Distribution and amino acid content of enkephalin-immunoreactive inputs onto juxtacellularly labelled bulbospinal barosensitive neurons in rat rostral ventrolateral medulla.

The activity of bulbospinal (presympathetic) vasomotor neurons of the rostral ventrolateral medulla is modulated pre- and postsynaptically by exogenously applied opioid agonists. To determine whether these neurons receive direct opioid inputs, we examined the relationship between bulbospinal barosensitive neurons and nerve terminals immunoreactive for enkephalin in the rostral ventrolateral medulla of rats. By light microscopy, we mapped the distribution of close appositions by enkephalin-immunoreactive varicosities on 10 bulbospinal barosensitive neurons labelled in vivo with biotinamide. We also examined four labelled neurons ultrastructurally for synapses by enkephalin-immunoreactive terminals and determined with post-embedding immunogold labelling whether these enkephalin-positive terminals contained amino acids. Enkephalin-immunoreactive varicosities closely apposed all bulbospinal barosensitive neurons. Maps of the dendritic distribution of appositions indicated that fast-conducting bulbospinal barosensitive neurons with myelinated axons (conduction velocity >3 m/s; n=3) received many appositions (up to 470/neuron); and slowly conducting neurons with unmyelinated axons (conduction velocity <0.90 m/s; n=3), substantially fewer. Ultrastructural analysis of three fast- and one slowly conducting bulbospinal barosensitive neurons revealed numerous synapses from enkephalin-immunoreactive terminals on cell bodies and dendrites. Enkephalin-positive terminals synapsing on bulbospinal barosensitive neurons contained one or more amino acid: GABA+glycine, glutamate alone or GABA+glutamate. Enkephalin-immunoreactive terminals located near biotinamide-labelled cells contained a similar variety of amino acids. In summary, enkephalin-immunoreactive terminals in the rostral ventrolateral medulla densely innervate lightly myelinated presympathetic neurons and more sparsely those with unmyelinated axons. Enkephalin is present in both excitatory (glutamate-immunoreactive) and inhibitory (GABA- and/or glycine-immunoreactive) terminals. The data suggest that endogenous enkephalin inhibits amino acid release from terminals that innervate bulbospinal barosensitive neurons of the rostral ventrolateral medulla.

Activity of 2-substituted lysophosphatidic acid (LPA) analogs at LPA receptors: discovery of a LPA1/LPA3 receptor antagonist.

The physiological implications of lysophosphatidic acid occupancy of individual receptors are largely unknown because selective agonists/antagonists are unavailable currently. The molecular cloning of three high-affinity lysophosphatidic acid receptors, LPA1, LPA2, and LPA3, provides a platform for developing receptor type-selective ligands. Starting with an N-acyl ethanolamide phosphate LPA analog, we made a series of substitutions at the second carbon to generate compounds with varying spatial, stereochemical, and electronic characteristics. Analysis of this series at each recombinant LPA receptor using a guanosine 5'-O-(3-[35S]thio)triphosphate (GTP[gamma35S]) binding assay revealed sharp differences in activity. Our results suggest that these receptors have one spatially restrictive binding pocket that interacts with the 2-substituted moieties and prefers small hydrophobic groups and hydrogen bonding functionalities. The agonist activity predicted by the GTP[gamma35S] binding assay was reflected in the activity of a subset of compounds in increasing arterial pressure in anesthetized rats. One compound with a bulky hydrophobic group (VPC12249) was a dual LPA1/LPA3 competitive antagonist. Several compounds that had smaller side chains were found to be LPA1-selective agonists.

Regulation of sympathetic tone and arterial pressure by the rostral ventrolateral medulla after depletion of C1 cells in rats.

