Inhibitory effect of tranilast on substance P-induced plasma extravasation in rat skin.

The effect of tranilast, an inhibitor for IgE-mediated mediator release from mast cells, on plasma extravasation induced by the intradermal injection of substance P in rats was examined. Tranilast (100 mg/kg, intraperitoneally) decreased plasma extravasation induced by substance P (10(-7)-10(-5) M). Tranilast decreased plasma extravasation induced by the amino-terminal peptide substance P1-9 (10(-6)-10(-4) M), which is active for rat mast cells, but not by the carboxy-terminal peptide substance P6-11 (10(-6)-10(-4) M), which is inactive for the mast cells. Therefore, tranilast prevents substance P-induced plasma extravasation most likely by inhibiting mast cell degranulation.     

NK1 receptors mediate tachykinin-induced plasma extravasation in the rat knee joint

The tachykinin receptor type that medicates tachykinin-induced plasma extravasation in the rat knee joint was identified by using selective antagonists as well as natural or synthetic agonists. Substance P (SP) and neurokinin (NK) A induced plasma extravasation with almost the same potency and the maximum response was obtained at 5 nmol/knee. NKB was about ten times less potent than SP or NKA. The NK₁ selective agonist, [Sar⁹, Met(O₂)¹¹]-SP, was about ten times more potent than SP, and the NK₂ selective agonist, [Nle¹⁰]-NKA_4–10, was about fifty times less potent than NK₁ agonist. The NK₃ agonist, Senktide, was totally ineffective at 0.5–50 nmol/knee. All responses induced by SP (5 nmol/knee), NKA (5 nmol/knee), NKB (50 nmol/knee), NK₁ agonsit (0.5 nmol/knee) or NK₂ agonist (25 nmol/knee) were significantly and profoundly inhibited by the NK₁ selective antagonist, RP67580, but not by the NK₂ selective antagonist, SR48968. Taken together, we conclude that tachykinin-induced plasma extravasation in the rat knee joint is mediated via NK₁ receptors.     

Chronically administered nicotine attenuates bradykinin-induced plasma extravasation and aggravates arthritis-induced joint injury in the rat.

We recently showed that acute administration of nicotine in the rat decreases bradykinin-induced plasma extravasation and that adrenal medullary-derived epinephrine, acting at a beta 2-adrenergic receptor, mediates the nicotine effect. Since agents which decrease bradykinin-induced plasma extravasation have been associated with increased joint injury in a rat model of chronic inflammation (experimental arthritis induced by subcutaneous injection of Mycobacterium butyricum) we examined the effect of chronic nicotine on both plasma extravasation and the severity of joint injury. In normal rats, bradykinin-induced plasma extravasation was decreased after nicotine administered both by repeated injection (10(-2) mg/kg, s.c., once per h for 4 h) and by continuous long-term infusion (subcutaneous mini-osmotic pump; 1.5 x 10(-3) mg/kg per h for 30 days). Nicotine-induced inhibition of bradykinin-induced plasma extravasation did not show tachyphylaxis. In rats with arthritis, chronic administration of nicotine also produced a decrease in bradykinin-induced plasma extravasation. This effect of chronic nicotine in the arthritic rats was antagonized by co-administration of hexamethonium (a nicotinic receptor antagonist), by surgical removal of the adrenal medulla, or by co-administration of ICI-118,551 (a beta 2-adrenoceptor antagonist). Chronic administration of nicotine decreased the latency to the onset of arthritis and, in a dose-dependent manner, led to an increase in the radiographic joint injury score.(ABSTRACT TRUNCATED AT 250 WORDS)     

Delta- and kappa-opioid agonists inhibit plasma extravasation induced by bradykinin in the knee joint of the rat.

