Characterization of the Sheep Round Window Membrane.

Intratympanic injection is a clinically used approach to locally deliver therapeutic molecules to the inner ear. Drug diffusion, at least in part, is presumed to occur through the round window membrane (RWM), one of the two openings to the inner ear. Previous studies in human temporal bones have identified a three-layered structure of the RWM with a thickness of 70-100 µm. This is considerably thicker than the RWM in rodents, which are mostly used to model RWM permeability and assess drug uptake. The sheep has been suggested as a large animal model for inner ear research given the similarities in structure and frequency range for hearing. Here, we report the structure of the sheep RWM. The RWM is anchored within the round window niche (average vertical diameter of 2.1 ± 0.3 mm and horizontal diameter of 2.3 ± 0.4 mm) and has a curvature that leans towards the scala tympani. The centre of the RWM is the thinnest (55-71 µm), with increasing thickness towards the edges (< 171 µm), where the RWM forms tight attachments to the surrounding bony niche. The layered RWM structure, including an outer epithelial layer, middle connective tissue and inner epithelial layer, was identified with cellular features such as wavy fibre bundles, melanocytes and blood vessels. An attached "meshwork structure" which extends over the cochlear aqueduct was seen, as in humans. The striking anatomical similarities between sheep and human RWM suggest that sheep may be evaluated as a more appropriate system to predict RWM permeability and drug delivery in humans than rodent models.

Reduced sensory stimulation alters the molecular make-up of glutamatergic hair cell synapses in the developing cochlea.

Neural activity during early development is known to alter innervation pathways in the central and peripheral nervous systems. We sought to examine how reduced sound-induced sensory activity in the cochlea affected the consolidation of glutamatergic synapses between inner hair cells (IHC) and the primary auditory neurons as these synapses play a primary role in transmitting sound information to the brain. A unilateral conductive hearing loss was induced prior to the onset of sound-mediated stimulation of the sensory hair cells, by rupturing the tympanic membrane and dislocating the auditory ossicles in the left ear of P11 mice. Auditory brainstem responses at P15 and P21 showed a 40-50-dB increase in thresholds for frequencies 8-32kHz in the dislocated ear relative to the control ear. Immunohistochemistry and confocal microscopy were subsequently used to examine the effect of this attenuation of sound stimulation on the expression of RIBEYE, which comprises the presynaptic ribbons, Shank-1, a postsynaptic scaffolding protein, and the GluA2/3 and 4 subunits of postsynaptic AMPA receptors. Our results show that dislocation did not alter the number of pre- or postsynaptic protein puncta. However, dislocation did increase the size of RIBEYE, GluA4, GluA2/3 and Shank-1 puncta, with postsynaptic changes preceding presynaptic changes. Our data suggest that a reduction in sound stimulation during auditory development induces plasticity in the molecular make-up of IHC glutamatergic synapses, but does not affect the number of these synapses. Up-regulation of synaptic proteins with sound attenuation may facilitate a compensatory increase in synaptic transmission due to the reduced sensory stimulation of the IHC.

High frequency bone conduction auditory evoked potentials in the guinea pig: Assessing cochlear injury after ossicular chain manipulation.

Permanent high frequency (>4 kHz) sensorineural hearing loss following middle ear surgery occurs in up to 25% of patients. The aetiology of this loss is poorly understood and may involve transmission of supra-physiological forces down the ossicular chain to the cochlea. Investigating the mechanisms of this injury using animal models is challenging, as evaluating cochlear function with evoked potentials is confounded when ossicular manipulation disrupts the normal air conduction (AC) pathway. Bone conduction (BC) using clinical bone vibrators in small animals is limited by poor transducer output at high frequencies sensitive to trauma. The objectives of the present study were firstly to evaluate a novel high frequency bone conduction transducer with evoked auditory potentials in a guinea pig model, and secondly to use this model to investigate the impact of middle ear surgical manipulation on cochlear function. We modified a magnetostrictive device as a high frequency BC transducer and evaluated its performance by comparison with a calibrated AC transducer at frequencies up to 32 kHz using the auditory brainstem response (ABR), compound action potential (CAP) and summating potential (SP). To mimic a middle ear traumatising stimulus, a rotating bur was brought in to contact with the incudomalleal complex and the effect on evoked cochlear potentials was observed. BC-evoked potentials followed the same input-output function pattern as AC potentials for all ABR frequencies. Deterioration in CAP and SP thresholds was observed after ossicular manipulation. It is possible to use high frequency BC to evoke responses from the injury sensitive basal region of the cochlea and so not rely on AC with the potential confounder of conductive hearing loss. Ongoing research explores how these findings evolve over time, and ways in which injury may be reduced and the cochlea protected during middle ear surgery.

