Cardiovascular effects of substance P and capsaicin microinjected into the nucleus tractus solitarii of the rat

This report deals with the effect of substance P (SP) and capsaicin on blood pressure and heart rate after administration into different sites of the nucleus tractus solitarii (NTS) of urethane-anesthetized rats. Microinjection of SP at 6 different coordinates throughout the NTS showed 3 sites where SP administration evoked changes in blood pressure and heart rate. The most sensitive sites where application of SP into the NTS evoked dose-dependent hypotension and bradycardia were at the level of the posterior tip of the area postrema (zero level) and at the level of the obex. Capsaicin evoked dose-dependent hypotension and bradycardia at the same sites. These results further support the possibility that SP may be a neurotransmitter or neuromodulator of baroreceptor afferents in the NTS.     

[H]substance P binding in the intermediolateral cell column and striatum of the rat

[³H]substance P binding was studied in the intermediolateral cell column and striatum in the rat using slide-mounted sections. The intermediolateral cell column had a single high affinity binding component with a dissociation constant,K_d = 1.45 nM and the number of sites,B_max= 18.1fmol per mg protein. The striatum had aK_d = 0.77nM and aB_max= 23.5fmol per mg protein. The relative potency of various substance P-like tachykinins in displacing [³H]substance P suggested that both these areas may contain a substance P-P (for physalaemin) receptor subtype. (d-Pro⁴,d-Trp^7,9)substance P(4–11), a substance P antagonist, has a relatively low affinity (micromolar range) in both these areas.     

Cardiovascular responses to microinjection of AMPA into the rostral commissural nucleus tractus solitarius of awake rats

Previous studies have shown that microinjection of NMDA or non-NMDA receptor agonists into the nucleus tractus solitarius of anesthetized rats produces bradycardia and hypotension. In the present study, we evaluated the autonomic components of the cardiovascular responses to the microinjection of alpha-amine-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) into the nucleus tractus solitarius of awake rats. AMPA (0.05 nmol 50 nl(-1)) was microinjected into the nucleus tractus solitarius before and at 2, 10, 30 and 180 min after intravenous injection of methylatropine (2 mg kg(-1)). The control response to the microinjection of AMPA into the lateral aspect of the commissural nucleus tractus solitarius consisted of bradycardia and hypotension, (-190+/-20 bpm and -49+/-7 mm Hg), which were effectively blocked at 2 min (-5+/-2 bpm and -4+/-5 mm Hg), 10 min (-3+/-2 bpm and -6+/-3 mm Hg) and 30 min (-9+/-4 bpm and -5+/-5 mm Hg) after methylatropine. Although the bradycardic and hypotensive responses remained significantly reduced 180 min after injection of methylatropine, the responses tended to normalize. The data show that microinjection of AMPA into the rostral commissural nucleus tractus solitarius of awake rats produced intense bradycardia essentially mediated by parasympathetic excitation, which was the cause of the concomitant hypotensive response.     

Cardiovascular effects of substance P peptides in the nucleus of the solitary tract

Microinjection of the neuropeptide substance P (SP) into the baroreceptor portions of the nucleus of the solitary tract (NTS) caused a dose-dependent decrease in blood pressure (BP) and heart rate (HR), consistent with the putative role for SP as a transmitter in the baroreceptor reflex arc. In contrast, SP elevated BP and HR when microinjected into the adjacent area postrema. Structure-activity studies of effects of SP in the NTS revealed that an aminoterminal heptapeptide fragment of SP could fully reproduce the depressor and bradycardic effects of SP. In contrast, a carboxyterminal hexapeptide fragment of SP significantly elevated both BP and HR. The structural requirements for aminoterminal fragment effects were quite specific in terms of peptide length and sensitivity to D-amino acid substitutions. These findings are consistent with a role for SP as a baroreceptor reflex transmitter and suggest, furthermore, that this action is mediated by the aminoterminal region of SP.     