This review describes experiments designed to determine the role of bulbospinal (BS) C1 cells in regulating the sympathetic outflow and blood pressure. This goal was achieved by analyzing the physiological consequences of destroying BS C1 cells. These cells were destroyed by suicide transport of an anti-dopamine-beta-hydroxylase antibody conjugated to saporin (anti-D beta H-SAP). Two to 3 weeks after spinal cord injection (T2-T6), the toxin destroyed 75-85% of BS C1 and C3 cells along with > 95% of BS noradrenergic neurons (A5, A6, A7). The toxin spared BS noncatecholaminergic cells. Under anesthesia, toxin-treated rats had a normal blood pressure and an apparently normal sympathetic nerve discharge (SNA, splanchnic), and intravenous clonidine caused a normal degree of sympathoinhibition. Inhibition of rostral ventrolateral medulla (RVLM) neurons by bilateral injection of muscimol caused the same hypotension and sympathoinhibition as in control rats. The baroreflex range was 41% attenuated by the toxin, but the MAP50 was unchanged. Sympathoexcitatory responses to stimulation of peripheral chemoreceptors with cyanide or to electrical stimulation of RVLM were severely depressed (60% to 80%) in toxin-treated rats. Rats in which A5 neurons were selectively destroyed had no deficit in the parameters tested. Unit recordings of BS RVLM neurons indicated that the toxin destroyed most barosensitive C1 neurons, but spared noncatecholaminergic lightly myelinated BS cells. In summary, the integrity of C1 neurons is not essential for the generation of SNA and the maintenance of BP under resting conditions, perhaps because these functions are performed primarily by noncatecholaminergic BS neurons. However, the deficits caused by treatment with anti-D beta H-SAP indicate that BS C1 neurons play a crucial role in several sympathoexcitatory responses mediated by the RVLM.

Preproenkephalin mRNA is expressed by C1 and non-C1 barosensitive bulbospinal neurons in the rostral ventrolateral medulla of the rat.

The autonomic regions of the thoracolumbar spinal cord receive a dense enkephalinergic (ENK) innervation from supraspinal sources, including the rostral ventrolateral medulla (RVLM). In the present study, we sought to determine whether the barosensitive bulbospinal (BSBS) neurons of the RVLM express preproenkephalin (PPE) mRNA. After injection of Fluoro-Gold (FG) into the upper thoracic spinal cord, neurons with PPE mRNA (PPE(+) neurons) were retrogradely labeled throughout the ventrolateral medulla. At the most rostral RVLM level, 29% of bulbospinal PPE+ cells were tyrosine hydroxylase-immunoreactive (TH-ir) and the latter constituted 19.4% of the bulbospinal TH-ir cells. We determined whether the bulbospinal PPE(+) RVLM neurons are barosensitive in two ways. First, we examined Fos production by FG-labeled RVLM neurons after 2 hours of hydralazine-induced hypotension (to 73 +/- 2 mm Hg) in conscious rats. Hydralazine (10 mg/kg i.v.) increased the number of Fos-ir neurons by two- to eightfold at all levels of the ventrolateral medulla examined. In the RVLM, 54% of bulbospinal PPE(+) neurons were Fos-ir, whereas such cells were more rarely found at caudal ventrolateral medullary levels. Second, we recorded individual BSBS RVLM units extracellularly in anesthetized rats and filled them juxtacellularly with biotinamide. Most biotinamide-filled neurons were PPE(+) (10 of 17), and the PPE(+) BSBS cells had a faster axonal conduction velocity than those without PPE mRNA (4.2 vs. 0.67 m/sec). Four of the 10 PPE(+) BSBS RVLM neurons were TH-ir. In summary, PPE mRNA is predominantly expressed by RVLM BSBS neurons with lightly myelinated spinal axons. PPE mRNA is present in most noncatecholaminergic BSBS neurons and also in approximately 20% of the bulbospinal C1 neurons. BSBS RVLM neurons most likely provide a major ENK input to sympathetic preganglionic neurons and PPE mRNA is the first identified positive phenotype of the non-C1 BSBS RVLM neurons.

Mu-opioid receptors are present in functionally identified sympathoexcitatory neurons in the rat rostral ventrolateral medulla.