Noise exposure of workers and the use of hearing protection equipment in New Zealand.

Hearing loss from occupational noise exposure is a significant occupational health problem, requiring effective health and safety strategies. Essential to this is an understanding of the noise exposure of workers and the use of hearing protection equipment (HPE). This study reports on data collected in New Zealand. Visits were made to companies in each economic sector. Personal dosimetry was used to assess individual noise exposure of 529 workers. Workers were also interviewed about their use of HPE. Overall, 40.4% of production workers had a daily noise exposure greater than 1 Pa(2)h, exceeding the New Zealand National Standard for occupational noise exposure without HPE. Of these, 88.5% reported to use HPE when working in noise; however, some observations suggested that workers do not consistently use the devices. These data add to the overall picture of noise exposure of workers in New Zealand and are especially useful in areas where data did not previously exist or were difficult to access.

Noise exposure induces up-regulation of ecto-nucleoside triphosphate diphosphohydrolases 1 and 2 in rat cochlea.

Extracellular ATP acting via P2 receptors in the inner ear initiates a variety of signaling pathways that may be involved in noise-induced cochlear injury. Nucleoside triphosphate diphosphohydrolase (NTPDase)1/CD39 and NTPDase2/CD39L1 are key elements for regulation of extracellular nucleotide concentrations and P2 receptor signaling in the cochlea. This study characterized the effect of noise exposure on regulation of NTPDase1 and NTPDase2 expression in the cochlea using a combination of real-time RT-PCR, immunohistochemistry and functional studies. Adult Wistar rats were exposed to broad band noise at 90 dB and 110 dB sound pressure level (SPL) for 72 h. Exposure to 90 dB SPL induced a small and temporary change of auditory thresholds (temporary threshold shift), while exposure to 110 dB SPL induced a robust and permanent change of auditory thresholds (permanent threshold shift). NTPDase1 and NTPDase2 mRNA transcripts were upregulated in the cochlea exposed to 110 dB SPL, while mild noise (90 dB SPL) altered only NTPDase1 mRNA expression levels. Changes in NTPDases expression did not correlate with levels of circulating corticosterone, implying that the up-regulation of NTPDases expression was not stress-related. Semi-quantitative immunohistochemistry in the cochlea exposed to 110 dB SPL localized the increased NTPDase1 and NTPDase2 immunostaining in the stria vascularis and up-regulation of NTPDase2 in the intraganglionic spiral bundle. In contrast, NTPDase1 was down-regulated in the cell bodies of the spiral ganglion neurones. Distribution of NTPDases was not altered in the cochlea exposed to 90 dB SPL. Functional studies revealed increased ectonucleotidase activities in the cochlea after exposure to 110 dB SPL, consistent with up-regulation of NTPDases. The changes in NTPDases expression may reflect adaptive response of cochlear tissues to limit ATP signaling during noise exposure.

Ensemble spontaneous activity in the guinea-pig cochlear nerve.

