Barbara J. Morley

Identification of the subunits of the nicotinic cholinergic receptors in the rat cochlea using RT-PCR and in situ hybridization.

There are two tissues in the adult mammalian cochlea that are post-synaptic to cholinergic efferent fibers: The outer hair cells (OHCs) and the dendrites of the afferent fibers of the type I spiral ganglion cells. The unusual nicotinic-like pharmacology of cochlear cholinergic responses and the unique embryonic development of cochlear tissues suggest that the inner-ear nicotinic cholinergic receptor (nAChR) may be different from nAChRs described previously at synapses in the mammalian brain, autonomic ganglia, or skeletal muscle. In this study, we determined the mRNA expression of the alpha2-7, alpha9, and beta2-4 subunits of the nicotinic acetylcholine receptor (nAChR) family in the rat cochlea. In micro-dissected tissue from the organ of Corti, spiral ganglion, and the membranous lateral wall, we found mRNA expression of the alpha7 and alpha9 subunits in the organ of Corti and alpha5-7, and beta2 and beta3 in the spiral ganglion using RT-PCR. Employing in situ hybridization with 35S-riboprobes, we localized alpha9 in hair cells regions and alpha6, alpha7 and beta2 in the type I cells of the spiral ganglion. No evidence of nAChR subunit mRNA expression was found in supporting cells, but beta2 was expressed in type II spiral ganglion cells, which are neither cholinergic nor cholinoceptive.

Expression of nicotinic acetylcholine receptor mRNA in the adult rat peripheral vestibular system

The mRNA expression of the neuronal nicotinic acetylcholine receptor subunits was determined in adult rat vestibular end-organs and in Scarpas ganglion (SCG) by in situ hybridization with [35S] riboprobes. Neurons in the SCG expressed the alpha 4-7 and beta 2-3 mRNAs, but not alpha 3 or beta 4 mRNAs. Not all SCG neurons expressed every mRNA found in SCG. The alpha 6 and beta 2-3 riboprobes labeled all neurons, but alpha 4, alpha 5, and alpha 7 mRNAs were selectively expressed in one or more subpopulations of SCG neurons. Vestibular sensory hair cells, in contrast, expressed only alpha 9 mRNA.

The Interpeduncular Nucleus

Publisher Summary The interpeduncular nucleus (IPN) is a phylogenetically conserved area of the brain. Anatomically, the IPN is unique in that it is primarily an unpaired structure, but receives a significant input from a paired structure, the habenula (Hb). Solely based on its anatomical connections it can be concluded that the IPN is an important integrative center for the limbic system. The IPN is highly vascularized and protrudes into the interpeduncular cistern. In some species, IPN cells having morphologic similarities to neurosecretory cells are identified. An important aspect of both the neurochemical and neuroanatomical understanding of the IPN is to determine whether important species differences exist. The observation that the biochemical measurements indicate extremely high concentrations of acetylcholine (ACh) and choline acetyltransferase (CAT) is in agreement with reports that the IPN contains a high concentration of the binding of [3H]ACh as well as other receptor ligands. Metabolic activity in the IPN is spared or increased during anesthesia, suggesting several possible behavioral functions related to sleep, to autonomic functions such as respiration, or to a generalized emotional state.

Differential expression of the α9 nicotinic acetylcholine receptor subunit in neonatal and adult cochlear hair cells

The expression of the alpha9 nicotinic acetylcholine receptor (nAChR) subunit was investigated in perinatal and adult rat cochleae using [35S] labeled cRNA in situ hybridization techniques. In the adult, alpha9 expression showed both longitudinal and radial gradients. The highest expression occurs over outer hair cells (OHCs) in basal regions, and particularly, OHCs in row 1. In contrast, expression over IHCs is lowest in basal regions and highest in apical regions. During embryonic and postnatal ages, the pattern of alpha9 expression differs. Expression of alpha9 was nearly equivalent over IHCs and OHCs. Additionally, the greater epithelial ridge, which is adjacent to IHCs before birth, shows a high level of alpha9 expression. These data are consistent with current models of efferent synaptogenesis and suggest that the expression of the alpha9 nAChR may be influenced by the arrival of efferent axons.

Identification of a glutamate/aspartate transporter in the rat cochlea.

The neurotransmitter at the synapses between hair cells and spiral ganglion cells in the cochlea is probably L-glutamate or a similar excitatory amino acid. Glutamate uptake by nerve terminals and glial cells is an important component of neurotransmission at glutamatergic synapses of the central nervous system, for providing a reservoir of transmitter or transmitter precursors and the termination of the released glutamate. Hair cell synapses are not surrounded by glial cells, therefore, the uptake mechanism for glutamate in the cochlea may be unique. cDNA was synthesized from total RNA isolated separately from the rat organ of Corti, spiral ganglia, and lateral wall tissues. The expression of a glutamate/aspartate transporter (GLAST) was detected by DNA amplification with the polymerase chain reaction. The other two members of glutamate transporters in this family were not detected by this method. A partial cDNA encoding to GLAST was identified by sequence analysis in a rat cochlear cDNA library. Data concerning the expression and the molecular structure of the glutamate transporter GLAST in the cochlea may provide important information regarding the neurotransmission process at the hair cell-afferent synapses.

Cholinergic receptors: dual roles in transduction and plasticity.

