Gary D. Housley

Ionic currents of outer hair cells isolated from the guinea‐pig cochlea.

1. Whole‐cell currents were measured in outer hair cells isolated from each turn of the organ of Corti of the guinea‐pig. 2. The slope input conductances at ‐70 mV of the cells ranged from 3.6 to 51 nS depending on the length of the cell. Shorter cells from the basal turns of the cochlea had the highest values. The membrane time constant of the cells varied from 3 to 0.2 ms from the apex to the base. 3. Irrespective of the position of the cells along the cochlea, three distinct currents were found. Each type of current was found in approximately the same proportion in all cells. 4. An outward K+ current was present which activated at potentials more positive than ‐35 mV. The current was sensitive to tetraethylammonium (30 mM), quinidine (100 microM) and nifedipine (50 microM). It could be removed by replacing external Ca2+ with Ba2+ or Mg2+. The current was also removed by substituting Nai+ or Csi+ for Ki+ pipette solution. This outwardly rectifying current appears similar to the calcium‐activated K+ current described in other hair cells. 5. The main current present at membrane potentials from ‐90 mV to ‐50 mV was a second voltage‐activated K+ current. It was 50% activated at ‐80 mV, and relaxed with a time constant of 20‐40 ms on hyperpolarization to ‐120 mV. Near rest the kinetics were essentially time‐dependent , but depended upon the external K+ concentration. The current was blocked by 5 mM external Cs+. 6. This current was highly selective for K+. Measured from reversal of the tail currents, the permeability ratio PK:PNa was approximately 30:1. Depolarization of the cell, presumed to lead to an elevation of intracellular calcium, produced a prolonged activation of the current. 7. A third current found in the cells was a cation current. By external ion replacement, the selectivity sequence was determined to be Ca2+ greater than Na+ approximately equal to K+ greater than choline+ greater than NMDG+ (respective permeabilities relative to Na: 2.9, 1.0, 0.99, 0.63 and 0.37). This current was reduced by external Ba2+ (3 mM) and by nifedipine (50 microM). The activation of this current appeared to depend upon raised levels of Cai2+. 8. These currents account for reported in vivo properties of cochlear outer hair cells as cells permeable to potassium at large negative resting potentials. The consequences for sound detection in the cochlea are briefly discussed.

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. J. Comp. Neurol. 407:11–32, 1999.

Direct measurement of the action of acetylcholine on isolated outer hair cells of the guinea pig cochlea

Acetylcholine has long been thought to be the neurotransmitter of the cochlear efferent system in mammals although the evidence is largely indirect. By using whole-cell recordings from isolated outer hair cells, we show that acetylcholine activates a large rapidly desensitizing outward potassium current. This corresponds to hyperpolarization of the membrane potential from rest. The half maximal dose for acetylcholine was 13.5 u.M with a cooperativity of 2. The response was not due to a conventional muscarinic action of acetylcholine for it was not blocked by 0.1 u.M atropine and muscarinic antagonists but it could be blocked by 0.1 uM curare, suggesting that it shared many properties of a nicotinic receptor. It was, however, inhibited by 10 uM strychnine. The potassium current activated by acetylcholine required external calcium and was characterized by a significant delay at room temperature. This points to the involvement of a second messenger system, possibly calcium itself.

Brain stem projections of the glossopharyngeal nerve and its carotid sinus branch in the rat.

Transganglionic transport of horseradish peroxidase or lectin-conjugated horseradish peroxidase from an application site in the cervical trunk of the glossopharyngeal (IXth cranial) nerve of the rat produced extraperikaryal reaction product characteristic of axon terminal processes in three regions of the brain stem: (1) the nucleus of the tractus solitarius, from approximately 2.5 mm rostral to the obex to approximately 3 mm caudal to the obex; (2) the spinal trigeminal nucleus at the level of obex; (3) the cuneate fasciculus, approximately 3 mm caudal to the obex. In contrast, labelling of the carotid sinus nerve, a branch of the glossopharyngeal nerve which conveys chemoreceptor and baroreceptor afferent fibers from the carotid bifurcation, revealed a restricted central projection to within 1 mm of the obex and corresponding to the intermediate region of the glossopharyngeal nerve projection to the nucleus of the tractus solitarius. Two distinct aggregations of label were observed: (1) rostral to the obex, within the lateral and dorsomedial subnuclei of the nucleus of the tractus solitarius; (2) caudal to the obex, within the commissural and ventrolateral subnuclei of the nucleus of the tractus solitarius. Between these two sites the density of labelling was reduced. Retrogradely labelled neurons were demonstrated in the inferior salivatory nucleus and in the nucleus ambiguus after application of lectin-conjugated horseradish peroxidase to the glossopharyngeal nerve. Of the labelled neurons in the nucleus ambiguus (approximately 100), 25% contributed fibers to the carotid sinus nerve. The concentration of extraperikaryal reaction product located rostral to the obex after labelling of the carotid sinus nerve closely matches descriptions of the region of afferent terminations from carotid and aortic baroreceptors in the cat. The concentration of label caudal to the obex may therefore correspond to the region of afferent terminations from carotid chemoreceptors. This study may therefore provide some basis for a separation of the central synapses of primary afferent fibers from the carotid baroreceptors and chemoreceptors in the rat. The labelled neurons of the nucleus ambiguus provide the anatomical substrate for centrifugal control of carotid chemoreceptor activity.