Agonists of the mu-opioid receptor (MOR) produce profound hypotension and sympathoinhibition when microinjected into the rostral ventrolateral medulla (RVL). These effects are likely to be mediated by the inhibition of adrenergic and other presympathetic vasomotor neurons located in the RVL. The present ultrastructural studies were designed to determine whether these vasomotor neurons, or their afferents, contain MORs. RVL bulbospinal barosensitive neurons were recorded in anesthetized rats and filled individually with biotinamide by using a juxtacellular labeling method. Biotinamide was visualized by using a peroxidase method and MOR was identified by using immunogold localization of an antipeptide antibody that recognizes the cloned MOR, MOR1. The subcellular relationship of MOR1 to RVL neurons with fast- or slow-conducting spinal axons was examined by electron microscopy. Fast- and slow-conducting cells were not morphologically distinguishable. Immunogold-labeling for MOR1 was found in all RVL bulbospinal barosensitive neurons examined (9 of 9). MOR1 was present in 52% of the dendrites from both types of cells and in approximately half of these dendrites the MOR1 was at nonsynaptic plasmalemmal sites. A smaller portion of biotinamide-labeled dendrites (16%) from both types of cells were contacted by MOR1-containing axons or axon terminals. Together, these results suggest that MOR agonists can directly influence the activity of all types of RVL sympathoexcitatory neurons and that MOR agonists may also influence the activity of afferent inputs to these cells. The heterogenous distribution of MORs within individual RVL neurons indicates that the receptor is selectively targeted to specific pre- and postsynaptic sites.

Regulation of sympathetic tone and arterial pressure by rostral ventrolateral medulla after depletion of C1 cells in rat.

1. In this study we examined whether the rostral ventrolateral medulla (RVLM) maintains resting sympathetic vasomotor tone and activates sympathetic nerve activity (SNA) after the depletion of bulbospinal C1 adrenergic neurones. 2. Bulbospinal C1 cells were destroyed ( approximately 84% loss) by bilateral microinjections (spinal segments T2-T3) of an anti-dopamine-beta-hydroxylase antibody conjugated to the ribosomal toxin saporin (anti-DH-SAP). 3. Extracellular recording and juxtacellular labelling of bulbospinal barosensitive neurones in the RVLM revealed that treatment with anti-DH-SAP spared the lightly myelinated neurones with no tyrosine hydroxylase immunoreactivity. 4. In rats treated with anti-DH-SAP, inhibition of RVLM neurones by bilateral microinjection of muscimol eliminated splanchnic SNA and produced the same degree of hypotension as in control rats. 5. Following treatment with anti-DH-SAP the sympathoexcitatory (splanchnic nerve) and pressor responses to electrical stimulation of the RVLM were reduced. 6. Treatment with anti-DH-SAP also eliminated the majority of A5 noradrenergic neurones. However, rats with selective lesion of A5 cells by microinjection of 6-hydroxydopamine into the pons showed no deficits to stimulation of the RVLM. 7. In summary, the loss of 84% of bulbospinal adrenergic neurones does not alter the ability of RVLM to maintain SNA and arterial pressure at rest in anaesthetized rats, but this loss reduces the sympathoexcitatory and pressor responses evoked by RVLM stimulation. The data suggest sympathoexcitatory roles for both the C1 cells and non-C1 cells of the RVLM and further suggest the C1 cells are critical for the full expression of sympathoexcitatory responses generated by the RVLM.

Role of presympathetic C1 neurons in the sympatholytic and hypotensive effects of clonidine in rats.

The rostral ventrolateral medulla (RVLM) may play an important role in the sympatholytic and hypotensive effects of clonidine. The present study examined which type of presympathetic RVLM neuron is inhibited by clonidine, and whether the adrenergic presympathetic RVLM neurons are essential for clonidine-induced sympathoinhibition. In chloralose-anesthetized and ventilated rats, clonidine (10 microg/kg iv) decreased arterial pressure (116 +/- 6 to 84 +/- 2 mmHg) and splanchnic nerve activity (93 +/- 3% from baseline). Extracellular recording and juxtacellular labeling of barosensitive bulbospinal RVLM neurons revealed that most cells were inhibited by clonidine (26/28) regardless of phenotype [tyrosine hydroxylase (TH)-immunoreactive cells: 48 +/- 7%; non-TH-immunoreactive cells: 42 +/- 5%], although the inhibition of most neurons was modest compared with the observed sympathoinhibition. Depletion of most bulbospinal catecholaminergic neurons, including 76 +/- 5% of the rostral C1 cells, by microinjection of saporin anti-dopamine beta-hydroxylase into the thoracic spinal cord (levels T2 and T4, 42 ng. 200 nl(-1). side(-1)) did not alter the sympatholytic or hypotensive effects of clonidine. These data show that although clonidine inhibits presympathetic C1 neurons, bulbospinal catecholaminergic neurons do not appear to be essential for the sympatholytic and hypotensive effects of systemically administered clonidine. Instead, the sympatholytic effect of clonidine is likely the result of a combination of effects on multiple cell types both within and outside the RVLM.

