An adenylyl cyclase signaling pathway predicts direct dopaminergic input to vestibular hair cells.

Adenylyl cyclase (AC) signaling pathways have been identified in a model hair cell preparation from the trout saccule, for which the hair cell is the only intact cell type. The use of degenerate primers targeting cDNA sequence conserved across AC isoforms, and reverse transcription-polymerase chain reaction (RT-PCR), coupled with cloning of amplification products, indicated expression of AC9, AC7 and AC5/6, with cloning efficiencies of 11:5:2. AC9 and AC5/6 are inhibited by Ca(2+), the former in conjunction with calcineurin, and message for calcineurin has also been identified in the trout saccular hair cell layer. AC7 is independent of Ca(2+). Given the lack of detection of calcium/calmodulin-activated isoforms previously suggested to mediate AC activation in the absence of Gαs in mammalian cochlear hair cells, the issue of hair-cell Gαs mRNA expression was re-examined in the teleost vestibular hair cell model. Two full-length coding sequences were obtained for Gαs/olf in the vestibular type II-like hair cells of the trout saccule. Two messages for Gαi have also been detected in the hair cell layer, one with homology to Gαi1 and the second with homology to Gαi3 of higher vertebrates. Both Gαs/olf protein and Gαi1/Gαi3 protein were immunolocalized to stereocilia and to the base of the hair cell, the latter consistent with sites of efferent input. Although a signaling event coupling to Gαs/olf and Gαi1/Gαi3 in the stereocilia is currently unknown, signaling with Gαs/olf, Gαi3, and AC5/6 at the base of the hair cell would be consistent with transduction pathways activated by dopaminergic efferent input. mRNA for dopamine receptors D1A4 and five forms of dopamine D2 were found to be expressed in the teleost saccular hair cell layer, representing information on vestibular hair cell expression not directly available for higher vertebrates. Dopamine D1A receptor would couple to Gαolf and activation of AC5/6. Co-expression with dopamine D2 receptor, which itself couples to Gαi3 and AC5/6, will down-modulate levels of cAMP, thus fine-tuning and gradating the hair-cell response to dopamine D1A. As predicted by the trout saccular hair cell model, evidence has been obtained for the first time that hair cells of mammalian otolithic vestibular end organs (rat/mouse saccule/utricle) express dopamine D1A and D2L receptors, and each receptor co-localizes with AC5/6, with a marked presence of all three proteins in subcuticular regions of type I vestibular hair cells. A putative efferent, presynaptic source of dopamine was identified in tyrosine hydroxylase-positive nerve fibers which passed from underlying connective tissue to the sensory epithelia, ending on type I and type II vestibular hair cells and on afferent calyces.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) and its receptor (PAC1-R) are positioned to modulate afferent signaling in the cochlea.

