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Biomedical subjects

A F Ryan

Publications and source records attributed to A F Ryan.

At least 37 records · Page 2Linked to original sources
PubMed1999

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.

Adenosine Triphosphate↗
PubMed1999

The bacterial Neo gene confers neomycin resistance to mammalian cochlear hair cells.

The aminoglycoside antibiotics are important agents in the treatment of bacterial infection. At pharmacological doses they also preferentially damage the sensory hair cells of the inner ear, leading to ototoxic hearing loss. However, it has been suggested that the mechanism of ototoxicity is different from that of bacterial toxicity. The bacterial neomycin phosphotransferase gene (Neo) confers resistance to this and related aminoglycoside antibiotics. To determine whether the Neo gene also confers resistance to vertebrate ototoxicity, the sensitivity of cochlear hair cells to neomycin was evaluated in mice with a targeted insertion of Neo. Organotypic cultures of the organ of Corti, isolated from neonatal wild-type mice and two strains of mice carrying the Neo gene, were cultured for 72 h in the absence (controls) or in the presence of 200 microM neomycin. Organs from wild-type mice showed no remaining outer hair cells and <25% of inner hair cells when incubated with neomycin. In contrast, organs from mice carrying the Neo gene showed no loss of hair cells after neomycin treatment.

Animals↗
PubMed1999

Effects of stimulus frequency and intensity on c-fos mRNA expression in the adult rat auditory brainstem.

Induction of the cellular fos gene (c-fos) is one of the earliest transcriptional changes observed following neuronal excitation. Although not an activity marker in the strict electrophysiological sense, many neurons in the central nervous system increase their c-fos expression after periods of sustained stimulation at physiological levels of intensity. In the present study, induction of c-fos mRNA expression was examined in the auditory brainstem after 1 hour of continuous free-field acoustic stimulation. Sprague-Dawley rats were exposed to pure tones of 2, 8, 16, or 32 kHz or half-octave noise bands centered on 2, 8, or 32 kHz at 80-120 dB SPL. Stimulation-induced c-fos mRNA expression was evident at all levels of the auditory brainstem, and this expression was intensity dependent. In some brain areas, induced expression manifested a clear tonotopic organization, i.e., in dorsal, posteroventral, and anteroventral cochlear nuclei, and in the medial nucleus of the trapezoid body. The inferior colliculus exhibited multiple tonotopic representations. The dorsal nucleus of the lateral lemniscus had a crude tonotopy. Although expression was present, tonotopy was not evident in periolivary nuclei or in the ventral or intermediate nuclei of the lateral lemniscus. Free-field diotic stimulation did not induce c-fos mRNA expression in the medial or lateral superior olivary nuclei. Expression was induced in the lateral superior olive by dichotic stimulation (after a unilateral cochlear ablation), and that expression was tonotopically organized. The results suggest that stimulation-induced c-fos mRNA expression can be an effective way of mapping neuronal activity in the central auditory system under both normal and pathological conditions.

Acoustic Stimulation↗
PubMed1999

Cochlear ablation alters acoustically induced c-fos mRNA expression in the adult rat auditory brainstem.

Expression of c-fos mRNA was studied in the adult rat brain following cochlear ablations by using in situ hybridization. In normal animals, expression was produced by acoustic stimulation and was found to be tonotopically distributed in many auditory nuclei. Following unilateral cochlear ablation, acoustically driven expression was eliminated or decreased in areas normally activated by the ablated ear, e.g., the ipsilateral dorsal and ventral cochlear nuclei, dorsal periolivary nuclei, and lateral nucleus of the trapezoid body and the contralateral medial and ventral nuclei of the trapezoid body, lateral lemniscal nuclei, and inferior colliculus. These deficits did not recover, even after long survivals up to 6 months. Results also indicated that neurons in the dorsal cochlear nucleus could be activated by contralateral stimulation in the absence of ipsilateral cochlear input and that the influence of the contralateral ear was tonotopically organized. Results also indicated that c-fos expression rose rapidly and persisted for up to 6 months in neurons in the rostral part of the contralateral medial nucleus of the trapezoid body following a cochlear ablation, even in the absence of acoustic stimulation. This response may reflect a release of constitutive excitatory inputs normally suppressed by missing afferent input or changes in homeostatic gene expression related to sensory deprivation. Instances of transient, surgery-dependent increases in c-fos mRNA expression in the absence of acoustic stimulation were observed in the superficial dorsal cochlear nucleus and the cochlear nerve root on the ablated side.

