Postnatal changes in TASK-1 and TREK-1 expression in rat brain stem and cerebellum

Developmental changes in expression of two-pore domain K+ channels, TASK-1 and TREK-1, were investigated in the juvenile (postnatal day 13; P13) and adult (P105) rat brain stem and cerebellum using immunohistochemistry. In the juvenile, extensive TASK-1-like immunoreactivity (TASK-1-LIR) was seen among glial cells in the white matter (e.g., radial glia), which showed marked reduction in the adult. In contrast, TASK-1-LIR in neurons including cerebellar Purkinje and granule cells, hypoglossal and facial motoneurons, and ventrolateral medulla neurons was increased in the adult. TASK-1-LIR in neuroglia surrounding peripheral axons of cranial nerves was persistent. TREK-1-LIR was similar between ages, although TREK-1-LIR was neuronal and present only in juvenile cerebellar external germinal layer. Present results suggest roles for TASK-1 and K+ homeostasis in neuro-glial interaction, neurogenesis, differentiation, migration, axon guidance, synaptogenesis and myelination.     

Tandem-pore K(+) channels display an uneven distribution in amphibian retina

Previous studies in retinal glial (Müller) cells have suggested that the dominant membrane currents are mediated by K(+) inward-rectifier (Kir) channels. After blockade of inwardly (Kir) and outwardly (KD and BK) conducting channels, a large K(+) conductance remains, but its nature has not been determined. Tandem-pore K(+) channels are likely candidates for this potassium conductance and the purpose of the present study was to determine, using immunocytochemistry, whether Müller cells express TASK-1, TASK-2, TREK-1 and/or TREK-2 potassium channel subunits. The results reveal that retinal glial cells express TASK-1 and TASK-2 subunits, but not TREK-1 or TREK-2 subunits. Furthermore, the distribution of TASK subunits differs from that of Kir channels and may contribute to the potassium conductance of Müller cells.     

Spatiotemporal patterns of neuronal programmed cell death during postnatal development of the gerbil cochlea

During early postnatal development, afferent neurons of the cochlear (spiral) ganglion progressively refine their projections to auditory hair cells so that, by hearing onset, most cochlear nerve fibers innervate a single hearing receptor. One mechanism that might contribute to these changes in cochlear innervation is the programmed cell death (apoptosis) of developing neurons within the spiral ganglion. In the present study, we used the TUNEL method and morphological criteria to identify apoptotic cells within the spiral ganglion of the Mongolian gerbil during the first week of postnatal life when afferent projections to the cochlea are actively refined in this species. The locations of individual apoptotic spiral ganglion cells were mapped onto three-dimensional reconstructions of the entire ganglion for an age-graded series of gerbils to produce the first high-resolution, spatiotemporal maps of apoptotic ganglion cell death for the postnatal cochlea. We observed a significant increase in apoptosis in the spiral ganglion from postnatal day (P) 4 through P6. During this time, the most intense apoptotic activity occurred in regions of the spiral ganglion providing innervation to the lower middle and apical turns of the cochlea. The time course and regional variation of programmed cell death within the developing gerbil spiral ganglion are discussed in terms of the postnatal refinement of cochlear innervation and its possible functional significance for hearing in gerbils.     

Localization of mRNA encoding the P2X2 receptor subunit of the adenosine 5′-triphosphate-gated ion channel in the adult and developing rat inner ear by in situ hybridization

