Neuronal expression of Fos protein in the hypothalamus of rats with heart failure.

We sought to identify the areas that have altered neuronal activity within the hypothalamus of rats with heart failure (HF) by mapping neuronal staining of c-Fos protein (Fos) 6-8 weeks following coronary artery ligation (HF group; n=17) or sham surgery (sham-operated control group, n=15). Fos-like immunoreactivity was observed in the paraventricular nucleus (PVN), supraoptic nucleus (SON), median preoptic nucleus (MnPO), anterior hypothalamus (AH) and posterior hypothalamus (PH) using a standard ABC immunocytochemical protocol. The rats in the HF group displayed infarcts averaging 34+/-2% of the outer circumference and 41+/-1% of the inner circumference of the left ventricular wall. Sham-operated control rats had no observable damage to the myocardium. Rats with chronic heart failure (n=5) but no manipulation (no surgery) had a similar number of Fos-staining cells in PVN SON, MnPO, AH and PH compared to sham-operated rats. Acute surgery for isolation of vagus nerves and anesthesia for 90 min increased the number of Fos positive cells in PVN, SON and MnPO of both sham-operated rats and rats with HF. Furthermore, rats with heart failure (n=5) had significantly higher number of Fos-staining cells in PVN (four times), SON (4.5 times) and MnPO (1.5 times) compared to sham-operated rats after acute surgery for isolation of the vagus. The number of Fos-staining cells remained unaltered in AH and PH in both groups of rats. However, in a third series of experiments vagotomy reduced the number of Fos-staining cells in the PVN, SON or MnPO of rats with HF (n=5) to those observed in sham-operated vagotomized rats. This study shows that: (1) there is augmented neuronal activity as indicated by increased number of Fos staining neurons in the PVN, SON and MnPO due to acute surgical stress in rats with HF, and (2) vagal afferents are responsible for the increased neuronal activity in PVN, SON and MnPO of rats with HF during acute surgical stress. These data support the conclusion that vasopressin producing neurons and autonomic areas within the hypothalamus influenced by vagal afferents are activated during HF and are sensitive to 'acute surgical stress' and may contribute to the elevated levels of vasopressin and sympatho-excitation commonly observed in heart failure.

Activation of renal afferent pathways following furosemide treatment. I. Effects Of survival time and renal denervation.

Three experiments were performed to determine whether renal afferent pathways were activated by the diuretic drug, furosemide. It was hypothesized that activated neurons of the renal afferent pathway would express the protein product Fos of the c-fos immediate early gene and be identified by immunocytochemical staining for Fos in the cell nucleus. In the first two experiments, rats were injected with either furosemide (5 mg) or vehicle solution (sterile isotonic saline) and sacrificed either 1.75 h (short-survival experiment) or 3.5 h (long-survival experiment) after injection. In both experiments, the furosemide-treated rats had significantly more Fos-positive cell nuclei than vehicle-treated rats in the subfornical organ (SFO), organum vasculosum lamina terminalis (OVLT), supraoptic nuclei (SON), and magnocellular region of the paraventricular nuclei (PVN) - areas previously shown to be activated by hypovolemia or peripheral angiotensin. In the short-survival experiment, the furosemide-treated rats had more Fos-positive cell nuclei in the nucleus of the solitary tract (NTS) and in the dorsal horn of the spinal cord at spinal levels T(11), T(12), and T(13). In contrast, furosemide treatment did not produce more Fos-positive cell nuclei in the NTS and dorsal horn of the spinal cord in the long-survival experiment. These results suggest that the activation of the SFO, OVLT, SON and PVN may be via a different mechanism than that of NTS or spinal cord dorsal horn. Based upon our previous work, we hypothesized that the NTS and spinal cord dorsal horn labeling was due to activation of sympathetic afferents originating in the kidney and labeling in forebrain structures was due to stimulation by angiotensin generated by renal renin release. To test this hypothesis, a third experiment was devised that was identical to the short-survival experiment, except that all rats had bilateral renal denervation surgery 1 week previously. In this experiment, furosemide administration increased the number of Fos-positive cells in the SFO, OVLT, SON and PVN, but not in the caudal thoracic spinal cord or NTS. These results together with the results of first two experiments lend support to our hypothesis that furosemide-induced neuronal activation in the thoracic spinal cord and NTS is due to activation of second- and/or third-order neurons of a renal sympathetic afferent pathway. Furosemide-induced activation in the SFO, OVLT, SON and PVN does not depend on renal innervation. It is hypothesized that activation in these forebrain regions depends on the action of angiotensin II that is generated after furosemide treatment. Our results indicate that both a hormonal pathway and a renal sympathetic afferent pathway conduct information from the kidney to the central nervous system (CNS) after furosemide treatment.

Altered number of diaphorase (NOS) positive neurons in the hypothalamus of rats with heart failure.

