Neural mechanisms involved in cardiovascular control during affective behaviour.

The changes in respiration and cardiovascular output seen during mammalian defensive behaviour are no longer considered to be the product of simple reflexes. Nuclei in the diencephalon (in particular, in the hypothalamus and amygdala) are involved in controlling defence reactions. In this article, K. M. Spyer describes the evidence illustrating the actions of these descending influences and discusses the potential for future modelling paradigms in relation to cardiovascular control.

Studying rhythmogenesis of breathing: comparison of in vivo and in vitro models.

In all mammalian species, breathing is controlled by a neuronal network within the lower brainstem. A component known as the ventral respiratory group produces rhythmic activity, which is transmitted to spinal motoneurons to produce a periodic contraction of respiratory muscles. A dispute about the mechanisms of 'normal' respiratory rhythm generation arose from the differences between experimental preparations that have been used to dissect the process. It is, therefore, essential to compare the various experimental approaches and to discuss the differences between experimental data. We conclude that the various preparations all have great value, but that they define different operational conditions of the network, including maturation of neurons and synaptic processes. We have taken note of these in formulating a 'maturational network-burster model' for rhythm generation that includes most features of the existing models of respiratory rhythm generation.

Coexpression of rat P2X2 and P2X6 subunits in Xenopus oocytes.

Transcripts for P2X(2) and P2X(6) subunits are present in rat CNS and frequently colocalize in the same brainstem nuclei. When rat P2X(2) (rP2X(2)) and rat P2X(6) (rP2X(6)) receptors were expressed individually in Xenopus oocytes and studied under voltage-clamp conditions, only homomeric rP2X(2) receptors were fully functional and gave rise to large inward currents (2-3 microA) to extracellular ATP. Coexpression of rP2X(2) and rP2X(6) subunits in Xenopus oocytes resulted in a heteromeric rP2X(2/6) receptor, which showed a significantly different phenotype from the wild-type rP2X(2) receptor. Differences included reduction in agonist potencies and, in some cases (e.g., Ap(4)A), significant loss of agonist activity. ATP-evoked inward currents were biphasic at the heteromeric rP2X(2/6) receptor, particularly when Zn(2+) ions were present or extracellular pH was lowered. The pH range was narrower for H(+) enhancement of ATP responses at the heteromeric rP2X(2/6) receptor. Also, H(+) ions inhibited ATP responses at low pH levels (

Localization of cardiac vagal preganglionic motoneurones in the rat: immunocytochemical evidence of synaptic inputs containing 5-hydroxytryptamine.

The origin of cardiac vagal preganglionic motoneurones in the rat is still controversial and knowledge of the chemistry of synaptic inputs onto these neurones is limited. In this investigation vagal preganglionic motoneurones innervating the heart were identified by the retrograde transport of cholera toxin conjugated to horseradish peroxidase (CT-HRP) combined with the immunocytochemical localization of 5-hydroxytryptamine. Injection of CT-HRP into the myocardium resulted in the retrograde labelling of neurones primarily in the ventral regions of the nucleus ambiguus (75.1%). Labelled neurones were also distributed in a narrow band through the reticular formation extending between the dorsal motor nucleus of the vagus nerve and the nucleus ambiguus (17.3%) as well as in the dorsal motor nucleus itself (7.6%). A combination of retrograde labelling with immunocytochemistry for 5-hydroxytryptamine revealed that the neuronal perikarya and the dendrites of cardiac vagal motoneurones in the nucleus ambiguus were often ensheathed in 5-hydroxytryptamine-immunoreactive axonal boutons. Electron microscopic examination of this material confirmed that there were synaptic specializations between these boutons and the cardiac vagal motoneurones. The identification of 5-hydroxytryptamine-containing synaptic inputs to this population of vagal motoneurones provides further detail towards the understanding of the regulation of heart rate by the parasympathetic nervous system.

In vivo modulation of nucleus tractus solitarius (NTS) neurones by activation of 5-hydroxytryptamine(2) receptors in rats.

