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Membrane currents influencing action potential latency in granule neurons of the rat cochlear nucleus.

Granule cells are the most numerous neurons in the cochlear nucleus, but, because of their small size, little information on their membrane properties and ionic currents is available. We used an in vitro slice preparation of the rat ventral cochlear nucleus to make whole-cell recordings from these cells. Under current clamp, some granule neurons fired spontaneous action potentials and all generated a train of action potentials on depolarization (threshold current, 10-35 pA). Hyperpolarization increased the latency to the first action potential evoked during a subsequent depolarization. We examined which voltage-gated currents might underlie this latency shift. In addition to a fast inward Na+ current, depolarization activated two outward potassium currents. A transient current was rapidly inactivated by membrane potentials positive to -60 mV, while a second, more slowly inactivating current was observed following the decay of the transient current. No hyperpolarization-activated conductances were observed in these cells. Modelling of the currents suggests that removal of inactivation on hyperpolarization accounts for the increased action potential latency in granule cells. Such a mechanism could account for the 'pauser'-type firing patterns of the fusiform cells which receive a prominent projection from the granule cells in the dorsal cochlear nucleus.

Animals↗

Expression of glycine receptor subunit mRNAs in the rat cochlear nucleus.

The distribution of glycine receptor subunit mRNAs in the cochlear nucleus of the adult rat was examined using radioactive in situ hybridization. Expression was compared among six cell types by counting silver grains over somata. Expression of the immature alpha2 subunit was not above the threshold for detection in any neurons. Levels of expression of mature subunits varied among different cell types. Spherical bushy, small cell cap/shell neurons and fusiform cells had high expression of glycine receptor mRNA for alpha1, alpha 3 and beta subunits. Octopus cells and corn cells had high expression for alpha 3 and beta subunits, and only moderate expression for alpha1 subunit. Granule cells located between the dorsal and ventral cochlear nucleus had moderate expression of alpha 3 and beta subunits and no detectable alpha1 expression. These patterns of expression predict differences in glycinergic pharmacological properties between the cochlear nucleus neurons.

Animals↗

Glycine receptor immunoreactivity in the ventral cochlear nucleus of the guinea pig.

Glycine appears to be a major inhibitory neurotransmitter in the cochlear nucleus. In order to determine more precisely the distribution of glycinergic synapses, we have studied the immunocytochemical distribution of the glycine postsynaptic receptor. Two monoclonal antibodies were used, Gly Rec Ab 2, which recognizes the 48kD polypeptide and Gly Rec Ab 7, which primarily recognizes the 93kD subunit of the glycine receptor complex. At the light microscopic level, glycine receptor immunoreactivity was found throughout the ventral cochlear nucleus with a punctuate distribution often found outlining large cell bodies. Indistinguishable patterns of staining were obtained with the two antibodies. Ultrastructural localization was done with Gly Rec Ab 7 because immunoreactivity remained after fixation with glutaraldehyde containing solutions. At the ultrastructural level, immunoreactivity was concentrated at postsynaptic sites on dendrites and cell bodies. In the anteroventral cochlear nucleus, neurons identified as spherical cells contained numerous inmunoreactive synapses on their cell bodies, whereas most immunoreactive synapses on stellate cells were on their proximal dendrites. In the posteroventral cochlear nucleus, neurons identified as octopus cells were immunoreactive on their cell bodies and proximal dendrites. In the granule cell layer, immunoreactivity was found only in the neuropile. Throughout the ventral cochlear nucleus, glycine receptor immunoreactivity was found postsynaptic to terminals containing flattened synaptic vesicles as well as those containing oval/pleomorphic synaptic vesicles.

Animals↗

[Response of cochlear nucleus neurons of the cat to complex tone].