Spectral analysis of electrical noise recorded from the round window (RW) of the cochlea is referred to as the ensemble spontaneous activity (ESA) of the cochlear nerve. The ESA is considered to represent the summed spontaneous activity of single fibers of the auditory nerve and changes in the spectral characteristics of the ESA have been observed in humans with tinnitus. Experiments were undertaken to determine the relationship of the ESA to auditory neurotransmission. The ESA consisted of energy centered at approximately 900 Hz, similar to the spectral peak of single auditory neuron discharges. The amplitude of the ESA was correlated with good auditory sensitivity in the 12-30 kHz region of the cochlea. Constant pure tones of 12-22 kHz suppressed the ESA reducing its amplitude in a frequency and intensity dependent manner implying that the ESA recorded at the RW is generated or dominated by neurons in the basal region of the cochlea. The ESA was significantly suppressed by round window perfusion of the P2X receptor agonist adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) (10 mM) the glutamate receptor antagonist 6-7-dinitroquinoxaline-2,3-dione (DNQX) (1 mM), and the sodium channel antagonist tetrodotoxin (TTX) (20 microM). Following intravenous furosemide injection (40 mg/kg) reduction and recovery of the ESA correlated with similar changes in the endocochlear potential (EP). Following DNQX and ATPgammaS an additional spectral peak at 200 Hz was often observed. This peak has been postulated to be a correlate of tinnitus in humans but had not previously been observed in a guinea-pig model of tinnitus. These data confirm the spectral characteristics of the ESA in guinea-pigs and show it is dependent on the sensitivity of the auditory nerve and intact auditory neurotransmission. In addition these experiments support the view that the ESA represents summed spontaneous neural activity in the cochlea and provide a platform for studies of the influence of ototoxic compounds on the spontaneous neural outflow of the cochlea as a model of tinnitus.

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.

Auditory evoked potentials as measures of plasticity in humans.

There is increasing evidence from animal studies for plasticity of auditory function. This has prompted research to determine whether such plastic changes occur in adults and children with hearing disorders. Behavioural measures such as speech perception scores do show improvements after hearing aid fitting and cochlear implantation. Several studies have also shown changes in cortical auditory evoked potentials after cochlear implantation and after auditory training. These studies indicate that improvements in speech perception ability are associated with changes in the central auditory system, particularly at the cortical level.

Transient expression of P2X(1) receptor subunits of ATP-gated ion channels in the developing rat cochlea.

The expression pattern of the ATP-gated ion channel P2X(1) receptor subunit was studied in the developing rat cochlea by riboprobe in situ hybridisation and immunohistochemistry. Embryonic (E12, E14, E16 and E18) and postnatal (P0, P2, P4, P6, P10 and adult) rat cochleae were examined. Both mRNA and protein localisation techniques demonstrated comparable P2X(1) receptor expression from E16 until P6 but this expression was absent at later developmental stages. P2X(1) receptor mRNA expression was localised within the otic capsule and associated mesenchyme (from E16 to P6), spiral limbus (from P0 to P6) and within the spiral ligament adjacent to the insertion of Reissner's membrane (from P2 to P6). P2X(1) receptor protein had a similar distribution based upon immunoperoxidase localisation. P2X(1) receptor-like immunoreactivity was detected in the otic capsule and the surrounding mesenchyme (from E16 to P6), spiral limbus (from P0) and epithelial cells of Reissner's membrane (from P2 to P6). The spiral ganglion neurones showed the earliest P2X(1) receptor expression (from E16 to P6). This became associated with immunolabelling of their afferent neurite projections to the base of the developing inner and outer hair cells (observed from E18 and peaking at P2). Immunolabelling of the efferent nerve fibres of the intraganglionic spiral bundle (from E18 to P6) within the spiral ganglion was also observed. The results suggest that ATP-gated ion channels assembled from P2X(1) receptor subunits provide a signal transduction pathway for development of afferent and efferent innervation of the sensory hair cells and purinergic influence on cochlear morphogenesis.

Vesicular storage of adenosine triphosphate in the guinea-pig cochlear lateral wall and concentrations of ATP in the endolymph during sound exposure and hypoxia.

