Gene expression of brain nitric oxide synthase and soluble guanylyl cyclase in hypothalamus and medulla of two-kidney, one clip hypertensive rats.

Nitric oxide may act at autonomic sites in the brain to regulate arterial blood pressure. Our goal was to determine whether gene expressions of the brain isoform of nitric oxide synthase and of the beta subunit of soluble guanylyl cyclase, the target of nitric oxide, were altered in discrete autonomic brain regions after induction of hypertension in rats. The two-kidney, one clip model was used to induce hypertension, and measurements were made 3 and 6 weeks after the left renal artery was clipped. Only experimental rats with blood pressures elevated by at least 25 mm Hg were used. Total RNA was purified from microdissected tissue blocks containing hypothalamus, dorsal medulla, rostral ventrolateral medulla, and caudal ventrolateral medulla. Changes in nitric oxide synthase and guanylyl cyclase mRNA were semiquantified in each region by use of reverse transcription-polymerase chain reactions in which known concentrations of deletion mutants of the two genes were coamplified as internal standards. Compared with controls, significant decreases and increases in nitric oxide synthase mRNA were found in the hypothalamus (x 2.2) and caudal ventrolateral medulla (x 6.4), respectively, of hypertensive rats 3 weeks after clipping. These alterations were reversed in hypertensive rats at 6 weeks; levels increased (x 4.6) in the hypothalamus and decreased (x 5.5) in the caudal ventrolateral medulla. Changes in guanylyl cyclase expression paralleled those for nitric oxide synthase in some but not all areas at both time points.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of preproadrenomedullin mRNA in the rat central nervous system and its modulation by physiological stressors.

Adrenomedullin (ADM), encoded by the preproadrenomedullin (ppADM) gene, exerts multiple effects in a wide variety of peripheral and central tissues. Although ADM-like immunoreactivity has been shown to be widely distributed throughout the rat central nervous system (CNS), the detailed distribution of ppADM gene expression in the CNS and its modulation by physiological stimuli remain unknown. In our study, in situ hybridization was used to localize ppADM mRNA in the rat brain and to quantify its levels after exposure to different stressors including lipopolysaccharide (LPS; 100 microg/kg, iv), restraint stress (2 cycles of 1 hour restraint/1 hour rest), and 24 hours of dehydration. In addition, Fos immunoreactivity was used to identify the activation of neurons in response to LPS. Our results show that ppADM mRNA is widely distributed throughout the rat CNS, with especially high levels in autonomic centers including the hypothalamic paraventricular nucleus (PVN), hypothalamic supraoptic nucleus (SON), locus coeruleus, ventrolateral medulla, and intermediolateral cell column of the spinal cord. Furthermore, LPS inhibits ppADM gene expression in the parvocellular PVN (pPVN), magnocellular PVN (mPVN), SON, dorsal motor nucleus of the vagus, and area postrema among examined regions; restraint stress reduces ppADM mRNA levels in the pPVN, mPVN, SON, nucleus of the solitary tract, dorsal motor nucleus of the vagus, area postrema, and subfornical organ; 24 hours of water deprivation decreases ppADM gene expression only in the mPVN and SON. Taken together, our results suggest that ADM is involved in the regulation of the hypothalamo-neurohypophysial system, the hypothalamo-pituitary-adrenal axis, and central autonomic functions.

Stress-induced activation of nitric oxide-producing neurons in the rat brain.