N-Glycolylneuraminic Acid Deficiency in Mice: Implications for Human Biology and Evolution

ABSTRACT Humans and chimpanzees share >99% identity in most proteins. One rare difference is a human-specific inactivating deletion in the CMAH gene, which determines biosynthesis of the sialic acid N-glycolylneuraminic acid (Neu5Gc). Since Neu5Gc is prominent on most chimpanzee cell surfaces, this mutation could have affected multiple systems. However, Neu5Gc is found in human cancers and fetuses and in trace amounts in normal human tissues, suggesting an alternate biosynthetic pathway. We inactivated the mouse Cmah gene and studied the in vivo consequences. There was no evidence for an alternate pathway in normal, fetal, or malignant tissue. Rather, null fetuses accumulated Neu5Gc from heterozygous mothers and dietary Neu5Gc was incorporated into oncogene-induced tumors. As with humans, there were accumulation of the precursor N-acetylneuraminic acid and increases in sialic acid O acetylation. Null mice showed other abnormalities reminiscent of the human condition. Adult mice showed a diminished acoustic startle response and required higher acoustic stimuli to increase responses above the baseline level. In this regard, histological abnormalities of the inner ear occurred in older mice, which had impaired hearing. Adult animals also showed delayed skin wound healing. Loss of Neu5Gc in hominid ancestors ∼2 to 3 million years ago likely had immediate and long-term consequences for human biology.

Purinergic signaling in special senses.

We consider the impact of purinergic signaling on the physiology of the special senses of vision, smell, taste and hearing. Purines (particularly ATP and adenosine) act as neurotransmitters, gliotransmitters and paracrine factors in the sensory retina, nasal olfactory epithelium, taste buds and cochlea. The associated purinergic receptor signaling underpins the sensory transduction and information coding in these sense organs. The P2 and P1 receptors mediate fast transmission of sensory signals and have modulatory roles in the regulation of synaptic transmitter release, for example in the adaptation to sensory overstimulation. Purinergic signaling regulates bidirectional neuron-glia interactions and is involved in the control of blood supply, extracellular ion homeostasis and the turnover of sensory epithelia by modulating apoptosis and progenitor proliferation. Purinergic signaling is an important player in pathophysiological processes in sensory tissues, and has both detrimental (pro-apoptotic) and supportive (e.g. initiation of cytoprotective stress-signaling cascades) effects.

Localization by kainic acid lesions of neurones transmitting the carotid chemoreceptor stimulus for respiration in rat.

1. An attempt has been made to test the hypothesis that in the nucleus of the tractus solitarius (NTS) in the rat, the most caudal region of synaptic terminals of the carotid sinus nerve, just caudal to the obex, represents mainly the site of synapse of chemoreceptor fibres from the carotid body. 2. Under halothane anaesthesia, the neurotoxin kainic acid was used to lesion this region and a second region, immediately rostral to obex, where terminals are thought to arise mainly from baroreceptor fibres of the carotid sinus nerve. 3. Measurements based on the distribution of fluorescent dye co‐injected with the kainic acid showed that the two groups of 100 nl microinjections were centered 0.82 mm apart and that the injectate spread through mean distances of 0.57 mm (caudal microinjections) and 0.52 mm (rostral microinjections). Nissl staining was used to determine cellular degeneration. The caudal lesions mostly involved ventrolateral and commissural subnuclei of NTS and the rostral lesions involved lateral and dorsolateral subnuclei. 4. Ventilatory sensitivity to hypoxia was tested under light halothane anaesthesia, 1 day after lesioning. To enhance the responses, the contralateral carotid sinus nerve was sectioned prior to experiments. Caudal lesions reduced the ventilatory response to inspired oxygen (20.9‐9.6% O2) by a mean of 67% and rostral lesions by 18% of the effect produced by carotid sinus nerve section on that side. Subsequent section of the carotid sinus nerve on the side of the NTS lesion confirmed that caudal lesions produced effects comparable to those of carotid body denervation; rostral lesions did not. 5. These results strongly support the hypothesis that chemoreceptor and baroreceptor afferent fibres in the carotid sinus nerve synapse at substantially separable sites in the nucleus of the tractus solitarius. The identification of the site in NTS caudal to the obex as the principal site of carotid chemoreceptor synapses places them close to but not upon respiratory premotor neurones of the same nucleus.

