Cardiovascular autonomic effects of transcutaneous auricular nerve stimulation via the tragus in the rat involve spinal cervical sensory afferent pathways.

BACKGROUND: Electrical stimulation on select areas of the external auricular dermatome influences the autonomic nervous system. It has been postulated that activation of the Auricular Branch of the Vagus Nerve (ABVN) mediates such autonomic changes. However, the underlying neural pathways mediating these effects are unknown and, further, our understanding of the anatomical distribution of the ABVN in the auricle has now been questioned. OBJECTIVE: To investigate the effects of electrical stimulation of the tragus on autonomic outputs in the rat and probe the underlying neural pathways. METHODS: Central neuronal projections from nerves innervating the external auricle were investigated by injections of the transganglionic tracer cholera toxin B chain (CTB) into the right tragus of Wistar rats. Physiological recordings of heart rate, perfusion pressure, respiratory rate and sympathetic nerve activity were made in an anaesthetic free Working Heart Brainstem Preparation (WHBP) of the rat and changes in response to electrical stimulation of the tragus analysed. RESULTS: Neuronal tracing from the tragus revealed that the densest CTB labelling was within laminae III-IV of the dorsal horn of the upper cervical spinal cord, ipsilateral to the injection sites. In the medulla oblongata, CTB labelled afferents were observed in the paratrigeminal nucleus, spinal trigeminal tract and cuneate nucleus. Surprisingly, only sparse labelling was observed in the vagal afferent termination site, the nucleus tractus solitarius. Recordings made from rats at night time revealed more robust sympathetic activity in comparison to day time rats, thus subsequent experiments were conducted in rats at night time. Electrical stimulation was delivered across the tragus for 5 min. Direct recording from the sympathetic chain revealed a central sympathoinhibition by up to 36% following tragus stimulation. Sympathoinhibition remained following sectioning of the cervical vagus nerve ipsilateral to the stimulation site, but was attenuated by sectioning of the upper cervical afferent nerve roots. CONCLUSIONS: Inhibition of the sympathetic nervous system activity upon electrical stimulation of the tragus in the rat is mediated at least in part through sensory afferent projections to the upper cervical spinal cord. This challenges the notion that tragal stimulation is mediated by the auricular branch of the vagus nerve and suggests that alternative mechanisms may be involved.

Temporal and spatial properties of local circuits in neocortex.

A large body of anatomical data has detailed many complexities of neocortical circuitry, and physiological studies have indicated some roles for this circuitry in the complex functions of the cortex. Until recently, however, we have little precise information about the spatio-temporal properties of synaptic connections between individual neocortical neurones. Studies of synaptic responses elicited in one neocortical neurone by action potentials in another, and parallel morphological studies that have identified these neurones and the synaptic connections between them, have now described these parameters for certain types of local circuit connection in the neocortex. Some of these studies confirmed previous observations and inferences, but others provided major surprises. Evidence indicates that the class of both the presynaptic and postsynaptic neurone together determine a wide range of synaptic properties, such as the type of postsynaptic receptors involved and the temporal pattern of transmitter release, so that each type of synapse displays unique properties. A role for retrograde diffusable messages in determining the temporal properties of these circuits is postulated.

Detection of angiotensin II mediated nitric oxide release within the nucleus of the solitary tract using electron-paramagnetic resonance (EPR) spectroscopy.

We previously identified an action of nitric oxide (NO) within the nucleus tractus solitarii (NTS) that attenuates the cardiac component of the baroreceptor reflex. In the present study we have tested the hypothesis that angiotensin II (AngII), acting on angiotensin type 1 receptors (AT1R), can release NO within the NTS and that its actions are mediated by soluble guanylate cyclase (sGC). Utilising cryogenic electron paramagnetic resonance (EPR), we have detected NO release in brainstem samples following AngII, but not saline, microinjections into the NTS. In these experiments, we confirmed that both AngII and a NO donor (diethylamine NONOate) in the NTS both depressed the baroreflex bradycardia. In additional studies, we showed that the latter effects were both sensitive to blockade of sGC using 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ). To initiate studies to resolve the cellular source of NO released by angiotensin II in the NTS, we performed immunohistochemical/electron microscopy studies on the distribution of AT1R. We found AT1R located on NTS neurones and blood vessels. Since a rise in intracellular calcium [Ca]i levels is prerequisite for nNOS activation, we imaged responses in [Ca]i in NTS neurones during exposure to AngII in vitro using confocal microscopy. Our data indicate a paucity of neurones showing changes in [Ca]i when exposed to AngII (200 nM). We suggest that AngII-induced release of NO is from non-neuronal sites. With the presence of AT1R on blood vessel endothelial cells we propose that AngII released NO in the NTS is due to activation of endothelial nitric oxide synthase located within the endothelium. The present study supports the novel concept that AngII can trigger NO release in the NTS by a mechanism of vascular-neuronal signalling that affects central neuronal networks regulating cardiovascular function.