Acoustic Stimulation↗
PubMed1999

Spatial representation of frequency in the rat dorsal nucleus of the lateral lemniscus as revealed by acoustically induced c-fos mRNA expression.

The conventional view, based largely on studies in cats, holds that the dorsal nucleus of the lateral lemniscus (DNLL) is tonotopically organized with a dorsal (low-frequency) to ventral (high-frequency) representation. Based on the topography of projections between the DNLL and inferior colliculus, it has been proposed that the rat DNLL has a concentric, inside-to-outside, tonotopic organization with high frequencies represented along the rind and low frequencies represented in the core. We used acoustic stimulation and c-fos mRNA expression to examine this issue. Results suggest that the rat DNLL does have a crude tonotopic organization and that this tonotopy has a concentric component. Following high-frequency stimulation, labeled neurons were found most frequently along the margins of DNLL, although they also tended to be more concentrated ventrally. Many fewer neurons labeled following middle-frequency stimulation, and these tended to be more uniformly distributed throughout the nucleus. Still fewer neurons labeled after low-frequency stimulation and these tended to be scattered mostly in the dorsal half of the nucleus. We conclude that: (i) many more neurons in the rat DNLL are responsive to high-frequency than to low-frequency acoustic stimulation; and (ii) that the frequency representation of the rat DNLL has both concentric and dorsal-to-ventral components.

Acoustic Stimulation↗
PubMed1999

Effects of a hair cell transcription factor, Brn-3.1, gene deletion on homozygous and heterozygous mouse cochleas in adulthood and aging.

The transcription factor Brn-3.1, is expressed in the inner ear hair cells throughout life and is necessary for the development of these cells. Mutant mice in which the Brn-3.1 encoding region has been deleted have no identifiable hair cells, greatly reduced numbers of spiral ganglion cells and are deaf. A mutation in the human homologue of this gene has been shown to be related to adult onset, sensorineural hearing loss (Vahava et al., 1998). The question whether haploinsufficiency in the mutant Brn-3.1 mouse with a mixed C57BL6/129Sv genetic background could affect the adult or aged cochlea was tested, therefore, by measuring the auditory brainstem responses and examining the cochlea's histologically at 2, 18 and 24 months of age. The heterozygotes had a comparable hearing to the wild-type animals and similar patterns of cochlear degeneration. Both groups showed an about 30 dB hearing loss beginning at 18 months of age, outer hair cell degeneration and loss of spiral ganglion neurons in the basal turn. There appeared to be no effect of Brn-3.1 haploinsufficiency on the mouse cochlea, implying that one intact copy of the gene is sufficient to maintain a normal cochlea.

Aging↗
PubMed1999

Absence of mRNA encoding estrogen receptor in the rat cochlea.

Based on changes in hearing thresholds and tinnitus that are co-related with the menstrual cycle, it has been suggested that the cochlea may respond directly to estrogen. For this to occur, the cochlea should express estrogen receptors. In situ mRNA hybridization was performed on normal female rat cochleas, using radiolabeled RNA probes complementary to mRNA encoding estrogen receptor, to determine whether estrogen receptors are present in the cochlea. Strong hybridization of the riboprobes to sections of uterus and hypothalamus indicated that the technique detected estrogen receptor mRNA. No hybridization to any cochlear tissues was observed. The results indicate that estrogen receptors are not expressed on cochlear cells, at least in rats. This in turn suggests that variation in cochlear responses during the estrus cycle are not the result of the direct effect of estrogen on the cochlea. Such variation may, however, be caused by systemic changes in fluid regulation induced by estrogen receptors at a distant site, or by other hormone receptors.

