Voltage-gated Ca2+ conductances in acutely isolated guinea pig dorsal cochlear nucleus neurons

Although it is known that voltage-gated Ca2+ conductances (VGCCs) contribute to the responses of dorsal cochlear nucleus (DCN) neurons, little is known about the properties of VGCCs in the DCN. In this study, the whole cell voltage-clamp technique was used to examine the pharmacology and voltage dependence of VGCCs in unidentified DCN neurons acutely isolated from guinea pig brain stem. The majority of cells responded to depolarization with sustained inward currents that were enhanced when Ca2+ was replaced by Ba2+, were blocked partially by Ni2+ (100 microM), and were blocked almost completely by Cd2+ (50 microM). Experiments using nifedipine (10 microM), omegaAga IVA (100 nM) and omegaCTX GVIA (500 nM) demonstrated that a variety of VGCC subtypes contributed to the Ba2+ current in most cells, including the L, N, and P/Q types and antagonist-insensitive R type. Although a large depolarization from rest was required to activate VGCCs in DCN neurons, VGCC activation was rapid at depolarized levels, having time constants <1 ms at 22 degrees C. No fast low-threshold inactivation was observed, and a slow high-threshold inactivation was observed at voltages more positive than -20 mV, indicating that Ba2+ currents were carried by high-voltage activated VGCCs. The VGCC subtypes contributing to the overall Ba2+ current had similar voltage-dependent properties, with the exception of the antagonist-insensitive R-type component, which had a slower activation and a more pronounced inactivation than the other components. These data suggest that a variety of VGCCs is present in DCN neurons, and these conductances generate a rapid Ca2+ influx in response to depolarizing stimuli.     

Responses to parallel fiber stimulation in the guinea pig dorsal cochlear nucleus in vitro

1. Parallel fibers of the guinea pig dorsal cochlear nucleus (DCN) were electrically stimulated at the pial surface of the nucleus in a brain-slice preparation. Extracellular field potentials produced by the parallel fibers and postsynaptic cells, and the response of single units were identified and characterized. Responses were compared with those reported for stimulation of parallel fibers in the cerebellum and to those seen with electrical stimulation of the auditory nerve. 2. Stimulation of the DCN parallel fibers generates a consistent set of extracellular field potentials. In layer 1 of the DCN, a short-latency triphasic wave (P1(1)-N1(1)-P2(1)) is followed by a slower negative wave (N2(1)). The onset phase of the N2(1) often exhibits a small positive notch (P2a1). In layer 2, an initial triphasic wave (P1(2)-N1(2)-P2(2)) is followed by a short-latency negative wave (N2(2)) and a slower positive wave (P3(2)). The N1(2) is approximately coincident with the N1(1), whereas the P3(2) is coincident with N2(1). The falling phase of the P3(2) is sometimes interrupted by a brief negative deflection (N3(2)). These field potentials are similar, but not identical to those reported for parallel fiber stimulation in the cerebellum in vivo (15). These responses differ substantially from those produced in the DCN by electrical stimulation of the auditory nerve (50). 3. Low-calcium solutions and pharmacologic manipulations were used to separate pre- and postsynaptic response components in the field potential records. When the slice is bathed in a low-calcium solution the P2a1, N2(1), N2(2), P3(2), and the brief late deflections are abolished. However, the P1(1)-N1(1)-P2(1) and P1(2)-N1(2)-P2(2) remain unaffected. A similar separation of pre- and postsynaptic components can be achieved with 100 microM adenosine or 0.5 mM kynurenic acid. It is concluded that the P1(1)-M1(1)-P2(1) wave is the compound action potential of the unmyelinated parallel fibers, whereas the longer-latency field potential components are generated postsynaptically. 4. The conduction velocity of the parallel fiber volley was measured to be 0.30 m/s at the pial surface, in a line approximately parallel to the strial axis of the nucleus. Mapping experiments reveal that the spread of the P1(1)-N1(1)-P2(1) is greatest along the strial axis, and more limited in the orthogonal direction. 5. Single units were recorded in layer 2. At a distance of 500-700 microns from the stimulating electrode, the latencies of single-unit discharges fall between 2.5 and 4 ms, at the time of the N2(2).(ABSTRACT TRUNCATED AT 400 WORDS)     