Adenosine A1 receptors reduce release from excitatory but not inhibitory synaptic inputs onto lateral horn neurons.

Although adenosine is an important neuromodulator in the CNS, its role in modulating sympathetic outflow at the level of the spinal cord has not been studied. Because very little is known about adenosine A1 receptors (A1Rs) in the spinal cord, we determined their location and role with particular reference to the control of sympathetic preganglionic activity and interneuronal activity in the rat. High levels of immunoreactivity for A1Rs were observed throughout the spinal cord. Immunostaining was dense in the intermediolateral cell column (IML) and intercalated nucleus, regions containing retrogradely labeled sympathetic preganglionic neurons (SPNs). Electron microscopy revealed A1R immunoreactivity (A1R-IR) within presynaptic terminals and (to a lesser extent) postsynaptic structures in the IML, as well as the luminal membrane of endothelial cells lining capillaries. Using double-labeling techniques, some presynaptic terminals were observed to synapse onto SPNs. To investigate the effects of activating these A1Rs, visualized whole-cell patch-clamp recordings were made from electrophysiologically and morphologically identified SPNs and interneurons. Applications of the A1R agonist cyclopentyladenosine (CPA) reduced the amplitude of EPSPs elicited by stimulation of the lateral funiculus, an effect blocked by the A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine. These effects were attributable to adenosine acting at a presynaptic site because CPA application increased the paired-pulse ratio. CPA did not affect evoked IPSPs. These data show that activating A1Rs reduces fast excitatory, but not inhibitory, transmission onto SPNs and interneurons in the IML and that A1Rs may play a protective role on neurons involved in the control of sympathetic outflow.

Neuronal P2X7 receptors are targeted to presynaptic terminals in the central and peripheral nervous systems.

The ionotropic ATP receptor subunits P2X(1-6) receptors play important roles in synaptic transmission, yet the P2X(7) receptor has been reported as absent from neurons in the normal adult brain. Here we use RT-PCR to demonstrate that transcripts for the P2X(7) receptor are present in extracts from the medulla oblongata, spinal cord, and nodose ganglion. Using in situ hybridization mRNA encoding, the P2X(7) receptor was detected in numerous neurons throughout the medulla oblongata and spinal cord. Localizing the P2X(7) receptor protein with immunohistochemistry and electron microscopy revealed that it is targeted to presynaptic terminals in the CNS. Anterograde labeling of vagal afferent terminals before immunohistochemistry confirmed the presence of the receptor in excitatory terminals. Pharmacological activation of the receptor in spinal cord slices by addition of 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP; 30 microm) resulted in glutamate mediated excitation of recorded neurons, blocked by P2X(7) receptor antagonists oxidized ATP (100 microm) and Brilliant Blue G (2 microm). At the neuromuscular junction (NMJ) immunohistochemistry revealed that the P2X(7) receptor was present in motor nerve terminals. Furthermore, motor nerve terminals loaded with the vital dye FM1-43 in isolated NMJ preparations destained after application of BzATP (30 microm). This BzATP evoked destaining is blocked by oxidized ATP (100 microm) and Brilliant Blue G (1 microm). This indicates that activation of the P2X(7) receptor promotes release of vesicular contents from presynaptic terminals. Such a widespread distribution and functional role suggests that the receptor may be involved in the fundamental regulation of synaptic transmission at the presynaptic site.

Differential expression of vesicular glutamate transporters by vagal afferent terminals in rat nucleus of the solitary tract: projections from the heart preferentially express vesicular glutamate transporter 1.