Pituitary adenylyl cyclase-activating polypeptide (PACAP), via its specific receptor pituitary adenylyl cyclase-activating polypeptide receptor 1 (PAC1-R), is known to have roles in neuromodulation and neuroprotection associated with glutamatergic and cholinergic neurotransmission, which, respectively, are believed to form the primary basis for afferent and efferent signaling in the organ of Corti. Previously, we identified transcripts for PACAP preprotein and multiple splice variants of its receptor, PAC1-R, in microdissected cochlear subfractions. In the present work, neural localizations of PACAP and PAC1-R within the organ of Corti and spiral ganglion were examined, defining sites of PACAP action. Immunolocalization of PACAP and PAC1-R in the organ of Corti and spiral ganglion was compared with immunolocalization of choline acetyltransferase (ChAT) and synaptophysin as efferent neuronal markers, and glutamate receptor 2/3 (GluR2/3) and neurofilament 200 as afferent neuronal markers, for each of the three cochlear turns. Brightfield microscopy giving morphological detail for individual immunolocalizations was followed by immunofluorescence detection of co-localizations. PACAP was found to be co-localized with ChAT in nerve fibers of the intraganglionic spiral bundle and beneath the inner and outer hair cells within the organ of Corti. Further, evidence was obtained that PACAP is expressed in type I afferent axons leaving the spiral ganglion en route to the auditory nerve, potentially serving as a neuromodulator in axonal terminals. In contrast to the efferent localization of PACAP within the organ of Corti, PAC1-R immunoreactivity was co-localized with afferent dendritic neuronal marker GluR2/3 in nerve fibers passing beneath and lateral to the inner hair cell and in fibers at supranuclear and basal sites on outer hair cells. Given the known association of PACAP with catecholaminergic neurotransmission in sympathoadrenal function, we also re-examined the issue of whether the organ of Corti receives adrenergic innervation. We now demonstrate the existence of nerve fibers within the organ of Corti which are immunoreactive for the adrenergic marker dopamine beta-hydroxylase (DBH). DBH immunoreactivity was particularly prominent in nerve fibers both at the base and near the cuticular plate of outer hair cells of the apical turn, extending to the non-sensory Hensen's cell region. Evidence was obtained for limited co-localization of DBH with PAC1-R and PACAP. In the process of this investigation, we obtained evidence that efferent and afferent nerve fibers, in addition to adrenergic nerve fibers, are present at supranuclear sites on outer hair cells and distributed within the non-sensory epithelium of the apical cochlear turn for rat, based upon immunoreactivity for the corresponding neuronal markers. Overall, PACAP is hypothesized to act within the organ of Corti as an efferent neuromodulator of afferent signaling via PAC1-R that is present on type I afferent dendrites, in position to afford protection from excitotoxicity. Additionally, PACAP/PAC1-R may modulate secretion of catecholamines from adrenergic terminals within the organ of Corti.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) and its receptor (PAC1-R) in the cochlea: evidence for specific transcript expression of PAC1-R splice variants in rat microdissected cochlear subfractions.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from the hypothalamus, named for its high potency in stimulating adenylyl cyclase in pituitary cells. PACAP acts through the specific receptor PAC1-R to modulate the action of neurotransmitters, and additionally, to regulate cell viability via autocrine/intracrine mechanisms. Evidence has now been obtained that PACAP and multiple splice variants of PAC1-R are expressed in the rat cochlea. mRNA for PACAP precursor protein is found by reverse transcription-polymerase chain reaction (RT-PCR) in microdissected cochlear lateral wall, organ of Corti, and spiral ganglion subfractions. A specific pattern of expression of mRNA for PAC1-R splice variants, which mediate the response to PACAP, has been revealed by RT-PCR and cloning for the cochlear subfractions. Transcript for the short form of PAC1-R is found in all three subfractions. Four additional splice variants -- hop1, hop2, hip, and a novel hop1 splice variant -- are expressed in the lateral wall. For the amino terminus splice region of PAC1-R, a new splice variant has been detected in the organ of Corti, representing a deletion of the first 7 of 21 amino acids detected in the PAC1-R very-short sequence. Overall, from message determinations in cochlear subfractions, there are five PAC1-R splice variants in the lateral wall, two in the organ of Corti and one in the spiral ganglion, indicating multiple possible responses to PACAP and/or mechanisms to modulate the response to PACAP in the cochlea. The variety of PAC1-R splice variants expressed may reflect the diversity in cell function between subfractions that is modulated by PACAP. The neuropeptide and its specific receptor have been immunolocalized in the lateral wall, the source of the largest number of cochlear PAC1-R splice variants. The receptor was targeted by primary antibodies which would elicit immunoreactivity for all splice variants of PAC1-R detected with RT-PCR, and evidence has been obtained with Western blot analysis suggesting that PAC1-R is glycosylated in vivo. Within the lateral wall, PACAP and PAC1-R were immunolocalized primarily to the stria vascularis, with immunoreactivity for both neuropeptide and receptor increasing from the basal to apical cochlear turns. Within the stria, PACAP immunoreactivity was localized to the basolateral extensions of marginal cells, while PAC1-R was clearly associated with tight junctions between the marginal cells close to the endolymphatic compartment. In addition, evidence was obtained that PAC1-R was associated with endothelial cells of the capillaries in the stria vascularis. The large number of splice variants expressed, coupled to the specificity in linkage between PAC1-R splice variants and G-protein-coupled second messenger pathways, could provide a mechanism to closely modulate tight junction integrity in the stria vascularis, impacting the endolymphatic potential.

Molecular identification of an N-type Ca2+ channel in saccular hair cells.