In in vivo experiments, 5-hydroxytryptamine (5-HT) and (+/-)-2,5-dimethoxy-4-iodoamphetamine HCl (DOI), a 5-HT(2) receptor agonist, were applied by ionophoresis to rat nucleus tractus solitarius (NTS) neurones identified by their vagal and cardiopulmonary afferent inputs to test whether the response of NTS cells to 5-HT(2) receptor activation was related to whether they received mono- or polysynaptic vagal inputs and their presumed function as defined by their afferent input. Cells were classified on the basis of the variability of the latency of the vagal-evoked spikes: this varied by less than 3 ms for Group 1, from 3 to 5 ms for Group 2, and more than 5 ms for Group 3. Both 5-HT and DOI inhibited most Group 1 cells (16/18) and inactive (without ongoing activity) cells (8/13) in Group 2. Cells inhibited by DOI were also inhibited by cardiopulmonary afferent stimulation, evoked by atrial phenylbiguanide administration. By contrast, application of 5-HT and DOI excited the majority of Group 3 cells (14/19) and Group 2 with ongoing activity (7/9). Cells excited by DOI were also activated by cardiopulmonary stimulation. Both actions of DOI were reversed by application of ketanserin (n=15). In conclusion, these data demonstrate that activation of 5-HT(2) receptors in the NTS produces different effects dependent on whether the neurones received mono- or polysynaptic vagal input and their response to cardiopulmonary afferent stimulation.

A novel influence of adenosine on ongoing activity in rat rostral ventrolateral medulla.

We have investigated whether exogenously applied adenosine modulates neuronal activity in a region of the central nervous system crucial for cardiovascular regulation. Extracellular recordings were made from neurons in the rostral ventrolateral medulla of the anaesthetized rat. Ionophoretic application of adenosine altered ongoing activity in 91% of neurons, evoking either a long-lasting depression or a short-lasting increase in firing rate. Both responses were blocked by application of the broad spectrum adenosine receptor antagonist 8-sulphophenyltheophylline, indicating that the responses were mediated by specific cell surface receptors. The adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine blocked the increase, and partially blocked the decrease in firing rate in response to adenosine. The GABA(A) receptor antagonist bicuculline also blocked the increase in firing rate in response to adenosine, suggesting that adenosine may inhibit release of GABA from axon terminals in this region. The adenosine A2a receptor agonist CGS 21680 produced a long-lasting depression of ongoing activity. These results suggest that A1 receptors mediate an increase in firing rate, whilst A1 and A2a receptors mediate decreases in firing rate in some rostral ventrolateral medulla neurons. Thus, adenosine has been shown to modulate the ongoing activity of neurons in the rostral ventrolateral medulla by acting at both A1 and A2a receptors. Accordingly, we suggest, and provide some evidence to support the idea, that adenosine acts as an important neuromodulator in this region of the central nervous system, possibly by modulating the presynaptic release of neurotransmitters such as GABA.

Central distribution of substance P, calcitonin gene-related peptide and 5-hydroxytryptamine in vagal sensory afferents in the rat dorsal medulla.

The central distribution of vagal afferents in the medulla containing either substance P, calcitonin gene-related peptide or 5-hydroxytryptamine was examined using a double-labelling technique and laser scanning confocal microscopy. Areas of the nucleus tractus solitarii, dorsal motonucleus of the vagus nerve and area postrema were scanned for double-labelled axon profiles. Analysis of this material revealed that all three neurochemicals were contained within the central terminals of vagal nerve sensory neurons. However, the distribution of vagal nerve afferents containing each of these putative transmitters differed. Afferents containing 5-hydroxytryptamine were detected mainly in the areas postrema and the adjacent nucleus tractus solitarii, with a smaller number in the ventral subnuclei of the solitary tract. In contrast afferents containing calcitonin gene-related peptide were found primarily in the medial and commissural regions of the nucleus tractus solitarii. Afferents containing substance P-immunoreactivity were surprisingly few in number and did not appear to be associated with any particular region. These results establish the presence of 5-hydroxytryptamine, substance P and calcitonin gene-related peptide in the central axons of vagal sensory afferents. Furthermore, the differential distribution of afferents immunoreactive for these neurochemicals seen in this study, together with previous demonstrations of the viscerotopic organization of vagal sensory afferents suggests a possible "chemical coding" for individual end organs.

Evidence for the involvement of purinergic signalling in the control of respiration.

