Expression of the P2X(2) receptor subunit of the ATP-gated ion channel in the cochlea: implications for sound transduction and auditory neurotransmission.

Extracellular ATP has multimodal actions in the cochlea affecting hearing sensitivity. ATP-gated ion channels involved in this process were characterized in the guinea pig cochlea. Voltage-clamped hair cells exhibited a P2 receptor pharmacology compatible with the assembly of ATP-gated ion channels from P2X(2) receptor subunits. Reverse transcription-PCR experiments confirmed expression of the P2X(2-1) receptor subunit mRNA isoform in the sensory epithelium (organ of Corti); a splice variant that confers desensitization, P2X(2-2), was the predominant subunit isoform expressed by primary auditory neurons. Expression of the ATP-gated ion channel protein was localized using a P2X(2) receptor subunit-specific antiserum. The highest density of P2X(2) subunit-like immunoreactivity in the cochlea occurred on the hair cell stereocilia, which faces the endolymph. Tissues lining this compartment exhibited significant P2X(2) receptor subunit expression, with the exception of the stria vascularis. Expression of ATP-gated ion channels at these sites provides a pathway for the observed ATP-induced reduction in endocochlear potential and likely serves a protective role, decoupling the "cochlear amplifier" in response to stressors, such as noise and ischemia. Within the perilymphatic compartment, immunolabeling on Deiters' cells is compatible with purinergic modulation of cochlear micromechanics. P2X(2) receptor subunit expression was also detected in spiral ganglion primary afferent neurons, and immunoelectron microscopy localized these subunits to postsynaptic junctions at both inner and outer hair cells. The former supports a cotransmitter role for ATP in a subset of type I spiral ganglion neurons, and latter represents the first characterization of a receptor for a fast neurotransmitter associated with the type II spiral ganglion neurons.

Distribution of the P2X2 receptor subunit of the ATP-gated ion channels in the rat central nervous system.

The distribution of the P2X2 receptor subunit of the adenosine 5'-triphosphate (ATP)-gated ion channels was examined in the adult rat central nervous system (CNS) by using P2X2 receptor-specific antisera and riboprobe-based in situ hybridisation. P2X2 receptor mRNA expression matched the P2X2 receptor protein localisation. An extensive expression pattern was observed, including: olfactory bulb, cerebral cortex, hippocampus, habenula, thalamic and subthalamic nuclei, caudate putamen, posteromedial amygdalo-hippocampal and amygdalo-cortical nuclei, substantia nigra pars compacta, ventromedial and arcuate hypothalamic nuclei, supraoptic nucleus, tuberomammillary nucleus, mesencephalic trigeminal nucleus, dorsal raphe, locus coeruleus, medial parabrachial nucleus, tegmental areas, pontine nuclei, red nucleus, lateral superior olive, cochlear nuclei, spinal trigeminal nuclei, cranial motor nuclei, ventrolateral medulla, area postrema, nucleus of solitary tract, and cerebellar cortex. In the spinal cord, P2X2 receptor expression was highest in the dorsal horn, with significant neuronal labeling in the ventral horn and intermediolateral cell column. The identification of extensive P2X2 receptor immunoreactivity and mRNA distribution within the CNS demonstrated here provides a basis for the P2X receptor antagonist pharmacology reported in electrophysiological studies. These data support the role for extracellular ATP acting as a fast neurotransmitter at pre- and postsynaptic sites in processes such as sensory transmission, sensory-motor integration, motor and autonomic control, and in neuronal phenomena such as long-term potentiation (LTP) and depression (LTD). Additionally, labelling of neuroglia and fibre tracts supports a diverse role for extracellular ATP in CNS homeostasis.

Barosensitive neurons in the rostral ventrolateral medulla of the rat in vivo: morphological properties and relationship to C1 adrenergic neurons.