Localization of expression of the adenosine 5′-triphosphate (ATP)-gated ion channel P2X₂ receptor subunit (P2X₂R) in the rat inner ear at different stages of development was achieved by using in situ mRNA hybridization. In the adult, P2X₂R mRNA was strongly expressed in many of the cells bordering the cochlear endolymphatic compartment. This included the interdental cells of the spiral limbus, all cells of the inner sulcus and organ of Corti, and cells of the spiral prominence. In the vestibular labyrinth, strong expression was noted in the transitional cells at the base of the crista ampullaris and in the sensory epithelium of the crista and maculae. During development, P2X₂R mRNA expression was evident in the precursors of these structures at the earliest period studied, embryonic day 12 (E12). Expression increased during the ontogeny in both the cochlear and the vestibular end organs. In addition, both the spiral and vestibular ganglia showed developmental expression. In contrast to the supporting cells of the organ of Corti, both inner and outer hair cells exhibited P2X₂R mRNA only after postnatal day 10 (P10) through P12, concomitant with the onset of hearing. P2X₂R expression levels in all cells fell from a maximum at P12–P18 to lower levels in the adult. In the adult, P2X₂R mRNA levels were modest in outer hair cells in the basal (high-frequency) encoding region of the cochlea, and inner hair cell labeling was low throughout the cochlea. Reissner's membrane, which maintains an electrochemical barrier between scala vestibuli and scala media, showed considerable expression of P2X₂R mRNA in early postnatal development, and expression was maintained at moderate levels in the adult cochlea. These data are consistent with a role for the P2X₂R subunit in the processes of labyrinthine development and the regulation of the electrochemical gradients supporting auditory and vestibular sensory transduction. J. Comp. Neurol. 393:403–414, 1998. © 1998 Wiley-Liss, Inc.     

Identification of native rat cerebellar granule cell currents due to background K channel KCNK5 (TASK-2)

The TWIK-related, Acid Sensing K (TASK-2; KCNK5) potassium channel is a member of the tandem pore (2P) family of potassium channels and mediates an alkaline pH-activated, acid pH-inhibited, outward-rectified potassium conductance. In previous work, we demonstrated TASK-2 protein expression in newborn rat cerebellar granule neurons (CGNs). In this study, we demonstrate TASK-2 functional expression in CGNs as a component of the pH-sensitive, volatile anesthetic-potentiated, standing-outward potassium conductance (I(K,SO)). Using excised, inside-out patch-clamp technique, we studied CGNs grown in primary culture. We identified four distinct, noninactivating single channel potassium conductances, Types 1-4. Types 1-3 have previously been attributed to TASK-1 (KCNK3), TASK-3 (KCNK9) and TASK-1/TASK-3 heteromers, and TREK-2 (KCNK10) 2P potassium channel function, respectively; however, the Type 4 conductance is currently unassigned. Previous studies demonstrated that Type 4 single channel activity is potentiated by extracellular, alkaline pH and cytoplasmic arachidonic acid (10-20 microM) and inhibited by cytoplasmic tetraethylammonium (TEA; 1 mM). We determined that heterologously expressed TASK-2 channels have single channel gating, conductance properties and pH sensitivity identical to the Type 4 conductance. Additionally, we found that TASK-2 single channel activity, like the Type 4 conductance is potentiated by cytoplasmic arachidonic acid (20 microM) and inhibited by cytoplasmic TEA (1 mM). We conclude that TASK-2 mediates the Type 4 single channel conductance in CGNs as a component of I(K,SO).     

NADPH-diaphorase histochemistry reveals an autonomic-like innervation in the postnatal hamster cochlea.

Previous studies used nicotinamide adenine diphosphate (NADPH)-diaphorase histochemistry as an indicator of nitric oxide synthase (NOS) expression in the adult mammalian cochlea. In this study, we investigated the early postnatal expression of diaphorase activity in the hamster cochlea. Two types of extrinsic fibers were intensely labeled as early as postnatal day 3 (P3) in the portion of the cochlear nerve that innervates the base of the modiolus. By P10, these fibers had reached the spiral ganglion and were projecting toward the organ of Corti. The perivascular type of fiber did not project into the organ of Corti; however, the nonperivascular type could be traced among the supporting cells below the outer hair cells. Spiral ganglion cell somata were also labeled as early as P3. The onset of diaphorase expression in the spiral ganglion cells corresponds to a critical period of synaptogenesis for these sensorineural cells. If NADPH-diaphorase activity is an indicator of NOS, then our results suggest that NO may play a role during postnatal cochlear development.     