We report new molecular evidence for the presence of an N-type (Ca(v)2.2, alpha1B) voltage-gated Ca(2+) channel in hair cells of the saccular epithelium of the rainbow trout. The Ca(v)2.2 amino-acid sequence shows 68% and 63% identity compared with chick and human Ca(v)2.2, respectively. This channel reveals features that are characteristic of an N-type Ca(2+) channel: an omega-conotoxin GVIA binding domain, G(betagamma) binding regions, and a synaptic protein interaction site. Immunohistochemical studies with a custom antibody show that immunoreactivity for the Ca(v)2.2 is concentrated in the basolateral and apical regions of hair cells. Whereas trout brain and saccular macula express an 11-amino-acid insert in the second G(betagamma) binding domain of the Ca(v)2.2 I-II loop, isolated hair cells appear not to express this variant. We constructed fusion polypeptides representing portions of the I-II loop, beta1 and beta2a auxiliary subunits, the II-III loop, and syntaxin, and examined their intermolecular interactions via immunoprecipitation and surface plasmon resonance. The I-II loop polypeptides bound both beta1 and beta2a subunits with a preference for beta1, and the II-III loop exhibited Ca(2+)-dependent syntaxin binding. We demonstrated syntaxin immunoreactivity near afferent endings in hair cells, at hair-cell apices, and in efferent endings on hair cells, the former two sites consistent with binding of syntaxin to Ca(v)2.2. The present molecular characterization of the Ca(v)2.2 channel provides novel biochemical evidence for an N-type channel in hair cells, and details molecular interactions of this channel that reflect hair-cell function, such as spontaneous activity and vesicular trafficking. The current work, to our knowledge, represents the first demonstration of a putative N-type channel in hair cells as documented by tissue-specific antibody immunoreactivity and hair-cell-specific cDNA sequence.

Cloning and characterization of alpha9 subunits of the nicotinic acetylcholine receptor expressed by saccular hair cells of the rainbow trout (Oncorhynchus mykiss).

alpha9/alpha10 Subunits are thought to constitute the nicotinic acetylcholine receptors mediating cholinergic efferent modulation of vertebrate hair cells. The present report describes the cloning and sequence analysis of subunits of the alpha9-containing receptor of a hair-cell layer from the saccule of the rainbow trout (Oncorhynchus mykiss). A major alpha9 subunit, termed alpha9-I, displayed typical features of a nicotinic alpha subunit, with total coding sequence of 572 amino acids including a 16 amino-acid signal peptide. It possessed an extended cytoplasmic loop between membrane-spanning regions M3 and M4, compared with mammalian homologs. Transcript for alpha9-I was robustly expressed in the saccular hair cell layer and less prominently in trout olfactory mucosa, spleen, pituitary gland, and liver, as determined by reverse transcription-polymerase chain reaction. alpha9-I cDNA was not detected in trout brain, skeletal muscle, retina, and kidney. The alpha9-I nicotinic receptor protein was immunolocalized, with an affinity-purified antibody directed against a trout alpha9-I epitope, to hair-cell and neural sites in the saccular hair-cell layer. Foci were found at basal and basolateral membrane sites on hair cells as well as on afferent nerve. Receptor clustering was observed in hair cells bordering non-sensory epithelium. Since in higher vertebrates the alpha9 is reported to associate with another nicotinic subunit, alpha10, we examined the possibility of expression of additional nicotinic subunits in trout saccular hair cells. Message for another nicotinic subunit, termed alpha9-II, was found to be expressed in the hair cells, although more difficult to amplify than alpha9-I. In contrast to alpha9-I, alpha9-II was expressed in brain, as well as in olfactory mucosa, less prominently in pituitary gland and liver, but not in spleen, skeletal muscle, retina, or kidney. The cloned alpha9-II had a total coding sequence of 550 amino acids, which included a 17-amino-acid signal peptide, and an extended M3-M4 loop. A third nicotinic subunit message, termed alpha9-III, was PCR-amplified from trout olfactory mucosa where it was strongly expressed. However, message for alpha9-III was not detected in hair cells. Message for alpha9-III was moderately expressed in trout brain, retina, and pituitary gland but not in trout spleen, skeletal muscle, liver, and kidney. Thus, alpha9-I and alpha9-II may together contribute to the formation of the hair-cell nicotinic receptor of teleosts, where no ortholog of alpha10 appears to exist. The current work is, to our knowledge, the first description of alpha9 coding sequences directly from a vertebrate hair cell source. Further, the generality of hair cell expression of subunits for the alpha9-containing nicotinic cholinergic receptor has been extended to fishes, suggesting a similar efferent mechanism across all vertebrate octavolateralis sensory systems.