We used an experimental model of neurogenic inflammation, plasma extravasation induced by bradykinin or capsaicin, to study the effect of receptor-selective opioid agonists on plasma extravasation. Plasma extravasation was induced in the knee joint of the rat by continuous perfusion of either bradykinin (160 ng/ml), an inflammatory mediator produced at sites of tissue injury, that produces plasma extravasation significantly dependent on the sympathetic postganglionic neuron, or capsaicin (5 mg/ml), a C-fiber excitotoxin, that induces plasma extravasation that is dependent on both primary afferents and sympathetic post-ganglionic neurons. When selective delta-((d-Pen2,5)-enkephalin) or kappa-(trans-3,4-dichloro-N-methyl-N[2-(- pyrolidinyl)cyclohexyl]benzeneacetamide; U50,488H) opioid agonists were perfused with bradykinin, plasma extravasation was significantly attenuated. Co-perfusion of the non-selective opioid antagonist naloxone (1 microM), reversed this opioid-induced inhibition of bradykinin-induced plasma extravasation. In contrast, co-perfusion of a selective mu-opioid agonist (Tyr-d-Ala-Gly-NMe-Phe-Gly-ol) did not reduce bradykinin-induced plasma extravasation. Tyr-d-Ala-Gly-NMe-Phe-Gly-ol was, however, able to completely inhibit the plasma extravasation produced by capsaicin. These results suggest that delta- and kappa-, but not mu-selective opioids inhibit bradykinin-stimulated plasma extravasation, while a mu-selective opioid inhibits primary afferent-dependent plasma extravasation. Therefore, inhibition of neurogenic plasma extravasation by receptor-selective opioids may depend on the relative contribution to plasma extravasation of unmyelinated afferent and sympathetic postganglionic neuron terminals. Our findings can also explain, in part, the variation in anti-inflammatory effects of receptor-selective opioids reported in different inflammatory conditions.     

Paf induces rat plasma extravasation and releases eicosanoids during anaphylaxis

The effects of anaphylaxis on vascular protein extravasation in selected tissues and on the release of prostaglandins in the peritoneal cavity were studied in sensitized rats. Extravasation of Evans blue dye was used as a measure of vascular permeability. Specific antigen challenge increased by 279, 297, 328, 250, and 192% the protein extravasation in the trachea, upper and lower bronchi, pancreas, and duodenum, respectively, but did not modify significantly the vascular permeability of the lung parenchyma, heart, liver, and kidney. Extravasation of Evans blue dye also was increased by 43-fold in the peritoneal cavity. Pretreatment of the animals with indomethacin (10 mg/kg) did not modify significantly the protein extravasation of the trachea, upper and lower bronchi, pancreas, and duodenum induced by anaphylaxis. Pretreatment with a mixture of mepyramine (3 mg/kg) and methysergide (2.5 mg/kg) reduced by 62, 66, and 40% the protein extravasation in the trachea, upper bronchi, and peritoneal cavity, respectively, in similar conditions. The PAF antagonist BN-52021 (5 mg/kg) very strongly reduced the protein extravasation elicited by anaphylaxis in the trachea, upper and lower bronchi, pancreas, and duodenum by 72, 87, 82, 67, and 85%, respectively, and by 53% in the peritoneal cavity. Anaphylaxis also increased the concentrations of thromboxane B₂ and leukotriene B₄ in the peritoneal exudates, but prostaglandin E₂ levels were not affected. Pretreatment with BN-52021 reduced by 29 and 75 % the level of thromboxane B₂ and leukotriene B₄ in the exudates. These results suggest that PAF, histamine, and serotonin mediate the protein extravasation associated with anaphylaxis, whereas prostaglandins are likely to play a minor role in this reaction.     