Nitric oxide (NO) is a gaseous neurotransmitter that may mediate a decrease in sympathetic output to the periphery. This implication predicts that NO-producing neurons in the brain are activated in animals experiencing increased levels of sympathetic activity. To test this prediction, we subjected three groups of experimental rats to differing levels of environmental stimulation for 1 hour: minimal stimulation, moderate stimulation, and restraint stress. NO-producing neurons were histochemically visualized in sections of the brain, and activation of these neurons was assessed according to the neuronal expression of the immediate early gene c-fos. Constitutive activation of NO-producing neurons was found in the hypothalamus (paraventricular and supraoptic nuclei), dorsal raphe nuclei, and spinal nucleus of the trigeminal nerve of minimally stimulated rats. When animals were subjected to a novel environment (moderate stimulation), additional NO-producing neurons were activated in the medial septum, medial amygdala, hypothalamic nuclei (lateral, periventricular, and posterior), colliculi, nucleus raphe obscurus, medial vestibular nucleus, nucleus of the tractus solitarius, and several components of the ventrolateral medulla. Restraint stress caused the activation of NO-producing neurons in all of these areas, often in increasing numbers, and the activation of additional NO-producing neurons in the diagonal band of Broca, lateral and medial preoptic areas, basomedial and basolateral amygdalar nuclei, hypothalamic nuclei (dorsomedial, retrochiasmatic supraoptic, and circularis), nucleus raphe pontus, lateral parabrachial nucleus, and pontine nuclei. Expressed as a proportion of NO-producing neurons per section, the largest percentages (>20%) of double-stained neurons were found in the basolateral amygdala (46%), hypothalamic paraventricular nucleus (35%), corpora quadrigemina (estimated at 40%), dorsal raphe (45%), nuclei raphe pontus (33%) and obscurus (63%), lateral parabrachial nucleus (22%), medial vestibular nucleus (25%), lateral division of the nucleus paragigantocellularis (26%), and lateral reticular nucleus (35%). Evidence from other studies increasingly supports the concept that NO plays a generalized role in autonomic regulation by decreasing sympathetic output. Our results show that more NO-producing neurons were activated during stress than during minimal or moderate levels of stimulation. Together, the evidence suggests that NO is a neurochemical messenger that is utilized by individual autonomic neurons as the organism responds to increased levels of sympathetic activity.

Somatostatin-like immunoreactivity in neurons, nerve terminals, and fibers of the cat spinal cord.

The distribution of somatostatin-like immunoreactivity (SS) was studied in the spinal cord of untreated cats and of cats that had received colchicine at all levels of the cord. In the dorsal horn small (less than 15 microns in diameter), round neurons were found in Rexed laminae II and III at all levels. At all levels laminae IV-VI contained smaller numbers of immunoreactive neurons that were medium (between 15 and 25 microns in diameter) to large (greater than 25 microns in diameter) in size. In addition, small numbers of medium-sized neurons were observed at the dorsal and dorsomedial borders of the gray and white matter in segments C1-5. In the sacral cord (S1-3), a group of medium-sized bipolar neurons was found in the dorsolateral funiculus. In transverse sections the processes of the neurons in these two latter groups travelled in a direction parallel to the border of the gray and white matter. In the intermediate and central gray matter, in addition to the immunoreactive neurons found in the region of the intermediolateral nucleus and nucleus intercalatus of lamina VII in segments C8 to L4 (Krukoff et al., '85a), lamina VII contained immunoreactive neurons at all levels with the largest number occurring in the thoracic cord. These neurons were medium to large in size and were generally multipolar with processes travelling in all directions. Multipolar small immunoreactive neurons were also found in the central gray region (lamina X) in the thoracic and upper lumbar cord. Finally, small numbers of neurons containing SS were found in the ventral horn of the cervical and upper thoracic cord. These multipolar neurons were medium to large in size. The distribution of nerve terminals and fibers containing SS was similar to that previously described in mice, rats, guinea pigs, and primates. Although the function of somatostatin in the spinal cord is not known, its presence in neurons with short processes suggests that it may act to modify local activity in the regions where it is found, including areas involved in sensory, visceromotor, and motor functions.

The hypothalamic paraventricular and lateral parabrachial nuclei receive collaterals from raphe nucleus neurons: a combined double retrograde and immunocytochemical study.

Retrograde tracer injections of fluorescein- and rhodamine-labelled latex microspheres centered in the parvicellular zone of the hypothalamic paraventricular nucleus and pontine lateral parabrachial nucleus revealed that 36% of the labelled neurons in the dorsal raphe nucleus send collaterals to both structures. These cells were organized in a well-distinguishable cluster within the dorsal raphe nucleus. By combining retrograde tracing with immunocytochemistry, it was found that less than 8% of the double-labelled cells stained positively for serotonin. Of the remaining raphe nuclei that were examined, only the median raphe nucleus contributed a minor nonserotoninergic projection to the paraventricular or lateral parabrachial nuclei. Few of the retrogradely labelled cells in the median raphe nucleus contained both tracers. These results suggest that nonserotoninergic and serotoninergic neurons in the dorsal raphe nucleus, via collateral branching, may simultaneously influence the activity of two central nervous system nuclei involved in autonomic control.

Activation by hypotension of neurons in the hypothalamic paraventricular nucleus that project to the brainstem.