Hyperpolarization-activated, cyclic AMP-gated, HCN1-like cation channel: the primary, full-length HCN isoform expressed in a saccular hair-cell layer.

The expression of transcript for hyperpolarization-activated, cyclic nucleotide-sensitive cation channel (HCN) isoforms underlying hyperpolarization-activated, inward current (I(h)) has been determined for a model hair-cell preparation from the saccule of the rainbow trout, Oncorhynchus mykiss. Based upon identification from homology to known vertebrate HCN cDNA sequence, cloning of PCR products amplified with degenerate primers indicated an expression frequency of 7:2:1 (HCN1:HCN2:HCN4) for the hair-cell sheet compared with 1:1:7 for brain. Full-length sequence has been obtained for the HCN1-like isoform representing the primary HCN transcript expressed in the hair-cell preparation. The channel protein is 938 amino acids in length with 93% amino acid identity for the region extending from the S1-S6 membrane spanning domains through the voltage-pore and cyclic nucleotide-binding domains, compared with HCN1 for rabbit, rat, mouse and human. The N- and C-terminal regions are less homologous, with 39-51% and 43-44% amino acid identities, respectively. Compared with other vertebrate HCN1, the hair-cell HCN1 contains additional consensus phosphorylation sites associated with unique repeats in the carboxy terminus. The HCN1-like transcript has been localized to hair cells of the saccular sensory epithelia by in situ hybridization. Previous electrophysiological studies have identified I(h) as the sole inwardly rectifying ion channel in a specific population of hair cells of the saccule of frogs [J Neurophysiol (1995) 73:1484] and fish [J Physiol (1996) 495:665]. I(h) is an important determinant of the resting membrane potential, and for this population of hair cells, is predicted to maintain the membrane potential within a voltage range allowing the voltage-gated calcium channels to open, permitting "spontaneous" release of transmitter. The molecular properties of the HCN1-like isoform underlying I(h) expressed in the saccular hair cells of the teleost, trout, may consequently impact spontaneous release of transmitter from hair cells of the saccule.

Muscarinic receptor subtypes are differentially distributed in the rat cochlea.

Five different genes encode the muscarinic acetylcholine receptors. The muscarinic receptor subtypes M1, M3, and M5 are typically coupled to activation of the Galpha(q/11)-phosphatidyl inositol pathway, whereas the M2 and M4 subtypes are typically linked to Galpha(i) and adenylyl cyclase inhibition. In order to localize muscarinic receptors in the rat cochlea, we applied polyclonal antibodies for subtypes M1, M2, M3, and M5, and monoclonal antibody for subtype M4 to paraffin sections. In the organ of Corti, outer hair cells exhibited strong immunoreactivity for M3 and weak immunoreactivity for M1. Deiters' cells were strongly immunoreactive to antibodies for the M1 and M2 subtypes, with weak staining observed for M3, and weaker yet for M5. Inner hair cells showed moderate immunoreactivity for the M1 subtype, weaker staining for the M5 subtype, and slight staining for the M3 subtype. Among the spiral ganglion neurons, weak to moderate immunoreactivity was detected for M3 and M5 subtypes and weak staining was observed for the M1 subtype. The efferent fibers of the intraganglionic spiral bundle were positive for M2 and M5. In the lateral wall, weak to moderate staining was detected for M5 in the stria vascularis corresponding in position to the basolateral extensions of marginal cells. Staining for M3 was observed associated with capillaries. Fibrocytes of the spiral ligament exhibited limited but selective subtype immunoreactivity. No immunoreactivity was detected in the cochlea for the M4 subtype. From the present findings we suggest that M3 is the primary muscarinic receptor subtype in outer hair cells mediating a postsynaptic response to the medial olivocochlear cholinergic efferent input. The muscarinic receptor subtypes M1, M3, and M5 appear to subserve the action of cholinergic lateral olivocochlear efferent stimulation on postsynaptic responses in type I afferents. Whether M1, M3, and M5 protein in inner hair cells indicates constitutive or vestigial expression remaining from development is unknown. M2 and M5 muscarinic receptors expressed presynaptically may modulate the efferent signal. Finally, expression by Deiters' cells of several muscarinic subtypes raises the possibility that cholinergic efferents couple to these non-sensory cells through muscarinic receptors.

Immunohistochemical localization of adenylyl cyclase isoforms in the lateral wall of the rat cochlea.