The response of the cochlear nucleus neurons in the anesthetized cat to complex tones has been studied for understanding the processing of tonal informations in that nucleus. Forty-eight cats were used in this study and data was obtained from 148 neurons with microelectrode technique. Two kinds of stimulus tones were applied and each of them consisted of pure tones (f1, f2, f3) and three complex tones (f1 + f2, f1 + f3, f2 + f3). The first group was composed of three pure tones of 670, 1250, 2400Hz and the second group was composed of those of 350, 500, 1100Hz, respectively. In 50 out of 148 cochlear nucleus neurons, we obtained some responses to complex tones. Response patterns to complex tones in 34 out of 50 cochlear nucleus neurons were similar to response patterns which had been obtained from auditory nerve. In 16 out of 50 neurons, however, the PST histogram pattern which had been obtained by complex tone stimuli did not show the same pattern as that of pure tone stimuli. For example, in same neuron, sustained type and pauser type responses had obtained by pure tones stimulus, but chopper type response had obtain by complex tone stimuli. And some neural activities were facilitated by the complex tones. From these results it can be speculated that the certain functions of the processing of the complex tones will be exist in the cochlear nucleus.

Acoustic Stimulation↗

Neural encoding of single-formant stimuli in the ventral cochlear nucleus of the chinchilla.

Responses of the principal unit types in the ventral cochlear nucleus of the chinchilla were studied with a single-formant stimulus set that covered fundamental frequency (f0) from 100 Hz to 200 Hz and formant center frequency (F1) from 256 to 782 Hz. Temporal coding for f0 and F1 was explored for 95 stimulus combinations of f0 (n = 5) and F1 (n = 19) in primarylike, onset and chopper unit categories. Several analyses that explored temporal coding were employed including: autocorrelation, interspike interval analysis, and synchronization to each harmonic of f0. In general, the representation of f0 is better in onset and chopper units than in primarylike units. Nearly all units in the cochlear nucleus showed a gain in phase locking to the envelope (f0) of the single-formant stimulus relative to the auditory nerve. The fundamental is represented directly in neural discharges of units in the cochlear nucleus with an interval code (also Cariani and Delgutte, 1996; Rhode, 1995). The formant is represented in the temporal domain in primarylike units, though some chopper and onset units also possess the ability to code F1 through discharge synchrony. Onset-I units, which are associated with the octopus cells, exhibited the strongest phase locking to f0 of any unit types studied. The representation of f0 and F1 in the temporal domain is weak or absent in some units. All-order-interspike interval distributions computed for populations of units show preservation of temporal coding for both f0 and F1. Results are in agreement with earlier amplitude modulation studies that showed nearly all cochlear nucleus unit types phase lock to the signal envelope better than auditory nerve fibers over a considerable range of signal amplitudes.

Acoustic Stimulation↗

The distribution of GABA and glycine immunostaining in the cochlear nucleus of the mustached bat (Pteronotus parnellii).

The distribution of neuronal elements immunoreactive for gamma-aminobutyric acid (GABA) and glycine in the cochlear nucleus of the mustached bat Pteronotus parnellii has been studied by means of the postembedding technique on serial semithin sections. Our goal has been to identify similarities and differences in the organization of putatively inhibitory circuits between a highly specialized echolocating bat and previously studied non-echolocating mammals. The results reveal a basically conserved pattern of putatively GABAergic and glycinergic elements in the bat cochlear nucleus, and subtle but distinct modifications in certain inhibitory circuits. As in other mammals, immunoreactive cells possibly representing local interneurons are most abundant in the dorsal cochlear nucleus. These include single-GABA-immunoreactive cells and double-labeled cells in the superficial layers and single-glycine-labeled cells in the deep layers. Coincident with the phylogenetic trend toward a reduced lamination of the dorsal cochlear nucleus in bats, there is a clear reduction in the numbers of local interneurons of the superficial layer. In contrast, the tuberculoventral system of the deep layer appears hypertrophied. As in other mammals, the ventral cochlear nucleus contains a few large single-glycine-immunoreactive cells and scattered double-labeled cells. Immunoreactive puncta are abundant throughout the cochlear nucleus complex with no trends indicating a differential strength of inhibitory inputs to regions representing the various harmonics of the echolocating signal.

Animals↗

Functional organization of ascending and descending connections of the cochlear nucleus of horseshoe bats.

