CNS projections to the pterygopalatine parasympathetic preganglionic neurons in the rat: a retrograde transneuronal viral cell body labeling study.

The retrograde transneuronal viral cell body labeling method was used to study the CNS nuclei that innervate the parasympathetic preganglionic neurons which project to the pterygopalatine ganglion. Small injections of a suspension of pseudorabies virus (PRV) were made in the pterygopalatine ganglion of rats and after 4 days their brains wer e processed for immunohistochemical detection of PRV. Some of the tissues were stained with a dual immunofluoresence method that permitted the visualization of PRV and neurotransmitter enzyme or serotonin immunoreactivity in the same cell. Retrograde cell body labeling was detected in the ipsilateral ventrolateral medulla oblongata in the region that has been termed the superior salivatory nucleus. This area was the same region that was retrogradely labeled after Fluoro-Gold dye injections in the pterygopalatine ganglion. Retrograde transneuronally infected cell bodies that provide putative afferent inputs to the pytergopalatine parasympathetic preganglionic neurons were mapped throughout the brain. In the medulla oblongata, transneuronally labeled neurons were seen in the nucleus tractus solitarii, dorsomedial part of the spinal trigeminal nucleus and gigantocellular reticular nucleus. In most experiments, some A1 catecholamine cells and serotonin neurons of the raphe magnus, raphe pallidus, raphe obscurus, and parapyramidal nuclei were labeled. In the pons, labeled cells were found in the parabrachial nucleus. A5 catecholamine cell group, and non-catecholamine part of the subcoeruleus region. In the midbrain, cell body labeling was located in the central gray matter and retrorubral field. In the diencephalon, labeling was found mainly in the hypothalamus. The areas included the lateral hypothalamic area, lateral preoptic area, dorsomedial and paraventricular hypothalamic nuclei, and ventral zona incerta. Contralateral second order cell body labeling was seen in the tuberomammillary nucleus of the hypothalamus. Some of these cells were histidine decarboxylase-immunoreactive. In the forebrain, the bed nucleus of the stria terminalis, substantia innominata, and an area of the cerebral cortex called the amygdalopiriform transition zone were labeled.

L-glutamate mapping of cardioreactive areas in the rat posterior hypothalamus.

The posterior hypothalamus has long been regarded as a CNS region that provides a sympatho-excitatory influence on the cardiovascular system and functions in thermoregulation as a heat-producing center. These ideas have been based on data derived from electrical stimulation and lesion experiments. These methods are now regarded as inadequate for accurate localization of CNS functions. In order to re-examine the function of the posterior hypothalamus, a chemical stimulation study was performed. Microinjections of the excitatory amino acid L-glutamate were made in the posterior hypothalamus of pentobarbital-anesthetized rats. This method was used in combination with autoradiography to localize [3H]glutamate, which was included with the injectate. No pressor responses were elicited from any site within the posterior hypothalamus. In contrast, chemical stimulation of the posterior periventricular hypothalamus produced large decreases in blood pressure (delta BP = 25 mm Hg) and in heart rate (delta HR = 30 bpm). Injections in the posterior hypothalamic nucleus elicited small reductions in blood pressure and heart rate. Injections in the dorsal hypothalamic area produced a similar small response. Injections ventral to the periventricular zone were also weakly reactive, but a significant elevation in rectal temperature was seen. To summarize, the most cardioresponsive area was within the periventricular zone caudal to the posterior hypothalamic nucleus and was situated near the fasciculus retroflexus.

Cardiovascular effects produced by L-glutamate stimulation of the lateral hypothalamic area.

L-Glutamate microinjections into the tuberal region of the lateral hypothalamic area (LHAt) caused a fall in blood pressure and heart rate in pentobarbital-anesthetized rats. The bradycardia was mediated by both beta-adrenergic and muscarinic mechanisms as demonstrated with pharmacological blockade. The hypotension was due to a decrease in cardiac output, not a decrease in total peripheral resistance. In addition, there was a reduction in coronary blood flow. If heart rate was held constant by pharmacological blockade or by electrical cardiac pacing, L-glutamate stimulation of the LHAt still caused a fall in blood pressure. When the electrically paced model was used, this hypotension was due to a fall in cardiac output. In contrast, with the pharmacological blockade of the heart, the hypotension was due to a decrease in the total peripheral resistance. The cardiac output reduction in the paced condition was not mediated solely by either beta-sympathetic or parasympathetic mechanisms as determined by pharmacological blockade. With heart rate held constant by either drugs or pacing, LHAt stimulation did not alter regional blood flow or resistance in any vascular bed, including the coronary circulation. We conclude that L-glutamate stimulation of the LHAt lowers the cardiac output and heart rate by both parasympathetic and beta-adrenergic mechanisms and elicits hypotension by lowering cardiac output in the naive and electrically paced model.