The enzymatic activity of adenylyl cyclase (AC) is attributable to nine isoforms with individual pharmacology and tissue distribution. Polyclonal antibodies for AC isoforms I-IV, VII and VIII were applied to sections of cochlear lateral wall, a tissue involved in ion transport contributing to the unique ion content of endolymph and electrical potential of scala media. Within the stria vascularis, immunoreactivity primarily to Ca(2+)/calmodulin-independent isoforms II, IV and VII was localized to sites consistent in position to the basolateral extensions of marginal cells. Little immunoreactivity was observed in the stria vascularis for Ca(2+)/calmodulin-dependent isoforms I, III and VIII. Within the spiral ligament, type II and type IV fibrocytes exhibited moderate staining for ACII, IV and VII, less staining for VIII and little for I and III. Immunoreactivity to ACII, IV, VII and VIII was observed in type I fibrocytes. The outer sulcus cells and root processes were highly immunoreactive for isoforms I and VIII, but not for III or the Ca(2+)/calmodulin-independent isoforms. The differential pattern of immunoreactivity in the lateral wall overall appears to reflect subfamily-specific expression with Ca(2+)/calmodulin-independent isoforms expressed in the stria vascularis and Ca(2+)/calmodulin-dependent isoforms expressed in the outer sulcus cells and root processes. cAMP-mediated modulation of ion transport by marginal cells is predicted to exhibit, in the microenvironment of basolateral membrane infoldings, pharmacological characteristics of the AC type II subfamily (II, IV and VII), including activation by protein kinase C (II and VII).

Autoradiographic demonstration of quinuclidinyl benzilate binding sites in the vestibular organs of the gerbil.

Gerbil vestibular tissues were isolated by microdissection and incubated in vitro with 3H-quinuclidinyl benzilate (3H-QNB). Control tissues were incubated in medium containing unlabeled atropine to differentiate non-specific from specific binding. Autoradiographic grain densities were determined by morphometric techniques and evaluated by two-tailed t-test. The label densities of sensory epithelia from experimental preparations of ampulla, utricle and saccule were found to be significantly higher than those in the adjacent endolymphatic compartment and also higher than those of adjacent stromal tissue comprising connective tissue, nerve fibers and capillaries. In contrast, no tissue region in atropine controls showed label density significantly above that of the endolymphatic compartment. Label density of ampullar sensory epithelium incubated with 3H-QNB alone was significantly higher than that of sensory epithelium from utricle or saccule. Grain density was greater in the peripheral regions of the ampullar crista compared to the vertex. Appreciable label was also present in nerve bundles beneath the sensory epithelium of the ampulla. The current study demonstrates the existence of putative muscarinic neurotransmitter/neuromodulator receptor sites in mammalian vestibular sense organs at locations corresponding to efferent innervation, with particularly significant concentrations in the ampulla.

Pulmonary venous air embolism following accidental patient laceration of a hemodialysis catheter.

As the number of patients at home with indwelling central venous catheters increases, more complications from their use will present to the emergency department. We report a case of pulmonary venous air embolism after a patient inadvertently severed the distal few centimeters of an indwelling central venous catheter.

Selective expression of serotonin receptor transcripts in the mammalian cochlea and its subdivisions.

Expression of serotonin receptor (5-HTR) mRNA has been determined in the mammalian cochlea and its subdivisions by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Specific primers targeting individual 5-HTRs 1-7 directed amplification of 5-HTR subtypes 1A, 1B, 2B, 2C, 3, 5B, and 6 from mouse cochlea cDNA. No evidence of expression was obtained for 5-HTRs 1D, 2A, 4 (L and S), 5A, and 7. The distribution of receptor mRNA within the cochlea was determined with application of RT-PCR to morphologically defined microdissected subfractions of the rat cochlea. Messages for 5-HTR subtypes 1A, 1B, 2B, and 6 were present in the organ of Corti, lateral wall, and spiral ganglion subfractions. Messages for 5-HTR subtypes 2C, 3 and 5B were found in the spiral ganglion, but not in the organ of Corti or lateral wall fractions. The existence of transcripts for 5-HTRs 1A, 1B, 2B and 6 in the organ of Corti is consistent with a role for these receptors in serotonin-mediated modulation of the mechanosensory signal.

Heating oil company responses to inquiries concerning carbon monoxide toxicity.