The ventrolateral medulla has a critical role in the generation and patterning of respiration via an extensive network of respiratory neurones. We have investigated the effects of activating purinergic P2 receptors within the ventrolateral medulla of the anaesthetised rat on the overall pattern of respiratory activity. In addition, using immunohistochemical techniques, we have identified the subtypes of P2X receptors in the ventrolateral medulla. Unilateral microinjection of ATP into the ventrolateral medulla reduced in a dose-dependent manner, or abolished, resting phrenic nerve discharge recorded as an indication of central inspiratory drive. ATP also elicited increases in blood pressure and variable changes in heart rate. These effects were mimicked by microinjection of the P2X receptor agonist alpha,beta-methylene ATP into the ventrolateral medulla. Whilst microinjection of suramin, a P2 receptor antagonist, had no effect on resting cardiorespiratory variables it blocked the respiratory and cardiovascular effects of ATP microinjected into the ventrolateral medulla. Immunohistochemical staining using IgG antibodies showed that P2X1, P2X2, P2X5 and P2X6, but not P2X3, P2X4 or receptor subunits were localised in the rostral ventrolateral medulla.Our results indicate that several P2X receptor subtypes are localised within areas of the ventrolateral medulla that are important for cardiorespiratory control (including the pre-Bötzinger and Bötzinger complexes), and that activation of these receptors can have profound effects on both the cardiovascular and the respiratory networks. Our pharmacological data suggest that different P2X subunits in this region may co-assemble to form hetero-oligomeric assemblies as well as homomultimers within this region.

Late vagal inhibition in neurons of the ventrolateral medulla oblongata in the rat.

Stimulation of cervical vagal afferents elicits long-lasting inhibitory effects in a variety of neuronal populations, although little is known concerning the cellular mechanisms that are involved in these effects. In the present study, the electrophysiological characteristics of responses elicited by cumulative activation of vagal afferents were examined in neurons of the rostral ventrolateral medulla oblongata, which play an important role in the coordination of cardiovascular and other visceral activities. The study has focused on the late-onset, slow inhibitory component of vagal responses, which is likely to affect the temporal modulation of postsynaptic effects. Vagal stimulation elicited four distinct response patterns in intracellularly penetrated neurons (n = 78): excitation, inhibition, excitation-inhibition and inhibition-inhibition. The late inhibitory component was encountered in 43 (55%) of the cells, including five putative medullospinal neurons. It was due to a postsynaptic hyperpolarization which reversed at potentials more negative than -83 mV. The voltage dependency, as well as the average onset latency (93+/-3.0 ms), duration (270+/-16.5 ms) and amplitude (1.3+/-0.2 mV as measured at resting membrane potentials), of late inhibition were clearly different from those of the short-latency inhibitory response. The differences in the voltage dependency and time-course of the short-latency responses and the late inhibition indicate that they are mediated by different central relays. In the majority of neurons, late inhibition could be elicited by stimulating only myelinated vagal afferents. The magnitude of the response was, however, significantly enhanced in 63% of the examined cells when the intensity of stimulation was raised to recruit further myelinated and non-myelinated fibres. This indicates that late vagal inhibition is often elicited by a cumulative activation of convergent afferent inputs. The intracellularly labelled vagally responsive neurons were present at all rostrocaudal levels of the rostral ventrolateral medulla, with an accumulation in the region of the lateral paragigantocellular nucleus. Neurons that exhibited late vagal inhibition were dominant in the juxtafacial region of this nucleus. Due to its slow time-course, late vagal inhibition may contribute to a tonic modulation of the activity of neurons in the rostral ventrolateral medulla oblongata. It is proposed that late vagal inhibition plays an important role in the temporal integration of sensory inputs in neurons of the rostral ventrolateral medulla oblongata. The time-course and strength of this modulatory effect are related to the level of activity in those visceral sensory inputs that converge onto the inhibitory interneurons that mediate late inhibition to rostral ventrolateral medulla oblongata neurons.

Vagal modulation of responses elicited by stimulation of the aortic depressor nerve in neurons of the rostral ventrolateral medulla oblongata in the rat.

