Biomedical subjects

W F Sewell

Publications and source records attributed to W F Sewell.

At least 19 recordsLinked to original sources

PubMed2005

Extracts of retina and brain that excite afferent fibers innervating hair cells contain a compound related to hydroxyphenylglycine-N-carbamoyl.

To identify new neurotransmitter and modulator candidates that might be important in transmission from sensory hair cells to afferent nerves, we examined extracts of neural tissue for compounds that excite afferent fibers innervating hair cells. Here, we describe the extraction and purification from retina and brain of a potent, unstable, excitatory compound with pharmacological activity similar to glutamate on afferent fibers innervating hair cells. This compound, however, was clearly distinguished from glutamate, other common amino acids, and known endogenous glutamate-receptor agonists. After derivatization and analysis by gas chromatography-mass spectrometry, the major compound found in highly purified neuroactive chromatographic fractions had the same gas chromatographic elution time and mass spectrum as the compound formed by derivatization of L-p-hydroxyphenylglycine-N-carbamoyl. Hydroxyphenylglycine-N-carbamoyl, however, did not copurify with the neuroactive compound and was not neuroactive. We thus hypothesize that the detected compound was produced from a precursor, structurally related to L-p-hydroxyphenylglycine-N-carbamoyl, that was a major component of the neuroactive chromatographic fractions. Because several compounds related to hydroxyphenylglycine are known to act on glutamate receptors, such a compound is an interesting candidate to be an endogenous glutamate-receptor ligand in the mammalian nervous system.

Afferent Pathways↗

PubMed2001

Morphology and cell type heterogeneities of the inner ear epithelia in adult and juvenile zebrafish (Danio rerio).

Although the zebrafish has become an important model for genetic analysis of the vertebrate auditory system, a comprehensive description of the zebrafish ear has been provided for embryonic and larval development only (Haddon and Lewis [1996] J. Comp. Neurol. 365:113). Here we describe the development of sensory maculae in juvenile fish and the morphology of the adult zebrafish ear. This description was obtained via three-dimensional reconstruction of serial sections and confocal microscopy of immunolabeled preparations and includes the Weberian ossicles and fluid spaces. Phalloidin staining, which labels actin filaments of stereocilia, was used to delineate the sensory epithelia, to visualize the distribution of hair cells, to estimate their density in different areas of the maculae, and to perform hair cell counts. Morphology of ciliary bundles in different regions of the lagena, saccule, utricle, macula neglecta, and cristae was characterized with an anti-acetylated tubulin antibody and by phalloidin staining. We have identified two antibodies characterized by region-specific staining patterns in the inner ear epithelia. Zn-1 antibody staining largely correlates with the presence of short-bundle hair cells in the peripheral regions of sensory epithelia. Zn-4 antibody, on the other hand, labels a zone of epithelial cells surrounding the sensory maculae. These analyses extend previous observations of cell-type heterogeneity in both sensory and nonsensory epithelia of the fish ear.

Age Factors↗

PubMed2001

Fast, but not slow, effects of olivocochlear activation are resistant to apamin.

Olivocochlear (OC) efferent suppression of auditory-nerve responses comprises a fast effect lasting tens of milliseconds and a slow effect building and decaying over tens of seconds. Both fast and slow effects are mediated by activation of the same alpha 9 nicotinic receptor. We have hypothesized that fast effects are generated at the OC synapse, but that slow effects reflect activation of calcium-activated potassium (K(Ca)) channels by calcium release from the subsurface cisternae on the basolateral wall of the hair cells. We measured in vivo effects of apamin, a blocker of small-conductance (SK) K(Ca) channels, and charybdotoxin, a blocker of large-conductance K(Ca) channels, perfused through scala tympani, on fast and slow effects evoked by electrical stimulation of the OC bundle in anesthetized guinea pigs. Apamin selectively and reversibly reduced slow-effect amplitude without altering fast effects or baseline amplitude of the auditory-nerve response, but only when perfused at concentrations of 100 microM. In contrast, the effects of charybdotoxin were noted at 30 nM, but were not specific, reducing both afferent and efferent responses. The very high concentrations of apamin needed to block efferent effects contrasts with the high sensitivity of isolated hair cells to apamin's block of acetylcholine's effects. The results suggest that in vivo fast OC effects are dominated by a conductance that is not apamin sensitive.