The aim of this study, conducted in anaesthetized rats, was to examine the morphology of barosensitive neurons in the rostral ventrolateral medulla and their immunoreactivity for a catecholamine synthesizing enzyme, tyrosine hydroxylase. Thirty neurons displaying inhibitory postsynaptic potentials following stimulation of the aortic depressor nerve were intracellularly labelled with Lucifer Yellow or Neurobiotin. Some of these neurons could be excited antidromically from the second thoracic segment of the spinal cord, with conduction velocities of spinal axons ranging from 1.9 to 7.2 m/s. The filled somas were found immediately caudal to the facial nucleus and ventral or ventromedial to compact formation of the nucleus ambiguus. Some dendrites reached the ventral medullary surface. Axons usually projected dorsomedially and then made a sharp rostral and/or caudal turn. The caudally projecting axon could, in some cases, be followed to the first cervical segment of the spinal cord. Seven cells issued fine axon collaterals on the ipsilateral side. These were identified mainly in two areas: in the rostral ventrolateral medulla (or immediately dorsomedial to that region), and within the dorsal vagal complex. Seven of 27 examined cells (26%) were tyrosine hydroxylase-immunoreactive and were classified as C1 adrenergic neurons. No clear relationship was found between the presence or absence of adrenergic phenotype and the morphology of filled cells. However, the amplitude of aortic nerve-evoked inhibitory postsynaptic potentials was significantly larger in tyrosine hydroxylase-positive neurons. Possible reasons for the low percentage of barosensitive cells with tyrosine hydroxylase immunoreactivity found in this study, in comparison with previously published estimates, are discussed. This is the first study describing the morphology of neurons in this part of the medulla identified as barosensitive in vivo, and directly demonstrating adrenergic phenotype in a subset of these neurons.

Localization of ATP-gated ion channels in cerebellum using P2x2R subunit-specific antisera.

The distribution of the P2x2 purinoceptor subunit protein, which forms ATP-gated ion channels by homo- and hetero-multimeric assembly, was examined in the adult rat and guinea-pig cerebellum using two novel antisera generated against separate 18 amino acid sequences located in the predicted extracellular domain of this subunit. These antisera, the first available for labelling the P2x2R subunit protein, were validated by selective labelling of a fusion protein containing the target amino acid sequences, and in cerebellum, by peptide specific block of immunoreactivity and by comparison with the distribution of P2x2R mRNA. P2x2R-like immunoreactivity was seen in Purkinje cells, specifically the soma and dendrites, neurons in the granular and molecular layers and deep cerebellar nuclei. The identification of P2x2R-like immunoreactivity within the cerebellar neural circuitry is consistent with a role for extracellular ATP acting as a fast neurotransmitter in motor learning and coordination of movement. Additionally, labelling of neuroglia and fibre tracts supports a diverse role for extracellular ATP in CNS homeostasis.

Morphological study of long axonal projections of ventral medullary inspiratory neurons in the rat.

The aim of this study was to examine medullary and spinal axonal projections of inspiratory bulbospinal neurons of the rostral ventral respiratory group (VRG) in the rat. A direct visualization of long (9.8-33 mm) axonal branches, including those projecting to the contralateral side of the medulla oblongata and the spinal cord, was possible due to intracellular labeling with neurobiotin and long survival times (up to 22 h) after injections. Seven of the nine labeled neurons had bilateral descending axons, which were located in discrete regions of the spinal white matter; ipsilateral axons in the lateral and dorsolateral funiculus, contralateral in the ventral and ventromedial funiculus. The collaterals issued by these axons at the mid-cervical level formed close appositions with dendrites of phrenic motoneurons, which had also been labeled with neurobiotin. None of these collaterals crossed the midline. The significance of this finding is discussed in relation to the crossed-phrenic phenomenon. Additional spinal collaterals were identified in the C1 and T1 segments. Within the medulla, collaterals with multiple varicosities were identified in the lateral tegmental field and in the dorsomedial medulla (in the hypoglossal nucleus and in the nucleus of the solitary tract). These results demonstrate that inspiratory VRG neurons in the rat have some features which have not been previously described in the cat, including frequent bilateral spinal projection and projection to the nucleus of the solitary tract. In addition, this study shows that intracellular labeling with neurobiotin offers an effective way of tracing long axonal projections, supplementing results previously obtainable only with antidromic mapping, and providing morphological details which could not be observed in previous studies using labeling with horseradish peroxidase.

Electrophysiological study of dorsal respiratory neurons in the medulla oblongata of the rat.