Stimulation of cervical vagal afferents inhibits central sympathetic outflows in part by inhibiting the ongoing activity of putative baroreceptive neurons in the rostral ventrolateral medulla oblongata. The aim of the present study was to examine the electrophysiological characteristics of vagal responses and their interactions with responses elicited by stimulation of the aortic nerve in neurons there. The study focused on the role of the long-lasting, late-onset vagal inhibition, which is likely to play an important role in the tonic inhibitory effects of vagal afferent stimulation. In vivo intracellular recordings were obtained from 33 neurons that received convergent inputs from aortic and vagal afferents. Sixty-four percent of these neurons exhibited a late inhibition following electrical stimulation of myelinated vagal afferents (mean onset latency of 100+/-5 ms). The average duration of late inhibition (294+/-19 ms) exceeded the duration of the cardiac cycle. As a consequence of this, sustained vagal stimulation diminished the effect of rhythmic baroreceptor inputs in neurons that exhibited late vagal inhibition. Simultaneous activation of aortic and vagal afferents significantly increased the magnitude of late inhibition, even in those neurons where stimulation of the aortic nerve alone did not elicit a response (n = 15). This suggested that the convergence between vagal and aortic afferent inputs occurred in inhibitory inteneurons antecedent to the recorded rostral ventrolateral medulla oblongata neurons. Focal stimulation of the caudal part of the nucleus of the solitary tract also elicited a late-onset inhibition in 73% of the neurons that responded to stimulation of the aortic nerve. This inhibition appeared to be similar to the late vagal inhibition, except for its shorter average onset latency (64+/-7 ms). Based on this observation, it is proposed that inhibitory inteneurons that mediate late inhibition to rostral ventrolateral medulla oblongata neurons may lie within the caudal part of the nucleus of the solitary tract. The present study established that activation of myelinated vagal afferents exerts a complex modulation over the ongoing and evoked activity of neurons that respond to stimulation of the aortic nerve. The complex interaction that occurs between aortic and vagal inputs in neurons of the rostral ventrolateral medulla may be implicated in long-term modulation of sympathetic outflows in response to changes in the activation of visceral receptors supplied by vagus afferents. The modulation elicited by late vagal inhibition may help to adjust cardiovascular outflows according to requirements set by the thoraco-abdominal visceral environment.

The localization of adrenoceptors and opiate receptors in regions of the cat central nervous system involved in cardiovascular control.

The distribution of adrenoceptors and opiate receptors in the nucleus of the tractus solitarius and the intermediolateral cell column of the thoracic spinal cord of the cat have been investigated using an in vitro autoradiographic technique. Specific binding of [3H]yohimbine and [3H]rauwolscine (alpha 2-adrenoceptor ligands) was seen within the intermediolateral cell column but no obvious binding of [3H]prazosin, an alpha 1-ligand, was observed. No evidence of a significant population of opiate receptors was obtained in the intermediolateral cell column. Within the nucleus of the tractus solitarius a marked binding of [3H]yohimbine and [3H]rauwolscine was accompanied, however, by a more restricted binding of [3H]naloxone and [3H]dihydromorphine indicating the presence of both alpha 2-adrenoceptors and opiate receptors. As with the intermediolateral cell column no evidence of [3H]prazosin binding was seen. These observations may have particular relevance for the physiology and pharmacology of cardiovascular control. In the case of the intermediolateral cell column it is consistent with evidence of a catecholamine innervation originating from the brainstem. With regard to the nucleus of the tractus solitarius the location of the receptor groups is discussed in the light of the anatomy and physiology of its afferent innervation.

Characterization of P2 receptors modulating neural activity in rat rostral ventrolateral medulla.

This study investigated the effects of ATP, and related compounds, on the activity of neurons within the rostral ventrolateral medulla, an area of fundamental importance in reflex control of the cardiovascular system. Extracellular recordings were made from single neurons in anaesthetized, paralysed and artificially ventilated rats. Ionophoretic application of alpha,beta-methylene-ATP, adenosine 5'-O-(2-thiodiphosphate), UTP, 2-methylthio-ATP and ATP altered the ongoing activity in the majority of neurons (>74% of neurons), generally causing increases in the firing rate. Nine of 11 cells with presumed spinal projection were excited by ATP and/or the P2X-selective agonist alpha,beta-methylene-ATP. Desensitization of the excitatory responses to alpha,beta-methylene-ATP was observed in four of 20 rostral ventrolateral medulla neurons. For the remainder of the rostral ventrolateral medulla neurons, the increase in firing rate evoked by alpha,beta-methylene-ATP, and by the other purine compounds tested, did not undergo desensitization. Suramin, a P2 receptor antagonist, blocked excitatory responses to adenosine 5'-O-(2-thiodiphosphate) or alpha,beta-methylene-ATP in five of 16 neurons. These results indicate that ATP can modulate the activity of neurons in the rostral ventrolateral medulla via actions at P2 purine receptors. The data suggest that both P2X and P2Y receptors are involved, and that the functional expression of these receptors within the rostral ventrolateral medulla is not uniform.