Action Potentials↗

PubMed2000

Calcitonin gene-related peptide suppresses hair cell responses to mechanical stimulation in the Xenopus lateral line organ.

The presence of calcitonin gene-related peptide (CGRP) in the efferent fibers of virtually every hair cell organ studied suggests it may serve some fundamental but heretofore unknown role in control of hair cell function. We examined the effects of CGRP on spontaneous and stimulus-evoked discharge patterns in an in vitro preparation of the lateral line organ of Xenopus laevis. Discharge patterns were determined by sinusoidally displacing the cupula with a glass micropipette driven with a piezoelectric device while recording afferent fiber activity. All afferent fibers had characteristic frequencies of 16-32 Hz. Responses synchronized to cupular displacements as small as 20 nm. CGRP suppressed responses of the lateral line organ to displacement while increasing spontaneous discharge rate. In the presence of CGRP, stimulus-response curves were shifted 10 dB toward higher displacement levels. The suppression of stimulus-evoked responses suggests a function for CGRP as an efferent neurotransmitter that is similar to that of cholinergic efferent transmission in other hair cell organs. The 10 dB shift toward larger displacements makes it comparable in magnitude with the effects of electrical stimulation of efferents in the mammalian cochlea. This suggests a significant role for CGRP in efferent modulation of the output of this mechanosensory organ.

Acoustic Stimulation↗

PubMed2000

Pharmacological characterization of the CGRP receptor in the lateral line organ of Xenopus laevis.

Calcitonin gene-related peptide (CGRP) is a neurotransmitter candidate colocalized with acetylcholine in efferent fibers innervating hair cell organs. We have used the Xenopus laevis lateral line organ to investigate the responses of a hair cell organ to the CGRP family of peptides. Two isoforms of CGRP, r alpha-CGRP and r beta-CGRP, and a human analog of alpha-CGRP, h(Tyro) alpha-CGRP, produced dose-dependent increases in afferent nerve fiber discharge rate with EC50 values of approximately 1 microM. Rate increases were 31.2, 18.9, and 10.3%, respectively. The peptide fragment rCGRP8-37 a selective CGRP1 receptor antagonist, competitively inhibited the response to r alpha-CGRP. Diacetoamidomethyl cysteine CGRP (r[Cys(ACM)2,7]alpha-CGRP), a CGRP2 agonist, did not change discharge rate. Rat amylin did not increase rate until very high concentrations, and then the change was less than 7%. Rat adrenomedullin produced no increase in rate. Responses to r alpha-CGRP developed after metamorphosis. No change in spontaneous discharge rate was observed until postmetamorphic day 6, and then it was only a fraction of the maximal response. This response progressively increased until postmetamorphic day 28, when it reached its maximal value. The most straightforward interpretation of our results is that the effect of CGRP is mediated by the CGRP1 receptor and that CGRP, of the peptides presently known to exist in the CGRP family, is the most likely endogenous peptide mediating these effects.

Adrenomedullin↗

PubMed2000

Contribution of glutamate receptors to spontaneous and stimulus-evoked discharge in afferent fibers innervating hair cells of the Xenopus lateral line organ.