Animals↗
PubMed1999

Transcript-specific mRNA trafficking based on the distribution of coexpressed myosin isoforms.

mRNAs encoding four myosin heavy chain (MHC) isoforms were localized in rat skeletal muscle fibers by in situ hybridization. The ratio of MHC transcript signal in the fiber core compared to the fiber periphery was quantified using image analysis. Two distinct patterns of subcellular localization were observed. Type 1 (beta-cardiac) and type 2A MHC mRNAs were located preferentially in the muscle fiber periphery, while type 2B and type 2X mRNAs were distributed homogeneously across the fiber cross section. Since most normal muscle fibers express only a single MHC isoform, this difference in mRNA distribution could reflect either variation in the localization of the synthetic apparatus across different fiber types or differences in the trafficking of different MHC transcripts. To examine the basis for the observed differential distribution in normal muscles, mRNA distribution was assessed in muscle fibers that coexpressed multiple isoforms of the fast MHCs (i.e. types 2A, 2X and 2B), which occurred either in the combination type 2A/2X or type 2X/2B. The quantitative mRNA distribution seen in muscle fibers expressing a single isoform was not significantly different compared to that observed for mRNAs coexpressed in the same fiber (p > 0.6). Given the size similarity and homology of our riboprobes, these data suggest that their subcellular localization may be determined by relatively small differences in the sequences of the mRNAs, perhaps by differential binding of RNA sequence motifs to cytoskeletal elements.

Animals↗
PubMed1998

GDNF protects the cochlea against noise damage.

Glial-derived neurotrophic factor (GDNF) was tested for its ability to prevent hearing and sensory cell loss in guinea pigs exposed to acoustic trauma. Hearing was measured prior to any treatment. Animals were exposed to damaging levels of noise either before or after local application of GDNF to one ear. Four weeks later, hearing and sensory cell loss was greater in the control ear than in the ear receiving GDNF before acoustic trauma or 2 h after trauma, but not 4 or 6 h after trauma. The results indicate that GDNF treatment in vivo can prevent cochlear sensory cell damage and hearing loss if present during or shortly after acoustic trauma.

Animals↗
PubMed1998

Four novel myosin heavy chain transcripts define a molecular basis for muscle fibre types in Rana pipiens.

1. Differential expression of myosin heavy chain (MHC) isoforms dramatically affects mechanical and energetic properties of skeletal muscle fibre types. As many as five different fibre types, each with different mechanical properties, have been reported in frog hindlimb muscles. However, only two frog MHC isoforms have previously been detected by SDS-PAGE and only one adult hindlimb MHC isoform has been cloned. 2. In the present study, four different fibre types (type 1, type 2, type 3 and tonic) were initially identified in adult Rana pipiens anterior tibialis muscle based on myosin ATPase histochemistry, size and location. Each fibre type exhibited unique reactivity to a panel of MHC monoclonal antibodies. Single fibre analysis using SDS-PAGE revealed that MHCs from immunohistochemically defined type 1, type 2 and type 3 fibres ran as three distinct isoform bands, while MHC of tonic fibres co-migrated with type 1 MHC. The combined data from immunohistochemistry and SDS-PAGE suggests that Rana fibre types are composed of four different MHCs. 3. Four novel MHC cDNAs were cloned and expression of the corresponding transcripts was measured in single immuno-identified fibres using specific polymerase chain reaction (PCR) primer pairs. Each of the four transcripts was found to be primarily expressed in a different one of the four fibre types. 4. Coexpression of MHC isoforms was observed only between types 1/2 and types 2/3 at both the protein and mRNA level. 5. These data provide a molecular basis for differentiation between frog fibre types and permit future molecular studies of MHC structure/function and gene regulation in this classic physiological system. 6. Comparison of sequence homology among amphibian, avian and mammalian MHC families supports the concept of independent evolution of fast MHC genes within vertebrate classes subsequent to the amphibian/avian/mammalian radiation.