Electrically evoked responses in onset chopper neurons in guinea pig cochlear nucleus

Extracellular recordings were obtained from single cochlear nucleus neurons in guinea pigs anesthetized with Nembutal and Hypnorm. Neurons were classified by their spontaneous firing rates and responses to acoustic stimuli. In addition, electrical shocks were applied to the midline at the level of the IVth ventricle and spike responses were recorded. Spikes were evoked by shocks only in neurons that were classified as onset choppers (O(c)). The shock-evoked spikes could be extinguished by acoustically evoked action potentials in the same neurons. In roughly 30% of the sample of O(c) neurons, quantitative aspects of the timing of this extinction were not compatible with the shock-evoked spike being antidromically conducted from O(c) output axons. Together with the presence of temporal jitter at high shock rates, the data suggest the possibility that at least some of the shock-evoked spikes may be generated by excitatory synaptic input to the O(c) neurons, most likely from the collaterals of the medial olivocochlear system (MOCS), whose axons pass close to the floor of the IVth ventricle. This excitatory synaptic input may operate to modulate the activity of O(c) neurons in addition to MOCS actions in the auditory periphery.     

Distribution and targets of the cartwheel cell axon in the dorsal cochlear nucleus of the guinea pig

Summary This investigation attempted to determine the mode of distribution and synaptic targets of the cartwheel cell axon in the guinea pig dorsal cochlear nucleus (DCoN). Antiserum against PEP-19, a putative calcium-binding neuropeptide, was employed at the light and electron microscopic levels. We show that in the hindbrain of the guinea pig, cerebellar Purkinje cells and DCoN cartwheel cells are the most densely immunoreactive neurons. The PEP-19 immunoreaction product is localized to all neuronal compartments of these cells. Primary targets of cartwheel cell axons are the DCoN pyramidal cells, the large efferent neurons of layer 2. These neurons receive numerous immunoreactive synaptic boutons on their cell bodies and apical and basal dendritic arbors. A PEP-19-immunoreactive axonal plexus, largely formed by cartwheel cell axons, highlights layer 3, co-extensively with the basal arbors of pyramidal cells. This plexus is oriented predominantly in the transstrial plane of the DCoN, in parallel with the sheetlike basal dendritic arbor of pyramidal neurons and with the isofrequency bands of primary cochlear nerve fibers. PEP-19-positive boutons contain pleomorphic synaptic vesicles and form symmetric synaptic junctions, indicative of inhibitory innervation. In addition, immunoreactive boutons, similar to those synapsing on pyramidal neurons, were observed on the cell bodies and main dendritic trunks of cartwheel neurons, indicating a system of recurrent collaterals. Furthermore, a small number of PEP-19-positive axons of unknown origin reach the caudal rim of the posteroventral cochlear nucleus. Within the territory of distribution of the cartwheel cell axon are the dendrites of at least two other types of DCoN neuron, the vertical cells of Lorente de Nó and the giant cells. These neurons may represent additional targets of the cartwheel cell axon, but this remains to be ascertained with specific methods. Our data demonstrate that the cartwheel neurons modulate the activity of pyramidal neurons and, therefore, play a key role in shaping the output of the DCoN superficial layers.     

Dendrodendritic connections between the cochlear efferent neurons in guinea pig

The outer hair cells of organ of Corti are innervated by the efferent neurons of medial olivocochlear neurons (MOC) of the brainstem which modify the cochlear auditory processing and sensitivity. Most of the MOC neurons are excited by a dominant ear and only a small portion of them is excited by both ears resulting in a binaural facilitation. The functional role of the feedback system between the organ of Corti and the cochlear efferent neurons is the protection of the ear from acoustic injury. The rapid impulse propagation in the bilateral olivocochlear system is suggestive of an electrotonic interaction between the bilateral olivocochlear neurons. The morphological background of the MOC pathway is not yet completely characterized. Therefore, we have labeled the bilateral cochlear nerves with different neuronal tracers in guinea pigs. In the anesthetized animals the cochlear nerves were exposed in the basal part of the modiolus and labeled simultaneously with different retrograde fluorescent tracers. By using confocal laser scanning microscope we could detect close appositions between the dendrites of the neurons of bilateral MOC. The distance between the neighboring profiles suggested close membrane appositions without interposing glial elements. These connections might serve as one of the underlying mechanisms of the binaural facilitation mediated by the olivocochlear system.     