To investigate the involvement of neuronal nitric oxide (NO) in the response of the brain to changes in blood pressure, we studied the activation of putative NO-producing neurons in the paraventricular nucleus of the hypothalamus (PVN) in rats whose mean arterial pressures (MAPs) were decreased by 40-50% with hemorrhage (HEM) or infusion of sodium nitroprusside (NP). Activation was assessed on the basis of expression of the immediate early gene, c-fos; putative NO-producing neurons were identified with the histochemical stain for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d); and the proportions of neurons projecting to the nucleus of the tractus solitarius (NTS) and/or caudal ventrolateral medulla (CVLM) were determined with retrograde tracing techniques. No differences were found for results obtained from HEM and NP animals. Three to four percent of activated PVN neurons projected to the NTS or CVLM. Conversely, approximately 33% and 16% of neurons projecting to the NTS and CVLM, respectively, were activated. About 43% of NADPH-d neurons in the PVN were activated. Of PVN neurons projecting to the NTS or CVLM, 38% and 32%, respectively, were NADPH-d positive. About 11% of NADPH-d PVN neurons projected to the NTS or CVLM. An average of 3 NADPH-d neurons per section were activated and projected to either target. Finally, 7 PVN cells per section sent collateral branches to the NTS and CVLM; 2 or 3 of these cells per section were also activated by decreases in arterial pressure. No NADPH-d cells were found that sent collateral branches to the NTS and CVLM. This study shows that decreases in MAP activate PVN neurons that project, singly and through collaterals, to the NTS and CVLM. A relatively high proportion of the singly projecting neurons is NADPH-d positive. These results support the contention that descending projections from the PVN to the brainstem play an important role in the physiological response to decreases in arterial pressure and suggest that NO may participate in this response.

Immune stress activates putative nitric oxide-producing neurons in rat brain: cumulative effects with restraint.

Immune and restraint stresses induce changes in the hypothalamo-pituitary-adrenal axis activity and autonomic function. In the hypothalamus, the paraventricular nucleus (PVN) plays an integral role, and nitric oxide (NO) is hypothesized to participate in this process. We used 1) intravenous injections of lipopolysaccharide (LPS, 125 microg/kg) to identify activated (Fos-positive) putative NO-producing neurons, 2) retrograde tracing to determine if autonomic medullary regions signal the PVN to mediate this activation, and 3) intravenous LPS injections plus restraint stress to determine if responses to restraint are altered by the presence of immune stress. At 2 hours after LPS injections, approximately 15% of putative NO-producing neurons were activated in the nucleus of the tractus solitarius (NTS) and ventrolateral medulla (VLM); about half of the putative NO neurons in the PVN were activated. In LPS + restraint rats, the percentage of activated putative NO neurons in the PVN was not significantly different from LPS-treated rats, but the numbers of putative NO neurons and activated NO neurons per section increased significantly. Retrogradely labeled neurons were found mostly in the middle NTS and VLM, and about 75% were activated. No neurons in the NTS or VLM were triple labeled. The results show that putative NO-producing neurons in the PVN, NTS, and VLM are activated by circulating LPS. However, the LPS-induced signaling to the PVN likely occurs through pathways other than the NO network of neurons in NTS or VLM. Finally, superimposition of restraint stress onto animals already exposed to immune stress stimulates the NO system in the PVN to a greater extent than either stress alone.

Segmental distribution of peptide- and 5HT-like immunoreactivity in nerve terminals and fibers of the thoracolumbar sympathetic nuclei of the cat.

The distribution of leucine-enkephalin, methionine-enkephalin, neurotensin, somatostatin, substance P, oxytocin, vasopressin, neurophysin II, and serotonin in nerve terminals and fibers of sympathetic autonomic areas of the thoracolumbar (T-L) spinal cord was studied immunohistochemically in cats. Densities of these immunoreactive terminals and fibers were estimated in the intermediolateral nucleus pars principalis (IMLp) and pars funicularis (IMLf), the nucleus intercalatus (IC), and the central autonomic area (CA). Results for leucine- and methionine-enkephalin-like immunoreactivity (ENK) were similar and immunoreactivity for vasopressin was not observed. The greatest numbers of terminals and fibers in the IMLp region contained ENK, neurotensin-(NT), and serotonin-like immunoreactivity (5HT); terminals and fibers containing substance P-(SP) and neurophysin II-like immunoreactivity (NP2) were intermediate in number, and those containing somatostatin-(SS) and oxytocin-like immunoreactivity (OXY) were generally sparse. In the IC and CA, terminals and fibers containing ENK and NT were dense, those containing SP were moderate, and those containing OXY, NP2, and 5HT were sparsely represented. In the IMLp, where the largest proportion of sympathetic preganglionic neurons (SPN) is found, the greatest concentration of terminals and fibers containing ENK was found in segments T1-T8; for NT these segments were T1-T5 and T11-L1, for SP-C8-T2 and T11-L1, for NP2-T4-T7 and L2 to L3, and for 5HT-T1-T5. Terminals and fibers containing SS and OXY were present in segments C8-T10 and segments C8, T2-T8, T13, and L2 to L3, respectively. These results indicate that while ENK, NT, SP, NP2, and 5HT fibers and terminals are widely distributed throughout the T-L cord, they may influence to a greater degree the SPN in segments where they are present in greater numbers. As SS and OXY were not found at all levels of the IMLp, their functions may be more organ specific.