The central projections and neurochemistry of vagal afferent neurones supplying the heart in the rat were investigated by injecting cholera toxin B-subunit into the pericardium. Transganglionically transported cholera toxin B-subunit was visualized in the medulla oblongata in axons and varicosities that were predominantly aggregated in the dorsomedial, dorsolateral, ventrolateral and commissural subnuclei of the caudal nucleus of the solitary tract. Unilateral vagal section in control rats prevented cholera toxin B-subunit labeling on the ipsilateral side of the nucleus of the solitary tract. Fluorescent and electron microscopic dual labeling showed colocalization of immunoreactivity for vesicular glutamate transporter 1, but only rarely vesicular glutamate transporters 2 or 3 with cholera toxin B-subunit in terminals in nucleus of the solitary tract, suggesting that cardiac vagal axons release glutamate as a neurotransmitter. In contrast, populations of vagal afferent fibers labeled by injection of cholera toxin B-subunit, tetra-methylrhodamine dextran or biotin dextran amine into the aortic nerve, stomach or nodose ganglion colocalized vesicular glutamate transporter 2 more frequently than vesicular glutamate transporter 1. The presence of other neurochemical markers of primary afferent neurones was examined in nucleus of the solitary tract axons and nodose ganglion cells labeled by pericardial cholera toxin B-subunit injections. Immunoreactivity for a 200-kDa neurofilament protein in many large, cholera toxin B-subunit-labeled nodose ganglion cells indicated that the cardiac afferent fibers labeled are mostly myelinated, whereas binding of Griffonia simplicifolia isolectin B4 to fewer small cholera toxin B-subunit-labeled ganglion cells suggested that tracer was also taken up by some non-myelinated axons. A few labeled nucleus of the solitary tract axons and ganglion cells were positive for substance P and calcitonin gene-related peptide, which are considered as peptide markers of nociceptive afferent neurones. These data suggest that the population of cardiac vagal afferents labeled by pericardial cholera toxin B-subunit injection is neurochemically varied, which may be related to a functional heterogeneity of baroreceptive, chemoreceptive and nociceptive afferent fibers. A high proportion of cardiac neurones appear to be glutamatergic, but differ from other vagal afferents in expressing vesicular glutamate transporter 1.

Properties of presynaptic P2X7-like receptors at the neuromuscular junction.

Adenosine triphosphate is released into the synaptic cleft of the neuromuscular junction during normal synaptic transmission, and in much greater quantities following injury and ischaemia. There is much data to suggest roles for presynaptic P2 receptors but little to demonstrate which specific receptor subunits are present. Here we show P2X7 receptor subunits on presynaptic motor nerve terminals from birth, but no evidence for P2X1, P2X2, P2X3, P2X4, P2X5 or P2X6 receptor subunits. Further, P2X receptor subunits are present as multimeric, membrane-inserted receptors. A selective agonist, 2'-3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP: 100 microM), triggers vesicle release from motor nerve terminals, which is blocked by P2X7RS-specific concentrations of periodate oxidised ATP (OxATP: 100 microM) and brilliant blue G (BBG: 1 microM), but not by suramin (100 microM). Vesicle release is enhanced in the absence of extracellular divalent cations and occurs through activation of the ion channel and not any associated large pore, as we failed to label nerve terminals with large membrane-impermeant molecules after addition of BzATP. We conclude that a P2X7-like receptor is present at mouse motor nerve terminals, and that their activation promotes vesicle release.

Neck muscle afferents influence oromotor and cardiorespiratory brainstem neural circuits.

Sensory information arising from the upper neck is important in the reflex control of posture and eye position. It has also been linked to the autonomic control of the cardiovascular and respiratory systems. Whiplash associated disorders (WAD) and cervical dystonia, which involve disturbance to the neck region, can often present with abnormalities to the oromotor, respiratory and cardiovascular systems. We investigated the potential neural pathways underlying such symptoms. Simulating neck afferent activity by electrical stimulation of the second cervical nerve in a working heart brainstem preparation (WHBP) altered the pattern of central respiratory drive and increased perfusion pressure. Tracing central targets of these sensory afferents revealed projections to the intermedius nucleus of the medulla (InM). These anterogradely labelled afferents co-localised with parvalbumin and vesicular glutamate transporter 1 indicating that they are proprioceptive. Anterograde tracing from the InM identified projections to brain regions involved in respiratory, cardiovascular, postural and oro-facial behaviours--the neighbouring hypoglossal nucleus, facial and motor trigeminal nuclei, parabrachial nuclei, rostral and caudal ventrolateral medulla and nucleus ambiguus. In brain slices, electrical stimulation of afferent fibre tracts lateral to the cuneate nucleus monosynaptically excited InM neurones. Direct stimulation of the InM in the WHBP mimicked the response of second cervical nerve stimulation. These results provide evidence of pathways linking upper cervical sensory afferents with CNS areas involved in autonomic and oromotor control, via the InM. Disruption of these neuronal pathways could, therefore, explain the dysphagic and cardiorespiratory abnormalities which may accompany cervical dystonia and WAD.

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.

Morphological and electrophysiological properties of neurones in the dorsal vagal complex of the rat activated by arterial baroreceptors.