Adenosine Triphosphatases↗
PubMed1998

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.

Adenosine Triphosphate↗
PubMed1998

Developmental expression of alpha 9 acetylcholine receptor mRNA in the rat cochlea and vestibular inner ear.

Expression of alpha9 acetylcholine receptor (AChR) mRNA was studied by in situ hybridization in the rat adult and developing cochlea and vestibular inner ear. Alpha9 AChR mRNA was first observed in cochlear hair cells (HCs) at embryonic day 18 (E18), increased markedly after birth, stayed high until postnatal day 10 (P10), and decreased to substantially lower adult levels by P14. High levels of alpha9 AChR mRNA expression were also noted in the developing nonneuronal structures of the inner sulcus, chondrocytes, and/or osteoblasts in the cochlear capsule and interscalar laminae. Both developing and adult bone marrow cells also expressed intense alpha9 AChR mRNA. In the vestibular system, alpha9 AChR mRNA was first observed in HCs at E16 in all sensory epithelia, increased to its highest levels by P0-P4, then decreased slightly to reach adult levels by P10. The results are consistent with the alpha9 AChR subserving efferent neurotransmission to both cochlear and vestibular HCs. The observation of alpha9 AChR mRNA in cochlear HCs 2 weeks prior to functional onset in the cochlea further suggests that expression of this gene is not related to HC activity. The observation of substantial nonneuronal expression of alpha9 AChR mRNA suggests that this receptor also has functions separate from its role in neurotransmission.

Animals↗
PubMed1998

Focal delivery of fibroblast growth factor-1 by transfected cells induces spiral ganglion neurite targeting in vitro.

Sensory cells in the cochlea of the rat transiently express acidic fibroblast growth factor (FGF-1) during the developmental period of terminal innervation in the sensory epithelium. To explore the potential role of FGF-1 in terminal innervation events, the response of cochlear ganglion neurons to FGF-1 was evaluated in culture. Explants from the spiral ganglion of postnatal day 5 rats were cultured in the presence of exogenous FGF-1, with or without heparin. FGF-1 in the culture medium produced a dose-dependent increase in the number and length of neurites produced by spiral ganglion neurons, a response that was enhanced by heparin. To assess the effects of FGF-1 produced by a focal, cellular source, additional explants were cocultured with 3T3 cell transfectants that secrete FGF-1. Neurites that came into contact with FGF-1 secreting cells branched, formed bouton-like terminal swellings on the surface of the transfectants, and stopped extending. The results suggest that FGF-1 may stimulate neurite extension into the sensory epithelium of the cochlea and that focal production of FGF-1 may contribute to the formation of contacts on sensory cells by developing neurites.

3T3 Cells↗
PubMed1998

Protein kinase C mediates P2U purinergic receptor inhibition of K+ channel in apical membrane of strial marginal cells.

Strial marginal cells (SMC) electrogenically secrete K+ via slowly activating K+ (I[sK]) channels, consisting of I(sK) regulatory and KvLQT1 channel subunits, and the associated short circuit current (I[sc]) is inhibited by agonists of the apical P2U receptor [Liu et al., Audit. Neurosci. 2 (1995) 331-340]. Measurements of relative K+ flux (JK) with a self-referencing K+-selective probe demonstrated a decrease in JK after apical perfusion of 100 microM ATP. On-cell macro patch recordings from the apical membrane of gerbil SMC showed a decrease of the I(sK) channel current (I[IsK]) by 88 +/- 8% during pipette perfusion of 100 microM ATP. The magnitude of the decrease of L(sc) by ATP was diminished in the presence of inhibitors of phospholipase C (PLC) and protein kinase C (PKC), U-73122 and GF109203X. Activation of PKC by phorbol 12-myristate 13-acetate (20 nM) decreased I(IsK) (gerbil: by 62 +/- 10%; rat: by 72 +/- 6%) in perforated-patch whole-cell recordings while the inactive analog, 4alphaPMA, had no effect. By contrast, elevation of cytosolic [Ca2+] by A23187 increased the whole-cell I(IsK). The expression of the isk gene transcript was confirmed and the serine responsible for the species-specific response to PKC was found to be present in the gerbil I(sK) sequence. These data provide evidence consistent with a direct effect of the PKC branch of the PLC pathway on the I(sK) channel of SMC in response to activation of the apical P2U receptor and predict that the secretion of endolymph in the human cochlea may be controlled by PKC in the same way as in our animal model.