The relative contributions of NMDA (N-methyl-D-aspartate) and non-NMDA glutamate receptors to spontaneous and stimulus-evoked transmission at the hair cell/afferent fiber synapse were determined in the Xenopus laevis lateral line organ. The non-NMDA receptor antagonist, CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), reversibly reduced both spontaneous and stimulus-evoked discharge rate with an EC(50) of 0.5 microM. NMDA receptor antagonism with the combination of chlorokynurenic acid (100 microM) and elevated magnesium (1.1 mM), or elevated magnesium alone, blocked responses to NMDA without significantly altering spontaneous or stimulus-evoked discharge rate or the responses to kainate. All non-NMDA receptor agonists tested increased discharge rate at low concentrations and, at higher concentrations, increased, then suppressed discharge rate. The EC(50)s were: domoic acid (2.4 mcM)<quisqualic acid (6 mcM)<kainic acid (18 mcM)<AMPA (82 mcM)<<glutamate (1150 mcM). NMDA and ibotenic acid also produced an increase in discharge followed by a suppression, but the suppressive phase of the response predominated and maximum increases in discharge rates were low compared to effects of the non-NMDA agonists. The EC(50)s were: NMDA (148 mcM)<ibotenic acid (463 mcM). The EC(50) for the suppression of afferent discharge that followed the initial excitatory effect was similar to the EC(50) for excitation. Perfusion with active concentrations of kainate, AMPA, or NMDA did not alter the threshold for electrical stimulation of these nerve fibers. We conclude that most of the postsynaptic signal normally seen in afferent fibers is mediated by non-NMDA receptors.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1999

Gentamicin blocks both fast and slow effects of olivocochlear activation in anesthetized guinea pigs.

The medial olivocochlear (MOC) efferent system, which innervates cochlear outer hair cells, suppresses cochlear responses. MOC-mediated suppression includes both slow and fast components, with time courses differing by three orders of magnitude. Pharmacological studies in anesthetized guinea pigs suggest that both slow and fast effects on cochlear responses require an initial acetylcholine activation of alpha-9 nicotinic receptors on outer hair cells and that slow effects require additional intracellular events downstream from those mediating fast effects. Gentamicin, an aminoglycoside antibiotic, has been reported to block fast effects of sound-evoked OC activation following intramuscular injection in unanesthetized guinea pigs, without changing slow effects. In the present study, we show that electrically evoked fast and slow effects in the anesthetized guinea pig are both blocked by either intramuscular or intracochlear gentamicin, with similar time courses and/or dose-response curves. We suggest that sound-evoked slow effects in unanesthetized animals are fundamentally different from electrically evoked slow effects in anesthetized animals, and that the former may arise from effects of the lateral OC system.

Acoustic Stimulation↗

PubMed1997

Unique postsynaptic signaling at the hair cell efferent synapse permits calcium to evoke changes on two time scales.

The cholinergic efferent fibers to the outer hair cells (OHCs) of the mammalian cochlea suppress sound-evoked activity of the auditory nerve on two time scales via one nicotinic receptor. A rapid action (tens of milliseconds) is responsible for modulating auditory nerve responses to acoustic stimulation. A slower action (tens of seconds) may protect the ear from acoustic overstimulation. The rapid action is likely caused by calcium influx through the nicotinic receptor that leads to opening of calcium-activated potassium (KCa) channels, but the mechanism of the slower action has not been explained. To investigate this mechanism, we perfused the cochlea with agents that alter intracellular calcium release and uptake. Both fast and slow effects were enhanced by perfusion of the cochlea with ryanodine, an agonist of calcium-induced calcium release (CICR). Antagonists of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), cyclopiazonic acid, and thapsigargin (1) selectively enhanced the magnitude of slow effects, (2) prevented the diminution of slow effects with continued efferent stimulation, and (3) spread the range of frequencies over which slow effects were observed. We propose that the slow effect is attributable to release of calcium from the subsurface cisterna of the OHC, perhaps triggered by CICR from the synaptic cisterna; the two time scales of efferent action may result from the unique arrangement of the two cisternae in the baso-lateral region of the OHC.

Animals↗

PubMed1997

Ribonucleases may limit recovery of ribonucleic acids from archival human temporal bones.