A Forward Genetics Screen in Mice Identifies Recessive Deafness Traits and Reveals That Pejvakin Is Essential for Outer Hair Cell Function

Deafness is the most common form of sensory impairment in the human population and is frequently caused by recessive mutations. To obtain animal models for recessive forms of deafness and to identify genes that control the development and function of the auditory sense organs, we performed a forward genetics screen in mice. We identified 13 mouse lines with defects in auditory function and six lines with auditory and vestibular defects. We mapped several of the affected genetic loci and identified point mutations in four genes. Interestingly, all identified genes are expressed in mechanosensory hair cells and required for their function. One mutation maps to the pejvakin gene, which encodes a new member of the gasdermin protein family. Previous studies have described two missense mutations in the human pejvakin gene that cause nonsyndromic recessive deafness (DFNB59) by affecting the function of auditory neurons. In contrast, the pejvakin allele described here introduces a premature stop codon, causes outer hair cell defects, and leads to progressive hearing loss. We also identified a novel allele of the human pejvakin gene in an Iranian pedigree that is afflicted with progressive hearing loss. Our findings suggest that the mechanisms of pathogenesis associated with pejvakin mutations are more diverse than previously appreciated. More generally, our findings demonstrate that recessive screens in mice are powerful tools for identifying genes that control the development and function of mechanosensory hair cells and cause deafness in humans, as well as generating animal models for disease.

Spatiotemporal definition of neurite outgrowth, refinement and retraction in the developing mouse cochlea

The adult mammalian cochlea receives dual afferent innervation: the inner sensory hair cells are innervated exclusively by type I spiral ganglion neurons (SGN), whereas the sensory outer hair cells are innervated by type II SGN. We have characterized the spatiotemporal reorganization of the dual afferent innervation pattern as it is established in the developing mouse cochlea. This reorganization occurs during the first postnatal week just before the onset of hearing. Our data reveal three distinct phases in the development of the afferent innervation of the organ of Corti: (1) neurite growth and extension of both classes of afferents to all hair cells (E18-P0); (2) neurite refinement, with formation of the outer spiral bundles innervating outer hair cells (P0-P3); (3) neurite retraction and synaptic pruning to eliminate type I SGN innervation of outer hair cells, while retaining their innervation of inner hair cells (P3-P6). The characterization of this developmental innervation pattern was made possible by the finding that tetramethylrhodamine-conjugated dextran (TMRD) specifically labeled type I SGN. Peripherin and choline-acetyltransferase immunofluorescence confirmed the type II and efferent innervation patterns, respectively, and verified the specificity of the type I SGN neurites labeled by TMRD. These findings define the precise spatiotemporal neurite reorganization of the two afferent nerve fiber populations in the cochlea, which is crucial for auditory neurotransmission. This reorganization also establishes the cochlea as a model system for studying CNS synapse development, plasticity and elimination.

Quinacrine staining of marginal cells in the stria vascularis of the guinea-pig cochlea: a possible source of extracellular ATP?

There is accumulating evidence for a purinergic humoral system involved in the control of cochlear function. Evidence of specific P2 purinoceptors on cochlear tissues implies a role for extracellular adenosine triphosphate (ATP) in the cochlea. To further this hypothesis a study was undertaken to determine if there was any specific source of purine compounds in cochlear tissues. Cochlear tissues (the sensory epithelium and lateral wall) from the guinea pig were incubated with the acridine derivative quinacrine dihydrochloride (5 x 10(-6) M in phosphate-buffered saline for 30 min at room temperature) which fluoresces on binding to high concentrations of ATP. Most cochlear tissues showed a diffuse green fluorescence slightly above the background level. However, a region of the marginal cells of the stria vascularis showed a specific punctate fluorescence. Optical sectioning of these cells by confocal microscopy revealed that the fluorescent structures in these marginal cells was confined to a region up to 10 microns from their endolymphatic surface. Similar cells studied by transmission electron microscopy showed membrane-bound vesicles located in the same region of the cell. These data imply that purine compounds are localized in discrete structures, perhaps vesicles, within the marginal cells which could serve as a source of extracellular ATP in the cochlea.