Sympathetic reflexes after depletion of bulbospinal catecholaminergic neurons with anti-DbetaH-saporin.

We examined the effects of destroying bulbospinal catecholaminergic neurons with the immunotoxin anti-dopamine beta-hydroxylase-saporin (anti-DbetaH-Sap) on splanchnic nerve activity (SNA) and selected sympathetic reflexes in rats. Anti-DbetaH-Sap was administered into the thoracic spinal cord with the retrograde tracer fast blue. After 3-5 wk, anti-DbetaH-Sap eliminated most bulbospinal C1 (>74%), C3 ( approximately 84%), A5 ( approximately 98%), and A6 cells. Noncatecholaminergic bulbospinal neurons of the rostral ventrolateral medulla and serotonergic neurons were spared. Under chloralose anesthesia, mean arterial pressure and heart rate of anti-DbetaH-Sap-treated rats (3-5 wk) were normal. Resting SNA was not detectably altered, but the baroreflex range and gain were reduced approximately 40% (P < 0.05). Phenyl biguanide-induced decreases in mean arterial pressure, heart rate, and SNA were unchanged by anti-DbetaH-Sap, but the sympathoexcitatory response to intravenous cyanide was virtually abolished (P < 0.05). Rats that received spinal injections of saporin conjugated to an anti-mouse IgG had intact bulbospinal C1 and A5 cells and normal physiological responses. These data suggest that C1 and A5 neurons contribute modestly to resting SNA and cardiopulmonary reflexes. However, bulbospinal catecholaminergic neurons appear to play a prominent sympathoexcitatory role during stimulation of chemoreceptors.

Nucleus of the solitary tract lesions enhance drinking, but not vasopressin release, induced by angiotensin.

Rats with chronic nucleus of the solitary tract lesions (NTS-X) drink water and release vasopressin (VP) in response to reduced blood volume despite an absence of neural signals from cardiac and arterial baroreceptors. The present study determined whether rats with NTS-X have a greater sensitivity to circulating ANG II, which may contribute to the drinking and VP responses to hypovolemia. In conscious control rats and rats with NTS-X, ANG II was infused intravenously for 1 h at 10, 100, or 250 ng. kg(-1). min(-1). At the two higher doses, ANG II stimulated more water intake with a shorter latency to drink in rats with NTS-X than in control rats. In contrast, infusion of ANG II produced comparable increases in plasma VP in the two groups. At the two higher doses, ANG II produced an enhanced increase in arterial pressure (AP) in rats with NTS-X, and the bradycardia seen in control rats was reversed to a tachycardia. Infusion of hypertonic saline, which did not alter AP or heart rate, produced comparable drinking and VP release in the two groups. These results demonstrate that chronic NTS-X increases the dipsogenic response of rats to systemic ANG II but has no effect on ANG II-induced VP release or the osmotic stimulation of these responses.

Evidence for glycinergic respiratory neurons: Bötzinger neurons express mRNA for glycinergic transporter 2.