Physiological studies on neurons in the dorsal cochlear nucleus of cat

Results reported here support the conclusion that an individual neuron in the dorsal cochlear nucleus (DCN) can exhibit pauser, buildup, and chopper patterns in response to tone pips. Fusiform cells have been previously identified as the principal cell exhibiting these patterns. Fusiform cells can also exhibit an onset response followed by suppression of spontaneous activity at their characteristic frequency (CF). Off CF only suppression is seen. These neurons are characterized by a restricted excitatory region near threshold. All these cells can exhibit nonmonotonic rate curves, narrow excitatory regions, and inhibitory sidebands. Nonmonotonicity occurred in 34% of pausers, 52% of buildup, 89% of onsets with a graded response, and 50% overall in the DCN cells. Chopper units occur as often as the other types combined in the DCN. Only 14% show nonmonotonic rate curves. Those with high-spontaneous activity also show inhibitory sidebands. Cells with a predominant buildup pattern occur most frequently in the fusiform cell layer, whereas pausers occur throughout the DCN below the molecular layer. Intracellular potentials often reflect the average response pattern. Sharply delimited response areas indicate that these cells may be useful for performing a spectral analysis. These cells show almost no phase locking suggesting that temporal encoding is an unlikely function. It is suggested that the effects of anesthetic on the function of the DCN is not as marked as previously indicated.     

Cytology, synaptology and immunocytochemistry of commissural neurons and their putative axonal terminals in the dorsal cochlear nucleus of the rat

The first binaural integration within the auditory system responsible for sound localization depends upon commissural neurons that connect the two symmetrical cochlear nuclei. These cells in the deep polymorphic layer of the rat dorsal cochlear nucleus were identified with the electron microscope after injection of the retrograde tracer, Wheat Germ Agglutinin conjugated to Horseradish Peroxydase, into the contralateral cochlear nucleus. Commissural neurons are multipolar or bipolar with an oval to fusiform shape. Few commissural neurons, most inhibitory but also excitatory, connect most of the divisions of the rat cochlear nuclei. The most common type is a glycinergic, sometimes GABAergic, moderately large cell. Its ergastoplasm is organized into peripheral stacks of cisternae, and few axo-somatic synaptic boutons are present. Another type of commissural neuron is a medium-sized, spindle-shaped cell, glycine and GABA-negative, with sparse ergastoplasm and synaptic coverage. A giant, rare type of commissural neuron is glycine-positive and GABA-negative, with short peripheral stacks of ergastoplasmic cisternae. It is covered with synaptic boutons, many of which contain round synaptic vesicles. Another rare type of commissural neuron is a moderately large cell, oval to fusiform in shape, immunonegative for both glycine and GABA, and contacted by many axo-somatic boutons. It contains large dense mitochondria and numerous dense core vesicles of peptidergic type. Some labelled boutons, mostly inhibitory and probably derived from commissural neurons, contact pyramidal, cartwheel, giant and tuberculo-ventral neurons. The prevalent inhibition of electrical activity in a cochlear nucleus observed after stimulation of the contralateral cochlear nucleus may be due to commissural inhibitory terminals which contact excitatory neurons such as pyramidal and giant cells. Other inhibitory commissural terminals which contact inhibitory neurons such as cartwheel and tuberculo-ventral neurons, may explain the stimulation of electrical activity in the DCN after contralateral stimulation.     