Segmental distribution of peptide-like immunoreactivity in cell bodies of the thoracolumbar sympathetic nuclei of the cat.

The distribution of leucine-enkephalin, methionine-enkephalin, neurotensin, somatostatin, substance P, oxytocin, vasopressin, and neurophysin II in cell bodies of sympathetic autonomic nuclei of the thoracolumbar (T-L) spinal cord was studied immunohistochemically in cats after intrathecal administration of colchicine. Neurons containing only enkephalin-, neurotensin-, somatostatin-, and substance P-like immunoreactivity (ENK, NT, SS, SP, respectively) were found in the intermediolateral nucleus pars principalis (IMLp) and pars funicularis (IMLf), the nucleus intercalatus (IC), and the central autonomic area (CA). The size, shape, location, and numbers of the peptide-positive neurons in the IMLp, IMLf, and IC suggested that they were sympathetic preganglionic neurons (SPN). This was confirmed by a combined retrograde tracing/immunohistochemical study showing that most of these neurons at the levels of the T-L cord known to provide preganglionic fibers to the stellate ganglion were SPN. On the other hand, the functional identification of the neurons in the CA is uncertain as neurons were not observed which were both retrogradely labelled and contained ENK, NT, SS, or SP. Immunoreactive neurons in each area were counted in ten sections from each segment from C8 to L4. In the IMLp, the SPN with ENK were greatest in number (up to 25) in segments T4-T7 and L2-L3. The maximum number of SPN containing NT was found in segments T4-T7 (45 neurons). Of the four peptides, neurons containing SS were found in the greatest number (up to 48 in segments T2-T6); neurons containing SP were found in the smallest number (15 or fewer per segment). Few SPN containing each of the four peptides were found in the IC; CA neurons with ENK and NT were also few in number. A comparison of the numbers of immunoreactive neurons in the IML with earlier estimates for the total numbers of SPN in the IML at each level showed that the proportions of IML neurons containing each of the four peptides were fairly consistent throughout the T-L cord, with some exceptions. These results suggest that the innervation of visceral organs is not obviously peptide-specific, although some organs may be innervated by a greater proportion of SPN containing one of these peptides. Finally, the presence of ENK, NT, SS, and SP in SPN suggests that these four peptides act as neurotransmitters in preganglionic pathways to sympathetic ganglia.

Distribution and morphology of vasopressin-, neurophysin II-, and oxytocin-immunoreactive cell bodies in the forebrain of the cat.

Experiments were done to provide a detailed map of the location and a description of morphological characteristics of vasopressin (AVP-IR)-, neurophysin II (NII-IR)- and oxytocin (OXY-IR)-immunoreactive neuronal perikarya in the forebrain of the cat. In addition, the location of cells in the forebrain retrogradely labeled following injections of tracers into the neurohypophysis was determined. The distribution of AVP-IR and NII-IR was similar in all cases studied. Most of the cells containing AVP-IR and OXY-IR were observed in the hypothalamic paraventricular (PVH) and supraoptic (SON) nuclei. In addition, AVP-IR and OXY-IR cell bodies were found in the regions of the nucleus of the diagonal band of Broca, the dorsal chiasmatic nucleus, the anterior hypothalamic-preoptic area, the periventricular area, the nucleus circularis, the perifornical area of the lateral hypothalamus, the accessory SON, the area of the tuber cinereum (Tca), and the medial nucleus of the amygdala. The density of AVP-IR cells was greater than that of OXY-IR cells in these regions. Several forebrain areas were also observed to contain only AVP-IR perikarya: the suprachiasmatic nucleus (Sc), the bed nucleus of the stria terminalis, and the region of the substantia innominata and ventral globus pallidus (SI/GP). In addition, the dorsomedial nucleus of the hypothalamus only contained OXY-IR perikarya. Most of the cells immunoreactive to AVP were multipolar and had spinelike processes over their somata and proximal dendrites. In addition, the majority of cells in the PVH and SON were round or oval, whereas those outside these nuclei were fusiform or triangular. The mean somal area of AVP-IR cells in the region of the SI/GP was significantly (P less than 0.05) larger than that of AVP-IR cells in all other regions examined, whereas the mean somal area of Sc AVP-IR cells was significantly (P less than 0.05) smaller than that of all other groups of AVP-IR cells examined. Most OXY-IR cells were similar morphologically to those immunoreactive to AVP, except that OXY-IR cell bodies and their appendages did not have spinelike processes. In addition, OXY-IR perikarya were generally of uniform size. OXY-IR cells in the PVH and accessory SON were significantly (P less than 0.05) larger than AVP-IR cells in the same regions, but were not different from AVP-IR cells in the lateral hypothalamus and SON.(ABSTRACT TRUNCATED AT 400 WORDS)