Adenosine Triphosphate↗
PubMed1998

Receptor-targeted delivery of an intracellular toxin to outer hair cells by fibroblast growth factor.

The presence and distribution of functional, high-affinity receptors for fibroblast growth factors (FGFs) in the neonatal organ of Corti were probed using the intracellular toxin saporin conjugated to basic FGF (FGF-2). FGFs that bind to high-affinity FGF receptors are internalized as part of the normal process of receptor inactivation. The receptor can thus be used for the targeted delivery of molecules conjugated to FGF into the cytoplasm. Incubation of postnatal day 5 (P5) rat organ of Corti cultures with FGF-saporin caused a dose dependent destruction of outer hair cells, Deiters cells and outer pillar cells. Inner hair cells and other cells were unaffected. Organ of Corti cultures at P0 and P10 showed much less damage than at P5. The results suggest that outer hair cells and adjacent supporting cells in the organ of Corti transiently express high-affinity FGF receptors, and that these receptors can mediate the intracellular delivery of bioactive molecules.

Animals↗
PubMed1998

Expression of mRNA encoding vasopressin V1a, vasopressin V2, and ANP-B receptors in the rat cochlea.

The expression of mRNAs encoding vasopressin V1a, V2, and ANP-B receptors in the rat cochlea was examined by PCR and in situ hybridization. After reverse-transcription of rat cochlear RNA, cDNA was amplified by PCR using pairs of primers specific to these receptors. After subcloning of the PCR products, clones with sequences identical to those cloned previously from the rat liver (V1a receptor), kidney (V2 receptor) and brain (ANP-B receptor) were obtained. The localization of expression of those receptors in the developing and adult rat cochlea was examined by in situ hybridization using 35S-labeled cRNA probes. The V1a and V2 receptors were expressed throughout the whole of the neonatal rat cochlea, while no expression was detected in the adult cochlea. The ANP-B receptor was expressed throughout the whole of the neonatal cochlea. In the adult cochlea, expression was observed in the spiral ganglion and the spiral ligament. These results suggest that vasopressin may play a role in the development of the cochlea, and that natriuretic peptide may play a role in the function of the spiral ganglion and the spiral ligament.

Aging↗
PubMed1998

Evidence for differential regulation of calcium by outer versus inner hair cells: plasma membrane Ca-ATPase gene expression.

The expression of mRNA encoding plasma membrane calcium ATPase (PMCA) subunit isoforms (1-4) and splice variants was examined in the adult and developing rat cochlea by PCR and in situ hybridization. High levels of PMCA mRNA expression were observed in the neurons of the spiral ganglion, and in hair cells. Spiral ganglion neurons expressed PMCA 1-3 beginning in embryonic development, reaching high levels shortly after birth, and continuing into adulthood. Inner hair cells expressed PMCA 1 at moderate levels from birth to the time of onset of cochlear function on postnatal day 12, and strongly from then until adulthood. Outer hair cells expressed PMCA 2 at high levels from shortly after birth through adulthood. The data suggest that the calcium clearance requirements of inner and outer hair cells are distinct. PMCA 2 is the isoform with the highest affinity for calmodulin, and has also been associated with high levels of inositol triphosphate. Its presence in outer hair cells suggests that regulation of the enzyme by calmodulin may be particularly important for this hair cell type. It further suggests that inositol phosphate may play a unique role in the outer hair cell.

Animals↗