Previous studies have revealed putative vesicular stores of adenosine triphosphate (ATP) in the marginal cells of the cochlear stria vascularis which may serve as a source of ATP for purinergic signalling. This study aimed to provide further evidence of ATP storage in the cochlea and to see whether ATP levels in the endolymph are affected by noise and hypoxia. Tissues from the lateral wall and organ of Corti of the guinea-pig cochlea were fractionated to obtain vesicular (VF) and mitochondrial (MF) fractions. Free and total ATP were then measured by the luciferase-luciferin reaction from which membrane-bound vesicular ATP was calculated. In the lateral wall, the VF contained 2.02+/-0.04 nmol ATP/mg protein (n = 5), significantly greater (p < 0.001; paired Student's t-test) than the concentration of ATP in the MF (0.36+/-0.05). In the organ of Corti, the VF contained 0.69+/-0.08 nmol ATP/mg protein (n = 4), significantly smaller than the amount in the VF of the lateral wall tissues (p < 0.001; non-paired Student's t-test). Small amounts of fumarase. an enzyme of the mitochondrial matrix, in the VF, excluded the possibility of mitochondrial ATP contamination. To investigate the effect of hypoxia and noise on the ATP concentrations in the endolymph, fluid samples were collected from the first (basal) cochlear turn of anaesthetized guinea-pigs. As a result of hypoxia (15 min, 13% F1O2), ATP concentrations (nM, mean +/- SEM) increased from 6.2+/-2.3 to 9.3+/-4.5 (n = 4), but the difference was not statistically significant. As a result of noise (15 min, 10 kHz, 110 dB SPL. broad band), the ATP levels increased significantly from 7.4+/-1.2 to 16.0+/-1.8 (p = 0.01; Student's t-test: n = 4). This study has demonstrated the presence of a vesicular store of ATP in the stria vascularis of the cochlea and described an increase in the ATP levels in the endolymph during noise exposure. The findings suggest that ATP is actively secreted from the vesicular store under conditions of metabolic stress. The presence of ATP under basal conditions supports a role for ATP in the sound transduction process during normal function.

Purinergic signalling: an experimental perspective.

Investigation of the multiple roles of extracellular nucleotides in the cochlea has developed from analysis of ATP-activated conductances in single sensory hair cells. Molecular probes such as radiolabelled ATP analogues and radiolabelled mRNA for ATP-gated ion channel subunits (P2X receptors) rapidly revealed the extensive nature of ATP signalling in this sensory organ. This has provided a foundation for physiological investigations which put extracellular nucleotides at the centre of homeostatic regulation of the driving force for sound transduction, modulation of mechanical tuning, control of cochlear blood flow and auditory neurotransmission. The purinergic signal transduction pathways associated with these processes have several novel features of significance to the broader field of purinergic neuroscience. In turn, these studies have benefited from the recent experimental advances in the field of purinergic signalling, a significant component of which is associated with the work of Professor Geoffrey Burnstock.

Immunohistochemical localization of adenosine 5'-triphosphate-gated ion channel P2X(2) receptor subunits in adult and developing rat cochlea.

Substantial in vitro and in vivo data support a role for extracellular adenosine 5;-triphosphate (ATP) and associated P2 receptors in cochlear function. However, the precise spatiotemporal distribution of the involved receptor protein(s) has not been determined. By using a specific antiserum and immunoperoxidase labeling, the tissue distribution of the P2X(2) subunit of the ATP-gated ion channel was investigated. Here, we describe the first extensive immunohistochemical mapping of P2X(2) receptor subunits in the adult and developing rat cochlea. In the adult, immunoreactivity was observed in most cells bordering on the endolymphatic compartment (scala media), particularly in the supporting cells. Hair cells were not immunostained by the P2X(2) antiserum, except for outer hair cell stereocilia. In addition, weak immunolabeling was observed in some spiral ganglion neurons. P2X(2) receptor subunit protein expression during labyrinthine ontogeny was detected first on embryonic day 19 in the spiral ganglion and in associated nerve fibers extending to the inner hair cells. Immunostaining also was observed underneath outer hair cells, and, by postnatal day 6 (P6), intense immunolabeling was seen in the synaptic regions of both types of hair cell. Supporting cells of the sensory epithelium were labeled at P0. This labeling became most prominent from the onset of cochlear function (P8-P12). Conversely, expression in the vascular stria declined from this time. By P21, the pattern of immunolabeling was similar to that found in the adult. The localization and timing of P2X(2) immunoreactivity suggest involvement of extracellular ATP and associated ATP-gated ion channels in important physiological events, such as inner ear ontogeny, sound transduction, cochlear micromechanics, electrochemical homeostasis, and auditory neurotransmission.

Apoptosis in the developing rat cochlea and its related structures.