A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections.

Pseudorabies virus (PRV) injections of various sympathetic ganglia and the adrenal gland were made in rats. These produced immunohistochemically detectable retrograde viral infections of ipsilateral sympathetic preganglionic neurons (SPNs) and transneuronal infections of the specific sets of second order neurons in the spinal cord and brain that innervate the infected SPNs. Five cell groups in the brain appear to regulate the entire sympathetic outflow: the paraventricular hypothalamic nucleus (PVH), A5 noradrenergic cell group, caudal raphe region, rostral ventrolateral medulla, and ventromedial medulla. In addition, local interneurons in laminae VII and X of the spinal cord are also involved. Other CNS areas also became transneuronally labeled after infections of certain sympathetic ganglia, most notably the superior cervical and stellate ganglia. These areas include the central gray matter and lateral hypothalamic area. The zona incerta was uniquely labeled after stellate ganglion infections. The cell body labeling was specific. This specificity was demonstrated in the PVH where the neurons of the parvocellular PVH that form the descending sympathetic pathway were labeled in a topographic fashion. Finally, we demonstrate that the retrograde transneuronal viral cell body labeling method can be used simultaneously with either neuropeptide transmitter or transmitter synthetic enzyme immunohistochemistry.

CNS cell groups regulating the sympathetic outflow to adrenal gland as revealed by transneuronal cell body labeling with pseudorabies virus.

The CNS cell groups that innervate the sympathoadrenal preganglionic neurons of rats were identified by a transneuronal viral cell body labeling technique combined with neurotransmitter immunohistochemistry. Pseudorabies virus was injected into the adrenal gland. This resulted in retrograde viral infections of the ipsilateral sympathetic preganglionic neurons (T4-T13) and caused retrograde transneuronal cell body infections in 5 areas of the brain: the caudal raphe nuclei, ventromedial medulla, rostral ventrolateral medulla, A5 cell group, and paraventricular hypothalamic nucleus (PVH). In the spinal cord, the segmental distribution of virally infected neurons was the same as the retrograde cell body labeling observed following Fluoro-gold injections in the adrenal gland except there was almost a 300% increase in the number of cells labeled and a shift in cell group distribution. These results imply there are local interneurons that regulate the sympathoadrenal preganglionic neurons. In the medulla oblongata, serotonin (5-HT)-, substance P (SP)-, thyrotropin-releasing hormone-, Met-enkephalin-, and somatostatin-immunoreactive neurons of the raphe pallidus and raphe obscurus nuclei and the ventromedial medulla were infected. In the ventromedial and rostral ventrolateral medulla, immunoreactive phenylethanolamine-N-methyltransferase, SP, neuropeptide Y, somatostatin, and enkephalin neurons were infected. The A5 noradrenergic cells were labeled, as were some somatostatin-immunoreactive neurons in this area. In the were infected. The A5 noradrenergic cells were labeled, as were some somatostatin-immunoreactive neurons in this area. In the hypothalamus, tyrosine hydroxylase- and SP-immunoreactive neurons of the dorsal parvocellular PVH were infected. Only a few immunoreactive vasopressin, oxytocin, Met-enkephalin, neurotensin, and somatostatin PVH neurons were labeled.

L-glutamate stimulation of the zona incerta in the rat decreases heart rate and blood pressure.

Microinjections of L-glutamate into the zona incerta of pentobarbital anesthetized rats caused decreases in blood pressure and heart rate. The bradycardic response was reduced by approximately 70% after i.v. administration of atropine methyl nitrate. After combined muscarinic and beta-adrenergic blockade the bradycardic response was reduced to 90% of the control value. This suggests that the bradycardia was mediated primarily by activating the vagal outflow. Blood pressure decreases elicited after pharmacological blockade of the heart with both atropine and timolol were approximately 50% of the control values. This indicates that the zona incerta is also capable of altering stroke volume and/or inhibiting the sympathetic outflow controlling the peripheral blood vessels. By using an injectate containing L-glutamate mixed with [3H]L-glutamate and subsequent analysis of autoradiographic tissue sections, we have determined that the most reactive site is the region of the ventral zona incerta.