Bötzinger (BOTZ) neurons in the rostral ventrolateral medulla fire during the late expiratory phase of the respiratory cycle. These cells inhibit phrenic motor neurons and several types of respiratory neurons in the medulla oblongata. BOTZ cells produce a fast, chloride-mediated inhibition of their target neurons, but the neurotransmitter used by these cells has not been determined. In the present study, we examine whether gamma-aminobutyric acid (GABA) or glycine could be the inhibitory neurotransmitter of BOTZ cells. In chloralose-anesthetized rats, we individually filled 20 physiologically characterized BOTZ neurons with biotinamide by using a juxtacellular labeling method. Medullary sections containing the labeled BOTZ neurons were processed for in situ hybridization by using digoxigenin-labeled riboprobes for glutamic acid decarboxylase isoform 67 (GAD67), a marker for GABAergic neurons, or for glycine transporter 2 (GLYT2), a marker for glycinergic neurons. All BOTZ cells examined contained GLYT2 mRNA (n = 10), whereas none had detectable levels of GAD67 mRNA (n = 10). For a positive control, 12 GABAergic neurons in the substantia nigra pars reticulata also were recorded and filled with biotinamide in vivo. Most of these cells, as expected, had detectable levels of GAD67 mRNA (11 out of 12). These results demonstrate that the juxtacellular labeling method can be combined with in situ hybridization to identify physiologically characterized cells with probable GABAergic or glycinergic phenotypes. Furthermore, these data suggest that BOTZ neurons use the neurotransmitter glycine and not GABA to provide widespread inhibition of respiratory-related neurons.

Thirst and salt appetite elicited by hypovolemia in rats with chronic lesions of the nucleus of the solitary tract.

Cardiac vagal afferents terminating in the nucleus of the solitary tract (NTS) are believed to participate in stimulating neurohypophysial secretion of vasopressin as well as increased ingestion of water and NaCl solution in response to decreased blood volume. However, we recently reported that chronic lesions of NTS, which eliminate neural input from cardiac and arterial baroreceptors, do not impair hypovolemia-induced vasopressin secretion in rats. In the present investigation we sought to determine whether those sensory signals were necessary for hypovolemia-induced thirst and salt appetite. Rats with chronic lesions of the NTS increased consumption of water and NaCl solution normally when plasma volume was reduced isosmotically by subcutaneous injection of polyethylene glycol solution. These results were obtained whether rats were allowed to drink water or 0.15 M NaCl in one-bottle tests or water and 0.5 M NaCl in two-bottle tests. The induction of thirst and salt appetite by hypovolemia despite the apparent loss of neural input to the brain from cardiac volume-sensitive receptors indicates that other signals generated by plasma volume deficits can stimulate these behavioral responses in rats.

Identification of C1 presympathetic neurons in rat rostral ventrolateral medulla by juxtacellular labeling in vivo.

The rostral ventrolateral medulla (RVLM) contains barosensitive, bulbospinal neurons that provide the main supraspinal excitatory input to sympathetic vasomotor preganglionic neurons. However, the phenotype of the critical RVLM cells has not been conclusively determined. The goal of the current study was to identify the proportion of electrophysiologically defined, putative, presympathetic RVLM neurons that are C1 cells. We used a juxtacellular labeling technique to individually fill spontaneously active, barosensitive, bulbospinal RVLM neurons with biotinamide following electrophysiological characterization in chloralose-anesthetized rats. To determine whether these neurons could be classified as C1 cells, the biotinamide-labeled cells were processed for detection of tyrosine hydroxylase. The majority of barosensitive bulbospinal RVLM neurons were tyrosine hydroxylase immunoreactive (TH-ir; 28 of 39). All of the barosensitive bulbospinal RVLM neurons with axonal conduction velocities in the C fiber range (<1 m/second) were TH-ir (n = 16), whereas faster conducting cells (1 to 7 m/second) were either lightly TH-ir (n = 12) or not detectably TH-ir (n = 11). Adjacent respiratory-related RVLM units labeled with biotinamide were not detectably TH-ir (n = 10). To verify that TH-ir cells were indeed adrenergic, a subset of barosensitive bulbospinal cells labeled with biotinamide were examined for phenylethanolamine N-methyltransferase immunoreactivity (PNMT-ir). Three slowly conducting cells had detectable PNMT-ir, and two fast-conducting cells had no detectable PNMT-ir. These results indicate that the majority of bulbospinal RVLM neurons with putative sympathoexcitatory function are C1 cells.

The kidneys stimulate vasopressin release during hemorrhage in rats with chronic NTS lesions.

