Understanding the neurobiology of sympathetic responses to changes in osmolality has important implications for body fluid and cardiovascular physiology. By stabilizing osmolality, vascular volume is preserved and thereby relatively normal levels of cardiac output and arterial pressure are maintained.

Hyperinsulinemia increases sympathetic nerve activity and contributes to cardiovascular dysfunction in obesity and diabetes. Neurons of the hypothalamic paraventricular nucleus regulate sympathetic nerve activity through mono- and poly-synaptic connections to preganglionic neurons in the spinal cord. The purpose of the present study was to determine whether hypothalamic paraventricular nucleus neurons mediate the sympathetic response to insulin. Hyperinsulinemic-euglycemic clamps were performed in α-chloralose-anesthetized, male Sprague-Dawley rats (280–420 g) by an infusion of insulin (3.75 mU/kg/min) and 50% dextrose (0.75–2.0 ml/h) for 120 min. At 90 min, insulin significantly increased lumbar sympathetic nerve activity without any change in renal sympathetic nerve activity, heart rate, or blood glucose levels. Inhibition of the hypothalamic paraventricular nucleus with bilateral injection of the GABAA receptor agonist muscimol completely reversed the sympathoexcitatory response. However, direct injection of insulin into the hypothalamic paraventricular nucleus did not alter lumbar sympathetic nerve activity and thereby suggests insulin activates neurons upstream of the hypothalamic paraventricular nucleus. Interestingly, the sympathetic response to insulin was eliminated by hypothalamic paraventricular nucleus injection of the melancortin 3/4 receptor antagonist SHU9119 but unaffected by the angiotensin II type 1 receptor antagonist losartan. A final set of experiments suggests activation of hypothalamic paraventricular nucleus neurons during hyperinsulinemia increases glutamatergic drive to the rostral ventrolateral medulla. Collectively, these findings indicate insulin activates a melanocortin-dependent pathway to the hypothalamic paraventricular nucleus that increases glutamatergic drive to the rostral ventrolateral medulla and alter cardiovascular function.

The present study sought to determine whether water deprivation increases Fos immunoreactivity, a neuronal marker related to synaptic activation, in sympathetic-regulatory neurons of the hypothalamic paraventricular nucleus (PVN). Fluorogold (4%, 50 nl) and cholera toxin subunit B (0.25%, 20-30 nl) were microinjected into the spinal cord (T1-T3) and rostral ventrolateral medulla (RVLM), respectively. Rats were then deprived of water but not food for 48 h. Water deprivation significantly increased the number of Fos-positive nuclei throughout the dorsal, ventrolateral, and lateral parvocellular divisions of the PVN (water deprived, 215 +/- 23 cells; control, 45 +/- 7 cells, P < 0.01). Moreover, a significantly greater number of Fos-positive nuclei were localized in spinally projecting (11 +/- 3 vs. 2 +/- 1 cells, P < 0.025) and RVLM-projecting (45 +/- 7 vs. 7 +/- 1 cells, P < 0.025) neurons of the PVN in water-deprived vs. control rats, respectively. The majority of these double-labeled neurons was found in the ventrolateral and lateral parvocellular divisions of the ipsilateral PVN. Interestingly, a significantly greater percentage of RVLM-projecting PVN neurons were Fos positive compared with spinally projecting PVN neurons in the ventrolateral (25.8 +/- 0.7 vs. 8.0 +/- 1.5%, respectively, P < 0.01) and lateral (23.4 +/- 2.1 vs. 5.0 +/- 0.9%, respectively, P > 0.01) parvocellular divisions. In addition, we analyzed spinally projecting neurons of the RVLM and found a significantly greater percentage were Fos positive in water-deprived rats than in control rats (26 +/- 3 vs. 3 +/- 1%, respectively; P < 0.001). Collectively, the present findings indicate that water deprivation evokes a distinct cellular response in sympathetic-regulatory neurons of the PVN and RVLM.

Elevated sympathetic outflow contributes to the maintenance of blood pressure in water-deprived rats. The neural circuitry underlying this response may involve activation of a pathway from the hypothalamic paraventricular nucleus (PVH) to the rostral ventrolateral medulla (RVLM). We sought to determine whether the PVH-RVLM projection activated by water deprivation is glutamatergic and/or contains vasopressin-or oxytocin-neurophysins. Vesicular glutamate transporter2 (VGLUT2) mRNA was detected by in situ hybridization in the majority of PVH neurons retrogradely labeled from the ipsilateral RVLM with cholera-toxin subunit B (CTB; 85% on average with regional differences). Very few RVLM-projecting PVH neurons were immunoreactive for oxytocin-or vasopressin-associated neurophysin. Injection of biotinylated dextran amine (BDA) into the PVH produced clusters of BDA-positive nerve terminals within the ipsilateral RVLM that were immunoreactive (ir) for the VGLUT2 protein. Some of these terminals made close appositions with tyrosine-hydroxylase-ir dendrites (presumptive C1 cells). In waterdeprived rats (n=4), numerous VGLUT2 mRNA-positive PVH neurons retrogradely labeled from the ipsilateral RVLM with CTB were c-Fos-ir (16-40% depending on PVH region). In marked contrast, few glutamatergic, RVLM-projecting PVH neurons were c-Fos-ir in control rats (n=3; 0-3% depending on PVH region). Most (94 ± 4%) RVLM-projecting PVH neurons activated by water deprivation contained VGLUT2 mRNA. In summary, the majority of PVH neurons that innervate the RVLM are glutamatergic and this population includes the neurons that are activated by water deprivation. One mechanism by which water deprivation may increase the sympathetic outflow is the activation of a glutamatergic pathway from the PVH to the RVLM.