Intracellular analysis of respiratory-modulated hypoglossal motoneurons in the cat.

Intracellular recordings from hypoglossal motoneurons in the brainstem of cats are described, along with postsynaptic potentials evoked by superior laryngeal, vagal and carotid sinus nerve stimulation. The study concentrates on hypoglossal motoneurons with respiratory-related discharge, which can be categorized into inspiratory, inspiratory/early-expiratory and expiratory patterns. Seven cells were labelled with horseradish peroxidase, their location and morphology are described. Stimulation of laryngeal receptors by balloon inflation or by water injection into the larynx, or mimicked by electrical stimulation of the superior laryngeal nerve results in enhanced postinspiratory activity in those cells (inspiratory/early-expiratory, expiratory) already receiving postinspiratory excitation; or actually produces a wave of postinspiratory depolarization in cells (inspiratory) previously quiescent during that period. It is concluded that the firing pattern of the respiratory-modulated hypoglossal motoneurons is unlikely to be static but depends on other factors, one of these being the level of ongoing, or previous laryngeal receptor stimulation.

Localisation of P2X2 receptor subunit immunoreactivity on nitric oxide synthase expressing neurones in the brain stem and hypothalamus of the rat: a fluorescence immunohistochemical study.

A large body of evidence suggests that nitric oxide (NO) and ATP act as neurotransmitters in the regulatory mechanisms concerning several autonomic functions at the level of both the hypothalamus and the brain stem. In the present study, we investigated whether neuronal NO synthase containing neurones also express P2X(2) receptor subunit of the ATP-gated ion channel via double-labelling fluorescence immunohistochemistry. Our data demonstrate that a high percentage of neuronal NO synthase-immunoreactive neurones are also P2X(2)-immunoreactive in the rostral ventrolateral medulla (98%) and supraoptic nucleus of the hypothalamus (92%). Significant numbers of neuronal NO synthase-immunoreactive neurones are also P2X(2)-immunoreactive in the subpostremal (48%) and commissural (65%) subdivisions of the nucleus tractus solitarius. In the caudal ventrolateral medulla and raphe obscurus, 96% and 89%, respectively, of neuronal NO synthase containing neurones also express P2X(2) receptor subunit. In contrast to the supraoptic nucleus, there was a lower percentage of co-localisation between NO synthase and P2X(2) receptor subunit in the paraventricular nucleus of the hypothalamus. In summary, this study demonstrates for the first time that there is a widespread co-localisation of neuronal NO synthase and P2X(2) receptor subunit in the hypothalamus and brain stem of the rat. Further studies are required to elucidate whether NO and ATP functionally interact within the hypothalamus and the brain stem.

Localization and action of adenosine A2a receptors in regions of the brainstem important in cardiovascular control.

In vitro autoradiography and central microinjections of a P1 adenosine A2a receptor antagonist have been employed to investigate a possible role for centrally located adenosine A2a receptors in modulation of the baroreceptor reflex. In vitro autoradiography using [125I]4-(2-[7-amino-2-[2-furyl][3,2,4]triazolol[2,3-a][1,3,5]tr iazin-5-yl-amino]ethyl)phenol ([125I]ZM241385), the high-affinity adenosine A2a receptor antagonist, revealed a heterogeneous distribution of adenosine A2a binding sites within the lower brainstem of the rat. Image analysis showed high levels of binding in rostral regions of both the nucleus tractus solitarius and the ventrolateral medulla. Intermediate levels of binding were observed in the commissural nucleus tractus solitarius and the dorsal vagal motor nucleus, with low levels of binding in caudal regions of the nucleus tractus solitarius and the ventrolateral medulla, and the hypoglossal nucleus. Unilateral microinjections of unlabelled ZM241385 into the nucleus tractus solitarius had no effect on baseline levels of arterial pressure, heart rate and phrenic nerve activity recorded in anaesthetized, artificially ventilated rats. However, microinjections of ZM241385 reduced the bradycardia evoked by stimulation of the ipsilateral aortic nerve. In contrast, ZM241385 had no effect on the depressor response or the reduction in phrenic nerve activity evoked by aortic nerve stimulation. Our results indicate that adenosine A2a binding sites are located in a number of brainstem regions involved in autonomic function, consistent with the idea that adenosine acts as a neuromodulator of a variety of cardiorespiratory reflexes. Specifically, the data support the hypothesis that adenosine A2a receptors located within the nucleus tractus solitarius are activated during baroreceptor stimulation and have an important modulatory role in the pattern of cardiovascular changes associated with this reflex.