Elimination of baroreceptor afferent input to the brain produced by chronic lesion of nucleus of the solitary tract (NTS) does not alter vasopressin (VP) release during hypotensive hemorrhage in conscious rats. To investigate whether the kidneys play a critical role in stimulating VP release during hemorrhage in chronic NTS-lesioned rats, we examined the effects of removing potential signals arising from the kidneys. In NTS-lesioned rats, nephrectomy or renal denervation, but not captopril injection, markedly attenuated (but did not abolish) hemorrhage-induced VP release. In contrast, none of these manipulations attenuated the VP response in NTS-intact rats. Hemorrhage increased plasma renin activity in control and NTS-lesioned rats, and this response was not altered by renal denervation. In rats with NTS lesions and renal denervation, hemorrhage induced the expression of Fos in hypothalamic magnocellular VP neurons in a pattern similar to that of hemorrhage in intact rats. Collectively, these results indicate that in chronic NTS-lesioned rats an afferent signal arising from the kidneys stimulates VP release during hemorrhage, possibly through renal nerves. However, with the NTS intact or after the selective removal of arterial baroreceptor inputs, such a role for the kidneys is not apparent. Furthermore, in the absence of the NTS and renal nerves, another signal generated by hypotensive hemorrhage continues to stimulate VP neurons.

Blood pressure regulation in baroreceptor-denervated rats.

1. Arterial baroreceptor denervation produces acute hypertension, but chronically denervated animals have an average arterial pressure that is similar to that of baroreceptor intact animals. 2. Although cardiopulmonary baroreceptors and renal compensations have been suggested to mediate the restoration of a normal average arterial pressure in sino-aortic denervated rats, such mechanisms are inconsistent with the available data. 3. At present the processes involved in the restoration and long-term maintenance of a normal average arterial pressure in chronic baroreceptor denervated animals are not known. An understanding of the regulation of arterial pressure that occurs in the absence of arterial baroreceptor reflexes may provide important new insights into the mechanisms underlying the long-term regulation of arterial pressure.

Sodium deprivation blunts hypovolemia-induced pituitary secretion of vasopressin and oxytocin in rats.

The present investigations determined the effects of dietary sodium deprivation on the neurohypophysial secretion of arginine vasopressin (AVP) and oxytocin (OT) by rats in response to nonhypotensive hypovolemia induced by subcutaneous injection of 30% polyethylene glycol solution. In rats fed either standard sodium-rich laboratory chow or sodium-deficient diet for 8 days, AVP secretion increased gradually in proportion to plasma volume deficits up to 22-28% while pituitary secretion of OT was not stimulated. However, when hypovolemia was more pronounced, secretion of both hormones was marked in rats fed standard chow, whereas rats fed sodium-deficient diet were significantly less responsive. These effects did not reflect a general insensitivity of the neurohypophysial system because sodium-deprived and chow-fed rats secreted AVP and OT equivalently in response to intravenous infusion of 1.5 M NaCl solution. Nor did they reflect a general insensitivity to hypovolemia because sodium-deprived rats drank substantial, above-normal volumes of water after colloid treatment. Instead, the results appear to reflect a specific inhibition of stimulatory baroreceptor inputs to AVP and OT neurons during dietary sodium deprivation in rats.

Chronic nucleus tractus solitarius lesions do not prevent hypovolemia-induced vasopressin secretion in rats.

The present study examined the hypothesis that hypovolemia stimulates vasopressin (VP) secretion by removing tonic inhibitory baroreceptor input. Serial hemorrhage (4 samples of 2 ml/300 g body wt taken every 10 min) increased plasma VP levels in conscious rats devoid of cardiac and arterial baroreceptor reflex responses due to chronic bilateral lesions of nucleus tractus solitarius (NTS). The VP response to hemorrhage was similar to that seen in control rats and chronic sinoaortic-denervated (SAD) rats. After subcutaneous injection of 30% polyethylene glycol, NTS-lesioned rats, SAD rats, and control rats had elevated VP levels that correlated with the induced depletion of plasma volume. Additionally, in alpha-chloralose-anesthetized control rats, chronic SAD rats, and chronic NTS-lesioned rats, bilateral vagotomy had minimal effects on basal VP levels, and vagotomy in chronic NTS-lesioned rats did not prevent hemorrhage-evoked increases in VP secretion. These results do not support the idea that hemorrhage-induced VP secretion occurs through reduction in tonic inhibitory baroreceptor input. Instead, neither cardiac nor arterial baroreceptor input appears to be necessary for hypovolemia-induced VP secretion in rats.