Parabrachial nucleus projection towards the hypothalamic supraoptic nucleus: electrophysiological and anatomical observations in the rat.

It has been proposed that the pontine parabrachial nucleus (PBN) participates in the regulation of body fluid balance and the release of vasopressin from the neurohypophysis, although the pathways mediating the latter response are uncertain. This study in the rat, utilizing anatomical and electrophysiological methods, describes a projection from the lateral PBN towards the hypothalamic supraoptic nucleus (SON). Rats received iontophoretic injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L, 2% solution). After 14-17 days, rats were sacrificed and their brains prepared for immunofluorescent visualization of projections to the SON region. PHA-L-labelled terminals were found primarily in perinuclear regions immediately dorsal to the SON. In contrast, injections within the medial PBN and the nearby Kölliker-Fuse nucleus did not reveal labelling in or around the SON. Extracellular recordings from 86 of 118 antidromically identified neurons in anaesthetized rats revealed a set of complex synaptic responses after stimulation in the PBN. Excitatory responses (in 82 of 86 cells) of short (less than 100 msec, 39/82 cells) and long (greater than 100 msec, 43/82) duration were observed in both vasopressin- and oxytocin-secreting cells of the SON, while 4/86 cells displayed a depressant response to PBN stimulation. In the adjacent perinuclear zone, 22/39 nonneurosecretory cells responded with an increase in their excitability consequent to an identical stimulus. These data suggest a predominantly facilitatory influence of lateral PBN neurons on SON neurosecretory cells in the rat, and that the PBN-SON projection is an indirect one that utilizes an interneuronal network located in the perinuclear zone adjacent to the SON.

Nitric oxide regulates body temperature, neuronal activation and interleukin-1 beta gene expression in the hypothalamic paraventricular nucleus in response to immune stress.

An immune challenge initiates a complex cascade of events in the body including important responses from the central nervous system. As nitric oxide (NO) has been implicated in the central regulation of neuroendocrine and autonomic responses, this study was performed to determine if NO regulates physiological responses, neuronal activation, and/or interleukin-1 beta (IL-1 beta) gene expression in the paraventricular nucleus of the rat hypothalamus (PVN) in response to intravenous endotoxin, lipopolysaccharide (LPS, 100 microg/kg). Intracerebroventricular injections of NO synthase (NOS) inhibitors (7-nitroindazole sodium salt for neuronal NOS, N(G)-nitro-L-arginine for neuronal NOS and endothelial NOS, and aminoguanidine for inducible NOS) in LPS-treated rats showed that inhibition of NOS eliminated the drop in body temperature and led to increased neuronal activation in the PVN as assessed by immunohistochemistry for Fos-like immunoreactivity. Activation of NO-producing PVN neurons was also increased in these rats suggesting that NO influences neuronal NOS activity in PVN neurons. Finally, increased IL-1 beta gene expression in the PVN of LPS-treated rats receiving N(G)-nitro-L-arginine showed that NO regulates brain IL-1 beta gene expression. The results obtained with the NOS inhibitors support the hypothesis that NO produced from eNOS in the brain participates in temperature regulation, and inhibits PVN neuronal activity and IL-1 beta gene expression during immune stress.

Hypotension induces Fos immunoreactivity in NADPH-diaphorase positive neurons in the paraventricular and supraoptic hypothalamic nuclei of the rat.