The ascending projections of the cochlear nucleus (CN) and the sources of descending inputs to the CN were investigated in horseshoe bats (Rhinolophus rouxi) by tracing the anterograde and retrograde transport of horseradish peroxidase (HRP or WGA-HRP) injected into the CN. The tracer was iontophoretically deposited into physiologically characterized regions of the cochlear nucleus (Feng and Vater, '85). We report the course and termination of pathways arising from the anteroventral (AVCN), posteroventral (PVCN), and dorsal (DCN) cochlear nucleus. The projection fields within the auditory brainstem centers (superior olivary complex [SOC]; lateral lemniscus complex [LLC]; and inferior colliculus [IC]) and their tonotopic organization according to the frequency representations at the injection sites are described. While the projection pattern is generally in accordance with other mammals, several species-characteristic features are noted: i) the lateral superior olive (LSO) receives tonotopically organized input from both the AVCN and PVCN; ii) the CN-projections to medial nuclear groups of the SOC located between the LSO and the medial nucleus of the trapezoid body do not support previously suggested homologies; iii) the ventral nucleus of the LLC can be subdivided into two divisions with distinct input patterns from the AVCN and PVCN, respectively.

Animals↗

Directed outgrowth from a subset of cochlear nucleus fibers in a collagen-gel matrix.

We have employed a collagen-gel co-culture system to evaluate the influence of the hindbrain floor-plate on initial axon outgrowth from the cochlear nucleus. After 2 days in vitro, cochlear nucleus explants exhibited directed outgrowth towards co-cultured floor plate explants. Comparisons with co-cultures of cochlear nucleus/forebrain or cochlear nucleus/midbrain explants or with cochlear nucleus explants cultured alone suggest that the floor-plate has a specific chemoattractant effect on the outgrowth of the pioneer fibers of the hindbrain auditory commissure. Fiber outgrowth was not directed towards a recombinant source of the chemoattractant molecule netrin suggesting that floor plate directed outgrowth in the cochlear nucleus is not solely dependent on netrin. In the present report, we present evidence for the first time that the floor-plate is a chemotropic source in pathfinding of second-order auditory fibers from the cochlear nucleus in the hindbrain.

Animals↗

Amino acid uptake and release in the guinea pig cochlear nucleus after inferior colliculus ablation.

This study attempts to determine if the neurons in the guinea pig inferior colliculus that project to the cochlear nucleus could use certain amino acid transmitters. The left inferior colliculus was ablated surgically to destroy projections descending to the cochlear nuclei. Nissl and silver stained sections of the brain stem indicated that this procedure destroyed most of the left inferior colliculus, but spared a small amount of collicular tissue ventrally and rostrally. Six to seven days after the ablation, degenerated fibers were present in the right inferior colliculus, in the left lateral lemniscus, and in the cochlear nucleus, bilaterally. Three to five days after the ablation, the uptake and electrically-evoked release of exogenous, radiolabeled D-aspartate, gamma-aminobutyrate, and glycine were measured in the three major subdivisions of the cochlear nucleus, the anteroventral, posteroventral, and dorsal divisions. These activities were compared to those in unlesioned controls. The ablation did not alter the uptake and release of the amino acids in the dorsal and posteroventral divisions of the cochlear nucleus. However, it lowered slightly (by 10-18%) the uptake and release of gamma-aminobutyrate and glycine in the anteroventral division, although the difference from the control group was not statistically significant. These findings suggest that most of the neurons in the inferior colliculus that project to the cochlear nucleus probably do not use glutamate, aspartate, gamma-aminobutyrate, or glycine as a transmitter. However, the possibility remains that a small proportion of the collicular projections to the anteroventral cochlear nucleus might use gamma-aminobutyrate or glycine as a transmitter.

Amino Acids↗

Uptake and release of D-aspartate in the guinea pig cochlear nucleus.

This study attempted to determine if L-glutamate (L-Glu) and/or L-aspartate (L-Asp) might be the transmitters of neurons that provide synaptic endings to the cochlear nucleus of the medulla. The uptake and release of D-[3H]aspartate (D-Asp), a putative marker for L-Glu and L-Asp, were measured in the guinea pig cochlear nucleus before and after destruction of the cochlear afferents by cochlear ablation. The cochlear nucleus was dissected into the anteroventral (AVCN), posteroventral (PVCN), and dorsal (DCN) cochlear nuclei. Subdivisions from unlesioned animals took up D-Asp, achieving concentrations in the tissues that were 13-20 times that in the medium. Subsequently, electrical stimulation evoked a Ca2+-dependent release of part of the D-Asp from each subdivision. Disarticulation of the middle ear ossicles, which attenuates acoustic stimulation, produced a modest inhibition of D-Asp release in each subdivision, but did not alter the uptake of D-Asp. Cochlear ablation strongly depressed both the uptake and the release of D-Asp in each subdivision, presumably as a result of destruction of the cochlear nerve endings in the cochlear nucleus. Nevertheless, after lesions, there was a preservation of the uptake and release of D-Asp in the DCN relative to the AVCN and PVCN. These residual activities in the DCN may be mediated by the axonal endings of the granule cells of the cochlear nucleus. The present findings support the hypothesis that the granule cells of the cochlear nucleus, as well as the cochlear nerve fibers, use L-Glu and/or L-Asp as transmitters.