Localization of TREK-1, a two-pore-domain K+ channel in the peripheral vestibular system of mouse and rat

The distribution of two-pore-domain (2P-domain) K(+) channels of the TREK subfamily was studied using immunocytochemistry in the peripheral vestibular system of mouse and rat. Using RT-PCR, the mRNA for TREK-1, but not for TREK-2 or TRAAK, were detected in mouse vestibular endorgans and ganglia. The TREK-1 channel protein was immunodetected in both nerve fibers and nerve cell bodies in the vestibular ganglion, both afferent fibers and nerve calyces innervating type I hair cells in the utricle and cristae. The post-synaptic localization in afferent calyces may suggest a neuroprotective role in glutamatergic excitotoxicity during ischemic conditions. In non-neuronal cells, TREK-1 was immunodetected in the apical membrane of dark cells and transitional cells, both of which are involved in endolymph K(+) secretion and recycling. TREK-1 may subserve some neuroprotective function in afferent nerve fibers as well as play a role in endolymph potassium homeostasis.     

Developmental regulation of neuron-specific P2X3 receptor expression in the rat cochlea

ATP-gated ion channels assembled from P2X3 receptor (P2X3R) subunits contribute to neurotransmission and neurotrophic signaling, associated with neurite development and synaptogenesis, particularly in peripheral sensory neurons. Here, P2X3R expression was characterized in the rat cochlea from embryonic day 16 (E16) to adult (P49-56), using RT-PCR and immunohistochemistry. P2X3R mRNA was strongly expressed in the cochlea prior to birth, declined to a minimal level at P14, and was absent in adult tissue. P2X3R protein expression was confined to spiral ganglion neurons (SGN) within Rosenthal's canal of the cochlea. At E16, immunolabeling was detected in the SGN neurites, but not the distal neurite projection within the developing sensory epithelium (greater epithelial ridge). From E18, the immunolabeling was observed in the peripheral neurites innervating the inner hair cells but was reduced by P6. However, from P2-8, immunolabeling of the SGN neurites extended to include the outer spiral bundle fiber tract beneath the outer hair cells. This labeling of type II SGN afferent fiber declined after P8. By P14, all synaptic terminal immunolabeling in the organ of Corti was absent, and SGN cell body labeling was minimal. In adult cochlear tissue, P2X3R immunolabeling was not detected. Noise exposure did not induce P2X3R expression in the adult cochlea. These data indicate that ATP-gated ion channels incorporating P2X3R subunit expression are specifically targeted to the afferent terminals just prior to the onset of hearing, and likely contribute to the neurotrophic signaling which establishes functional auditory neurotransmission.     

Localization of the tandem pore domain K+ channel TASK-1 in the rat central nervous system

Recently, a new family of potassium channels with two pore domains in tandem and four transmembrane segments has been identified. Seven functional mammalian channels have been reported at this time. These channels give rise to baseline potassium currents because they are not gated by voltage and exhibit spontaneous activity at all membrane potentials. Although the physiological role of these ion channels has yet to be determined, three mammalian members of this family (TREK-1, TASK-1, TASK-2) are activated by volatile anesthetics and may therefore contribute to the central nervous system (CNS) depression produced by volatile anesthetics. In this study we used northern blot analysis and immunohistochemical localization to determine the expression of TASK-1 subunits in the CNS. TASK-1 immunoreactivity was prominently found in astrocytes of the hippocampus, in the median eminence, in the choroid plexus, and the granular layer, Purkinje cell layer, and molecular layer of the cerebellum. In the spinal cord, strong TASK-I immunoreactivity was seen in ependymal cells lining the central canal and in white matter. These findings suggest a role for the TASK-1 channel in the production of cerebrospinal fluid and function of hypothalamic neurosecretory cells.     

Time course of auditory impairment in mice lacking the electroneutral sodium bicarbonate cotransporter NBC3 (slc4a7)