Increase of plasma neuropeptide Y-like immunoreactivity following chronic hypoxia in the rat

In an attempt to understand the changes of circulating neuropeptide Y (NPY) during hypoxia, the plasma level of NPY was investigated by radioimmunoassay. Exposure of rats to hypobaric hypoxia at an altitude of 18,000 ft for 4 weeks causes an increase of pulmonary pressure and an elevation of plasma NPY-like immunoreactivity (NPY-LI). However, the systemic blood pressure was not elevated by this chronic hypoxia. Also, plasma noradrenaline (NA) estimated by chromatographic analysis (HPLC-ECD) was not markedly raised. Failure of bretylium and guanethidine, sympathetic neuron blockers, in reducing the plasma NPY-LI level of these rats ruled out the participation of adrenergic nervous terminals. Adrenal medulla seems responsible for this elevation of plasma NPY-LI because this magnitude disappeared in adrenalectomized rats. These data suggest that chronic hypoxia induced an elevation of circulating NPY from the adrenal gland of rats.     

Bi-directional modulation of 5-hydroxytryptamine-induced plasma extravasation in the rat knee joint by nociceptin

The role of nociceptin, the endogenous ligand for the opioid receptor-like (ORL1) receptor, in nociceptive processing is controversial. Most studies demonstrate hyperalgesia following supraspinal administration, analgesia following intrathecal and peripheral administration at higher doses, and hyperalgesia following intrathecal and peripheral application at lower doses. The present study investigates the effect of nociceptin on synovial plasma extravasation and its ability to modulate 5-hydroxytryptamine-induced synovial plasma extravasation using the rat knee joint model of inflammation. Nociceptin alone does not alter synovial plasma extravasation from baseline. Nociceptin at concentrations up to 1 nM enhances 5-hydroxytryptamine-induced synovial plasma extravasation (up to 50%) and nociceptin at concentrations above 100 nM inhibits 5-hydroxytryptamine-induced synovial plasma extravasation (down to 45%). The novel, selective ORL1 receptor antagonist J-113397 potently inhibits the pro-inflammatory effect of nociceptin, but only partly inhibits, at higher concentrations, the anti-inflammatory effects of nociceptin.These findings demonstrate a dose-dependent bi-directional effect of nociceptin on inflammatory processes and may indicate a target for novel therapeutics.     

Neutrophils contribute to sympathetic nerve terminal-dependent plasma extravasation in the knee joint of the rat

Infusion of bradykinin or 6-hydroxydopamine into the knee joint of the rat activates sympathetic postganglionic nerve terminals and increases plasma extravasation, a major sign of acute inflammation. Since bradykinin attracts and activates neutrophils in vivo and since neutrophils can release factors leading to plasma extravasation, we evaluated the contribution of the neutrophil to bradykinin-induced plasma extravasation. We report that perfusion of bradykinin into the rat knee joint produces a prolonged increase in plasma extravasation which is markedly reduced not only by sympathectomy (chronic pretreatment with systemic 6-hydroxydopamine) but also by depletion of circulating polymorphonuclear leukocytes (intravenous infusion of hydroxyurea combined with intraperitoneal glycogen). Depletion of polymorphonuclear leukocytes also reduced the plasma extravasation induced by intra-articular infusion of 6-hydroxydopamine, which acutely activates sympathetic postganglionic terminals. We next tested whether attraction of neutrophils into the joint, in the absence of bradykinin, was sufficient to enhance plasma extravasation. Although the classical neutrophil attractant glycogen attracted neutrophils into the knee joint, it did not increase plasma extravasation. Co-infusion of bradykinin and glycogen into the knee joint, however, provoked plasma extravasation that was significantly greater than that produced by bradykinin alone. We hypothesize, therefore, that bradykinin not only attracts neutrophils but also activates them, by an as yet undefined mechanism that requires the sympathetic terminal. The activated neutrophils release factors that lead to plasma extravasation. The next series of studies evaluated the role of the sympathetic nervous system in neutrophil attraction in vivo by bradykinin and glycogen. Since quantification of neutrophil attraction was not possible in the knee joint, we performed these studies in the peritoneal cavity, a site where neutrophils are readily attracted.(ABSTRACT TRUNCATED AT 250 WORDS)     

5-Hydroxytryptamine-induced plasma extravasation in the rat knee joint is mediated by multiple prostaglandins