This study physiologically identifies and anatomically describes arterial baroreceptive neurones in the nucleus tractus solitarii of the rat. Neurones were recorded using neurobiotin-containing whole cell patch electrodes in a working heart-brainstem preparation and characterized physiologically as arterial baroreceptive in response to stimulation of the aortic arch and/or ipsilateral carotid sinus. Fifteen of 84 neurones tested were arterial baroreceptive, 7 of 8 were morphologically identified as located in the solitary tract nucleus (NTS), and 1 of 8 was located in the dorsal vagal nucleus. The seven NTS neurones had a resting membrane potential of -52 +/- 3.6 mV and a membrane input resistance of 233 +/- 38 M omega. Action potential height was 62 +/- 4.2 mV, width at half amplitude 1.46 +/- 0.38 ms, and duration of after-hyperpolarization 1.7 +/- 2.33 ms. In six of eight neurones labelled there was an invariant excitatory synaptic input (latency 3.95 +/- 0.3 ms) to stimulation of the solitary tract. Labelled somata were dorsomedial or medial to the solitary tract from -0.3 mm to +1.5 mm with regard to obex. Neurones had three to eight primary dendrites, and branches often entered the solitary tract and also extended across the ipsilateral NTS. Axons, which were mostly unmyelinated with boutons of the en passant variety, could ramify within the NTS while the main axon exited the NTS (n = 4/6), in the direction of the ipsilateral ventral medulla (n = 5/6). This is the first morphological and localisation data of physiologically characterised arterial baroreceptive NTS neurones in the rat. By comparing labelled cells with each other as well as with other unidentified cells, we conclude that NTS arterially baroreceptive neurones are morphologically and physiologically heterogenous.

Single axon fast inhibitory postsynaptic potentials elicited by a sparsely spiny interneuron in rat neocortex.

Many of the different morphological types of interneurons in mammalian neocortex are presumed to be inhibitory, but to date, conclusive functional data have been lacking. Using paired intracellular recordings in slices of adult rat somatosensory cortex, we present a sparsely spiny, burst firing interneuron that elicits in a simultaneously recorded pyramid a fast inhibitory postsynaptic potential, reversing at -78 mV. Neither inhibitory postsynaptic potential time course, nor paired pulse depression (inter-spike interval 15-120 ms), was affected by addition of the GABAB antagonist/partial agonist 2-OH-Saclofen (250 microM), but increasing extracellular [Ca2+] enhanced inhibitory postsynaptic potential amplitude at low firing rates and increased paired pulse depression at higher rates. Light microscopic examination of the biocytin-filled neurons revealed the presynaptic cell to be a sparsely spiny interneuron and the postsynaptic to be a small pyramidal neuron, both in layer II. Ultrastructural examination of 16 terminals of the presynaptic interneuron revealed that they formed symmetric contacts with unlabelled neurons, four with neuronal somata, 10 with dendritic shafts and two with spine shafts. This, therefore, is the first report of the properties of a single axon inhibitory postsynaptic potential in neocortex resulting from action potentials in an electro-physiologically and morphologically identified interneuron. We propose that at least some of the sparsely spiny, burst firing interneurons inhibit pyramidal neurons via GABAA receptors.

Innervation of burst firing spiny interneurons by pyramidal cells in deep layers of rat somatomotor cortex: paired intracellular recordings with biocytin filling.

Intracellular recordings were obtained from a class of neuron defined electrophysiologically as burst firing interneurons in layers V and VI in slices of adult rat somatomotor cortex. Four of these cells were recovered histologically. These four cells had resting membrane potentials between -68 and -80 mV, a mean input resistance of 77 +/- 16.2 M omega (measured from the voltage deflection produced by a 100 ms, 0.5 nA hyperpolarizing pulse delivered from a membrane potential of -80 mV) and responded to injections of depolarizing current from membrane potentials negative of -70 to -75 mV with an initial burst of action potentials followed by a complex afterhyperpolarization. In response to injection of larger (0.5-1.5 nA) hyperpolarizing current pulses from membrane potentials between -60 and -70 mV, 15 of 20 burst firing cells (three of four recovered histologically) that were tested displayed delayed inward rectification, and in all 20 cells of this type, responses to large negative current pulses were followed by a rebound depolarization that could initiate action potentials. Filling of four of these cells with biocytin and subsequent histological processing revealed that they were bitufted with sparsely to medium spiny dendrites and extensive local axon ramifications. These neurons are similar to low threshold spiking cells [Kawaguchi (1993) J. Neurophysiol. 69, 416-431]. Ultrastructural examination of the axons of three cells revealed that of 53 labelled terminals studied, the majority formed synaptic contacts with dendritic shafts. Filling neurons with biocytin during paired intracellular recordings resulted in three well labelled interneurons, each of which was postsynaptic to a simultaneously recorded pyramidal neuron. In these pairs both cells were identified, but the presynaptic axon was poorly labelled in one. In one of the two pairs in which the pre- and postsynaptic neurons were fully recovered, light microscopic assessment indicated that the axon of the presynaptic pyramid formed 12 close appositions with dendrites of the postsynaptic interneuron. Six of these appositions were examined at the electron microscopic level and were identified as possible synaptic contacts. In the other pair three of six close appositions observed at the light level were verified as possible synaptic connections at the ultrastructural level. These correlated electrophysiological and anatomical studies provide the first evidence for connections from pyramid to burst firing interneurons in the neocortex and indicate that these connections can be mediated by multiple synaptic contacts. The accompanying paper describes the functional properties of these connections.