Receptive field for dorsal cochlear nucleus neurons at multiple sound levels

Neurons in the dorsal cochlear nucleus (DCN) exhibit nonlinearities in spectral processing, which make it difficult to predict the neurons' responses to stimuli. Here, we consider two possible sources of nonlinearity: nonmonotonic responses as sound level increases due to inhibition and interactions between frequency components. A spectral weighting function model of rate responses is used; the model approximates the neuron's rate response as a weighted sum of the frequency components of the stimulus plus a second-order sum that captures interactions between frequencies. Such models approximate DCN neurons well at low spectral contrast, i.e., when the SD (contrast) of the stimulus spectrum is limited to 3 dB. This model is compared with a first-order sum with weights that are explicit functions of sound level, so that the low-contrast model is extended to spectral contrasts of 12 dB, the range of natural stimuli. The sound-level-dependent weights improve prediction performance at large spectral contrast. However, the interactions between frequencies, represented as second-order terms, are more important at low spectral contrast. The level-dependent model is shown to predict previously described patterns of responses to spectral edges, showing that small changes in the inhibitory components of the receptive field can produce large changes in the responses of the neuron to features of natural stimuli. These results provide an effective way of characterizing nonlinear auditory neurons incorporating stimulus-dependent sensitivity changes. Such models could be used for neurons in other sensory systems that show similar effects.     

Age-related changes in glycine receptor subunit composition and binding in dorsal cochlear nucleus

Age-related hearing loss, presbycusis, can be thought of, in part, as a slow progressive peripheral deafferentation. Previous studies suggest that certain deficits seen in presbycusis may partially result from functional loss of the inhibitory neurotransmitter glycine in dorsal cochlear nucleus (DCN). The present study assessed age-related behavioral gap detection changes and neurochemical changes of postsynaptic glycine receptor (GlyRs) subunits and their anchoring protein gephyrin in fusiform cells of young (7–11 months) and aged (28–33 months) Fischer brown Norway (FBN) rats. Aged rats showed significantly (20–30 dB) elevated auditory brainstem-evoked response thresholds across all tested frequencies and worse gap detection ability compared to young FBN rats. In situ hybridization and quantitative immunocytochemistry were used to measure GlyR subunit message and protein levels. There were significant age-related increases in the α₁ subunit message with significant age-related decreases in α₁ subunit protein. Gephyrin message and protein showed significant increases in aged DCN fusiform cells. The pharmacologic consequences of these age-related subunit changes were assessed using [³H] strychnine binding. In support of the age-related decrease of α₁ subunit protein levels in DCN, there was a significant age-related decrease in the total number of GlyR binding sites with no significant change in affinity. These age-related changes may reflect an effort to reestablish a homeostatic balance between excitation and inhibition impacting on DCN fusiform cells by downregulation of inhibitory function in the face of an age-related loss of peripheral input. Age-related decrease in presynaptic glycine release results in altered subunit composition and this may correlate with loss of temporal coding of the aged fusiform cell in DCN. The previously reported role for gephyrin in retrograde intracellular receptor subunit trafficking could contribute to the α₁ decrease in the face of increased message.     

Fine structure and neurotransmitter cytochemistry of neurons in the rat ventral cochlear nucleus projecting to the ipsilateral dorsal cochlear nucleus

The neural tracer wheat germ agglutinin conjugated to horse radish peroxidase was injected into the rat dorsal cochlear nucleus and acoustic stria. Some labelled neurons in the ipsilateral ventral cochlear nucleus were found as a result. These neurons were studied at the ultrastructural level, and their axo-somatic synaptic profile and glycine immunoreactivity were determined. Most neurons were glycine negative and classified as type I multipolar neurons. The latter showed a different synaptic profile from that of neurons projecting to the contralateral inferior colliculus or cochlear nucleus. This suggests the presence of differing populations of multipolar cells based on their synaptic profile. Few labelled multipolar neurons of type II were found, which appeared glycine negative and, rarely, glycine positive. The latter show an ultrastructure and axo-somatic profile similar to that of glycinergic commissural neurons in the dorsal and ventral cochlear nucleus. In particular, about one-third of boutons contained round synaptic vesicles, which are believed to contain an excitatory neurotransmitter. The ultrastructural analysis of the synaptic boutons in the cochlear nucleus confirms the presence of numerous cases of colocalization of glycine and GABA where flat and pleomorphic synaptic vesicles are mixed. The present study is in accordance with previous tract-tracing light microscopic studies which have indicated that large glycinergic neurons in the ventral cochlear nucleus act as broad-band inhibitory neurons in microcircuits of the dorsal cochlear nucleus and contralateral cochlear nucleus.     