OBJECTIVE AND DESIGN: This study investigated whether prostaglandins (PGs) are involved in 5-hydroxytryptamine (5-HT)-induced synovial plasma extravasation. MATERIALS AND METHODS: Male Sprague-Dawley rat knee joints were perfused with 5-HT and synovial capillary Evans Blue dye leakage was measured using spectrophotometry. Cyclooxygenase (COX) inhibitors and PG receptor subtype-selective antagonists were tested for the ability to reduce 5-HT-induced synovial plasma extravasation. RESULTS: 5-HT-induced plasma extravasation was inhibited by indomethacin. The COX-1 selective inhibitor SC-560 and the COX-2 selective inhibitor NS-398 were equally effective, indicating that both isoforms are involved. Antagonists selective for EP1, EP2 and DP receptor subtypes significantly attenuated the 5-HT-induced plasma extravasation. However, antagonists selective for FP, IP and TP subtypes failed to reduce 5-HT-induced plasma extravasation. CONCLUSIONS: These results demonstrate that multiple, but selective, subtypes of PGs mediate synovial plasma extravasation produced by 5-HT, and suggest that PGs act downstream of 5-HT in the inflammatory cascade.     

Receptor mediation and nociceptin inhibition of bradykinin-induced plasma extravasation in the knee joint of the rat

Objective and design

The aim was to investigate the signaling mechanisms and regulation of bradykinin (BK)-induced inflammation in rat knee joint.

Materials and methods

Knee joints of anesthetized rats were perfused with BK (0.1–1.0 μM), and synovial plasma extravasation (PE) was evaluated by spectrophotometrical measurement of Evans Blue leakage. To examine the signaling pathway, B1 antagonist [des-Arg10]-HOE140 (0.1–1.0 μM) and B2 antagonist HOE140 (0.05–1.0 μM), calcitonin gene-related peptide (CGRP) antagonist CGRP8-37 (0.5–1.0 μM), prostaglandin E2 antagonist AH-6809 (0.1–1.0 μM), and histamine H1 antagonist mepyramine (0.1–1.0 μM) were used. Nociceptin (0.0001–1.0 μM) and antagonist J-113397 were tested for modulation of BK-induced PE. The analyses were compared side-by-side with 5-hydroxytryptamine-induced PE.

Results

BK perfusion dose-dependently induced PE, which was blocked by HOE140, CGRP8-37, AH-6809, and mepyramine. It was also inhibited by nociceptin, which could be reversed by antagonist J-113397. In contrast, 5-hydroxytryptamine-induced PE was biphasically regulated by nociceptin and was not antagonized by CGRP8-37.

Conclusions

BK-induced PE is mediated by B2 receptors and may involve CGRP, prostaglandin, and histamine pathways. BK-induced PE is inhibited by nociceptin through the activation of ORL1 receptors. There are differences between BK- and 5-hydroxytryptamine-induced inflammation in signaling and modulation.     

Hypothalamic neuropeptide Y and plasma leptin after long-term high-fat feeding in the rat

The ingestion of fat by rodents affects the level of neuropeptide Y (NPY) in the hypothalamus and we hypothesized that they might be linked via leptin, the adipose tissue hormone. The influence of fat intake on leptin and NPY levels was studied in rats fed on either a high-fat (HF) or a low fat diet (LF) for 5 months. Ingestion of the HF diet increased fat deposition (+48%; P < 0.01), leptinemia (+189%; P < 0.001) and reduced NPY levels in the arcuate nucleus (-35%; P < 0.01) and in the paraventricular nucleus (-22%; P < 0.01). However, although leptin levels reflected the amount of relative fat deposition (r = 0.62; P < 0.01), we found no evidence for a direct relationship between plasma leptin and NPY levels in the hypothalamus. These results suggest that the long-term effects of fat intake on NPY concentrations in the hypothalamus and plasma leptin are associated with different regulatory mechanisms.     