Double staining for Fos and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-D) was used to study the distribution of activated neurons that synthesize nitric oxide in the paraventricular (PVN) and supraoptic nuclei (SON) following hypotensive stimulation in conscious rats. Fos was detected in many magno- and parvocellular NADPH-D positive neurons in response to haemorrhage or drug-evoked hypotension using i.v. infusions of sodium nitroprusside. However, quantitative analysis did not reveal any differences in the number of Fos positive PVN neurons following either mode of stimulation. These results suggest that a subpopulation of hypothalamic NADPH-D positive neurons is activated following hypotensive challenge. This activation of NADPH-D neurons may occur indirectly through other CNS structures that influence the excitability of hypothalamic SON and PVN. Furthermore, the lack of a difference in activated neurons within the PVN following either haemorrhage or nitroprusside infusion suggests that while a drop in blood pressure causes activation of neurons that produce nitric oxide, a decrease in blood volume, which accompanies haemorrhage, does not.

Gamma(2)-melanocyte-stimulating hormone suppression of systemic inflammatory responses to endotoxin is associated with modulation of central autonomic and neuroendocrine activities.

Central autonomic and neuroendocrine activities are important components of the host response to bacterial inflammation. We demonstrate that intravenous infusion of gamma(2)-melanocyte-stimulating hormone (gamma(2)-MSH), a potent autonomic regulating peptide, prevents lipopolysaccharide (LPS)-induced hypotension and tachycardia, and modulates the ACTH response to endotoxin. In the hypothalamic paraventricular nucleus, a major neuroendocrine and autonomic center, gamma(2)-MSH inhibits LPS-induced increases in CRF mRNA levels, but does not suppress LPS-augmented arginine vasopressin heteronuclear RNA expression. In the locus coeruleus, a brainstem noradrenergic center, gamma(2)-MSH inhibits LPS-induced increases in tyrosine hydroxylase mRNA levels. Gamma(2)-MSH inhibits LPS-induced IL-1beta gene expression in the brain, pituitary and thymus, and prevents increases in plasma NO levels. These findings reveal that gamma(2)-MSH attenuates systemic inflammatory responses to endotoxin and suggest that modulation of central autonomic and neuroendocrine activities by gamma(2)-MSH contributes to its anti-inflammatory effects.

Differential neuronal activation in the hypothalamic paraventricular nucleus and autonomic/neuroendocrine responses to I.C.V. endotoxin.

The paraventricular nucleus (PVN) of the hypothalamus is a key site for regulating neuroendocrine and autonomic activities. To study the role of the PVN activation in brain inflammation-induced autonomic/endocrine responses, lipopolysaccharide (LPS; 0.5 or 5 microg) was administered i.c.v. and rats were killed 1, 3 or 6 h after the injection. I.c.v. LPS-0.5 microg did not cause changes in mean arterial pressure (MAP) over 6 h, whereas LPS-5 micro induced a temporary decrease in MAP approximately 30 min after the injection. LPS at either dose increased heart rate. Whereas induction of Fos-like immunoreactivity was confined to the dorsal medial parvocellular division (mpd) of the PVN with the lower dose, labeling was found throughout the PVN with the higher dose. At 3 h, LPS-5 microg also stimulated increases in arginine vasopressin (AVP) heteronuclear RNA levels in the posterior magnocellular and dorsal parvocellular divisions of the PVN at 3 h, and activation of catecholaminergic neurons in the hypothalamus and brainstem. Increases in tyrosine hydroxylase (TH) mRNA levels were found in the locus coeruleus at 6 h. LPS at both doses elevated plasma ACTH levels and corticotropin-releasing factor gene expression in the mpd of the PVN. I.c.v. LPS induced IL-1beta mRNA in the meninges and ventricular ependymal lining at 1 h, and in the periventricular PVN at 3 h. Induction of IL-1beta mRNA was found in the lung at 1 h, and a significant increase in plasma LPS binding protein occurred at 3 h. These findings suggest that PVN activation induced by the lower dose of LPS is related primarily to increases in activity of the HPA axis, whereas the higher dose of LPS more widely activates autonomic regulatory centers including the PVN and also stimulates changes in sympathetic output and hypothalamic AVP synthesis. Activation of the PVN by i.c.v. LPS likely occurs through both central and systemic routes. Differential neuronal activation in the PVN is functionally related to autonomic/endocrine responses elicited by brain inflammation.

Neurons in the rat medulla oblongata containing neuropeptide Y-, angiotensin II-, or galanin-like immunoreactivity project to the parabrachial nucleus.