The regional distributions and possible functions of nicotinic acetylcholine receptors (nAChRs) in the developing and adult auditory rat brain are reviewed. The predominant nAChR in the auditory brainstem is the alpha7 homomeric receptor. alpha7 mRNA and protein are expressed in selected regions of the cochlear nucleus (CN), inferior colliculus (IC), medial superior olive, lateral superior olive, ventral nucleus of the lateral lemniscus and superior paraolivary nucleus. Peak expression of mRNA and protein occurs by the second postnatal week in most auditory brainstem areas. In contrast, the alpha3 and beta4 nicotinic subunits are expressed in the embryo and early in postnatal development in the CN and IC, but not other brainstem nuclei. Of particular interest is the octopus cell region of the posteroventral cochlear nucleus (PVCN). alpha3 and beta4 are down-regulated in the octopus cell region about postnatal day 10, which is the age that alpha7 is at peak expression. NAChRs play important roles in transduction and in regulating intracellular calcium. The ability of the alpha7 receptor to synchronize synaptic activity and stabilize synapses makes it a prime candidate as a mechanism underlying homeostatic plasticity in the auditory system.

Purification and characterization of the α-bungarotoxin binding protein from rat brain

The α-bungarotoxin (BGT) binding protein from rat brain has been purified and its polypeptide chain composition has been examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polypeptide chains with Mrs of 55,000, 53,500 and 49,000 have been identified as constituents of the protein. The affinity ligand [3H]maleimidobenzyl trimethylammonium bromide ([3H]MBTA), used to identify the ligand binding site on neuromuscular junction acetylcholine receptors (NMJ AChRs), binds to the 55,000 dalton polypeptide chain. Using a technique where ligands are bound to the protein while the protein is immobilized on α-cobratoxin-Sepharose 4B, it was established that the brain BGT binding protein, like NMJ AChRs, possesses two binding sites for BGT. These experiments reinforce previous evidence that the brain BGT binding protein is closely related but not identical to NMJ AChRs.

Developmental mRNA expression of the α10 nicotinic acetylcholine receptor subunit in the rat cochlea

A recently discovered alpha10 subunit of the nicotinic acetylcholine receptor (nAChR) family is believed to form a heteromeric receptor with the alpha9 nAChR subunit in auditory hair cells. In the present study, the alpha10 nAChR subunit expression in the developing and adult rat inner ear was analyzed by PCR and localized using isotopic in situ hybridization. Unlike the alpha9 subunit, the alpha10 subunit was not detected at embryonic day 18 (E18). From E21 through postnatal day 15 (P15), the alpha10 subunit was localized over both inner hair cell (IHC) and outer hair cell (OHC) regions, but in the mature cochlea detectable levels of alpha10 mRNA were found only over the OHC region. From E21 through adult ages, there was also a small but consistent basal to apical gradient of alpha10 expression; that is, higher levels in basal regions and lower levels in apical regions. Previously, we detected the alpha9 nAChR subunit over IHCs as early as E18 and throughout adult ages with a clear basal-apical gradient of expression. Our studies raise the question of whether the alpha9 and alpha10 subunits are differentially regulated during embryonic and postnatal development.

Single-unit responses to cholinergic agents in the rat inferior colliculus

Single units were recorded from the inferior colliculi of adult male rats anesthetized with urethane. Units were driven with tonal stimuli, and changes in unit responses to the tones were monitored during iontophoretic application of cholinergic drugs. The cholinergic agonists acetylcholine and carbamylcholine potentiated responses in about 50% and suppressed responses in about 35% of units tested. Cholinergic antagonists typically produced effects when delivered alone. Both d-tubocurarine and atropine methyl nitrate excited over 80% of units tested, while mecamylamine and scopolamine inhibited the majority of tested units. Dihydro-beta-erythroidine was generally ineffective. Alpha-bungarotoxin was generally ineffective when delivered alone, but blocked agonist effects. Post-stimulus time histogram (PSTH) patterns, response-intensity functions and response areas were examined for changes during drug delivery. Cholinergic agents did not differentially affect either time periods within the PSTH or frequency bands of response areas, but were especially effective for those intensities producing larger response rates. Taken together with evidence from biochemical studies, our results suggest the presence of a functional cholinergic input into the inferior colliculus which acts to modulate acoustic processing.

Analysis of γ-aminobutyric acidA receptor subunits in the mouse cochlea by means of the polymerase chain reaction

Abstract: Thirteen GABAA receptor subunits were examined in the cochlea of the CBAJ mouse by PCR analysis. Total RNA was extracted from the auditory organs of 14–18‐day‐old animals, and mRNA was isolated using oligo‐dT cellulose. After reverse transcription, resulting cDNA was amplified by PCR with primers specific for nucleotide sequences representing GABAA subunits. PCR products corresponding to subunits α1‐αB, β1,‐β3, and γ2 were amplified, whereas those representing γ1, γ3, and δ were not amplified above background. These results provide the most direct evidence to date that GABAA receptors composed of the detected subunits are expressed in the mammalian cochlea, lending new support to previous studies implicating GABA as a co‐chlear transmitter. The pronounced expression of α2 and α6 sub‐units suggests type II and “cerebellar‐type” benzodiazepine pharmacology in the cochlea.