Cardiovascular responses to hypocretin-1 in nucleus ambiguus of the ovariectomized female rat

Objective: Several studies reported that the levels of proinflammatory cytokines such as TNF-, IL-1β, IL-6, and IL-8 are elevated in the cerebrospinal fluid (CSF) of patients after subarachnoid hemorrhage (SAH). Cytokines in CSF may contribute to the development of vasospasm and cerebral ischemia. In the present study, we investigated the possible cytotoxic effects of these cytokines on cultured cerebral microvascular endothelial cells. Method: The effects of TNF-, IL-1β, IL-6, and IL-8 were tested using cell viability assay, DNA fragmentation analysis (DNA laddering), Western blot analysis (Anti-poly-(ADP-ribose) polymerase [PARP] antibody), and caspase-3 activity. Results: TNF- and IL-1β, but not IL-6 or IL-8, caused cell detachment in a dose-dependent manner (p<0.05). TNF- (200 pg/ml) and IL-1β (150 pg/ml) produced DNA ladders at 24–72 h. TNF- but not IL-1β cleaved the PARP from 116- to 85-kDa fragments and enhanced caspase-3 activity at 24–72 h after incubation with endothelial cells. Caspase-3 inhibitor at 10 μmol/l significantly prevented TNF--induced cell detachment (p<0.05). Discussion: TNF- induces apoptosis in cultured cerebral endothelial cells through the cleavage of caspase-3. IL-1β decreases the adherent cells, produces DNA ladders, but fails to cleave PARP or increase caspase-3 activity. IL-1β may induce apoptosis in cerebral endothelial cells through different pathway from that of TNF-.     

Enkephalins, substance P and acetylcholine microinjected into the nucleus ambiguus elicit vagal bradycardia in rats.

Little is known about putative transmitters in the nucleus ambiguus (NA) mediating parasympathetic control of the heart, although Met-enkephalin (m-ENK), Leu-enkephalin (l-ENK), substance P (SP) and acetylcholine (Ach) have been detected in the cell bodies and fibers of this nucleus. The effects of these substances on arterial pressure (AP) and heart rate (HR) were studied by microinjecting them (4-20 nl) into the NA. Experiments were done in 26 spinal (high cervical) rats that were anesthetized with urethane and artificially ventilated. L-Glutamate (GLU) was microinjected into the right NA to identify the location of cell bodies from which decreases in HR and AP could be elicited. m-ENK, l-ENK, SP or Ach was then microinjected into these sites. Microinjection of 1 nmol of GLU elicited significant decreases in HR (-72.2 +/- 9.7 bpm, n = 15) which were not accompanied by significant decreases in mean AP. Microinjection of m-ENK (15-200 pmol; n = 7), l-ENK (15-200 pmol; n = 6), SP (0.9-15 pmol; n = 7) and Ach (2.0-20 pmol; n = 7) into the NA decreased HR in a dose-dependent manner but did not affect AP. The magnitudes of HR responses to m-ENK, l-ENK, SP and Ach were smaller but of longer duration than the changes in HR to microinjection of GLU. These results suggest a physiological role for GLU, enkephalins, SP and Ach in the vagal control of HR mediated by the NA.     

Excitatory actions of substance P in the rat lateral posterior nucleus

The lateral posterior nucleus (LP) receives inputs from both neocortex and superior colliculus (SC), and is involved with integration and processing of higher‐level visual information. Relay neurons in LP contain tachykinin receptors and are innervated by substance P (SP)‐containing SC neurons and by layer V neurons of the visual cortex. In this study, we investigated the actions of SP on LP relay neurons using whole‐cell recording techniques. SP produced a graded depolarizing response in LP neurons along the rostro‐caudal extent of the lateral subdivision of LP nuclei (LPl), with a significantly larger response in rostral LPl neurons compared with caudal LPl neurons. In rostral LPl, SP (5–2000 nm ) depolarized nearly all relay neurons tested (> 98%) in a concentration‐dependent manner. Voltage‐clamp experiments revealed that SP produced an inward current associated with a decreased conductance. The inward current was mediated primarily by neurokinin receptor (NK)₁ tachykinin receptors, although significantly smaller inward currents were produced by specific NK₂ and NK₃ receptor agonists. The selective NK₁ receptor antagonist RP67580 attenuated the SP‐mediated response by 71.5% and was significantly larger than the attenuation of the SP response obtained by NK₂ and NK₃ receptor antagonists, GR159897 and SB222200, respectively. The SP‐mediated response showed voltage characteristics consistent with a K⁺ conductance, and was attenuated by Cs⁺, a K⁺ channel blocker. Our data suggest that SP may modulate visual information that is being processed and integrated in the LPl with inputs from collicular sources.     