The relationship between unmyelinated afferent type and neurogenic plasma extravasation in normal and reinnervated rat skin.

Electrophysiological experiments have been carried out on rats to examine the relationship between plasma extravasation into skin and antidromic activation of individual unmyelinated afferent fibres supplying cutaneous receptors, particularly polymodal nociceptors. Normal animals and ones in which the skin had been reinnervated after peripheral nerve transection 26-52 weeks before were studied. In the normal animals 12 of 18 (66%) polymodal nociceptor afferents showed evidence of plasma extravasation after antidromic stimulation of the nerve filament from which they were initially recorded. Antidromic stimulation of other types of unmyelinated unit gave no detectable signs of plasma extravasation. The results from the animals with regenerated nerves were similar with 15 of 21 (71%) of the polymodal nociceptor afferents showing clear signs of plasma extravasation after antidromic activation and the other types of unit giving no signs of such a response. These results show that while antidromic stimulation of a cutaneous nerve after intravascular administration of Evans Blue dye can be used to get a general view of the extent of innervation by polymodal nociceptors, it does not necessarily show the entire distribution of these receptors. It is likely that the ability or lack of ability of a polymodal nociceptor afferent to enhance vascular permeability, and so bring about plasma extravasation, is related to its neuropeptide content, and thus it follows that this might be a useful criterion for separating this broad class of cutaneous receptor into sub-groups.     

Synaptic actions of neuropeptide FF in the rat parabrachial nucleus: interactions with opioid receptors

The pontine parabrachial nucleus (PBN) receives both opioid and Neuropeptide FF (NPFF) projections from the lower brain stem and/or the spinal cord. Because of this anatomical convergence and previous evidence that NPFF displays both pro- and anti-opioid activities, this study examined the synaptic effects of NPFF in the PBN and the mechanisms underlying these effects using an in vitro brain slice preparation and the nystatin-perforated patch-clamp recording technique. Under voltage-clamp conditions, NPFF reversibly reduced the evoked excitatory postsynaptic currents (EPSCs) in a dose-dependent fashion. This effect was not accompanied by apparent changes in the holding current, the current-voltage relationship or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced inward currents in the PBN cells. When a paired-pulse protocol was used, NPFF increased the ratio of these synaptic currents. Analysis of miniature EPSCs showed that NPFF caused a rightward shift in the frequency-distribution curve, whereas the amplitude-distribution curve remained unchanged. Collectively, these experiments indicate that NPFF reduces the evoked EPSCs through a presynaptic mechanism of action. The synaptic effects induced by NPFF (5 microM) could not be blocked by the specific mu-opioid receptor antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (1 microM), but application of delta-opioid receptor antagonist Tyr-Tic-Phe-Phe (5 microM) almost completely prevented effects of NPFF. Moreover, the delta-opioid receptor agonist, Deltorphin (1 microM), mimicked the effects as NPFF and also occluded NPFF's actions on synaptic currents. These results indicate that NPFF modulates excitatory synaptic transmission in the PBN through an interaction with presynaptic delta-opioid receptors. These observations provide a cellular basis for NPFF enhancement of the antinociceptive effects consequent to central activation of delta-opioid receptors.     

Differential effects of phosphoramidon and captopril on NK1 receptor-mediated plasma extravasation in the rat trachea