Recently, we have demonstrated a decreased neuronal isoform of nitric oxide synthase (nNOS) message in the hypothalamus of rats with heart failure (HF). The purpose of this study was to determine the changes in NADPH-diaphorase (a commonly used marker for neuronal NOS activity) positive neurons in specific hypothalamic sites of rats with HF. After a standard histochemical protocol, NOS positive neurons were measured in paraventricular nucleus (PVN), supraoptic nucleus (SON), median preoptic area (MePO), subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT) and lateral hypothalamus (LH) of rats with coronary artery ligation (HF group; n=8) and sham-operated control rats (n=9). A total of 4 months after coronary ligation, the rats in the HF group displayed infarcts greater than at least 35% of the left ventricular wall (n = 8). Sham-operated rats had no observable damage to the myocardium. Rats with HF had a significantly lower number of NOS positive cells in the PVN (36% less) compared to sham rats. The number of NOS positive cells remained unaltered in the SON, MePO and LH in rats with HF. Conversely there was an increased number of NOS positive cells in the SFO (42% greater) and OVLT (100% greater). These data support the conclusion that the NO system within the hypothalamus involved in controlling autonomic outflow is altered during HF and may contribute to the elevated levels of vasopressin and sympatho-excitation commonly observed in HF.

Decreased gene expression of neuronal nitric oxide synthase in hypothalamus and brainstem of rats in heart failure.

Nitric oxide may act at autonomic sites in the brain to regulate sympathetic outflow. Our goal was to determine whether gene expression of the neuronal isoform of nitric oxide synthase (nNOS) is altered in discrete autonomic brain regions of rats in the chronic phase of heart failure compared to sham-operated control rats. Experiments were performed in rats 4 to 5 weeks after left coronary artery ligation. Histological data indicated that there was a 39% outer and a 45% inner infarct of the left ventricular myocardium in the heart failure group. The myocardium in sham-operated rats showed no observable damage. Total RNA was purified from microdissected brain tissue blocks containing hypothalamus, dorsal pons, dorsal medulla, rostral ventrolateral medulla, and caudal ventrolateral medulla. Changes in nNOS mRNA were semiquantified in each region using reverse transcription-polymerase chain reactions in which known concentrations of deletion mutant of the gene were coamplified as an internal standard. Compared with controls, significant decreases in nNOS mRNA levels were found in hypothalamus (19%), dorsal pons (43%) and dorsal medulla (34%) of rats with heart failure. There were no statistically significant differences in nNOS mRNA levels in rostral or caudal ventrolateral medulla between the control and heart failure groups. Concomitant with the changes nNOS gene expression in central sites, the plasma concentration of norepinephrine was significantly elevated in rats with heart failure compared to sham-operated control rats. Our results show that heart failure is associated with decreases in nNOS gene expression in at least three regions of the brain and with increased sympathetic outflow to the periphery. The decreased NO production that is likely associated with the decreases in nNOS gene expression may lead to the increased sympathetic drive seen in chronic heart failure.

Increased norepinephrine turnover in the median preoptic nucleus following reduced extracellular fluid volume.

The role of noradrenergic input to fluid balance regulatory systems in the anterior hypothalamus was studied by examination of norepinephrine (NE) turnover during reduction of systemic extracellular fluid volume. Extracellular fluid volume was decreased iso-osmotically by subcutaneous polyethylene glycol (PEG), known to increase thirst and vasopressin secretion. NE turnover was assessed by measuring the decline of NE concentration in brain micropunches after administration of alpha-methyl tyrosine in PEG- or sham-treated groups. Several hypothalamic areas were investigated, including the median preoptic area (MnPO), preoptic area (POA), paraventricular nucleus, supraoptic nucleus (SON), subfornical organ, ventromedial hypothalamus, and posterior hypothalamus. Volume-depleted animals showed significantly increased NE turnover in the MnPO, an important area for integration of fluid balance information. The POA and the SON also showed trends toward increased NE turnover. All other areas showed no difference in NE turnover between volume-depleted and normal animals. These results are consistent with previous findings that NE innervation to the MnPO is important in the control of fluid balance and also support the hypothesis that basal forebrain NE projections facilitate thirst and vasopressin secretion.

Facilitation of baroreflex-induced bradycardia by stimulation of specific hypothalamic sites in the rat.

Hypothalamic stimulation generally inhibits baroreflex-induced bradycardia. However, we have noted discrete areas of the rat hypothalamus which facilitate reflex bradycardia. The effects of hypothalamic stimulation on baroreflex-induced changes in heart rate were investigated in urethane-anesthetized rats (1.2 g/kg, i.p.; n = 6) instrumented with femoral arterial and venous catheters. Bipolar electrodes (250 micron diameter) were implanted stereotaxically in the hypothalamus. Baroreflex-induced bradycardia was elicited by phenylephrine (PE) injection (8-20 micrograms/kg). Responses to stimulation (STIM) (50-150 microA, 80 Hz, 0.5 ms), PE, and Stim + PE were studied for 1 min. In the ventral medial and anterior hypothalamus, STIM caused transient increases in blood pressure and no changes in heart rate. Peak blood pressure was lower during STIM + PE than during PE (144 +/- 5 vs 164 +/- 3 mm Hg; P less than 0.05). However, STIM + PE resulted in a lower heart rate compared to PE (194 +/- 22 22 vs 270 +/- 17 bpm; P less than 0.05). At 1 min, the heart rate in STIM + PE rats remained lower than in PE rats (205 +/- 37 vs 319 +/- 16 bpm; P less than 0.05). Atropine administration indicated that the facilitation was primarily parasympathetic in nature. These results identify specific hypothalamic regions which facilitate baroreflex-induced bradycardia by parasympathetic mechanisms.