Animals↗

Excitatory/inhibitory response types in the cochlear nucleus: relationships to discharge patterns and responses to electrical stimulation of the auditory nerve.

We have studied the response properties of single units in the cochlear nucleus of unanesthetized decerebrate cats. The purpose of the study was to compare the properties of cochlear nucleus units as described in two commonly used classification schemes. Units were first classified according to their receptive-field properties based on the relative prominence of excitatory and inhibitory responses to tones and noise. Units were then classified on the basis of their discharge patterns to short tone bursts at their best frequencies (BFs). Our results show that systematic relationships exist between the receptive-field properties and discharge patterns of cochlear nucleus units. Type I units give only excitatory responses to tones and noise. They are characterized by primary-like and chopper discharge patterns. Some units in the anteroventral cochlear nucleus have prepotentials in their spike waveforms. Prepotential units most often show primary-like discharge patterns, but prepotential units characterized by nonprimary-like discharge patterns are also found. Most prepotential units lack detectable inhibitory sidebands (type I), but two of the nonprimary-like prepotential units encountered in this study had inhibitory sidebands (type III). Type III units also give excitatory responses to BF tones, but they have inhibitory sidebands. Most type III units give chopper discharge patterns, and these units can be recorded throughout the cochlear nucleus. Some type III units in the dorsal cochlear nucleus give complex discharge patterns that can be described as a composite of the pauser pattern and other patterns. The complexity of these responses seems to increase as the amount of inhibition at BF increases. Type I/III units give excitatory responses to tones and noise, but have little or no spontaneous activity so they cannot be tested directly for inhibitory responses. Type I/III units typically show chopper discharge patterns. One group of type I/III units have rate-level functions with sloping saturation, suggesting that these may receive a predominance of input from low spontaneous rate auditory nerve fibers. Type II units are nonspontaneous and give excitatory responses to tones, but give weak or no responses to noise. While type II units are homogeneous as a group in terms of their response maps. BF rate-level functions, and responses to noise, they show a variety of discharge patterns in response to short tone bursts at BF.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Multichannel cochlear nucleus stimulation.

Stimulation of the cochlear nucleus (CN) has been used on a limited basis for rehabilitation of a select group of patients with bilateral acoustic neuromas. These patients were implanted with an electrode placed on the surface of the CN after resection of their tumors. Animal studies have demonstrated greater efficiency of a penetrating CN electrode in activating the central auditory system than a surface electrode. The objective of this work was to study the electrically evoked middle latency response generated by stimulation through a penetrating multichannel CN electrode in an animal model. Six pigmented guinea pigs underwent implantation with a penetrating multichannel CN electrode. Threshold, latency, and input-output functions of electrically evoked middle latency responses with different stimulation pads were studied. There were systematic differences in the latency and amplitude of the input-output functions depending on the site of stimulation within the CN. The results support the hypothesis that discrete activation of neuronal subpopulations within the CN is possible with a penetrating multichannel microelectrode.

Animals↗

Glutamate regulates IP3-type and CICR stores in the avian cochlear nucleus.