There has been controversy whether the dorsal respiratory group (DRG), identified in the cat and several other species as a concentration of mainly inspiratory neurons located in the ventrolateral subnucleus of the solitary tract, also exists in the rat. The aim of this study was to re-examine this question by systematically exploring this region with extracellular microelectrodes, in anesthetized and artificially ventilated rats. One-hundred and forty-two units were recorded which fired in phase with central respiratory cycles (determined by recording from the phrenic nerve) and/or lung inflations. One-hundred and nineteen recordings were thought to be from neuronal cell bodies (confirmed in some cases by excitatory responses to microelectrophoretic administration of DL-homocysteic acid), while the remaining 23 were from lung vagal afferents. Most neurons in the former group (87/119) were inspiratory. Out of 96 neurons tested for spinal projections only 14 (12 inspiratory, 2 expiratory) responded antidromically following stimulation at C3 segment. These results confirm the existence of the DRG in the rat and demonstrate that neurons located in this region have firing patterns generally similar to those previously described in the cat. The main difference is the relative paucity in the rat of neurons projecting spinally below the C2 level, which indicates that most DRG neurons in this species do not project directly to phrenic and intercostal motoneurons, but to other, as yet unidentified, neuronal groups within the brainstem or upper cervical segments.

A comparative study of pre-sympathetic and Bötzinger neurons in the rostral ventrolateral medulla (RVLM) of the rat.

The aim of this study was to investigate the degree of functional and anatomical overlap between two major neuronal subpopulations in the rostral ventrolateral medulla: pre-sympathetic (sympathoexcitatory) neurons, and expiratory neurons of the Bötzinger complex. Extracellular recordings were made with dye-filled microelectrodes in pentobarbital anesthetized, paralyzed and artificially ventilated adult Wistar rats. Tests applied included stimulation of baroreceptor afferents, activation of peripheral chemoreceptors and lung stretch receptors, changes in central respiratory drive with hyper- or hypoventilation, nociceptive stimulation, and antidromic stimulation from the T2 segment of the spinal cord or medulla oblongata at obex level. The two groups of neurons showed different patterns of spontaneous activity and generally different responses to these stimuli. The recording positions showed some overlap, but the majority of Bötzinger neurons were dorsolateral to pre-sympathetic neurons. There was a large overlap between the location of pre-sympathetic neurons and the lateral part of the C1 adrenergic group, but only a small overlap between these adrenergic neurons and Bötzinger neurons. These results indicate that the anatomically adjacent pre-sympathetic and Bötzinger expiratory neurons form two functionally distinct neuronal subpopulations.

Pre-sympathetic neurones in the rostral ventrolateral medulla of the rat: electrophysiology, morphology and relationship to adjacent neuronal groups.

The activity of preganglionic sympathetic neurones largely depends on synaptic excitation from antecedent reticulospinal neurones located in the rostral ventrolateral medulla (RVLM). Our study, conducted in anaesthetized rats, showed that all RVLM pre-sympathetic neurones display a substantial synaptic noise and their action potentials are usually preceded by fast EPSPs. No evidence was found for presence of gradual depolarizations (autodepolarizations) between individual spikes. Therefore our results are consistent with the "network" hypothesis for the generation of sympathetic vasomotor tone. Axons of some pre-sympathetic neurones intracellularly labelled with Neurobiotin or Lucifer Yellow had collaterals arborizing in several medullary regions. Thus these neurones have synaptic inputs not only to preganglionic sympathetic neurones, but also to other, yet unidentified cells in the brainstem. Finally, our results show that anatomically adjacent RVLM pre-sympathetic and Bötzinger respiratory neurones from two functionally distinct neuronal subpopulations, and that some pre-sympathetic neurones have an adrenergic phenotype.

Opioids and pain.

1. The central nervous system in mammals is able to react to painful stimuli at many levels that are involved in transmission, modulation and sensation of pain. Endogenous opioid peptides and their receptors are located at key points in pain pathways, and response to pain can be modulated by local application of opioids at many sites. Mechanisms of opioid analgesia at peripheral, spinal, medullary and midbrain levels are only incompletely understood; forebrain systems are even less appreciated. Local circuits in the spinal dorsal horn play a critical role in processing nociceptive afferent input and in mediating the actions of descending pain modulating systems. 2. The opioid receptors, recently cloned, exert their effects by activating G protein coupled effector systems, such as ion channels and second messenger systems. Although the receptor most commonly associated with pain relief is the mu-receptor, specific delta- and kappa-agonists can also mediate antinociception at spinal and supraspinal sites. Acute effects of opioids on target neurons are inhibitory, but excitatory effects have also been reported. 3. Noxious stimulation increases neuronal activity and modulates expression of genes, including immediate-early genes and neuropeptide (i.e. opioid) genes at spinal and supraspinal levels of the somatosensory system. Opioid drugs and endogenously released opioid peptides can modulate signal transduction mechanisms and intracellular processes that lead to alterations in protein phosphorylation and gene expression. These effects of opioids at the cellular level may underlie the mechanisms of pre-emptive analgesia and neuroplastic changes such as tolerance, dependence, sensitization, hyperalgesia, adaptation, addiction, and modulation of pain memories.