CA1 pyramid-pyramid connections in rat hippocampus in vitro: dual intracellular recordings with biocytin filling.

In adult rat hippocampus, simultaneous intracellular recordings from 989 pairs of CA1 pyramidal cells revealed nine monosynaptic, excitatory connections. Six of these pairs were sufficiently stable for electrophysiological analysis. Mean excitatory postsynaptic potential amplitude recorded at a postsynaptic membrane potential between -67 and -70 mV was 0.7 +/- 0.5 mV (0.17-1.5 mV), mean 10-90% rise time was 2.7 +/- 0.9 ms (1.5-3.8 ms) and mean width at half-amplitude was 16.8 +/- 4.1 ms (11.6-25 ms). Cells were labelled with biocytin and identified histologically. For one pair that was fully reconstructed morphologically, excitatory postsynaptic potential average amplitude was 1.5 mV, 10-90% rise time 2.8 ms and width at half-amplitude 11.6 ms (at -67 mV). In this pair, correlated light and electron microscopy revealed that the presynaptic axon formed two synaptic contacts with third-order basal dendrites of the postsynaptic pyramid, one with a dendritic spine, the other with a dendritic shaft. In the four pairs tested, postsynaptic depolarization increased excitatory postsynaptic potential amplitude and duration. In two, D-2-amino-5-phosphonovalerate (50 microM) reduced the amplitude and duration of the excitatory postsynaptic potential. The remainder of the excitatory postsynaptic potential now increased with postsynaptic hyperpolarization and was abolished by 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione (n = 1). Paired-pulse depression was evident in the four excitatory postsynaptic potentials tested. This depression decreased with increasing inter-spike interval. These results provide the first combined electrophysiological and morphological illustration of synaptic contacts between pyramidal neurons in the hippocampus and confirm that connections between CA1 pyramidal neurons are mediated by both N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptors.

Single axon excitatory postsynaptic potentials in neocortical interneurons exhibit pronounced paired pulse facilitation.

In slices of adult rat somatosensory/motor cortex, paired recordings were made from pyramidal and non-pyramidal neurons. Single axon excitatory postsynaptic potentials evoked in the non-pyramidal neuron by action potentials in the pyramidal neuron were large and fast and demonstrated large fluctuations in amplitude, with coefficients of variation between 0.1 and 1.25. Excitatory postsynaptic potential amplitude distributions included a large number of apparent failures of transmission as well as some extremely large events. This contrasted dramatically with the relatively narrow distribution of amplitudes for pyramid-pyramid connections in neocortex. Excitatory postsynaptic potentials increased in amplitude with postsynaptic membrane hyperpolarization. Very small changes in the coefficient of variation when mean amplitudes increased substantially were consistent with the increase being due to a change in quantal amplitude. These excitatory postsynaptic potentials displayed profound paired pulse facilitation. Moreover, third and fourth spikes in a presynaptic burst also evoked large responses. This facilitation was associated with a decrease in the proportion of apparent failures in transmission and a change in the shape of the excitatory postsynaptic potential amplitude distribution, both indicative of an increase in the probability of transmitter release. However a large change in the mean amplitude was not associated with a similar change in the inverse square of the coefficient of variation. The result of this third test, taken in isolation, might therefore suggest that quantal amplitude had increased with paired-pulse facilitation. However, of the three tests applied, this last is the most heavily model-dependent and produced a result inconsistent with the results of the other two tests. The possibility is therefore discussed that both the shape of the excitatory postsynaptic potential amplitude distribution and the failure of coefficient of variation analysis to detect an apparently presynaptic change might result from the release at these synapses being poorly fit by a simple model. Based on a more complex model of synaptic release proposed by Faber and Korn [Faber and Korn (1991) Biophys. J. 60, 1288-1294] and a hypothesis proposed by Scharfman et al. [Scharfman et al. (1990) Neuroscience 37, 693-707], two hypotheses arising from the present study are discussed: (i) that branch point failure contributes to the pattern of synaptic activation at these connections; and (ii) that both presynaptic pyramidal firing pattern and axonal geometry contribute to the selection of the type of postsynaptic neurone preferentially activated.(ABSTRACT TRUNCATED AT 400 WORDS)

Properties of single axon excitatory postsynaptic potentials elicited in spiny interneurons by action potentials in pyramidal neurons in slices of rat neocortex.