We sought to confirm the identity of the tachykinin receptor subtype that mediates plasma extravasation in the rat trachea, and assess the respective contributions of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) in regulating this tachykinin-induced response. To achieve these aims, we determined the relative potencies of several natural tachykinins and receptor-selective synthetic agonists, both before and after inhibiting NEP with phosphoramidon and ACE with captopril. We also determined the effects of these peptidase inhibitors, and the NK-1 receptor antagonist L-703,606, on the plasma extravasation produced by capsaicin, which releases tachykinins endogenously from sensory nerve endings. We found that the rank order of potency for producing plasma extravasation in the rat trachea was NK-1 receptor agonist ([Sar⁹, Met(O₂)¹¹] SP)>substance P>neurokinin A> neurokinin B. The NK-2 ([Nle¹⁰]NKA (4–10)) and NK-3 ([MePhe⁷]NKB) receptor agonists were without effect. We observed no change in the relative potencies of these peptides after giving rats phosphoramidon or captopril, which suggests that the different peptide potencies are not simply the consequence of different rates of enzymatic degradation. Nevertheless, the responses to substance P and neurokinin A were clearly potentiated in rats given phosphoramidon, indicating that NEP effectively degrades tachykininsin vivo. No significant potentiation was evident for any peptide in rats given captopril. Similarly, the plasma extravasation produced by capsaicin was potentiated in rats given phosphoramidon, but not in those given captopril. Pretreating rats with L-703,606 abolished the response to capsaicin. We conclude from these observations that NK-1 receptors mediate tachykinin-induced plasma extravasation in the rat trachea, and that NEP regulates this response with little or no contribution from ACE.     

Sensory neurobiological analysis of neuropeptide modulation of meal size

Gerry Smith's emphasis on the meal as the functional unit of ingestion spurred experiments designed to (1) identify oral and postoral stimuli that affect meal size, and (2) identify peripheral and central neural mechanisms involved in the processing of sensory signals generated by these stimuli. His observations that gut-brain peptides can limit meal size were important in formulating the idea that neuropeptides involved in the control of food intake modulate the peripheral and central neural processing of meal-stimulated sensory signals. This focus on meal size continues to foster the development of hypotheses and the design of experiments that characterize the sites and modes of action of feeding modulatory neuropeptides. These investigations have focused attention on the gut-brain neuraxis as a critical sensory pathway in the control of ingestive behavior, and have revealed important integrative properties of peripheral and central neurons along this axis. The neuromodulatory function of peptides that alter food intake is supported by their ability to recruit the activation of neurons at multiple central nodes of the gut-brain axis and to affect the neural processing and behavioral potency of meal-related gastrointestinal signals important in the negative feedback control of meal size. This sensory neurobiological perspective may also be applied to determine whether feeding modulatory neuropeptides affect the neural and behavioral potency of oral positive feedback signals that promote ingestion.     

The role of substance P and calcitonin gene-related peptide in neurogenic plasma extravasation and vasodilatation in the rat

Immunohistochemistry combined with retrograde tracing has been used to show that of the afferent neurons supplying the dorsomedial surface of the hind paw, approximately 30% contain substance P and 50% calcitonin gene-related peptide immunoreactivity. Stimulation of the saphenous nerve causes plasma extravasation and antidromic vasodilatation in this area of skin. The roles of calcitonin gene-related peptide and substance P released from peripheral afferent endings in mediating these effects were examined using immunoneutralization. In pilot experiments, the binding of radiolabelled peptide to the immunoglobulin fraction of calcitonin gene-related peptide and substance P antisera was characterized in quasi-physiological conditions. Systemic administration of either substance P or calcitonin gene-related peptide antibodies caused a significant decrease (P less than 0.05) in plasma extravasation measured by the Evans Blue method in response to topical application of mustard oil (0.5%) to the skin, or of capsaicin (5 microM) to the saphenous nerve. Topical application of mustard oil also produced a 52.9 +/- 5.1% increase in skin red cell flux. This increase was significantly decreased by both substance P and calcitonin gene-related peptide antibodies. The results suggest that both peptides are involved in mediating neurogenic inflammatory responses.     

Sensory interactions in the anterior ectosylvian cortex of cats

Sensory interactions, namely, the responses of single cells to stimulations originating from the two sides of the body or from the two visual fields, or from more than one sensory modality (namely, visual, auditory and somatosensory), were evaluated within the anterior ectosylvian cortex (AEC) of cats. Results showed that responses of single neurons to a stimulus of one modality can be enhanced or inhibited by the presentation of another stimulus of either the same or another modality. This facilitatory or inhibitory modulation seems to depend upon temporal and/or spatial relationships between the stimuli. These results, taken together with those previously obtained in our laboratory and by others, suggest that neurons in the AEC may be involved in integrating inputs from various modalities and possibly linking sensory input with action.     