Mammalian development involves proliferation and programmed cell death (apoptosis). This study was undertaken to analyse the spatial and temporal organisation of apoptosis in developing rat cochlear and associated tissues using in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labelling of DNA fragments (TUNEL), and light and electron microscopy. Embryonic (E12-E19 days) and postnatal rats (P0-P21 days) were studied. Fixed tissues were stained for apoptosis using TUNEL technique and the cytomorphology of apoptosis was confirmed by light and electron microscopy. Apoptotic cells were detected predominantly during the embryonic and early postnatal development of the cochlea. Apoptosis occurred in embryonic precursors of the cochlear duct epithelium, mainly in the region of its outgrowth between E12 and E16. In the periotic mesenchyme, apoptosis occurred in areas committed to develop into the middle ear cavity (peaking at E16) and perilymphatic compartments (peaking around E18-E19). Apoptosis in the VIIIth nerve (statoacoustic) ganglion was detected throughout the embryonic and early postnatal periods, peaking at E18-E19, around the time when the cochlear neural connections are being established. At later postnatal days, apoptosis was seen only occasionally in cochlear tissues, predominantly in tissues lining the middle ear cavity and sporadically in cells of the otic capsule. Therefore, apoptosis appears to occur in areas of remodeling, in areas of cavitation and in areas of differentiation. These findings provide a template for studying the molecular mechanisms involved in the development of the rat inner ear.

Evidence for alternative splicing of ecto-ATPase associated with termination of purinergic transmission.

Ectonucleotidases provide the signal termination mechanism for purinergic transmission, including fast excitatory neurotransmission by ATP in the CNS. This study provides evidence for ectonucleotidase expression in the rat cochlea, brain and other tissues. In addition to detection of rat ecto-ATPase and ecto-ATPDase in these tissues, we identify a novel ecto-ATPase splice variant arising from the loss of a putative exon (193 bp) in the C-terminal coding region. This is the first evidence of alternative splicing in the ecto-ATPase gene family. Splicing of the 193-bp putative exon containing a stop codon extends the open reading frame and provides translation of an additional 50 amino acids compared with the isoform isolated earlier from the rat brain (rEATPase(A); GenBank accession #Y11835). The splice variant (rEATPase(B); GenBank accession #AF129103) encodes 545 amino acids with a predicted protein molecular mass of 60 kDa. rEATPase(B) contains a long cytoplasmic tail (62 amino acids) with three potential protein kinase CK2 phosphorylation sites not present in rEATPase(A). Co-expression of two ecto-ATPase isoforms with different regulatory sites suggests that the extracellular ATP signal levels may be differently influenced by intracellular feedback pathways.

P2X receptor-mediated changes in cochlear potentials arising from exogenous adenosine 5'-triphosphate in endolymph.

Our previous studies have determined the presence of adenosine 5'-triphosphate (ATP) in the cochlear fluids and shown that extracellular ATP introduced into the endolymphatic compartment of the guinea pig cochlea has a significant dose-dependent suppressive effect on both endocochlear potential (EP) and cochlear microphonic (CM), which is mediated via P2 receptors. In the present study, the influence of P2 receptor agonists and antagonists on this suppressive effect was investigated to characterise the subtypes of P2 receptor mediating the ATP-induced effect on cochlear function. Using a double-barreled pipette attached to a pressure injector, small volumes (2-10 nl) of ATP (0.01-1 mM) and P2 receptor agonists or P2 receptor antagonists in artificial endolymph were introduced into the scala media of the first (basal) and third turns of the guinea pig cochlea, while the EP and CM were monitored. ATP and P2 receptor agonists (5x10(-14)-1x10(-11)cibacron blue. Neither adenosine nor uridine 5'-triphosphate (2x10(-13)-2x10(-11) moles) nor the P2 receptor antagonists on their own had any effect on EP and CM. The ATP effect on the potentials was greater at the third cochlear turn when compared to the first turn. These results provide evidence that in the endolymphatic compartment of the guinea pig, the extracellular ATP effect on cochlear function is likely mediated through an interaction with P2 receptors which assemble as ATP-gated ion channels.

ATP-gated ion channel expression in primary auditory neurones.

Extracellular ATP acts via ionotropic P2X receptors to mediate fast neurotransmission in the central and autonomic nervous systems. Recent data, including identification of P2X2 receptor mRNA expression by spiral ganglion neurones, suggests that purinergic signalling may influence auditory neurotransmission via ATP-gated ion channels assembled from these subunits. Expression of the P2X2 receptor was localized to the region of the spiral ganglion neurone synapses with the inner hair cells using a P2X2 receptor specific antiserum. Whole-cell patch clamping of neurones cultured from post-natal day 3-5 spiral ganglia demonstrated a heterogeneity of ATP-activated conductances, consistent with the functional expression of P2X2 receptor subunit isoforms along with possible co-expression of additional P2X receptor subunits. These data provide substantive support for a purinergic transmission element at the peripheral auditory synapse.