In slices of adult rat somatomotor cortex, paired intracellular recordings determined the properties of a novel class of excitatory connection, that of presynaptic pyramidal axon collaterals onto burst firing, spiny inhibitory interneurons. Single axon excitatory postsynaptic potentials were brief in time course and displayed conventional voltage relations, increasing in amplitude with membrane hyperpolarization with no change in time course. Excitatory postsynaptic potential amplitude distributions were not skewed. Paired pulse facilitation was profound at interspike intervals < 50 ms, but not altered by raising extracellular [Ca2+] from 2.5 to 5 mM, despite an apparent increase in release probability. Raising presynaptic firing frequency did however produce an increase in excitatory postsynaptic potentials elicited by first spikes that was associated with a decline in excitatory postsynaptic potentials elicited by second and third spikes in brief trains of presynaptic spikes. That this pattern of synaptic activity may result from low probabilities of transmitter release is discussed. It is proposed that while raising Ca2+ and increasing presynaptic firing both increase release probability, repetitive presynaptic firing raises probability more effectively than does raising extracellular [Ca2+]. However, concomitant exhaustion of readily releasable transmitter at higher firing rates may partially obscure this effect. It is concluded that the major differences in the firing rate- and firing pattern-dependent properties of pyramid-pyramid and pyramid-interneuron connections are due to the typically lower release probability at synapses onto interneurons. The accompanying paper describes the morphology of these connections.

P2X(2) receptor immunoreactivity in the dorsal vagal complex and area postrema of the rat.

Adenosine 5'-triphosphate (ATP) can function as a fast synaptic transmitter through its actions on ionotropic (P2X) and metabotropic (P2Y) receptors in neuronal tissue. The ionotropic receptors have been classified into seven subtypes (P2X(1)-P2X(7)) by molecular cloning. However, they are difficult to distinguish pharmacologically owing to an absence of specific agonists and antagonists. In this study we used neuroanatomical methods to determine the origin and neurochemical phenotype of the P2X(2) subtype of purinoceptor in the dorsal medulla of the rat. Using immunohistochemistry we observed dense networks of P2X(2) receptor immunoreactive labelled fibres and terminals in the dorsal vagal complex and area postrema, as well as labelled cell bodies in the dorsal vagal nucleus and the area postrema. The P2X(2) receptor was localized presynaptically in vagal afferent fibres and terminals in the nucleus tractus solitarius at the ultrastructural level by combining injections of an anterograde tracer (biotin dextran amine) into the nodose ganglion with pre-embedding immunogold visualization of P2X(2) immunoreactivity. Terminals immunoreactive for the P2X(2) receptor in the nucleus tractus solitarius were found to contain glutamate, but not GABA immunoreactivity by post-embedding immunogold-labelling techniques. In cell bodies in the area postrema, dual immunofluorescence also indicated that P2X(2) receptor immunoreactive cells are glutamatergic but not GABAergic. The P2X(2) receptor was localized to vagal preganglionic neurons in the dorsal vagal nucleus that were identified by prior intraperitoneal injections of the retrograde tracer FluoroGold. No cells immunoreactive for the P2X(2) receptor were observed in the nucleus tractus solitarius. The localization of P2X(2) receptor immunoreactivity presynaptically in vagal afferent terminals indicates that the receptor may be involved in modulating transmitter release from vagal afferent fibres. Furthermore, the presence of the P2X(2) receptor in vagal preganglionic cells and in glutamatergic cells of the area postrema implies that it may, respectively, play a role in regulation of vagal efferent cell activity and modulation of excitatory outputs from the area postrema to other brain regions.

Properties of solitary tract neurones responding to peripheral arterial chemoreceptors.