In vivo modulation of vagal-identified dorsal medullary neurones by activation of different 5-Hydroxytryptamine(2) receptors in rats.

1. In in vivo experiments, DOI (a 5-HT(2) receptor agonist), MK-212 (a 5-HT(2C) receptor agonist), and BW-723C86 (a 5-HT(2B) receptor agonist) were applied by ionophoresis to neurones in the rat nucleus tractus solitarius (NTS) receiving vagal afferent input. 2. The majority of the putative 'monosynaptically' vagal activated cells were inhibited by both MK-212 (4/6) and DOI (2/4), but unaffected by BW-723C86 (12/14). In contrast, 'polysynaptically' activated NTS cells were excited by both BW-723C86 (13/19) and DOI (9/10). Inactive 'intermediate' cells were inhibited by BW-723C86 (9/12), MK-212 (5/6) and DOI (3/4), whilst active cells of this group were excited by BW-723C86 (7/13) and DOI (5/5). 3. The selective 5-HT(2B) receptor antagonist LY-202715 significantly reduced the excitatory actions of BW-723C86 on 'intermediate' and 'polysynaptic' cells (13/13), but not the inhibitory effects observed on inactive Group 2 cells (n=5) whereas the selective 5-HT(2C) receptor antagonist RS-102221 reversed the inhibitory effects of MK-212 and DOI on 'monosynaptic and 'intermediate' neurones. 4. Cardio-pulmonary afferent stimulation inhibited two of four putative 'monosynaptically' activated calls and all four inactive intermediate cells. These were also inhibited by DOI and MK-212. In contrast, cardio-pulmonary afferents excited all five active intermediate cells and all six putative 'polysynaptically' activated NTS cells, while all were also previously excited by BW-723C86 and/or DOI. 5. In conclusion, these data demonstrate that neurones in the NTS are affected differently by 5-HT(2) receptor ligands, in regard of their vagal postsynaptic location, the type of cardio-pulmonary afferent they receive and the different 5-HT(2) receptors activated.

Role of brainstem adenosine A1 receptors in the cardiovascular response to hypothalamic defence area stimulation in the anaesthetized rat.

1. The role of centrally located adenosine A1 receptors in the cardiovascular changes associated with the hypothalamic defence response has been investigated by in vitro autoradiography and the intraventricular application of an A1 receptor antagonist. 2. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), a highly selective adenosine A1 antagonist and its vehicle, ethanol, were administered directly into the posterior portion of the fourth ventricle of alpha-chloralose anaesthetized, paralysed and artificially ventilated rats. 3. DPCPX (0.01 to 0.3 mg kg-1) caused a dose-dependent decrease in the magnitude of the evoked pressor response (from -13 to -23 mmHg) elicited on hypothalamic defence area stimulation at a dose 10 fold lower than that required to produce an equivalent effect following systemic administration whilst ethanol, the vehicle, had no effect. 4. In vitro autoradiography revealed a heterogeneous distribution of adenosine A1 binding sites in the lower brainstem of rats. Image analysis showed the ventrolateral medulla to have the highest density of A1 receptors. Intermediate levels of binding were seen in caudal regions of the nucleus tractus solitarii and the hypoglossal nucleus. 5. These data imply that a proportion of the cardiovascular response to hypothalamic defence area stimulation are produced by the activation of adenosine A1 receptors localized close to the surface of, or adjacent to, the fourth ventricle in the immediate vicinity of the injection site.

The effect of 5-HT and selective 5-HT receptor agonists and antagonists on rat dorsal vagal preganglionic neurones in vitro.