STUDY OBJECTIVE: Carbon monoxide (CO) is the leading cause of poisoning fatalities in the United States. We conducted a survey to sample the quality of the information available to the public from Connecticut heating oil distributors regarding these risks. METHODS: An observational, cross-sectional telephone survey of oil distributors in Connecticut was conducted, using a scripted set of questions regarding CO poisoning risk from oil-burning furnaces. We first asked whether such a risk existed and then elicited explanations for confirmation or denial of that risk. We then inquired as to the proper response to a home CO detector alarm. Of 282 calls made, responses were obtained from 91 distributors. Reasons for lack of response included incorrect phone listings, duplicate listings, and unwillingness of the person answering to respond to the questions asked. RESULTS: Nearly one fourth of distributors responding (23%) denied any risk of CO poisoning from oil burners, 43% of these claiming that the odor would provide protective warning before toxic exposure; another 33% stated that CO is a risk only with gas heaters. Of the 77% who confirmed the possibility of CO toxicity, half (49%) minimized the risk, also invoking a "warning odor." In case of CO alarm, only 14% of responders recommended turning off the furnace until the source is found. Five percent did not recommend having ambient CO levels checked at the time of the alarm. CONCLUSION: Heating oil companies in Connecticut do not uniformly provide correct information regarding CO risks. In our survey a majority of distributors responding offered misplaced "reassurance." As such, they are a potential area for public health intervention and an untapped resource for educating the public on these risks.

Thrombophlebitis and pseudothrombophlebitis in the ED.

The patient presenting to the emergency department (ED) with a painful swollen lower extremity is considered to have deep venous thrombosis (DVT) until this diagnosis can be ruled out. This clinical presentation, however, is far from specific and the differential diagnosis includes symptomatic Baker's cyst, also known as pseudothrombophlebitis syndrome (PTP). This article presents two cases of PTP and reviews the literature relevant to diagnosis of DVT and PTP. Ultrasonography is now the diagnostic test of choice for both DVT and PTP, being safe, accurate, noninvasive, and rapid, and should ideally be available for use in the ED.

Expression of adenylyl cyclase type I in cochlear inner hair cells.

Expression of calcium/calmodulin-activated adenylyl cyclase type I (ACI) mRNA has been determined in the cochlea and in an organ-of-Corti subdissected tissue fraction by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Amplification products of predicted size were obtained from the mouse cochlea and rat organ of Corti with nucleotide sequences corresponding to respective ACI brain transcripts. In addition, ACI template was detected in a rat inner hair cell cDNA library by PCR. Immunoreactivity to ACI has been localized within the organ of Corti to the inner hair cell, with diaminobenzidine staining found in both the cell body and in the stereocilia. Evidence, thus, has been obtained that both ACI transcript and protein are expressed in the inner hair cell, the primary mechanosensory receptor cell of the cochlea. We hypothesize that ACI is activated by calcium influx through a calcium/calmodulin interaction and that this adenytyl cyclase isoform may have a role in modulation of receptoneural afferent transmission and/or mechanosensory transduction in the cochlea.

Quantitative analysis of dopamine receptor messages in the mouse cochlea.

Dopamine receptor isoforms were examined in the cochlea of the CBA(J) mouse by RT-PCR analysis and nucleotide sequencing, utilizing primers specific for known dopamine receptor isoforms. Cochlear cDNA sequences corresponding to dopamine D2(long) and D3 receptors were amplified, whereas those representing D1A, D1B, D2(short), and D4 were not detected. Utilizing quantitative competitive PCR analysis, relative levels of dopamine receptor transcripts were found to be 0.002, 0.014, 0.016, and 1.000 for D2(long) cochlea, D3 cochlea, D3 brain, and D2(long) brain, respectively. In the context of previously published findings, the current work provides key quantitative evidence necessary to establish that dopamine is a neurotransmitter in the auditory inner ear.

Calcium channel subunits in the mouse cochlea.