Cardiovascular effects of noradrenaline microinjection in the bed nucleus of the stria terminalis of the rat brain

The bed nucleus of the stria terminalis (BST) is a limbic structure involved in regulating the hypothalamic-pituitary-adrenal axis as well as in central cardiovascular control. We report here on cardiovascular effects caused by microinjection of noradrenaline (NA) in the BST of the rat brain and the peripheral mechanisms involved in their mediation. Injection of NA (3, 7, 10, 15, 30, or 45 nmol in 100 nl) in the BST of unanesthetized rats caused long-lasting dose-related pressor and bradycardiac responses. No responses were observed when the dose of 10 nmol NA was microinjected into surrounding structures, such as the anterior commissure, the stria terminalis, the fornix, and the internal capsule, indicating a predominant action at the BST. Additionally, microinjection of 50 nmol tyramine, an indirectly acting sympathomimetic amine, caused similar pressor response, indicating local NA release in the BST. Responses to NA microinjection in the BST were markedly reduced in urethane-anesthetized rats, favoring the idea of a central action without significant leakage to the peripheral circulation. The pressor response was potentiated by i.v. pretreatment with the ganglion blocker pentolinium and blocked by i.v. pretreatment with the selective V(1)-vasopressin antagonist dTyr(CH(2))(5)(Me)AVP, suggesting its mediation by vasopressin release into circulation. The bradycardiac response to NA microinjected into the BST was also abolished by pretreatment with the vasopressin antagonist, indicating its reflex origin. In conclusion, results indicate that microinjection of NA into the BST evokes pressor responses, which are mediated by acute vasopressin release.     

Peptidergic innervation of the cremaster nucleus. I. A sexually dimorphic population of substance P-containing intraspinal neurons exists in the substance P pathway to the rat cremaster nucleus.

The cremaster nucleus (CN) lies in the lumbar spinal cord and is sexually dimorphic: the male CN contains three times as many motoneurons as the female. The substance P (SP) innervation of the CN is also sexually dimorphic with males receiving a very prominent innervation which is greatly diminished in females. These investigations examined SP-containing neurons located in the ventral half of lamina IV and the lateral aspects of laminae V, VII, and IX, in lumbar spinal levels 1,2. SP-containing intraspinal neurons in these laminae are at least three times as numerous in males than females. This provides the first demonstration of a sexually dimorphic population of spinal neurons which is not motor or preganglionic in nature. These SP-containing interneurons are found within, or adjacent to, the SP-containing fibers which constitute the massive SP pathway to the male CN. Processes of these SP-containing neurons were observed to contribute to the formation of the SP pathway to the male CN. The immunohistochemically demonstrable presence of these lumbar 1,2, laminae IV-IX, SP-containing neurons validates former studies which suggested their existence (Gibson et al., Brain Research, 301 (1984) 243-251; Uda et al., Neurosci. Lett., 57 (1985) 185-190).     

Cardiovascular responses to glutamate microinjection in the dorsomedial periaqueductal gray of unanesthetized rats

The periaqueductal gray area (PAG) is a mesencephalic area involved in cardiovascular modulation. Glutamate (L‐Glu) is an abundant excitatory amino acid in the central nervous system (CNS) and is present in the rat PAG. Moreover, data in the literature indicate its involvement in central blood pressure control. Here we report on the cardiovascular effects caused by microinjection of L‐Glu into the dorsomedial PAG (dmPAG) of rats and the glutamatergic receptors as well as the peripheral mechanism involved in their mediation. The microinjection of L‐Glu into the dmPAG of unanesthetized rats evoked dose‐related pressor and bradycardiac responses. The cardiovascular response was significantly reduced by pretreatment of the dmPAG with a glutamatergic M‐methyl‐D‐aspartate (NMDA) receptor antagonist (LY235959) and was not affected by pretreatment with a non‐NMDA receptor antagonist (NBQX), suggesting a mediation of that response by the activation of NMDA receptors. Furthermore, the pressor response was blocked by pretreatment with the ganglion blocker pentolinium (5 mg/kg, intravenously), suggesting an involvement of the sympathetic nervous system in this response. Our results indicate that the microinjection of L‐Glu into the dmPAG causes sympathetic‐mediated pressor responses in unanesthetized rats, which are mediated by glutamatergic NMDA receptors in the dmPAG. © 2012 Wiley Periodicals, Inc.     