Neurons of the avian cochlear nucleus, nucleus magnocellularis (NM), are activated by glutamate released from auditory nerve terminals. If this stimulation is removed, the intracellular calcium ion concentration ([Ca2+]i) of NM neurons rises and rapid atrophic changes ensue. We have been investigating mechanisms that regulate [Ca2+]i in these neurons based on the hypothesis that loss of Ca2+ homeostasis causes the cascade of cellular changes that results in neuronal atrophy and death. In the present study, video-enhanced fluorometry was used to monitor changes in [Ca2+]i stimulated by agents that mobilize Ca2+ from intracellular stores and to study the modulation of these responses by glutamate. Homobromoibotenic acid (HBI) was used to stimulate inositol trisphosphate (IP3)-sensitive stores, and caffeine was used to mobilize Ca2+ from Ca2+-induced Ca2+ release (CICR) stores. We provide data indicating that Ca2+ responses attributable to IP3- and CICR-sensitive stores are inhibited by glutamate, acting via a metabotropic glutamate receptor (mGluR). We also show that activation of C-kinase by a phorbol ester will reduce HBI-stimulated calcium responses. Although the protein kinase A accumulator, Sp-cAMPs, did not have an effect on HBI-induced responses. CICR-stimulated responses were not consistently attenuated by either the phorbol ester or the Sp-cAMPs. We have previously shown that glutamate attenuates voltage-dependent changes in [Ca2+]i. Coupled with the present findings, this suggests that in these neurons mGluRs serve to limit fluctuations in intracellular Ca2+ rather than increase [Ca2+]i. This system may play a role in protecting highly active neurons from calcium toxicity resulting in apoptosis.

Adenylyl Cyclase Inhibitors↗

Giant cells of the dorsal cochlear nucleus of mice: intracellular recordings in slices.

1. In slices of the murine cochlear nuclear complex, intracellular recordings were made from five giant cells that were identified by intracellular labeling with biocytin. Giant cells form one of the two output pathways of the dorsal cochlear nucleus (DCN). Understanding how neuronal circuits and intrinsic electrical properties interact to control the firing of giant cells is a step toward understanding what acoustic information is conveyed through these cells. 2. Cell bodies of the labeled giant cells lay in the deep layer of the DCN. Dendrites, widespread both along the isofrequency axis and along the tonotopic axis, occupied mainly the deep layer, but some distal ends strayed into the molecular layer. Axons of giant cells were large, varying between 1 and 2 microns diam, and left through the dorsal acoustic stria. They were not observed to branch in the cochlear nuclei. 3. Giant cells fired large, overshooting action potentials that were followed by two afterhyperpolarizations. The first brought the membrane potential below rest, independent of the strength of injected current. The more variable second one produced either an undershoot or an inflection in the membrane potential between action potentials. 4. In each of the five labeled giant cells, shocks to the nerve root or to the anteroventral cochlear nucleus (AVCN) evoked a monosynaptic excitatory postsynaptic potential and two tandem inhibitory postsynaptic potentials (IPSPs) in the first 10 ms. Later IPSPs followed after latencies of between 10 and 50 ms. Monosynaptic excitation was usually cut short by the inhibition. 5. Strychnine, at 1 microM, blocked all IPSPs in the one giant cell tested, indicating that inhibitory input to this giant cell from circuits intrinsic to the cochlear nuclear complex was glycinergic. 6. The location of afferents was mapped for two giant cells. Both excitatory and inhibitory inputs to giant cells could be driven by the local application of glutamate to many loci in the AVCN and posteroventral cochlear nucleus, indicating that the ventral cochlear nucleus VCN contains interneurons that are monosynaptically or polysynaptically connected to giant cells. 7. An interpretation consistent with the results is that giant cells are excited by auditory nerve fibers and are inhibited by tuberculoventral cells. Giant cells may also be excited by granule or T stellate cells.

Animals↗

The distribution of N-acetylgalactosamine in the cochlear nucleus of the gerbil revealed by lectin binding with soybean agglutinin.

A horseradish peroxidase conjugated lectin from Glycine max (soy bean agglutinin; SBA) was used to characterise the distribution of N-acetylgalactosamine in the cochlear nucleus of the mongolian gerbil. SBA bound differentially to a variety of structures within the cochlear nucleus. Specific SBA labelling was associated with large non-granule neurones of variable size and shape throughout the cochlear nucleus. Compared to adjacent Nissl-stained sections 80% of the non-granule cells in the dorsal cochlear nucleus (DCN) and more than 90% of the non-granule cells in the ventral cochlear nucleus (VCN) bound SBA. The variation in location, size and shape as well as the high percentage of the labelled neurones suggest that cells of several, if not all, non-granule cell types, which have been described for the cochlear nucleus according to the usual Nissl schemes, are SBA positive. Granule cells did not bind SBA because all SBA-labelled cells had diameters above 10 microns. Diffuse labelling, not systematically associated with cells or fibres, was high in the molecular and fusiform cell layers of the DCN and that part of the granule cell area located close to the surface of the VCN. Darkly labelled granules (up to 2 microns diameter) were prominent in the area of the VIIIth nerve root. After long SBA incubations, they were also present in VCN and to a lesser degree in DCN. The results are discussed with respect to findings in other brain areas and the possible co-localisation of gamma aminobutyric acid (GABA), parvalbumin and N-acetylgalactosamine.