Use of sinoaortic denervation to study the role of baroreceptors in cardiovascular regulation.

To assess the role of arterial baroreceptors in cardiovascular regulation, many studies have used rats in which baroreceptor afferents have been surgically destroyed. However, interpretation of studies using sinoaortic-denervated (SAD) rats is complicated by variability in the extent of baroreceptor denervation. We have compared cardiovascular regulation in rats with total sinoaortic cardiovascular regulation in rats with total sinoaortic denervation, as assessed by the abolition of reflex changes in heart rate (HR) to increases and decreases in arterial pressure (AP), with rats that underwent the same denervation procedure but still had residual (although markedly blunted) reflex changes in HR to changes in AP. In totally SAD rats, the lability of AP was greatly exaggerated compared with sham-denervated rats, although the average AP was equivalent. In contrast, partially SAD rats had elevated AP, and although AP was more labile than in sham-denervated rats, it was less labile than in totally SAD rats. In addition, cardiovascular responses elicited by elimination of neural activity in the nucleus tractus solitarius (NTS) were qualitatively different between the two groups of rats; destruction of the NTS increased AP similarly in partially SAD rats and sham-denervated rats, whereas this treatment did not alter AP in totally SAD rats. Thus there are marked differences in SAD rats with no residual arterial baroreceptor reflex function compared with SAD rats with even a small degree of residual baroreceptor reflex function. These studies highlight the importance of carefully characterizing SAD rats used in studying the role of the baroreceptor reflex in cardiovascular regulation.

PNMT-containing neurons of the C1 cell group express c-fos in response to changes in baroreceptor input.

The immunocytochemical detection of Fos, the protein product of the immediate-early gene c-fos, was used as a marker for activated neurons to examine whether the C1 neurons in the rat rostral ventrolateral medulla (RVLM) respond to changes in baroreceptor afferent activity. After hydralazine-induced hypotension or sinoaortic denervation, two treatments that reduce baroreceptor afferent activity, numerous Fos-positive neurons were observed in the RVLM. The number of Fos-positive neurons in the RVLM was counted in brain stem sections from hydralazine-treated rats that had been previously injected with Fluorogold into the upper thoracic spinal cord to label spinally projecting RVLM neurons as well as stained for phenylethanolamine-N-methyltransferase (PNMT) as a marker of C1 neurons. The results indicate that approximately 80% of the C1 neurons expressed Fos in response to hydralazine injection; this was true of spinally projecting C1 neurons as well as those C1 neurons that were not labeled with Fluorogold. Furthermore, in hydralazine-treated rats, the majority of Fluorogold-labeled Fos-positive neurons contained PNMT. These results suggest that C1 neurons are sensitive to baroreceptor afferent input and support a role of these neurons in cardiovascular regulation.

Nucleus tractus solitarius and control of blood pressure in chronic sinoaortic denervated rats.

To determine the role of the nucleus tractus solitarius (NTS) in the tonic maintenance of arterial pressure (AP) following chronic baroreceptor denervation, the present study examined the effect of inhibition of the NTS on AP in chronic sinoaortic denervated (SAD) and control rats. One to two weeks after complete SAD (no residual arterial baroreceptor reflexes) mean AP was not significantly different from that of control rats. Bilateral microinjections of muscimol and lidocaine into the NTS markedly increased AP in alpha-chloralose-anesthetized control rats. However, microinjections of these neuroinhibitory drugs had no effect on AP in SAD rats. Similarly, 1 h after bilateral destruction of the NTS conscious control rats were hypertensive, while AP in SAD rats was not changed. Plasma levels of vasopressin (VP), which were also elevated in control rats 1 h after NTS lesions, were not significantly altered in SAD rats. These results demonstrate that inhibition of the NTS has no effect on AP or plasma levels of VP in chronic SAD rats. This suggests neither the NTS nor afferents to the NTS supply a tonic inhibitory influence on AP after chronic baroreceptor denervation.