An electrophysiological and morphological study of esophageal motoneurons in rats.

Previous anatomic studies have shown that motoneurons supplying the striated musculature of the esophagus form a tightly grouped cluster in the rostral portion of the nucleus ambiguus, known as the compact formation. This study, conducted in anesthetized rats, presents the first in vivo intracellular and extracellular recordings from this group of motoneurons, which were identified by antidromic stimulation directly from the esophagus (latency 7-68 ms). The motoneurons were silent at rest, and those impaled intracellularly (n = 44) showed no respiratory modulation of their membrane potential. Intracellular labelling with Lucifer Yellow (n = 3) or Neurobiotin (n = 15) revealed multipolar somas with longitudinally oriented dendritic trees mainly confined to the compact formation. No axon collaterals were found. When swallowing-like activity was induced by muscarine applied to the dorsal medullary surface, the motoneurons displayed bursting activity, with the majority of bursts occurring during expiration. These results show that antidromic stimulation of esophageal motoneurons with an electrode inserted into the esophagus provides a simple way of identifying these motoneurons. In the absence of pharmacological stimulation, these motoneurons receive no respiratory-modulated synaptic input, in contrast to adjacent motoneurons in the semicompact formation (supplying the upper airways), which are known to display respiratory activity. However, some synchronization of respiratory and swallowing-like activity was observed after pharmacological activation of the swallowing pattern generator in the dorsal medulla.

Criteria for intracellular identification of pre-sympathetic neurons in the rostral ventrolateral medulla in the rat.

Previous studies revealed that a relatively small group of reticulospinal neurons located in the rostral ventrolateral medulla (RVLM) plays a key role in the generation of resting vasomotor tone and in reflex control of arterial blood pressure. These medullary pre-sympathetic neurons have been extensively studied with extracellular microelectrodes, but so far few attempts have been made to examine their intracellular properties in vivo. This report, based on intracellular recordings from 8 RVLM pre-sympathetic neurons in anaesthetised rats, sets out criteria for intracellular identification of such neurons. We propose that two features are sufficient to classify RVLM neurons as pre-sympathetic during intracellular recording: inhibitory response to stimulation of the aortic depressor nerve with short bursts of pulses applied at low frequency; and antidromic stimulation from the upper thoracic segments. Cardiac oscillations in the membrane potential or responses during large changes in blood pressure can be due to movement artefact, and are therefore not reliable as a means of demonstrating baroreceptor input. Further intracellular studies of these neurons will undoubtedly result in further progress in understanding their function.

Properties of presympathetic neurones in the rostral ventrolateral medulla in the rat: an intracellular study "in vivo'.

1. Intracellular recordings were made in pentobarbitone-anaesthetized rats from sixty-eight neurones located in the rostral ventrolateral medulla (RVLM), which responded with inhibition (latency, 33.6 +/- 9.3 ms) after stimulation of the aortic depressor nerve with short bursts of pulses. This inhibition was due to chloride- and voltage-dependent IPSPs. 2. Seventeen neurones could be excited antidromically after stimulation in the T2 spinal segment (conduction velocity 1.9-8.5 m.s-1) and were classified as RVLM presympathetic vasomotor neurones. 3. "Spontaneously' active neurones (n = 29) displayed a largely irregular pattern of firing, with no clear relationship between the level of the membrane potential and cycles of phrenic nerve activity at end-tidal CO2 < 5.0%. Cardiac cycle-related shifts of the membrane potential were not considered indicative of baroreceptor input as they could be due to movement artifacts. 4. All neurones displayed large synaptic activity (EPSPs and IPSPs, peak-to-peak amplitude > 5.0 mV). The depolarizing IPSPs observed during injection of chloride and/or negative current consisted of a phasic and a tonic component. 5. The on-going activity of these neurones resulted from synaptic inputs, with individual action potentials usually preceded by identifiable fast EPSPs. 6. No evidence was found for the presence of gradual depolarizations (autodepolarizations) between individual action potentials, and therefore under these experimental conditions the activity of RVLM presympathetic neurones did not depend on intrinsic pacemaker properties. 7. These results are consistent with the "network' hypothesis for the generation of sympathetic vasomotor tone.

Postnatal maturational changes in rat pelvic autonomic ganglion cells: a mixture of steroid-dependent and -independent effects.