Mice with a targeted disruption of the gene encoding the stilbene-insensitive electroneutral sodium bicarbonate cotransporter (NBC3; slc4a7) exhibit cochlear and retinal degeneration. To establish the progressive nature of sensory cells loss in slc4a7-/- deficient mice, we studied the morphology of cochleas of slc4a7-/- and slc4a7+/+ mice from postnatal day two (P2) to ninety (P90). Cell death was evaluated in slc4a7-/- cochleas using the TUNEL technique and caspase-3 immunoreactivity. The time course of NBC3 expression in the cochlea was assessed by immunohistochemistry using an antibody against NBC3. Between P2 and P8, slc4a7-/- mice cochlea exhibit normal morphology. There was a normal complement of inner and outer hair cells from the hook to the apical region. At P15, slc4a7-/- mice cochlea inner and outer hair cells were still present at the hook region, and vacuoles were seen underneath Hensen's cells. At P21, inner and outer hair cells were degenerated in this region. Between P30 and P90, there was a pronounced loss of hair cells and spiral ganglia neurons. Morphological analysis of the spiral ligament showed a progressive loss of type II and IV fibrocytes beginning at day 21. Transmission electron microscopy observations at P30 and P90 revealed that type II and IV fibrocytes showed shrinkage and vacuolization. In addition, hair cells were deteriorated with evidence of shrinkage and picnotic nuclei. TUNEL staining showed apoptotic cells at P8 in the organ of Corti at the basal region of the cochlea. At P15, caspase-3 immunoreactivity was present in supporting cells of the organ of Corti. NBC3 mild immunoreactivity was detected in the organ of Corti at P11. There was an increase in the expression of NBC3 in the spiral ligament between P17 and P19. From P21 to P90, NBC3 expression was confined to the spiral ligament and inner and outer sulcus cells. The vestibular sensory epithelia from slc4a7-/- mice were normal from P2 to P90. Damage of the sensory epithelia at the high frequency zone of the cochlea suggests that NBC3 may play an important physiological role in this region.     

The resting transducer current drives spontaneous activity in prehearing Mammalian cochlear inner hair cells

Spontaneous Ca(2+)-dependent electrical activity in the immature mammalian cochlea is thought to instruct the formation of the tonotopic map during the differentiation of sensory hair cells and the auditory pathway. This activity occurs in inner hair cells (IHCs) during the first postnatal week, and the pattern differs along the cochlea. During the second postnatal week, which is before the onset of hearing in most rodents, the resting membrane potential for IHCs is apparently more hyperpolarized (approximately -75 mV), and it remains unclear whether spontaneous action potentials continue to occur. We found that when mouse IHC hair bundles were exposed to the estimated in vivo endolymphatic Ca(2+) concentration (0.3 mm) present in the immature cochlea, the increased open probability of the mechanotransducer channels caused the cells to depolarize to around the action potential threshold (approximately -55 mV). We propose that, in vivo, spontaneous Ca(2+) action potentials are intrinsically generated by IHCs up to the onset of hearing and that they are likely to influence the final sensory-independent refinement of the developing cochlea.     

Localization of the tandem pore domain K+ channel KCNK5 (TASK-2) in the rat central nervous system

Tandem pore domain K+ channels (2P K+ channels) are responsible for background K+ currents. 2P K+ channels are the most numerous encoded K+ channels in the Caenorhabditis elegans and Drosophila melanogaster genomes and to date 14 human 2P K+ channels have been identified. The 2P K+ channel TASK-2 (also named KCNK5) is sensitive to changes in extracellular pH, inhibited by local anesthetics and activated by volatile anesthetics. While TASK-1 has been shown to be involved in controlling neuronal cell excitability, much less is known about the cellular expression and function of TASK-2, originally cloned from human kidney. Previous studies demonstrated TASK-2 mRNA expression in high abundance in human kidney, liver, and pancreas, but only low expression in mouse brain or even absent expression in human brain was reported. In this study we have used immunohistochemical methods to localize TASK-2 at the cellular level in the rat central nervous system. TASK-2 immunoreactivity is prominently found in the rat hippocampal formation with the strongest staining observed in the pyramidal cell layer and in the dentate gyrus, and the Purkinje and granule cells of cerebellum. Additional immunofluorescence studies in cultured cerebellar granule cells demonstrate TASK-2 localization to the neuronal soma and to the proximal regions of neurites of cerebellar granule cells. The superficial layers of spinal cord and small-diameter neurons of dorsal root ganglia also showed strong TASK-2 immunoreactivity. These results suggest a possible involvement of TASK-2 in central mechanisms for controlling cell excitability and in peripheral signal transduction.     

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.