Messages for subunits of voltage-gated calcium channels were examined in the cochlea of the CBAJ mouse by PCR analysis. Total RNA was extracted from the auditory organs of 16-18-day-old animals. After reverse transcription, resulting cDNA was amplified by PCR with primers targeted to nucleotide sequences corresponding to 12 different calcium channel subunits. PCR products representing subunit gene expression were strongly and consistently amplified for alpha1C, alpha1D, alpha1E, alpha2delta, beta1, beta3, and beta4 but not for alph1A, alpha1B, alpha1S, beta2, or gamma. The chosen primers amplified cochlear cDNA to yield an overall pattern of bands different from that of any tissue studied thus far, in particular with respect to the alpha2delta and beta1 subunits; the alpha2delata product was found to be significantly shorter than the corresponding brain and skeletal muscle isoforms. Nucleotide sequencing confirmed the identity of mouse cochlear subunit cDNAs. The results suggest that L-type and presumptive R-type calcium channels are expressed in the mammalian cochlea and that the alpha2delta subunits may be coded by a characteristic splice-variant mRNA.

Analysis of nicotinic acetylcholine receptor subunits in the cochlea of the mouse.

The present study was designed to catalogue and compare nicotinic receptor subunit messages in the mammalian cochlea. Fourteen nicotinic acetylcholine receptor subunit messages were examined by polymerase chain reaction (PCR) analysis and nucleotide sequencing. Total RNA was extracted from the auditory organs of 14- to 18-day-old CBAJ mice, and mRNA was purified using oligo-dT cellulose. After reverse transcription, resulting cDNA was amplified by PCR with the use of primers specific for the nucleotide sequences representing the following nicotinic receptor subunits: muscle types alpha 1, beta 1, gamma, delta and epsilon and neuronal types alpha 2,3,4,5,6,7 and beta 2,3,4. cDNA from cochlear tissue corresponding to the muscle-type receptor subunit beta 1 and to neuronal-type receptor subunits alpha 2,4,5,6 and beta 2,3 was amplified, whereas cDNA for muscle types alpha 1, gamma, delta and epsilon and neuronal types alpha 3,7 and beta 4 was not. All PCR products were homologous in nucleotide sequence to the corresponding reference cDNAs from which the primers were designed. The current results indicate that nicotinic acetylcholine receptor (nAChR) subunits that are similar or identical to the stated muscle and neuronal types are expressed in the murine cochlea. The presence of messages corresponding to the muscle-type beta 1 and neuronal-type nAChR subunits may be correlated with the atypical cholinergic response of cochlear hair cells to agonists and antagonists.

Cytochemical localization of adenylyl cyclase activity within the sensory epithelium of the trout saccule.

Adenylyl cyclase, the enzyme of synthesis of cAMP, the second messenger molecule mediating signal transduction in response to sensory, neurotransmitter and hormonal stimuli, has been localized in the sensory epithelium of the rainbow trout (Salmo gairdneri R.) saccule by cytochemical detection of enzyme activity. In the sensory receptor cell, or hair cell, reaction product has been visualized in the stereocilia in close association with the outer cell membrane and also at the apical surface of the cuticular plate. A diffuse distribution of precipitate was observed within the cytoplasm of terminal endings of nerve fibers presumed to be efferent on the basis of characteristic synaptic specializations including presynaptic vesicles and a postsynaptic cistern lying within the hair cell. Occasionally, reaction product was observed to be associated with the external cell membrane of these nerve terminals. There appeared to be little or no adenylyl cyclase activity associated with the plasma membrane at the base of the hair cell or in presumptive afferent nerve endings. However, a subpopulation of nerve fiber endings which exhibited both efferent and afferent synaptic specializations contained precipitate. A concentration of adenylyl cyclase activity in hair cell stereocilia and efferent nerve terminals in the sensory epithelium is suggestive of a role for cAMP in second messenger action at these sites, possibly related to mechanosensory transduction and efferent neuromodulation, respectively.

Immunohistochemical localization of GABAA receptors in the mammalian crista ampullaris.

The distribution of GABAA receptor-like immunoreactivity in the hamster, rat, and mouse crista ampullaris was determined by use of a monoclonal antibody to the beta 2 and beta 3 subunits of the GABAA receptor. In the crista ampullaris, punctate staining was seen associated with the calyces surrounding vestibular type I hair cells. Afferent nerve fibers approaching the hair cell layer were often observed to be immunoreactive. Hair cells, supporting cells, and cells in the transitional and dark cell regions were not immunoreactive. The distribution of staining of calyces appeared to be relatively uniform in all regions (crest and slope) of the crista. In addition, cell bodies located in the vestibular ganglion were immunoreactive. The association of GABAA receptor-like immunoreactivity with the afferent nerve calyx and cell body of the vestibular ganglion cells suggests that GABA may act to modify afferent nerve transmission at the calyceal afferent nerve ending.