Despite the highly integrated pattern of response evoked by peripheral chemoreceptor stimulation, limited information exists regarding the neurones within the nucleus of the solitary tract that mediate this reflex. Using a working heart-brainstem preparation, we describe evoked synaptic response patterns, some intrinsic membrane properties, location, morphology and axonal projections of physiologically characterised 'chemoreceptive' neurones located in the solitary tract nucleus in the rat. From 172 whole cell recordings, 56 neurones were identified as chemoreceptive since they responded to aortic injections of low doses of sodium cyanide (2-5 microg). Chemoreceptive neurones had a mean resting membrane potential of -52+/-1 mV and input resistance was 297+/-15 M(Omega) (n=56). Synaptic responses evoked included excitatory synaptic potentials alone, excitatory-inhibitory post-synaptic potential complexes, inhibitory synaptic potentials alone and central respiratory modulated synaptic potentials. Synaptic response latency data were obtained by stimulating electrically the solitary tract: the mean excitatory synaptic latency was 5.2+/-0.4 ms (range 2.5-8.0 ms; n=17). Chemoreceptive neurones showed a heterogeneity in their intrinsic membrane properties: neurones displayed either steady state, augmenting or adapting firing responses to depolarising current injection and, in some neurones, either delayed excitation or rebound activity following hyperpolarising pulses. Eleven chemoreceptive neurones were labelled and provided the first morphological data of these cells. Labelled somata were detected dorsomedial or medial to the solitary tract spanning the obex. Neurones typically had three to eight primary dendrites which often entered the solitary tract as well as extending across the ipsilateral region of the nucleus of the solitary tract. Axons were mostly unmyelinated with boutons of the en passant variety and often ramified within the solitary tract nucleus as well as coursed towards the ipsilateral ventral medulla. In summary, this study provides new data on the neurophysiological, anatomical and morphological properties of nucleus of the solitary tract neurones responding to arterial chemoreceptors in the rat.

Properties of interneurones in the intermediolateral cell column of the rat spinal cord: role of the potassium channel subunit Kv3.1.

Sympathetic preganglionic neurones located in the intermediolateral cell column (IML) are subject to inputs descending from higher brain regions, as well as strong influences from local interneurones. Since interneurones in the IML have been rarely studied directly we examined their electrophysiological and anatomical properties. Whole cell patch clamp recordings were made from neurones in the IML of 250 microM slices of the thoracic spinal cord of the rat at room temperature. Action potential durations of interneurones (4.2+/-0.1 ms) were strikingly shorter than those of sympathetic preganglionic neurones (9.4+/-0.2 ms) due to a more rapid repolarisation phase. Low concentrations of tetraethylammonium chloride (TEA) (0.5 mM) or 4-aminopyridine (4-AP) (30 microM) affected interneurones but not sympathetic preganglionic neurones by prolonging the action potential repolarisation as well as decreasing both the afterhypolarisation amplitude and firing frequency. Following recordings, neurones sensitive to TEA and 4-AP were confirmed histologically as interneurones with axons that ramified extensively in the spinal cord, including the IML and other autonomic regions. In contrast, all cells that were insensitive to TEA and 4-AP were confirmed as sympathetic preganglionic neurones. Both electrophysiological and morphological data are therefore consistent with the presence of the voltage-gated potassium channel subunit Kv3.1 in interneurones, but not sympathetic preganglionic neurones. Testing this proposal immunohistochemically revealed that Kv3.1b was localised in low numbers of neurones within the IML but in higher numbers of neurones on the periphery of the IML. Kv3.1b-expressing neurones were not sympathetic preganglionic neurones since they were not retrogradely labelled following intraperitoneal injections of Fluorogold. Since Kv3.2 plays a similar role to Kv3.1 we also tested for the presence of Kv3.2 using immunohistochemistry, but failed to detect it in neuronal somata in the spinal cord. These studies provide electrophysiological and morphological data on interneurones in the IML and indicate that the channels containing the Kv3.1 subunit are important in setting the firing pattern of these neurones.

Properties of solitary tract neurons receiving inputs from the sub-diaphragmatic vagus nerve.

