of arterial pressure and RSNA. In sham rats, HS infusion (3 M NaCl, 0.18 ml/100 g bw, iv) induced a transient (</=30 min) hypertension (peak at 10 min; 9+/-5 mm Hg) and a fall in RSNA (-32+/-7% of baseline at 10 min). A1-lesions increased the duration of the pressor response induced by HS infusion (16+/-2 mm Hg at 60 min) and abolished the fall in RSNA (-6+/-8% of baseline at 10 min). Catecholaminergic lesions extensions were confirmed by immunocytochemistry. Unilateral renal denervation reduced the renal vasodilatation induced by HS infusion (112+/-7% in denervated rats versus 127+/-4% in sham, 20 min after HS). These results suggest that A1 noradrenergic neurons are involved in the sympathoinhibition and consequent renal vasodilatation to acute changes in the extracellular fluid compartment.

Patterning of somatosympathetic reflexes reveals nonuniform organization of presympathetic drive from C1 and non-C1 RVLM neurons

To determine the organization of presympathetic vasomotor drive by phenotypic populations of rostral ventrolateral medulla (RVLM) neurons, we examined the somatosympathetic reflex (SSR) evoked in four sympathetic nerves together with selective lesions of RVLM presympathetic neurons. Urethane-anesthetized (1.3 g/kg ip), paralyzed, vagotomized and artificially ventilated Sprague-Dawley rats (n = 41) were used. First, we determined the afferent inputs activated by sciatic nerve (SN) stimulation at graded stimulus intensities (50 sweeps at 0.5-1 Hz, 1-80 V). Second, we recorded sympathetic nerve responses (cervical, renal, splanchnic, and lumbar) to intensities of SN stimulation that activated A-fiber afferents (low) or both A- and C-fiber afferents (high). Third, with low-intensity SN stimulation, we examined the cervical SSR following RVLM microinjection of somatostatin, and we determined the splanchnic SSR in rats in which presympathetic C1 neurons were lesioned following intraspinal injections of anti-dopamine-β-hydroxylase-saporin (anti-DβH-SAP). Low-intensity SN stimulation activated A-fiber afferents and evoked biphasic responses in the renal, splanchnic, and lumbar nerves and a single peak in the cervical nerve. Depletion of presympathetic C1 neurons (59 ± 4% tyrosine hydroxylase immunoreactivity profiles lesioned) eliminated peak 2 of the splanchnic SSR and attenuated peak 1, suggesting that only RVLM neurons with fast axonal conduction were spared. RVLM injections of somatostatin abolished the single early peak of cervical SSR confirming that RVLM neurons with fast axonal conduction were inhibited by somatostatin. It is concluded that unmyelinated RVLM presympathetic neurons, presumed to be all C1, innervate splanchnic, renal, and lumbar but not cervical sympathetic outflows, whereas myelinated C1 and non-C1 RVLM neurons innervate all sympathetic outflows examined. These findings suggest that multiple levels of neural control of vasomotor tone exist; myelinated populations may set baseline tone, while unmyelinated neurons may be recruited to provide actions at specific vascular beds in response to distinct stressors.

Lesions of medullary catecholaminergic neurons increase salt intake in rats

Several findings suggest that catecholaminergic neurons in the caudal ventrolateral medulla (CVLM) contribute to body fluid homeostasis and cardiovascular regulation. From the CVLM other areas in central nervous system involved in cardiovascular regulation and hydroelectrolyte balance can be activated. Therefore, the aim of the present study was to investigate the effects of lesions of these neurons on 0.3M NaCl and water intake induced by subcutaneous injection of furosemide (FURO)+captopril (CAP) or 36 h of water deprivation/partial hydration with only water (WD/PR). Male Wistar rats (320-360 g) were submitted to medullary catecholaminergic neuron lesions by microinjection of anti-dopamine-beta-hydroxylase-saporin (anti-DbetaH-saporin; 6.3 ng in 60 nl) into the CVLM (SAP-rats). Sham rats received microinjections of free saporin (1.3 ng in 60 nl) in the same region. In SAP-rats, the 0.3M NaCl intake was increased after FURO+CAP (6.8+/-1.0 ml/2h, vs. sham: 3.7+/-0.7 ml/2h) as well as after WD/PR (11.1+/-1.3 ml/2h vs. sham: 6.1+/-1.8 ml/2h). Conversely, in SAP-rats, the water intake induced by FURO+CAP (14.8+/-1.3 ml/2h, vs. sham: 14.1+/-1.6 ml/2h) or by WD/PR (3.6+/-0.9 ml/2h, vs. sham: 3.2+/-1.1 ml/2h) was not different from sham rats. Immunohistochemical analysis indicates that microinjections of anti-DbetaH-saporin produced extensive destruction within the A1 cell groups in the CVLM. These results suggest an inhibitory role for medullary catecholaminergic neurons on sodium appetite.