Influence of renal nerves on noradrenergic responses to changes in arterial pressure.

Renal denervation has been shown previously to lower the increased arterial pressure as well as the increased hypothalamic and peripheral noradrenergic activity found in neurogenic and Goldblatt models of experimental hypertension. In the present study conscious Wistar rats with or without renal nerves were subjected to 60 min of saline infusion (controls), hypotension (intravenous sodium nitroprusside), or hypertension (intravenous phenylephrine HCl). Changes in the turnover of norepinephrine (NE) in the anterior hypothalamus, posterior hypothalamus, kidney, intestine, and skeletal muscle were assessed by measuring the decline of NE concentration 90 min after administration of alpha-methyl tyrosine. There was a significant increase in NE turnover in the posterior hypothalamus and all peripheral organs examined in the nitroprusside-infused group with intact renal nerves. In renal-denervated animals, acute hypotension produced similar changes in NE turnover in peripheral organs, but no significant change was observed in the posterior hypothalamus. In the acutely hypertensive group with intact renal nerves, there was no significant change in NE turnover in the hypothalamic sections or the peripheral organs; however, the turnover of NE was significantly decreased in both the anterior and posterior hypothalamus of the renal-denervated hypertensive group. Overall these studies suggest the presence of an interaction between inhibitory influences from baroreceptor afferents and excitatory influences from renal afferents on noradrenergic activity in the hypothalamus and changes in noradrenergic activity in hypothalamic structures may not be directly related to changes in sympathetic outflow.

Effect of renal denervation on arterial pressure in rats with aortic nerve transection.

The role of renal nerves in influencing the control of arterial pressure was studied in Wistar rats with aortic depressor nerve (ADN) transection. Renal denervation prevented or reversed the normal increase in arterial pressure seen after ADN transection. This effect was not due to an effect on the renin-angiotensin system, as the elevated arterial pressure after ADN section in rats with renal nerves intact was shown to be due to increased alpha-adrenergic activity. Food and water intake and urine output decreased significantly in both renal-denervated and sham-denervated rats after ADN section, suggesting that a pressure diuresis mechanism was not responsible for preventing the rise in pressure in renal-denervated rats. In another study, the concentration of norepinephrine in skeletal muscle and hypothalamus at 0 and 8 hours after inhibition of tyrosine hydroxylase with alpha-methyltyrosine was used as an index of norepinephrine turnover. Norepinephrine turnover in skeletal muscle was increased significantly over control values by ADN transection in sham renal-denervated rats, but was not significantly different from controls in renal-denervated rats with ADN section. In the hypothalamus, there was a significant difference between the turnover of norepinephrine in the two groups of ADN-sectioned rats. The results taken together suggest that renal denervation prevents the arterial pressure response to ADN transection by altering the central mechanisms governing sympathetic outflow. It is suggested that this effect may be due to elimination of information carried by afferent renal fibers.

Noradrenergic mechanisms in brain and peripheral organs after aortic nerve transection.

Noradrenergic mechanisms were studied in the hypothalamus, midbrain, medulla, kidney, duodenum, and skeletal muscle of Wistar rats at 3 or 13 days after either bilateral transection of the aortic depressor nerve (ADN) or a sham operation. The rate of decline of tissue norepinephrine (NE) concentration after inhibition of tyrosine hydroxylase with alpha-methyltyrosine was used as an index of NE turnover. Three days after ADN transection, arterial pressure and heart rate were elevated significantly, and NE turnover was increased in all three brain areas, kidney, and skeletal muscle but not in duodenum. The largest change occurred in skeletal muscle, where the time required for tissue NE concentration to decline to 50% of control decreased from 9.0 to 2.5 h. In rats 13 days after ADN transection, arterial pressure was significantly higher than in sham-operated controls, but heart rate was similar to control values. NE turnover was slightly increased in hypothalamus but was not significantly different in muscle and kidney when compared to sham-operated controls. These results suggest that 3 days after ADN transection in the rat, arterial pressure is elevated as a result of increased activity of noradrenergic neurons in the hypothalamus and brain stem, which is translated into increased sympathetic nerve activity to peripheral organs, particularly skeletal muscle. The normal turnover of NE in peripheral organs 13 days after ADN transection suggests that mechanisms other than increased sympathetic activity are responsible for maintaining the elevated arterial pressure.