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.

Modulation of cochlear blood flow by extracellular purines.

Humoral adenosine 5'-triphosphate (ATP), adenosine and uridine 5'-triphosphate (UTP) have been shown to have a role in controlling local blood flow in a variety of tissues. The presence of P1 and P2 receptors in the cochlea, and particularly the highly vascular region, the stria vascularis, implies a vasoactive role for these compounds in the inner ear. To test the effect of extracellular purines and pyrimidines on cochlear blood flow, cochleae from anaesthetised guinea-pigs were perfused with ATP (1 microM-10 mM), adenosine (1 microM-10 mM) and UTP (1 mM) in artificial perilymph while blood flow through the cochlea was measured. An acute perilymphatic perfusion technique was established via tubing placed through a hole in the bone overlying scala tympani of the first cochlear turn, with an outlet hole in scala vestibuli of the fourth turn. Blood flow was measured by placing the probe of a laser Doppler blood perfusion monitor on the bone overlying the stria vascularis in the third cochlear turn. ATP and adenosine produced a significant dose dependent increase in cochlear blood flow (28.8-229.0% and 35.8-258.1%, respectively). The effect of ATP (100 microM) on cochlear blood flow was reduced in the presence of reactive blue 2 (1 mM) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (1 mM). The blood flow response to adenosine (10 microM) was reduced in the presence of 1,3-dimethylxanthine (theophylline, 100 microM), but not with either 3,7-dimethyl-1-propargylxanthine (10 microM) or 8-cyclopentyl-1,3-dipropylxanthine (10 microM). UTP did not produce any change in the cochlear blood flow. To determine if the ATP effect was also mediated by adenosine derived from ectonucleotidase activity, the perilymphatic compartment was perfused with either ATP plus theophylline (100 microM) or with the non-metabolisable form of ATP, adenosine 5'-O-(3-thiophosphate) (ATPgammaS, 100 microM). The effect of ATP on cochlear blood flow was unaffected with the inclusion of theophylline while ATPgammaS produced an increase in cochlear blood flow similar to the one observed with ATP. These findings indicate that extracellular ATP and its metabolite adenosine have a modulatory role in cochlear blood flow possibly mediated by both P1 and P2 receptors.

Fluorescence imaging of Na+ influx via P2X receptors in cochlear hair cells.

The adenosine 5'-triphosphate (ATP)-activated membrane conductance, mediated by P2X receptors, was examined in isolated guinea-pig cochlear inner and outer hair cells. Photo-activated release of caged-ATP elicted a 30-ms latency inwardly rectifying non-selective cation conductance, blocked by the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10-100 microM), consistent with the direct activation of ATP-gated ion channels. A K(Ca) conductance in the inner hair cells (IHC), activated by the entry of Ca2+ through the ATP-gated ion channels, was blocked by including 10 mM 1,2-his(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the internal solution. Real-time confocal slit-scanning fluorescence imaging of Na+ influx through the ATP-gated ion channels was performed using the dye Sodium Green with simultaneous whole-cell recording of membrane currents. The Na+ entry was localized to the endolymphatic surface, with the increase in [Na+]i detected within approximately 200 ms of the onset of the inward current response. Within 600 ms Na+ had diffused throughout the cell cytoplasm with the exception of the subnuclear region of the outer hair cells. Correlation of voltage-clamp measurements of Na+ entry with regional increases in Na+-induced fluorescence demonstrated ATP-induced increases in intracellular Na+ in excess of 45 mM within 4 s. These data provide direct evidence for the Na+ permeability of the ATP-gated ion channels as well as independent evidence for the localization of P2X receptors at the endolymphatic surface of the sensory hair cells. The localization of the ATP-gated ion channels to the apical surface of the hair cells supports an ATP-mediated modulation of 'silent' K+ current across the cochlear partition which could regulate hearing sensitivity by controlling the transcellular driving force for both mechanoelectrical and electromechanical transduction in hair cells.