Functional organization of the dorsal cochlear nucleus of the horseshoe bat (Rhinolophus rouxi) studied by GABA and glycine immunocytochemistry and electron microscopy

Unique among mammals, the dorsal cochlear nucleus (DCN) of horseshoe bats consists of two functionally and anatomically distinct subdivisions: a laminated ventral portion that processes the frequency range below the constant frequency (CF) component of the echolocation signal and a nonlaminated dorsal portion that is specialized for processing the CF-signal range (76 kHz and higher). Using conventional transmission electron microscopy and postembedding immunocytochemistry for the inhibitory neurotransmitters GABA and glycine on semithin-alternating sections, we present further evidence that the ventral laminated subdivision of DCN conserves the main elements of microcircuitry and GABA/glycine labeling patterns typical for the mammalian DCN: (i) the main cell types and synaptic inventory of the granule cell/cartwheel cell system of the superficial layers are present as well as (ii) the tuberculoventral cell system of the deep layers. The nonlaminated dorsal subdivision lacks the granule cell/cartwheel cell system and is composed of a mixture of fusiform projection neurons with tuberculoventral cell analogues. Thus the inhibitory tuberculoventral system known to play an important role in temporal and spectral processing in VCN is conserved throughout the DCN of horseshoe bats, whereas functional components of cerebellar-like circuits are reduced in a specialized region that processes the dominant biosonar component. Accepted: 12 January 2001     

Heterogeneous biophysical properties of frog dorsal medullary nucleus (cochlear nucleus) neurons

The cochlear nucleus (CN) in mammals, or its counterpart in birds, has multiple subdivisions each containing distinct morphological and functional (i.e., temporal discharge patterns and biophysical properties) cell types that project to different auditory nuclei in the brain stem in parallel. The analogous structure in frogs, the dorsal medullary nucleus (DMN), is a single phylogenetically older structure with no subdivision. Similar to the CN, the DMN has complex cytoarchitecture and contains neurons with diverse morphological phenotypes, but whether these cell types possess distinct biophysical characteristics, like their counterparts in mammals and avians, is unclear. Here we show that DMN neurons in young adult northern leopard frogs (Rana pipiens pipiens) possess heterogeneous biophysical properties. There are four major biophysical phenotypes on the basis of the unit's response (i.e., its temporal firing pattern) to depolarizing currents: onset, phasic-burst, sustained-chopper, and adapting. These cells have distinct membrane input resistances and time constants, spike shapes, current-voltage relationships, first-spike latencies, entrainment characteristics, and ionic compositions (i.e., low-threshold potassium current, I(kl), and hyperpolarization-activated current, I(h)). Furthermore, these phenotypes correspond to cells' dendritic morphologies, and they bear similarities and differences to those found in the mammalian CN. The similarities are remarkable considering that amphibians are a distinct evolutionary lineage from birds and mammals.     

Response of guinea pig vestibular nucleus neurons to clicks

Responses of single neurons in the vestibular nuclei to clicks were studied by extracellular recording in anaesthetised guinea pigs. Eighty-four neurons in the ipsilateral vestibular nuclei were activated with an average latency of 1.75±0.30 ms, which is about 0.9 ms longer than the mean latency of activation of click-sensitive vestibular afferents to intense clicks. The threshold of clicks for evoking the response of these neurons was around 70 dB above the auditory brainstem response threshold. Earlier studies have indicated that click-sensitive vestibular afferents are tilt-sensitive and likely to originate from saccular receptors, and in the present study nine of the click-sensitive vestibular nucleus neurons were tilt-sensitive, suggesting that these central neurons receive monosynaptic input from the corresponding saccular afferents. Recording sites were marked by means of iontophoretic injection of FCF green dye; they were located in the lateral portion of the descending vestibular nucleus and the caudal and ventral regions of the lateral vestibular nucleus.     