Messenger ribonucleic acid (mRNA) for actin was detected in celloidin-embedded archival human temporal bone sections with reverse transcription polymerase chain reaction (RT-PCR). Actin mRNA was detected in 10% of sections analyzed. One possible reason for this modest detection incidence is enzymatic degradation of RNA by exogenously introduced ribonucleases (RNases). We have identified steps of the temporal bone processing protocol for archival storage in which exogenous RNases could be introduced to the tissue, and have verified that the bone sections are exposed to these enzymes. We have demonstrated that implementing precautions to minimize exogenous RNase contamination during processing improves recovery of intact RNA. This study indicates that although gene expression analysis of archival human temporal bones may be limited by enzymatic degradation of RNA, simple modification of processing protocol can improve yield of informative data.

Actins↗

PubMed1995

A novel cholinergic "slow effect" of efferent stimulation on cochlear potentials in the guinea pig.

This report documents slow changes in cochlear responses produced by electrical stimulation of the olivocochlear bundle (OCB), which provides efferent innervation to the hair cells of the cochlea. These slow changes have time constants of 25-50 sec, three orders of magnitude slower than those reported previously. Such "slow effects" are similar to classically described "fast effects" in that (1) they comprise a suppression of the compound action potential (CAP) of the auditory nerve mirrored by an enhancement of the cochlear microphonic potential (CM) generated largely by the outer hair cells; (2) the magnitude of suppression decreases as the intensity of the acoustic stimulus increases; (3) they share the same dependence on OCB stimulation rate; (4) both are extinguished upon cutting the OCB; and (5) both are blocked with similar concentrations of a variety of cholinergic antagonists as well as with strychnine and bicuculline. These observations suggest that both fast and slow effects are mediated by the same receptor and are produced by conductance changes in outer hair cells. Slow effects differ from fast effects in that (1) fast effects are greatest for acoustic stimulus frequencies between 6 and 10 kHz, whereas slow effects peak for frequencies from 12 to 16 kHz, and (2) fast effects persist over long periods of OCB stimulation, whereas slow effects diminish after 60 sec of stimulation. The time course of the slow effects can be described mathematically by assuming that each shock-burst produces, in addition to a fast effect, a small decrease in CAP amplitude that decays exponentially with a time constant that is long relative to the intershock interval. The long time constant of the slow effect compared to the fast effect suggests that it may arise from a distinct intracellular mechanism, possibly mediated by second-messenger systems.

Acoustic Stimulation↗

PubMed1993

Flavin adenine dinucleotide is a major endogenous fluorophore in the inner ear.

When illuminated with visible light, hair cells can exhibit autofluorescence (Lewis et al. [1982] Science 215, 1641-1643) concentrated in the basal pole near the synapses (Sento and Furukawa [1987] J. Comp. Neurol. 258, 352-367). The autofluorescence is enhanced by formaldehyde. The level of fluorescence is high enough to interfere with fluorescence microscopy of hair cells and to suggest that the fluorescent substance might have a particular role in hair-cell function. To identify this substance, we extracted a substance with formaldehyde-enhanced fluorescence from the inner ears of goldfish and purified it chromatographically. The substance copurified with FAD and had the same fluorescence emission spectrum. Two further results supported the identity of the endogenous fluorescent substance with FAD. First, as is the case with flavins, the autofluorescence in inner ear tissue examined within a few hours after fixation was reduced by addition of dithionite. Second, as is the case with the formaldehyde-enhanced fluorophore, the fluorescence of FAD was enhanced by formaldehyde. FAD accounted for 90% of flavins in goldfish inner ears; its concentration in the sensory epithelium was estimated to be about 30 nmol/g tissue weight, one of the highest tissue concentrations known. The FAD is probably associated with an unidentified flavoprotein concentrated in the basal, synaptic region of the hair cell.

Acoustic Maculae↗

PubMed1992

Neurite regeneration from single primary-auditory neurons in vitro.