Androgens have potent effects on the maturation and maintenance of a number of neural pathways involved in reproductive behaviors in males. Most studies in this area have focused on central pathways, but androgen receptors are expressed by many peripheral neurons innervating reproductive organs, and previous studies have demonstrated structural and chemical changes in these neurons at puberty and after castration. We have performed the first electrophysiological comparison of pelvic autonomic ganglion neurons in male rats before and after puberty and following pre- or postpubertal castration. Studies were performed in vitro on intact ganglia with hypogastric and pelvic nerves attached to allow synaptic activation of sympathetic or parasympathetic neurons, respectively. Pelvic ganglion neurons underwent many changes in their passive and active membrane properties over the pubertal period, and some of these changes were dependent on exposure to circulating androgens. The most pronounced steroid-dependent effects were on membrane capacitance (soma size) in sympathetic neurons and duration of the action potential afterhyperpolarization in tonic neurons. Our study also showed that rat pelvic ganglion cells and their synaptic inputs were more diverse than previously reported. In conclusion, this study demonstrated that rat pelvic ganglion neurons undergo considerable postnatal changes in their electrophysiological properties. The steroid dependence of some of these changes indicates that circulating androgens may influence reproductive behaviors at many locations within the nervous system not just in the brain and spinal cord.

P2 receptor excitation of rodent hypoglossal motoneuron activity in vitro and in vivo: a molecular physiological analysis.

The role of P2 receptors in controlling hypoglossal motoneuron (XII MN) output was examined (1) electrophysiologically, via application of ATP to the hypoglossal nucleus of rhythmically active mouse medullary slices and anesthetized adult rats; (2) immunohistochemically, using an antiserum against the P2X2 receptor subunit; and (3) using PCR to identify expression of P2X2 receptor subunits in micropunches of tissue taken from the XII motor nucleus. Application of ATP to the hypoglossal nucleus of mouse medullary slices and anesthetized rats produced a suramin-sensitive excitation of hypoglossal nerve activity. Additional in vitro effects included potentiation of inspiratory hypoglossal nerve output via a suramin- and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS)-sensitive mechanism, XII MN depolarization via activation of a suramin-sensitive inward current, decreased neuronal input resistance, and a slow-onset theophylline-sensitive reduction of inspiratory output likely resulting from hydrolysis of extracellular ATP to adenosine and activation of P1 receptors. Immunohistochemically, P2X2 receptors were detected in inspiratory XII MNs that were labeled with Lucifer yellow. These data, combined with identification of mRNA for three P2X2 receptor subunit isoforms within the hypoglossal nucleus (two of which have not been localized previously in brain) and the previous demonstration that P2X receptors are ubiquitously expressed in cranial and spinal motoneuron pools, support not only a role of P2 receptors in modulating inspiratory hypoglossal activity but a general role of P2 receptors in modulating motor outflow from the CNS.

Purinergic regulation of sound transduction and auditory neurotransmission.

In the cochlea, extracellular ATP influences the endocochlear potential, micromechanics, and neurotransmission via P2 receptors. Evidence for this arises from studies demonstrating widespread expression of ATP-gated ion channels (assembled from P2X receptor subunits) and G protein-coupled receptors (P2Y receptors). P2X2 receptor subunits are localized to the luminal membranes of epithelial cells and hair cells lining scala media. These ion channels provide a shunt pathway for K+ ion egress. Thus, when noise exposure elevates ATP levels in this cochlear compartment, the K+ conductance through P2X receptors reduces the endocochlear potential. ATP-mediated K+ efflux from scala media is complemented by a P2Y receptor G protein-coupled pathway that provides coincident reduction of K+ transport into scala media from the stria vascularis when autocrine or paracrine ATP signalling is invoked. This purinergic signalling likely provides a basis for a reactive homoeostatic regulatory mechanism limiting cochlear sensitivity under stressor conditions. Elevation of ATP in the perilymphatic compartment under such conditions is also likely to invoke purinergic receptor-mediated changes in supporting cell micromechanics, mediated by Ca2+ influx and gating of Ca2+ stores. Independent of these humoral actions, ATP can be classified as a putative auditory neurotransmitter based on the localization of P2X receptors at the spiral ganglion neuron-hair cell synapse, and functional verification of ATP-gated currents in spiral ganglion neurons in situ. Expression of P2X receptors by type II spiral ganglion neurons supports a role for ATP as a transmitter encoding the dynamic state of the cochlear amplifier.