Spinal origin of sympathetic preganglionic neurons in the rat.

The segmental distribution of sympathetic preganglionic neurons (SPNs) and dorsal root ganglion cells (DRGs) was studied after Fluoro-gold injections into the major sympathetic ganglia and adrenal gland in rats. A quantitative assessment of the segmental and nuclear locations was made. Four general patterns of innervation were apparent: (1) a large number of SPNs (1000-2000/ganglion) innervate the sympathetic ganglia which control head or thoracic organs and a relatively small number of SPNs (100-400/ganglion) innervate the sympathetic ganglia controlling the gut, kidney, and pelvic organs; this difference in density of innervation probably relates to the level of fine control that can occur in these end organs by the SPNs; (2) the reverse pattern is seen in the DRG labeling where a large number of DRGs were labeled after Fluoro-gold injections into the preaortic ganglia (celiac, superior, and inferior mesenteric) and a small number were labeled after injections into the cervical sympathetic ganglia; (3) the intermediolateral cell column is the main source of SPNs except for the inferior mesenteric ganglion which is innervated predominantly by SPNs originating in the central autonomic nucleus (75%); the lateral funiculus is a source of SPNs mainly for the cervical sympathetic ganglia; and (4) each sympathetic ganglion and the adrenal gland receives a multisegmental SPN and DRG input with one segment being the predominant source of the innervation. The adrenal gland shows an intermediate position in terms of the density of SPN input (approximately 800 cells) and dorsal root input (approximately 300 cells); it has a widespread segmental input (T4-T12) with the T8 segment being the major source.

Descending noradrenergic pathways involved in the A5 depressor response.

The objective of the present study was to analyze the anatomical basis of the A5 depressor response and to test if the putative neurotransmitter noradrenaline is involved in the response. Two approaches were used; one was neuroanatomical and the other was pharmacological. First, the retrograde transport method in which two fluorescent markers (Fast blue and rhodamine microspheres) was used in combination with the indirect immunofluorescence technique to establish that A5 catecholamine neurons project to both the spinal cord and the region of the nucleus tractus solitarii (NTS). Second, we analyzed the effects of 6-hydroxydopamine (6-OHDA) lesions of the spinal cord and/or NTS area on the A5 depressor response. This response was elicited by a 80-nl microinjection of L-glutamate (500 mM) into the A5 region in pentobarbital anesthetized rats; it was characterized by a decrease in blood pressure and heart rate. After destruction of various noradrenergic terminal fields we have found that intraspinal injections of 6-OHDA caused a 30% reduction in the blood pressure component of the A5 depressor response and a transient depression of the bradycardic response. This result suggests that only a small portion of the A5 depressor response depends on the descending A5 spinal pathway. Injections of 6-OHDA into the NTS region caused a transient depression of the A5 depressor response, and by 7-14 days postinjection, the response returned to normal. After combined 6-OHDA injections into the spinal cord and NTS area, the blood pressure and heart rate components of the A5 depressor response were reduced to 80% of the control level at 3 days postinjection. By 14 days, even with severe depletion of noradrenaline in the spinal cord (96%) and a moderate depletion of noradrenaline in the NTS (50%), the A5 response was restored to about 80% of its original magnitude, suggesting some type of functional recovery occurs in this system. Third, the blood pressure decrease elicited by L-glutamate stimulation of the A5 cell group was unaffected by pharmacological blockade of the heart. In addition, this response appeared to be normal in rats that had both their autonomic supply to the heart blocked pharmacologically and their spinal cord noradrenaline levels depleted (14 days after intraspinal 6-OHDA injections). These data suggest that the major A5 depressor response operates mainly by inhibition of the sympathetic outflow involved in control of total peripheral resistance and that this system is controlled by a descending spinal pathway which probably does not use noradrenaline as a neurotransmitter.

LH-RH analogue acts as substance P antagonist by inhibiting spinal cord vasomotor responses.