Neurons of the VIIIth cranial nerve in lower vertebrates precisely reconnect with their targets after sustaining injury. It is not known, however, whether the regenerating neurites are guided entirely by external cues or may also be directed by intrinsic mechanisms. To address this issue, single adult primary-auditory neurons were dissected from goldfish and placed in an in vitro environment, devoid of the normal complement of satellite cells, neighboring neurons, and synaptic targets, to observe their patterns of growth. Because acutely isolated neurons showed little neurite outgrowth, neurite regeneration was enhanced by focally crushing the VIIIth cranial nerve 2 to 24 h prior to removal for tissue culture. Neurons that regenerated under identical culture conditions showed growth patterns that could be categorized into three separate groups based on both their morphology and growth patterns. They either 1) remained unbranched (54%), 2) bifurcated or trifurcated into major branches directly from the myelinated stump (V-shaped) (19%), or 3) bifurcated from a regenerated process (Y-shaped), sometimes with a third, smaller branch (27%). Unbranched and V-shaped neurites grew at a constant elongation rate, while Y-shaped neurites grew variably, with alternating retractions and elongations. Neurite elongation was completed in a uniform time period of approximately 15 days despite the differences in elongation rate, maximum length, and latency to growth onset. The neurite branching morphology and manner of growth revealed in this study indicated that adult regenerating neurons can reproduce some elements of the final branching patterns in the absence of extrinsic cues, a capability which may ultimately contribute to the fidelity of reconnection seen in vivo.

Animals↗

PubMed1991

Neurotransmitter release from hair cells and its blockade by glutamate-receptor antagonists.

To assess the mechanism by which glutamate-receptor antagonists block afferent discharge at the hair cell synapse, we examined the effects of these and other agents on sound-evoked excitatory post-synaptic potentials (EPSPs) and on spontaneous miniature post-synaptic potentials (MEPSPs) in auditory-nerve fibers of the goldfish (Carassius auratus) saccule. A quantal analysis of synaptic transmission under conditions in which the probability of transmitter release was reduced by cobalt, an agent that can block transmitter release, supports Furukawa's (Jpn. J. Physiol. 36, 1059-1077, 1986) conclusion that transmitter release at this synapse is quantal. Cobalt reduced the rate of occurrence of spontaneous MEPSPS without reducing their amplitude. The glutamate-receptor antagonists, gamma-D-glutamyl glycine (DGG) and 5-aminophosphonovaleric acid (APV) both reduced the amplitude of sound-evoked EPSPs much more than that of the spontaneous MEPSPs. The glutamate-receptor agonists, L-glutamate, kainate, and quisqualate, produced a depolarization of the afferent nerve fiber, a decrease in the amplitude of the EPSP and an increased tendency for an EPSP to generate an action potential.

2-Amino-5-phosphonovalerate↗

PubMed1991

Effects of calcitonin gene-related peptide and efferent nerve stimulation on afferent transmission in the lateral line organ.

1. Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide immunolocalized in efferent fibers innervating hair-cell organs, including the lateral line organ of Xenopus laevis. CGRP, applied in nanomolar concentrations, increased the spontaneous discharge rate in afferent fibers innervating hair cells of the lateral line organ. 2. The increase in spontaneous discharge rate with application of CGRP was associated with an increase in the rate of occurrence of spontaneous excitatory postsynaptic potentials (EPSPs) and with little change in the amplitude of the EPSPs. 3. Prolonged (several hundred seconds) application of CGRP produced an increase in afferent fiber discharge rate that returned to control values in the continued presence of the peptide. 4. Efferent fibers were electrically stimulated to look for a non-cholinergic effect on spontaneous afferent discharge that might be attributed to CGRP. Electrical stimulation of the efferent fibers produced a rapid (100 ms) suppression of discharge rate followed by a rapid (100 ms) increase in discharge rate. However, both the rapid suppression and rapid excitation were likely to be mediated by the release of acetylcholine, because they were blocked by the application of the cholinergic blocking agents curare and atropine as well as by strychnine. 5. In almost one-half of the preparations examined, electrical stimulation of efferent fibers also produced a slowly developing increase in afferent discharge that could persist for several minutes after termination of the shocks. 6. This slow excitation by efferent stimulation was not blocked by concentrations of curare that blocked the rapid effects of efferent stimulation. Thus the slow effect is likely to be mediated by a receptor different from that for the rapid cholinergic effects. One possibility is that the excitation is mediated by the release of CGRP from the efferent nerve fibers.