Recombinant neutral endopeptidase attenuates substance P-induced plasma extravasation in the guinea pig skin.

To determine whether exogenously administered neutral endopeptidase (NEP; enkephalinase, EC 3.4.24.11) inhibits the substance P-induced increase in vascular permeability in the skin, we examined the effects of recombinant human NEP on plasma extravasation induced by intradermal injection of substance P in guinea pig skin. Injection of substance P (2.5 X 10(-8) M) induced significant plasma extravasation in the skin (53 +/- 4 mm2 of Evans blue extravasation; mean +/- 1 SEM). In vitro incubation of substance P with recombinant human NEP prior to injection prevented the substance P-induced plasma extravasation in the skin in a dose-dependent fashion. Intradermal preinjection of recombinant human NEP partially inhibited plasma extravasation induced by subsequent injection of substance P (52 +/- 9% of the control without NEP). The H1 and H2 histamine antagonists pyrilamine and cimetidine, and a muscarinic antagonist, atropine, had no effects on substance P-induced responses. Two products of substance P degradation by NEP containing the carboxy-terminal portion, substance P7-11 and substance P8-11, were also without effect. These findings suggest that recombinant human NEP can attenuate substance P-induced increases in vascular permeability in guinea pig skin and, therefore, may be useful in treating dermatologic disorders in which abnormal responses to substance P or other neuropeptides cleaved by NEP may occur.     

Hypotension activates neuropeptide Y-containing neurons in the rat medulla oblongata.

The present study was designed to determine whether neurons within cardiovascular control nuclei of the rat brainstem that become activated following a hypotensive insult also possess the capacity to utilize neuropeptide Y. Adult male Wistar-Kyoto rats were injected with glyceryl trinitrate (10 mg/kg, i.p.) or vehicle, and 4 h later anaesthetized (pentobarbitone, 60 mg/kg, i.p.) and transcardially perfused. The brains were removed and processed by standard two-colour peroxidase immunohistochemistry. Activated cells were determined by incubation with a primary antibody to Fos protein, which was followed by a second incubation with a primary antibody to neuropeptide Y for double labelling of Fos-positive cells. Compared to vehicle, glyceryl trinitrate-induced hypotension caused a marked induction of Fos protein in the caudal one-third of the nucleus tractus solitarius (bregma -14 to -13.3 mm), which tailed off rapidly in more rostral sections. Following hypotension, significant populations of activated cells were also observed in the rostral and caudal ventrolateral medulla. In the caudal nucleus tractus solitarius and the posterior part of the medial nucleus tractus solitarius, respectively, 15 of 104 and 40 of 120 Fos-positive cells exhibited cytoplasmic neuropeptide Y immunoreactivity following hypotension, compared to seven of 40 and 15 of 40 in vehicle-treated rats, indicating a significant (two- to three-fold) increase in double-labelled cells following systemic glyceryl trinitrate (P < 0.05, unpaired t-test). In contrast, in the anterior part of the medial nucleus tractus solitarius, the number of double-labelled cells did not change following hypotension. An increase in double-labelled cells was also observed in the rostral ventrolateral medulla (2.5-fold increase compared to vehicle) and caudal ventrolateral medulla (5.8-fold increase compared to vehicle) following hypotension. These data indicate that, in the rat, neuropeptide Y-containing neurons are involved in the central response to a hypotensive challenge. The primary regions where neuropeptide Y-containing neurons appear to be activated are the caudal one-third of the nucleus tractus solitarius and the caudal ventrolateral medulla/rostral ventrolateral medulla, which are key nuclei associated with the integration of the baroreceptor heart rate reflex and sympathetic vasomotor outflow.