Intrathecal injections of the luteinizing hormone-releasing hormone (LH-RH) analogue, [D-pGlu1-D-Phe2-D-Trp3,6]-LH-RH caused dose-dependent decreases in mean arterial blood pressure in anesthetized rats similar to those seen with [D-Pro4-D-Trp7,9]-substance P4-11. Similarly, intrathecal injections of either peptide reversed the pressor response elicited by application of kainic acid on the ventral surface of the medulla oblongata. Both analogues also had similar IC50 values (approximately 10(-5) M) for the inhibition of specific [3H]substance P binding in tissue-sections of the intermediolateral cell column. However, the LH-RH analogue failed to block substance P (SP)-induced contractions of the isolated guinea pig ileum and did not affect tail-flick withdrawal time in a thermal nociceptive test, whereas the SP analogue acted as an antagonist in both of these bioassays. These results suggest that [D-pGlu1-D-Phe2-D-Trp3,6]-LH-RH may act as an antagonist which can inhibit the sympathetic vasomotor outflow and potentially be a substance P-physalaemin (SP-P) receptor antagonist in the central nervous system but without effect on peripheral SP-P receptors.

Substance P mechanisms of the spinal cord related to vasomotor tone in the spontaneously hypertensive rat.

This report deals with substance P (SP) mechanisms involved in regulation of vasomotor tone at the spinal cord levels in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Our results indicate the following. (1) Intrathecal injections of the SP antagonist, D-Pro,D-Trp-SP cause dose-dependent decreases in mean arterial pressure and heart rate in Sprague-Dawley rats, WKYs and SHRs; the maximal blood pressure decreases are equal to those seen after cervical spinal cord transection. (2) Intrathecal injections of this antagonist into the L1 spinal level in WKYs or SHRs that had previously had their C8 spinal cords transected caused a rise in blood pressure and heart rate, suggesting that intrinsic spinal SP mechanisms are probably not involved in vasomotor tone. (3) The intermediolateral cell column region (IML) of 16-week-old WKYs and SHRs has a single high affinity and saturable binding component with approximately the same dissociation constant (Kd = 1.21 nM for WKYs; Kd = 1.25 nM for SHRs); the SHRs showed a higher number of sites (Bmax = 24.5 fmol/mg protein) than WKY rats (Bmax = 19.9 fmol/mg protein). The Kd and Bmax obtained from IML sections from 4-week-old WKYs and SHRs exhibit no differences, although their binding values with 2 nM [3H]SP are higher than those obtained from the 16-week-old animals. (4) D-Pro4,D-Trp7,9-SP4-11 has the same relatively low (micromolar range) potency for displacing [3H]SP binding in the IML of WKYs and SHRs. (5) SHRs (16 weeks old) contain 20% more SP immunoreactivity in the IML than WKY rats (834 +/- 36 pg/mg protein vs 694 +/- 50 pg/mg protein); 4-week-old rats do not show such differences. The potential significance of these results is discussed in relation to the control of vasomotor tone.

Changes in regional blood flow and cardiac output after L-glutamate stimulation of A5 cell group.

Changes in regional blood flow and cardiac output were measured by the reference organ method in pentobarbital-anesthetized rats with radioactive microspheres (15 microns) before and after chemical stimulation of the A5 cell group with the excitatory amino acid L-glutamate an agent that excites cell bodies but not fibers of passage. This stimulation caused a decrease in mean arterial pressure, heart rate, cardiac output, and calculated stroke volume. The limb skeletal muscles showed a large increase in blood flow and decrease in vascular resistance, whereas the trunk musculature showed no change in flow or resistance. The blood flow of the entire gastrointestinal tract decreased. Blood flow in the skin decreased with no change in resistance. The cardiac muscle of the ventricles showed a decrease in flow without a change in resistance. The ipsilateral half of the brain showed a decrease in blood flow, while the contralateral side showed no change. The kidneys exhibited no change in blood flow and a decrease in resistance. A5 stimulation in guanethidine-sympathectomized rats caused no change in regional blood flow. In contrast, an increase in cardiac output was observed, and the possible interpretations for this change are discussed. Rats treated with intraventricular injections of 6-hydroxydopamine showed no changes in regional blood flow or cardiac output, indicating that catecholamine neurons are involved in these responses.

Substance P antagonist inhibits vasomotor responses elicited from ventral medulla in rat.

Application of kainic acid to the ventral medulla in rats leads to increases in blood pressure and heart rate. These responses can be attenuated or reversed by infusion of the substance P antagonist--(D-Pro2,D-Trp7,9)-substance P into the spinal subarachnoid space. Kainic acid elicits normal pressor responses from rats that have been treated intracisternally with the serotonin neurotoxin 5,7-dihydroxytryptamine. These results suggest that the substance P-like neurons of the ventral medulla may play a role in maintaining vasomotor tone.