Cardiovascular responses to noradrenaline microinjection in the ventrolateral periaqueductal gray of unanesthetized rats

The periaqueductal gray area (PAG) is a mesencephalic area involved in cardiovascular modulation. Noradrenaline (NA), a neurotransmitter involved in central blood pressure control, is present in the rat PAG. We report here on the cardiovascular effects caused by NA microinjection into the ventrolateral PAG (vlPAG) of unanesthetized rats and the peripheral mechanism involved in their mediation. NA microinjection in the vlPAG of unanesthetized rats evoked dose-related pressor and bradycardiac responses. No significant cardiovascular responses were observed in urethane-anesthetized rats. The pressor response was potentiated by pretreatment with the ganglion blocker pentolinium (5 or 10 mg/kg, intravenously). Pretreatment with the vasopressin antagonist dTyr(CH2)5 (Me)AVP (50 microg/kg, intravenously) blocked the pressor response evoked by the NA microinjection into the vlPAG. Additionally, circulating vasopressin content was found to be significantly increased after NA microinjection in the vlPAG. The results suggest that activation of noradrenergic synapses within the vlPAG modulates vasopressin release in unanesthetized rats.     

Interaction between cholinergic neurons and substance P or calcitonin gene-related peptide terminals of the rat sacral intermediolateral nucleus: double immunostaining at the light and electron microscopic levels

The relationships both between cholinergic neurons and substance P (SP) and between cholinergic neurons and calcitonin gene-related peptide (CGRP) terminals were examined in the rat sacral intermediolateral nucleus at the light and electron microscopic levels by means of double-immunostaining methods. Cholinergic neurons were labeled by a monoclonal antibody to choline acetyltransferase (CAT) with the avidin-biotin technique and stained bluish-green by indolyl-beta-galactoside reaction products with beta-galactosidase as a marker. On the same sections, SP or CGRP fibers were labeled by polyclonal antisera to SP or CGRP after application of the peroxidase-antiperoxidase (PAP) method and stained brown by the p-dimethylaminoazobenzene (DAB) reaction. After embedding in Epon, light and electron microscopic sections were examined. At the light microscopic level, CGRP-like immunoreactive (CGRP-I) fibers and SP-like immunoreactive (SP-I) fibers were found to pass through the lateral edge of the dorsal horn and then into the dorsal region of the sacral intermediolateral nucleus. In addition, SP-I fibers also extend from the dorsolateral funiculus into the entire sacral intermediolateral region. At the electron microscopic level, many axosomatic and axodendritic synapses were found between CAT-I structures and SP-I terminals in the intermediolateral nucleus, whereas most of the CGRP-I terminals in this area made axodendritic synapses with CAT-I dendrites. These results indicate that cholinergic neurons in the sacral intermediolateral nucleus receive direct synaptic input from SP-I and CGRP-I terminals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood pressure modulation by substance P in the rat nucleus tractus solitarius

Substance P in a dose of 0.1-10 ng injected into the nucleus tractus solitarii (NTS) of the rat caused hypotension, bradycardia and apnea whereas a dose of 100 ng led to no response. A substance P antagonist injected into the NTS abolished the cardiovascular responses to substance P. The antagonist alone increased blood pressure and heart rate. The data suggest a role for substance P in the cardiovascular regulation by the NTS.     

A substance P projection from the superior colliculus to the parabigeminal nucleus in the rat and hamster

Immunocytochemical staining with antisera directed against substance P (SP) demonstrated the existence of numerous immunoreactive neurons throughout the mediolateral and rostrocaudal extents of the stratum griseum superficiale (SGS) of the superior colliculus (SC) of both rat and hamster. In both of these species, very dense SP-like immunoreactivity (SPLI) was also visible in the parabigeminal nucleus. Combination of retrograde tracing with True blue or Fluorogold and immunocytochemistry demonstrated that SP-positive SC neurons projected to the parabigeminal nucleus in both hamster and rat. Retrogradely labelled and double-labelled cells were most numerous in the rostromedial portion of the SC and rare in the caudal portion of the colliculus. Destruction of the superficial layers of the SC resulted in a virtually complete loss of SPLI in the ipsilateral parabigeminal nucleus in both species. SPLI was also visible in two other targets of the superficial SC laminae: the intergeniculate leaflet and the ventral lateral geniculate nucleus. Ablation of the dorsal SC laminae did not reduce SPLI in either of those nuclei. Our results thus indicate that at least some tectoparabigeminal neurons in hamster and rat contain SPLI and further that the SC appears to the sole source of SP-positive input to this nucleus.     