1. Whole-cell patch-clamp recordings were made from 142 visually identified rat dorsal vagal preganglionic neurones (DVMs). Applications of 5-hydroxytryptamine (5-HT, 20 microM, 2 min) elicited a slow depolarization (8.2 +/- 0.5 mV, n = 59) in 95% of the cells tested, accompanied by an increase in excitability. In (68%) of DVMs the depolarization was associated with an increase in apparent membrane resistance (Rmt 22.7 +/- 2.2%). These depolarizations and increases in Rm (14.3 +/- 2.6%, n = 8) were maintained in a medium which blocked synaptic transmission. 2. The response to 5-HT was associated with a reversal potential (Erev) of -91 +/- 1 mV at an extracellular K+ concentration (LK+]o) of 4.2 mM. This correlated well with the K+ equilibrium potential (Ek = -89 mV). 3. The depolarizing effect of 5-HT was attenuated by the 5-HT2A/2C receptor antagonists, ketanserin (1 microM), LY 53,857 (1 microM) and the 5-HT1A/2A receptor antagonist, spiperone (1 microM). The 5-HT1A receptor antagonist, pindobind 5-HT1A (5 microM), had no effect on the depolarizing response to 5-HT. 4. The effect of 5-HT was mimicked by the 5-HT2A/2C receptor agonist, alpha-methyl-5-HT (50 microM), the 5-HT1 receptor agonist, 5-carboxamidotryptamine (20 microM) and the putative 5-HT4 agonist, 5-methyoxytryptamine (5 microM). The selective 5-HT4 receptor antagonist, GR113808, had no effect on the depolarizing effect of 5-HT or 5-MEOT on DVMs. 5. The 5-HT3 antagonists, MDL 72222 (10 microM) and ICS-205-930 (1 and 10 microM), partially reduced the effect of 5-HT. The 5-HT3 receptor agonist, 2-methyl-5-HT (100-300 microM), excited a proportion of neurones tested (56%) by evoking a depolarizing and/or an increase in postsynaptic potentials (p.s.ps). 6. These results are consistent with direct, postsynaptic actions of 5-HT on DVMs via 5-HT2A receptors, being mediated, in part, by the reduction of K+ conductance.

Action of adenosine receptor antagonists on the cardiovascular response to defence area stimulation in the rat.

1. The action of adenosine in the mediation of the cardiovascular changes associated with the defence reaction has been investigated in the rat using two A1 receptor antagonists. 2. Cumulative doses of 1,3 dipropyl-cyclopentylxanthine (DPCPX) (0.3-3 mg kg-1) and ethanol (0.03-0.25 ml) and bolus doses of DPCPX (3 mg kg-1) and 8-sulphophenyltheophylline (8-SPT) (20 mg kg-1) were given into alpha-chloralose, paralysed and artificially ventilated rats. Recordings were made of arterial blood pressure and heart rate. 3. Ethanol, the vehicle for DPCPX, failed to modify the magnitude of the defence response; however, cumulative doses of DPCPX produced a dose-dependent decrease in the HDA (hypothalamic defence area)-evoked increase in arterial blood pressure, accompanied by a similar fall in the magnitude of the evoked heart rate response. 4. The evoked rise in arterial blood pressure was reduced significantly by intravenous injection of DPCPX (3 mg kg-1) but not 8-SPT (20 mg kg-1), a purely peripherally acting adenosine antagonist. 5. These results suggest that adenosine acting at A1 receptors located in the central nervous system, is involved in the HDA-evoked pressor response. Whilst the site of action of the A1 receptors is not known, possible locations are discussed.

Chemosensitivity of medullary inspiratory neurones: a role for GABA(A) receptors?

This study tested the hypothesis that during hypercapnia partial removal of a tonic GABA-mediated inhibition contributes to the increase in activity of the ventrolateral medulla (VLM) inspiratory neurones. Extracellular recordings were taken from 22 inspiratory neurones in the VLM of rats anaesthetised with pentobarbitone and artificially ventilated. It was found that during hypercapnia, changes in the discharge pattern (i.e. an increase in the discharge frequency during the neurone's normally active phase) and firing frequency of the VLM inspiratory neurones were similar to those evoked by GABA(A) receptor antagonist bicuculline methiodide (BMI, 10 mM, 20 nA), applied ionophoretically in conditions of normocapnia. During hypercapnia BMI (20 nA) failed to evoke a further increase in firing of these neurones. This suggests that CO2-evoked activation of VLM inspiratory neurones may involve a withdrawal in part of a tonic GABA(A) receptor-mediated inhibition. This disinhibition may play a role in the hypercapnia-induced increase in ventilatory activity.