Acetylgalactosamine↗

Quantitative inter-strain comparison of the distribution of choline acetyltransferase activity in the rat cochlear nucleus.

The distribution of choline acetyltransferase activity in the cochlear nucleus of Sprague-Dawley albino rats was quantitatively compared to those in two strains of pigmented rats, Long Evans hooded and Brown Norway, using microdissection and radiometric assay techniques. Although activities tended to be, on the whole, higher in the albino rats, the differences were fairly minor. The relative distributions of choline acetyltransferase activity were generally similar among the 3 rat strains, not only among regions, but also within regions. Stain for acetylcholinesterase activity in the cochlear nucleus also had a similar appearance among the 3 rat strains. These chemical results are consistent with previous anatomical and physiological studies suggesting that auditory differences between albino and pigmented animals may not be as great in the cochlear nucleus as in the superior olivary complex.

Animals↗

Fine structure, synaptology and immunocytochemistry of large neurons in the rat dorsal cochlear nucleus connected to the inferior colliculus.

Neurons in the rat dorsal cochlear nucleus that project to the inferior colliculus (pyramidal and giant) were retrograde labelled with wheat germ agglutinin conjugated to horseradish peroxydase. Both cell types showed a similar ultrastructural feature, particularly the rough endoplasmic reticulum was well developed and sometimes surrounded the nucleus. The synaptological profile was similar in pyramidal and giant cells. Axo-somatic terminals covered 40-70% of the perimeter of pyramidal cells and 35-60% of the perimeter of giant neurons. Giant neurons featured bipolar or multipolar shape and different orientation but they possessed a similar synaptic profile. Most axo-somatic terminals contained flat and pleomorphic synaptic vesicles, some pleomorphic vesicles. Few terminals contained round vesicles. These cells were consistently immuno-negative for both glycine and GABA and variably positive for glutamate. The immunoelectron microcopic study of thin sections showed that glycine immunoreactivity was constantly present in terminals enriched with flat vesicles, which often did not show GABA immunoreactivity. Few anterograde labelled boutons containing flat vesicles were in contact with the proximal dendrites and the cell bodies of pyramidal and giant neurons. The origin of these terminals is discussed. No other cells of the dorsal cochlear nucleus, in particular cartwheel and tuberculo-ventral neurons, were in contact with labelled boutons. The present results suggest that descending inhibitory collicular projections are essentially directed to the large excitatory neurons of the dorsal cochlear nucleus.

Animals↗

Accessing ampli-tonotopic organization of rat auditory cortex by microstimulation of cochlear nucleus.

Auditory brainstem implants (ABI) that electrically stimulate the surface of cochlear nucleus have been clinically used for the rehabilitation of deaf patients with bilateral vestibular schwannomas. The change of pitch perception with an active electrode location is not as clear in ABIs as in cochlear implants, a factor which might play a role in poorer speech performance in ABIs. The objective of present work was to develop an animal ABI model that could provide physiological data for future ABI development and optimization. The experimental system included a penetrating microelectrode array for microstimulation of the cochlear nucleus and a surface microelectrode array for mapping evoked potentials over the auditory cortex. We first obtained tone-evoked cortical activation patterns, which represented a place code of the frequency and intensity of test tones, i.e., the ampli-tonotopic organization, and compared the patterns with those evoked by cochlear nuclear microstimulation. Our experimental results demonstrated that microstimulation of both the dorsal and ventral cochlear nucleus (DCN and VCN) could access the cortical ampli-tonotopic organization as acoustic stimuli did. We also found that the cortical dynamic range was wider for the DCN than VCN stimulation and for the low-frequency than for the high-frequency pathway. The present results have great implications for improved ABI performance.

Action Potentials↗