Response properties of neurons in the central nucleus and external and dorsal cortices of the inferior colliculus in guinea pig

The inferior colliculus (IC) represents a mid-brain structure which integrates information from many ascending auditory pathways, descending corticotectal projections and intercollicular pathways. The processing of information is different in each of the three main subdivisions of the IC--the central nucleus (CNIC), the dorsal cortex (DCIC) and the external cortex (ECIC)--which may be distinguished morphologically as well as by different inputs and outputs. To assess the differences in information processing we compared the response properties of single neurons in individual subnuclei of the IC in anesthetized guinea pigs. In comparison with DCIC and ECIC neurons, the CNIC neurons as a group were characterized by a sharper frequency tuning (as expressed by Q10 values), a lower average threshold, a shorter average first-spike latency of response to tones at the characteristic frequency (CF), a higher occurrence of non-monotonic rate/level functions and a higher rate of spontaneous activity. CNIC neurons and DCIC neurons reacted to tones at the CF more frequently by a sustained type of response than did ECIC neurons. The difference between the parameters of DCIC neuronal activity and ECIC neuronal activity was found to be smaller. The frequency tuning (expressed in Q10 values), spontaneous activity and dominance of monotonic rate/level functions were very similar in both structures; ECIC neurons expressed a higher average threshold and a shorter average first-spike latency than did DCIC neurons. Responsiveness expressed as the average maximal firing rate to tones at the CF was significantly higher in the CNIC than in the ECIC. The results give additional support to the idea that the CNIC is a part of a fast, frequency-tuned, low threshold and intensity-sensitive ascending pathway, whereas the other two subdivisions are involved in additional processing of information that involves feedback loops and polysensory pathways.     

Functional organization of the dorsal cochlear nucleus of the horseshoe bat (Rhinolophus rouxi) studied by GABA and glycine immunocytochemistry and electron microscopy.

Unique among mammals, the dorsal cochlear nucleus (DCN) of horseshoe bats consists of two functionally and anatomically distinct subdivisions: a laminated ventral portion that processes the frequency range below the constant frequency (CF) component of the echolocation signal and a nonlaminated dorsal portion that is specialized for processing the CF-signal range (76 kHz and higher). Using conventional transmission electron microscopy and postembedding immunocytochemistry for the inhibitory neurotransmitters GABA and glycine on semithin-alternating sections, we present further evidence that the ventral laminated subdivision of DCN conserves the main elements of microcircuitry and GABA/glycine labeling patterns typical for the mammalian DCN: (i) the main cell types and synaptic inventory of the granule cell/cartwheel cell system of the superficial layers are present as well as (ii) the tuberculoventral cell system of the deep layers. The nonlaminated dorsal subdivision lacks the granule cell/cartwheel cell system and is composed of a mixture of fusiform projection neurons with tuberculoventral cell analogues. Thus the inhibitory tuberculoventral system known to play an important role in temporal and spectral processing in VCN is conserved throughout the DCN of horseshoe bats, whereas functional components of cerebellar-like circuits are reduced in a specialized region that processes the dominant biosonar component.     

Projection of primary vestibular afferent fibres to the cochlear nucleus in the guinea pig

After tracing the superior branch of the vestibular nerve and the macula sacculi by means of the neuronal tracers horseradish peroxidase (HRP) and wheat germ conjugated horseradish peroxidase (WGA-HRP), a conspicuous fibre bundle running into the cochlear nucleus could be observed. The HRP-labeled axons travel caudally through the descending vestibular nucleus, enter the cochlear nucleus at a level caudal to subgroup y and terminate at cells situated between the dorsal and posteroventral cochlear nucleus. Considering recent electrophysiological studies, it is reasonable to imply that these fibres are involved with the transduction of acoustic stimuli.