Animals↗

PubMed1990

Synaptic potentials in afferent fibers innervating hair cells of the lateral line organ in Xenopus laevis.

Spontaneous synaptic activity in afferent fibers innervating hair cells of the lateral line organ was monitored with micropipettes at visually identified locations near the peripheral terminations of the fibers. Spontaneous action potentials as well as spontaneous excitatory post-synaptic potentials (EPSPs) were observed. Some action potentials were preceded by EPSPs. Most EPSPs did not reach threshold to generate action potentials. The frequency of occurrence of spontaneous EPSPs was related to the number of neuromasts innervated by a fiber. Single EPSPs were most readily observed in fibres innervating only a few neuromasts. Perfusion of the synapse with solutions containing cobalt reversibly diminished discharge rate by reducing the occurrence of EPSPs. Manganese produced similar effects on discharge rate. The blockage of spontaneous EPSPs by cobalt, an agent that can block voltage-dependent transmitter release, supports the hypothesis that spontaneous discharge in afferent fibers of the lateral line organ is due to voltage-dependent release of transmitter from the hair cells.

Action Potentials↗

PubMed1990

Purification of a low-molecular-weight excitatory substance from the inner ears of goldfish.

The neurotransmitter released by the hair cell has not been identified; little is known about other neuroactive substances that may be important in hair-cell organ function. To identify neuroactive substances in hair cell tissue, we have examined substances in extracts of the inner ears of goldfish that can excite afferent fibers innervating hair cells. The extracts contain an unidentified low-molecular-weight (LMW) excitatory substance that is a candidate to be the hair-cell neurotransmitter. The LMW excitatory substance has been highly purified by a sequential combination of (1) treatment with cation-exchange resin; (2) gel-permeation chromatography; (3) gradient-elution cation-exchange chromatography; (4) isocratic-elution cation-exchange chromatography; and (5) high-performance anion-exchange chromatography. Based upon its separation behavior in these purification steps, the LMW excitatory substance may be a small, zwitterionic compound with titratable anionic and cationic groups.

Animals↗

PubMed1989

Pharmacological alterations of the activity of afferent fibers innervating hair cells.

To determine whether some of the substances that may be present in hair-cell sensory organs could affect neural activity in afferent fibers, we examined 56 compounds for the ability to alter the discharge rate of afferent fibers innervating hair cells in the lateral line organ of Xenopus laevis, the African clawed frog. These compounds included amino acids, glutamyl dipeptides, standard neurotransmitter candidates, and other constituents of tissues and body fluids. Substances found to be excitatory included some neutral amino acids (alanine, serine, threonine, asparagine, glutamine, and proline), ATP, carnosine, histidine, and barium chloride. Compounds that suppressed discharge included the aromatic amino acids (phenylalanine, tryptophan, and tyrosine), serotonin, and gamma-glutamyl dipeptides. GABA and acidic amino acids (glutamate, aspartate, and cysteine sulfinate) produced a brief excitation followed by a suppression of discharge rate. Several of these substances were active at sufficiently low concentrations that their presence in body fluids may affect afferent fiber discharge rate under normal or pathological conditions.

Amino Acids↗

PubMed1987

A possible neurotransmitter role for CGRP in a hair-cell sensory organ.

We report that calcitonin gene-related peptide (CGRP) increases the discharge rate of afferent fibers innervating hair cells in the lateral line organ of Xenopus laevis. We have localized CGRP-like immunoreactivity in small, presumably efferent, fibers innervating the lateral line organ. In addition to providing evidence for a neurotransmitter role for CGRP in a sensory system, these results may help explain the non-cholinergic excitatory effect seen with efferent stimulation in this and other hair cell organs such as the inner ear.

Action Potentials↗