Vagal afferents ascending from the gastrointestinal tract synapse on neurons in the nucleus of the solitary tract. Although these neurons constitute a significant proportion of solitary tract cells their firing behaviour and synaptic properties are not documented. Since gastrointestinal tract afferent termination sites overlap with regions mediating cardiorespiratory reflexes the possibility of convergence with afferents mediating cardiovascular and respiratory reflexes was proposed. Here we describe some electrophysiological and morphological properties of solitary tract neurons orthodromically driven from the subdiaphragmatic vagus nerves and assess possible convergent inputs from cardiorespiratory afferents. Whole-cell recordings of solitary tract neurons responding to electrical stimulation of the sub-diaphragmatic vagus nerves (0.1-1 ms; 1-10 V; 2-20 Hz) were made in a working heart-brainstem preparation of rat. Baroreceptors were stimulated by raising pressure in the aorta or carotid sinus, whereas aortic injection of sodium cyanide (0.05% solution 25-50 microl) was used to activate peripheral chemoreceptors. Phrenic nerve activity and heart rate were monitored continuously. Of 88 solitary tract neurons tested, 39 responded with an evoked excitatory synaptic potential following stimulation of the sub-diaphragmatic vagus nerves. Resting membrane potential and input resistance of sub-diaphragmatic vagus nerve driven solitary tract neurons were 53.2 +/- 0.5 mV and 291 +/- 17 Mohms, respectively (mean +/- S.E.M.). Response latencies to sub-diaphragmatic vagus nerve stimulation were divided into two groups: <20 ms (16.0 +/- 2 ms, n = 7; mean +/- S.E.M.) and >20 ms (77.3 +/- 5 ms, n = 32). One additional neuron displayed an evoked inhibitory postsynaptic potential (latency 175 ms). Nineteen neurons showed ongoing activity which consisted of either irregular single action potential firing (0.5-10 Hz; n = 12) or burst discharge (n = 7). Of 33 neurons tested, 17 showed spike frequency adaptation during injection of positive current, whereas 19 of 38 cells displayed rebound excitation following release from hyperpolarized potentials. There was no correlation between these properties and synaptic latencies. Ninety-one per cent of neurons tested displayed synaptic depression following paired pulse stimulation of the sub-diaphragmatic vagus nerve over intervals up to 500 ms. Stimulation of either baroreceptors (n = 31) or chemoreceptors (n = 36) failed to elicit a synaptic response in all sub-diaphragmatic vagus nerve-driven solitary tract neurons. Neurobiotin-labelled solitary tract neurons (n = 10) were from both latency groups and were located medial to the solitary tract at the level of area postrema, -0.3 mm to +1 mm from the obex. One cell was located in commissural subnucleus at midline, seven cells dorsal to the tractus solitarius and three ventral and medial to it. Soma sizes were 23 +/- 9.6 x 14 +/- 4.9 microm (range: 50 x 16 microm to 15 x 7 microm). The number of primary dendrites varied from three to five, secondary from one to eight and tertiary zero to four. Labelled axons were found in seven cells which ramified extensively in the solitary tract nucleus (n = 3) and/or branched extensively in the dorsal vagal motonucleus (n = 3) and/or projected towards the ventrolateral medulla (n = 3). We conclude that solitary tract neurons receiving signals from the sub-diaphragmatic vagus nerves (most likely from gastrointestinal tract structures) appear to be a distinct pool of neurons. There was a heterogeneity in terms of both their ongoing activity and projection targets but despite this, there were three consistent properties. First, sub-diaphragmatic vagus nerve evoked predominantly excitatory synaptic responses in solitary tract neurons; second, neurons exhibited lasting paired pulse depression following activation of sub-diaphragmatic vagus nerves; and third, sub-diaphragmatic vagus nerve-driven solitary tract neurons were

Differential co-localisation of the P2X7 receptor subunit with vesicular glutamate transporters VGLUT1 and VGLUT2 in rat CNS.

Presynaptic P2X(7) receptors are thought to play a role in the modulation of transmitter release and have been localised to terminals with the location and morphology typical of excitatory boutons. To test the hypothesis that this receptor is preferentially associated with excitatory terminals we combined immunohistochemistry for the P2X(7) receptor subunit (P2X(7)R) with that for two vesicular glutamate transporters (VGLUT1 and VGLUT2) in the rat CNS. This confirmed that P2X(7)R immunoreactivity (IR) is present in glutamatergic terminals; however, whether it was co-localised with VGLUT1-IR or VGLUT2-IR depended on the CNS region examined. In the spinal cord, P2X(7)R-IR co-localised with VGLUT2-IR. In the brainstem, co-localisation of P2X(7)R-IR with VGLUT2-IR was widespread, but co-localisation with VGLUT1-IR was seen only in the external cuneate nucleus and spinocerebellar tract region of the ventral medulla. In the cerebellum, P2X(7)R-IR co-localised with both VGLUT1 and VGLUT2-IR in the granular layer. In the hippocampus it was co-localised only with VGLUT1-IR, including in the polymorphic layer of the dentate gyrus and the substantia radiatum of the CA3 region. In other forebrain areas, P2X(7)R-IR co-localised with VGLUT1-IR throughout the amygdala, caudate putamen, striatum, reticular thalamic nucleus and cortex and with VGLUT2-IR in the dorsal lateral geniculate nucleus, amygdala and hypothalamus. Dual labelling studies performed using markers for cholinergic, monoaminergic, GABAergic and glycinergic terminals indicated that in certain brainstem and spinal cord nuclei the P2X(7)R is also expressed by subpopulations of cholinergic and GABAergic/glycinergic terminals. These data support our previous hypothesis that the P2X(7)R may play a role in modulating glutamate release in functionally different systems throughout the CNS but further suggest a role in modulating release of inhibitory transmitters in some regions.