Projections from the medulla to the parabrachial complex of the rat were examined for their content of neuropeptide Y-, angiotensin II- or galanin-like immunoreactivity using combined retrograde tracing and immunohistochemical techniques. Rhodamine-labelled latex microspheres were stereotaxically injected into discrete nuclei of the parabrachial complex. After survival of two to five days, colchicine (100 micrograms in 10 microliters saline) was injected into the cisterna magna. One day later, rats were perfused and the brainstems were prepared for visualization of the retrograde tracer and immunoreactivity of one of the three peptides. Retrograde labelling verified that the area postrema, nucleus of the tractus solitarius, caudal spinal nucleus of the trigeminal nerve, parvocellular reticular nucleus, and ventrolateral medulla including the rostral ventrolateral medulla and nucleus paragigantocellularis project to the lateral parabrachial and Kölliker-Fuse nuclei. While most projections were primarily ipsilateral, a small proportion of the projections from the ventrolateral medulla was bilateral. Neurons containing neuropeptide Y-like immunoreactivity were found in the caudal and intermediate nucleus of the tractus solitarius, dorsal to the lateral reticular nucleus and in the nucleus paragigantocellularis. After bilateral microsphere injections into the lateral parabrachial and Kölliker-Fuse nuclei, double-labelled neurons were found dorsal to the lateral reticular nucleus of caudal and intermediate medullary levels, at the ventral surface of the medulla at intermediate levels and in the nucleus paragigantocellularis at rostral levels. Neurons with angiotensin II-like immunoreactivity were observed at the dorsomedial border of the caudal and intermediate nucleus of the tractus solitarius, in the area postrema and in the lateral reticular nucleus and nucleus paragigantocellularis. Of these neurons, small numbers in the nucleus of the tractus solitarius and ventrolateral medulla also projected to the lateral parabrachial and Kölliker-Fuse nuclei. Neurons containing galanin-like immunoreactivity were found in the caudal nucleus of the tractus solitarius, the area postrema, the spinal trigeminal nucleus, the raphe nuclei (pallidus and obscurus), the nucleus paragigantocellularis and dorsal to the lateral reticular nucleus. Of these cells, double-labelled neurons were found in the commissural and medial subdivisions of the caudal nucleus of the tractus solitarius and in the rostral ventrolateral medulla including the ventral surface and the nucleus paragigantocellularis. The results suggest that neuropeptide Y, angiotensin II and galanin may serve as neurochemical messengers in pathways from the medulla to the parabrachial complex. The location of double-labelled neurons suggests that the information relayed by these neurons is related to autonomic activity.

Effects of renal denervation and reinnervation on ganglionic gene expression of neurotransmitter proteins and c-fos in rat.

To investigate the molecular basis for reno-renal interactions, Northern blot analyses of sympathetic ganglia were used to study the changes in levels of mRNA encoding tyrosine hydroxylase (TH), neuropeptide Y (NPY), and c-fos at 4, 14, 21, and 56 days after denervation of the left kidney, and of c-fos mRNA at 1 and 4 h after denervation. Ganglia included in the study were right and left paravertebral chain ganglia (PVG, T11 to L2), celiac-mesenteric plexus (CMP), and right and left superior cervical ganglia (SCG). Levels of TH mRNA in the left PVG and CMP were decreased at 4 and 14 days compared to controls. Levels were elevated at 21 days and similar to control levels at 56 days. In the right PVG, TH mRNA levels were elevated at 4 and 14 days, diminished from this elevated level at 21 days, and similar to control levels at 56 days. No differences were found in TH mRNA levels of left or right SCG compared to controls. In long-term experiments (days), no differences in NPY or c-fos mRNA levels were found in any of the ganglia from experimental rats compared to controls. Levels of c-fos mRNA in the left PVG and CMP were decreased at 1 hour compared to control levels. By 4 h, differences in mRNA levels were no longer apparent. In the right PVG, c-fos mRNA levels were elevated at 1 hour and no longer different from control levels at 4 h. No differences were found in c-fos mRNA levels of left or right SCG compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiovascular responses to subseptic doses of endotoxin contribute to differential neuronal activation in rat brain.