Orexinergic Activation of Medullary Premotor Neurons Modulates the Adrenal Sympathoexcitation to Hypothalamic Glucoprivation

Glucoprivation activates neurons in the perifornical hypothalamus (PeH) and in the rostral ventrolateral medulla (RVLM), which results in the release of adrenaline. The current study aimed to establish 1) whether neuroglucoprivation in the PeH or in the RVLM elicits adrenaline release in vivo and 2) whether direct activation by glucoprivation or orexin release in the RVLM modulates the adrenaline release. Neuroglucoprivation in the PeH or RVLM was elicited by microinjections of 2-deoxy-d-glucose or 5-thio-d-glucose in anesthetized, euglycemic rats. Firstly, inhibition of neurons in the PeH abolished the increase in adrenal sympathetic nerve activity (ASNA) to systemic glucoprivation. Secondly, glucoprivation of neurons in the PeH increased ASNA. Thirdly, in vivo or in vitro glucoprivation did not affect the activity of RVLM adrenal premotor neurons. Finally, blockade of orexin receptors in the RVLM abolished the increase in ASNA to neuroglucoprivation in the PeH. The evoked changes in ASNA were directly correlated to levels of plasma metanephrine but not to normetanephrine. These findings suggest that orexin release modulates the activation of adrenal presympathetic neurons in the RVLM.

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.

Asymmetrical changes in lumbar sympathetic nerve activity following stimulation of the sciatic nerve in rat.

Noxious stimulation of the leg increases hind limb blood flow (HBF) to the ipsilateral side and decreases to the contralateral in rat. Whether or not this asymmetrical response is due to direct control by sympathetic terminals or mediated by other factors such as local metabolism and hormones remains unclear. The aim of this study was to compare responses in lumbar sympathetic nerve activity, evoked by stimulation of the ipsilateral and contralateral sciatic nerve (SN). We also sought to determine the supraspinal mechanisms involved in the observed responses. In anesthetized and paralyzed rats, intermittent electrical stimulation (1 mA, 0.5 Hz) of the contralateral SN evoked a biphasic sympathoexcitation. Following ipsilateral SN stimulation, the response is preceded by an inhibitory potential with a latency of 50 ms (N=26). Both excitatory and inhibitory potentials are abolished following cervical C1 spinal transection (N=6) or bilateral microinjections of muscimol (N=6) in the rostral ventrolateral medulla (RVLM). This evidence is suggestive that both sympathetic potentials are supraspinally mediated in this nucleus. Blockade of RVLM glutamate receptors by microinjection of kynurenic acid (N=4) selectively abolished the excitatory potential elicited by ipsilateral SN stimulation. This study supports the physiological model that activation of hind limb nociceptors evokes a generalized sympathoexcitation, with the exception of the ipsilateral side where there is a withdrawal of sympathetic tone resulting in an increase in HBF.

Noxious somatic stimuli diminish respiratory-sympathetic coupling by selective resetting of the respiratory rhythm in anaesthetized rats

Noxious somatic stimulation evokes respiratory and autonomic responses. The mechanisms underlying the responses and the manner in which they are co‐ordinated are still unclear. The effects of activation of somatic nociceptive fibres on lumbar sympathetic nerve activity at slow (2–10 Hz) and fast frequency bands (100–1000 Hz) and the effects on respiratory–sympathetic coupling are unknown. In anaesthetized, artificially ventilated Sprague–Dawley rats under neuromuscular blockade, ensemble averaging of sympathetic activity following high‐intensity single‐pulse stimulation of the sciatic nerve revealed two peaks (∼140 and ∼250 ms) that were present at similar latencies whether or not slow or fast band filtering was used. Additionally, in the slow band of both lumbar and splanchnic sympathetic nerve activity, a third peak with a very slow latency (∼650 ms) was apparent. In the respiratory system, activation of the sciatic nerve decreased the expiratory period when the stimulus occurred during the first half of expiration, but increased the expiratory period if the stimulus was delivered in the second half of the expiratory phase. The phase shifting of the respiratory cycle also impaired the respiratory–sympathetic coupling in both splanchnic and lumbar sympathetic nerve activity in the subsequent respiratory cycle. The findings suggest that noxious somatosympathetic responses reduce the co‐ordination between respiration and perfusion by resetting the respiratory pattern generator.

In renovascular hypertension, TNF-α type-1 receptors in the area postrema mediate increases in cardiac and renal sympathetic nerve activity and blood pressure

AIMS Neuroinflammation is a common feature in renovascular, obesity-related, and angiotensin II mediated hypertension. There is evidence that increased release of the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α) contributes to the development of the hypertension, but the underlying neural mechanisms are unclear. Here, we investigated whether TNF-α stimulates neurons in the area postrema (AP), a circumventricular organ, to elicit sympathetic excitation, and increases in blood pressure (BP). METHODS AND RESULTS In rats with renovascular hypertension, AP neurons that expressed TNF-α type-1 receptor (TNFR1) remained constantly activated (expressed c-Fos) and injection of TNFR1 neutralizing antibody into the AP returned BP (systolic: ∼151 mmHg) to normotensive levels (systolic: ∼108 mmHg). Nanoinjection of TNF-α (100 pg/50 nL) into the AP of anaesthetized normotensive rats increased BP (∼16 mmHg) and sympathetic nerve activity, predominantly to the heart (∼53%), but also to the kidneys (∼35%). These responses were abolished by prior injection of a TNFR1 neutralizing antibody (1 ng/50 nL) within the same site. TNFR1 were expressed in the somata of neurons activated by TNF-α that were retrogradely labelled from the rostral ventrolateral medulla (RVLM). CONCLUSION These findings indicate that in renovascular hypertension, blocking TNFR1 receptors in the AP significantly reduces BP, while activation of TNFR1 expressing neurons in the AP by TNF-α increases BP in normotensive rats. This is mediated, in part, by projections to the RVLM and an increase in both cardiac and renal sympathetic nerve activity. These findings support the notion that proinflammatory cytokines and neuroinflammation are important pathological mechanisms in the development and maintenance of hypertension.