Cardiovascular responses to microinjection of L-glutamate into the NTS in AV3V-lesioned rats

The excitatory amino acid L-glutamate injected into the nucleus of the solitary tract (NTS) in unanesthetized rats similar to peripheral chemoreceptor activation increases mean arterial pressure (MAP) and reduces heart rate. In this study, we investigated the effects of acute (1 day) and chronic (15 days) electrolytic lesions of the preoptic-periventricular tissue surrounding the anteroventral third ventricle (AV3V region) on the pressor and bradycardic responses induced by injections of L-glutamate into the NTS or peripheral chemoreceptor activation in unanesthetized rats. Male Holtzman rats with sham or electrolytic AV3V lesions and a stainless steel cannula implanted into the NTS were used. Differently from the pressor responses (28+/-3 mm Hg) produced by injections into the NTS of sham-lesioned rats, L-glutamate (5 nmol/100 nl) injected into the NTS reduced MAP (-26+/-8 mm Hg) or produced no effect (2+/-7 mm Hg) in acute and chronic AV3V-lesioned rats, respectively. The bradycardia to l-glutamate into the NTS and the cardiovascular responses to chemoreflex activation with intravenous potassium cyanide or to baroreflex activation with intravenous phenylephrine or sodium nitroprusside were not modified by AV3V lesions. The results show that the integrity of the AV3V region is essential for the pressor responses to L-glutamate into the NTS but not for the pressor responses to chemoreflex activation, suggesting dissociation between the central mechanisms involved in these responses.     

Cardiovascular response to renin substrate microinjection into the central nucleus of the amygdala of rats

Central nucleus of the amygdala is involved in cardiovascular regulation. Although most components of the renin-angiotensin system have been found to be distributed in amygdala, renin expression in brain has remained controversial. This work was undertaken to elucidate the extent of renin presence in this nucleus. A cannula was implanted bilaterally into the central nucleus of the amygdala. Mean arterial pressure and heart rate were directly measured via indwelling femoral artery cannula post bilateral intra central nucleus of the amygdala microinjection of renin substrate. Renin substrate microinjection dose-dependently increased mean arterial pressure and heart rate, whereas captopril, saralasin and losartan pretreatment inhibited these effects. The results suggest the presence of local renin or similar proteases in this nucleus.     

Colocalization of substance P or enkephalin in serotonergic neuronal afferents to the hypoglossal nucleus in the rat

The serotonergic innervation of the hypoglossal nucleus originates from the caudal raphe nuclei. Non-serotonergic neurons in the caudal raphe nuclei also project to the hypoglossal nucleus. We employed a triple-fluorescence technique to determine whether the substance P- or the enkephalin-containing neurons in the caudal raphe nuclei that projected to the hypoglossal nucleus also contained serotonin. Rhodamine latex microspheres were injected into the hypoglossal nucleus, and then serotonin and peptide dual-immunofluorescence was performed to colocalize perikarya containing serotonin, substance P, and rhodamine microspheres; or perikarya containing serotonin, enkephalin, and rhodamine microspheres. Our results demonstrate that most substance P-containing neuronal afferents to the hypoglossal nucleus colocalize serotonin. In contrast, few enkephalin-containing neuronal afferents to the hypoglossal nucleus also contain serotonin. These data suggest that substance P projections to the hypoglossal nucleus are a subset of serotonergic projections and that limited overlap exists between the populations of enkephalinergic and serotonergic neuronal afferents to the hypoglossal nucleus. Either substance P- or enkephalin-containing somata account for a very small proportion of non-serotonergic caudal raphe projections to the hypoglossal nucleus. Finally, these data demonstrate the medial tegmental field origins of the substance P projections and the enkephalin projections to the hypoglossal nucleus. J. Comp. Neurol. 391:491–505, 1998. © 1998 Wiley-Liss, Inc.     