The contribution of cardiovascular activity in the early central responses to systemic inflammation was assessed in rats following intravenous administration of subseptic doses of lipopolysaccharide (LPS). LPS at 12.5 microg/kg increased heart rate (HR) but did not alter mean arterial pressure (MAP), and induced interleukin-1 beta (IL-1 beta) gene expression at 1 h in circumventricular organs (CVOs), choroid plexus, meninges, blood vessels, and pituitary gland. IL-1 beta mRNA levels were attenuated at 2 h in most regions studied. LPS at 50 microg/kg caused a biphasic change in MAP, increased HR, increased levels of arginine vasopressin heteronuclear RNA in the hypothalamic paraventricular nucleus (PVN), and induced IL-1 beta gene expression in the nucleus of the solitary tract (NTS) at 1 h. LPS (both doses) induced Fos-like immunoreactivity (FLI) in the area postrema, organum vasculosum of the lamina terminalis, NTS, preoptic area, supraoptic nucleus, and PVN at 1 h. In the PVN, neurons with FLI were found primarily in the dorsal and dorsal medial parvocellular divisions after 12.5 microg/kg of LPS whereas neurons with FLI were found throughout the PVN after 50 microg/kg of LPS. After 2 h, FLI was widespread throughout the brain. Plasma ACTH levels were elevated at 1 and 2 h in response to both doses of LPS, and levels of CRF mRNA were increased after 2 h in the parvocellular PVN. Our results reveal that central responses to increasing doses of LPS show different patterns which are related to activation of distinct immune and viscerosensory pathways, and that cardiovascular responses contribute to early neuronal activation as LPS concentrations are increased.

Increased gene expression of neuronal nitric oxide synthase in brain of adult spontaneously hypertensive rats.

Neuronal nitric oxide is hypothesized to participate in regulation of autonomic function by decreasing sympathetic output to the periphery. This hypothesis predicts that gene expression of neuronal nitric oxide synthase is increased during states of heightened sympathetic activity. To test the hypothesis, we measured gene expression in the spontaneously hypertensive rat (SHR), a genetic model of hypertension in which sympathetic activity is correlated with increasing pressure. SHRs and two strains of control rats (Wistar-Kyoto [WKY] and Sprague-Dawley [SD]) at 4 weeks (pre-hypertensive) and 14 weeks (established hypertension) of age were used to measure gene expression in hypothalamus, dorsal pons, dorsal medulla, rostral ventrolateral medulla, and caudal ventrolateral medulla. Semi-quantitative reverse transcription-polymerase chain reactions and in situ hybridization were used to measure changes in neuronal nitric oxide synthase mRNA. No significant differences were found in any of the areas studied among the three strains of rats in the 4-week rats. At 14 weeks significant increases in gene expression were found in the hypothalamus (73% compared to WKYs, 104% compared to SDs), dorsal medulla (31% and 45%), and caudal ventrolateral medulla (24% and 27%) of SHRs. In situ hybridization revealed that neurons expressing the synthase gene in the hypothalamus were found primarily in the paraventricular (both parvo- and magnocellular divisions) and supraoptic nuclei. These data show that gene expression of neuronal nitric oxide synthase is increased in central autonomic centers in animals with increased sympathetic activity and they support the hypothesis that nitric oxide plays an important role in maintenance of homeostatic balance through modulation of sympathetic activity.

Electrical stimulation of the central nucleus of the amygdala induces fos-like immunoreactivity in the hypothalamus of the rat: a quantitative study.

The effect of electrical stimulation of an important forebrain autonomic structure, the central nucleus of the amygdala (CNA), on c-fos expression in three hypothalamic nuclei was studied in rat with immunocytochemistry to reveal the protein (Fos) encoded by the immediate early gene (IEG). Image analysis was used to quantify the Fos immunoreactive neurons within the supraoptic (SON), paraventricular (PVN), and arcuate (AN) nuclei. Stimulation for 60 min induced a statistically significant increase of the number of Fos immunoreactive neurons in all three nuclei ipsilateral to the CNA stimulation site. Double immunocytochemical staining (Fos and vasopressin or Fos and oxytocin) was employed to evaluate the participation of different subpopulations of neurons within the SON and PVN in response to CNA stimulation. In the SON, the increased number of Fos immunoreactive nuclei following the stimulation was observed in the vasopressin and oxytocin-secreting cells within this nucleus. In the PVN, the increase in the number of Fos immunoreactive neurons was predominantly within the parvocellular compartment. These studies demonstrate that IEG expression in hypothalamic neurons can be evoked as a result of afferent stimulation from the CNA. Activation of peptide- and hormone-containing neurons within the SON, PVN and AN, through mono- or multisynaptic pathways, may play a role in hormonal and autonomic responses.