Substance P and substance K in the rat hypothalamus following monosodium glutamate lesions of the arcuate nucleus.

Adult rats treated neonatally with monosodium glutamate (MSG) exhibit lesions in the arcuate nucleus of the hypothalamus. Following MSG lesioning, dopamine content in median eminence/arcuate nucleus (ME/AN) tissue extracts declined by 60-70%. Substance P (SP) content as determined by radioimmunoassay was significantly decreased in the paraventricular nucleus (PVN) (531 +/- 30 pg, mean +/- SEM) compared to controls (871 +/- 110 pg) but was unchanged in ME/AN extracts. Substance K (SK) content decreased to 257 +/- 20 pg in the PVN of lesioned animals compared to controls (367 +/- 31 pg) and the ME/AN content of SK was also significantly decreased (236 +/- 36 pg compared to control levels of 619 +/- 65 pg). The CRF-41 content of the PVN and ME/AN was unchanged by MSG lesioning, indicating that these areas are not affected by MSG. The partial depletion of SP and SK in the PVN following MSG treatment provides evidence that at least some of the neurokinin content of the PVN may originate in cell bodies of the arcuate nucleus. However, the lack of response of ME/AN SP to MSG treatment may suggest that the arcuate nucleus is not the major source of SP in the median eminence.     

Marginal topography of neurons expressing the substance P receptor in the rat suprachiasmatic nucleus

Neurons expressing the substance P (SP) receptor (NK1 receptor) in the suprachiasmatic nucleus of the hypothalamus (SCN) have been topographically identified using radioactive in situ hybridization histochemistry. In the anterior hypothalamic area, clustered labeled neurons of small size and exhibiting low levels of gene expression are observed exclusively at the dorsolateral margin of the SCN, straddling cytoarchitectural boundaries of the nucleus. The marginal topography of neurons putative target of a SP-containing retinal input to the ventral SCN indicates that their dendrites bearing the receptor extend towards the retinorecipient part of the nucleus, where they can be modulated by overlapping inputs from the intergeniculate leaflet and the raphé. Eventual interactions between glutamatergic and putative tachykininergic retinal pathways for a coherent photic control of circadian rhythms may therefore occur mainly via intrinsic neuronal connections between their distinct target populations. In addition, since glutamate and SP induce electrophysiological responses in ventrolateral neurons with no interactive effect, neurons integrating both chemical signals, subsequently to their modulation by several influences, may be not located within the ventrolateral SCN. Alternatively but not exclusively, marginal neurons could be a target of SP-containing neurons within the SCN or nearby the nucleus, or from ascending projections from the raphé where serotonin and SP colocalize. The marginal topography of neurons expressing the SP receptor supports the view of the involvement of neurons located in the vicinity of the nucleus in the regulation of circadian rhythms.     

Monoamine oxidase in the intermediolateral nucleus of the thoracic spinal cord of the rat. A histochemical study

We examined monoamine oxidase (MAO) activity in the intermediolateral nucleus (IML) of the rat thoracic spinal cord by histochemistry with tyramine as a common substrate for both MAO types A and B. Light microscopy showed MAO activity in neuronal cell bodies, processes, and varicosities. Electron microscopic examination showed both MAO-positive and -negative neuronal cell bodies. In the stained cell bodies, histochemical reaction products were localized in the cytoplasm showing a selective association with mitochondrial outer membranes. MAO-positive axon terminals were often found in contact with MAO-negative neurons but only occasionally with MAO-positive neurons. MAO histochemistry in the IML was also performed using serotonin (a MAO type A preferential substrate) and beta-phenylethylamine (a MAO type B preferential substrate). Light microscopy identified MAO activity for serotonin in a plexus of varicosities but not in any neuronal cell bodies. The activity for beta-phenylethylamine was detected frequently in neuronal cell bodies but rarely in varicosities. Our findings indicate that two groups of IML neurons can be chemically distinguished, one contains MAO type B while the other lacks both MAO types A and B. In addition, many axon terminals contain MAO type A but only a few fibers include MAO type B in the IML.