Bushy Cells Research Articles

Reciprocal developmental regulation of presynaptic ionotropic receptors.

Activation of ionotropic glycine receptors potentiates glutamate release in mature calyceal nerve terminals of the rat medial nucleus of the trapezoid body, an auditory brainstem nucleus. In young rats, glycine and its receptors are poorly expressed. We therefore asked whether GABA (gamma-aminobutyric acid) might play a larger role than glycine in the regulation of glutamate release in the absence of glycine receptors. Indeed, in rats younger than postnatal day 11 (P11), and before the onset of hearing, calyces expressed high levels of ionotropic GABA(A) receptors but few glycine receptors. Isoguvacine, a selective agonist at GABA(A) receptors, strongly enhanced excitatory postsynaptic currents in young rats but had little effect in rats older than P11. Down-regulation of presynaptic GABA(A) receptors did not reflect global changes in receptor expression, because the magnitude of GABA and glycine responses was similar at P13 in the parent-cell bodies of the calyces, the bushy cells of the cochlear nucleus. In outside-out patches excised from the nonsynaptic face of calyces, GABA and glycine evoked single-channel currents consistent with the properties of postsynaptic GABA(A) and glycine receptors. Inhibitory GABA(B) receptors were present on the calyx at all developmental stages examined. Thus, GABA initially acts on two receptor subtypes, both promoting and inhibiting glutamate release. With age, the former role is transferred to the glycine receptor during the period in which postsynaptic glycinergic transmission is acquired.

Temporal coding of the pitch of complex sounds by presumed multipolar cells in the ventral cochlear nucleus

Extensive studies of the encoding of fundamental frequency ( f 0) in the auditory nerve indicate that f 0 can be represented by either the timing of the neuronal discharges or the mean discharge rate as a function of characteristic frequency. It is therefore of considerable interest to examine what happens to this information at the next level of the auditory pathway, the cochlear nucleus. Both physiologically and anatomically the cochlear nucleus is considerably more heterogenous than the auditory nerve. There are two main cell types in the ventral division of the cochlear nucleus; bushy and multipolar. Bushy cells give rise to primary-like responses whereas multipolar cells may be characterised by either onset or chopper type responses. Physiological studies have suggested that onset and chopper units may be good at representing the f 0 of complex sounds in their temporal discharge properties. However, in these studies the pitch-producing sounds were usually characterised by highly modulated envelopes and it was not possible to tell if the units were simply responding to the modulation or the temporal fine structure. In this paper we examine the ability of onset and chopper units to encode the f 0 of complex sounds when the modulation cue has been greatly reduced. These stimuli were steady-state vowels in the presence of background noise, and iterated rippled noise (IRN). The response of onset units to the vowel f 0 in the presence of background noise was varied but many still maintained a strong response. In contrast, the majority of chopper units showed a greater reduction in their response to vowel f 0 in the presence of background noise. In keeping with the vowel study, the responses of both types of unit to the delay of the IRN was reduced in comparison with their response to more highly modulated stimuli. Increasing anatomical, pharmacological and physiological evidence would seem to argue against onset units playing a direct role in pitch perception. However, some units, identified as sustained choppers, may be able to represent the pitch of complex sounds in their temporal discharges.

Ultrastructural basis of synaptic transmission between endbulbs of Held and bushy cells in the rat cochlear nucleus.

Auditory nerve fibres make large excitatory synaptic contacts, the endbulbs of Held, with bushy cells in the anteroventral cochlear nucleus (AVCN). We have used serial-section electron microscopy to reconstruct seven endbulbs of Held in contact with three different AVCN bushy cells from a 25-day-old rat, as a basis for interpreting our previous physiological results at this connection. Four endbulbs of Held contacting the same bushy cell were completely reconstructed. The number of separate synaptic specializations within these endbulbs varied from 85 to 217, with a mean of 155. Detailed measurements were obtained from high magnification segments of four endbulbs contacting three different bushy cells. Large variability was found in the size of synaptic specializations within an individual endbulb. The size of postsynaptic densities (PSDs) varied between endbulbs (mean PSD area 0.03, 0.07, 0.07 and 0.18 microm(2); n = 4 endbulbs). The number of morphologically docked vesicles at individual specializations within the same endbulb varied considerably (between 1 and 102). The mean number of morphologically docked vesicles per specialization differed between endbulbs (mean numbers of docked vesicles per specialization = 2.1, 3.7, 5.3, 14.8; n = 4 endbulbs). Despite these large differences, the density of docked vesicles per square micron of PSD was similar between endbulbs (54, 80, 81, 83 docked vesicles per microm(2); n = 4 endbulbs). Within an endbulb, a linear relationship was found between the number of docked vesicles and PSD area, and between PSD area and the number of undocked vesicles within 150 nm of the active zone. The ratio of undocked vesicles (< 150 nm) to docked vesicles ranged from 2 to 5 in different endbulbs (n = 4 endbulbs). These structural observations are discussed in relation to the functional properties of synaptic transmission between endbulbs of Held and bushy cells in the AVCN.

Summation of spatiotemporal input patterns in leaky integrate-and-fire neurons: application to neurons in the cochlear nucleus receiving converging auditory nerve fiber input.

The response of leaky integrate-and-fire neurons is analyzed for periodic inputs whose phases vary with their spatial location. The model gives the relationship between the spatial summation distance and the degree of phase locking of the output spikes (i.e., locking to the periodic stochastic inputs, measured by the synchronization index). The synaptic inputs are modeled as an inhomogeneous Poisson process, and the analysis is carried out in the Gaussian approximation. The model has been applied to globular bushy cells of the cochlear nucleus, which receive converging inputs from auditory nerve fibers that originate at neighboring sites in the cochlea. The model elucidates the roles played by spatial summation and coincidence detection, showing how synchronization decreases with an increase in both frequency and spatial spread of inputs. It also shows under what conditions an enhancement of synchronization of the output relative to the input takes place.

Maturation of Synaptic Transmission at End-Bulb Synapses of the Cochlear Nucleus

Neurons of the avian nucleus magnocellularis transmit phase-locked action potentials of the auditory nerve in a pathway that contributes to sound localization based on interaural timing differences. We studied developmental changes in synaptic transmission that enable the end-bulb synapse to function as a synaptic relay. In chick, although the auditory system begins to function early in embryonic development, maturation of audition around the time of hatching suggested that synaptic transmission in the cochlear nucleus of young chicks may undergo further developmental changes. Synaptic physiology was investigated via patch-clamp recordings from bushy cells in brainstem slices during stimulation of auditory nerve fibers at 35 degrees C. Compared with embryonic synapses (embryonic day 18), post-hatch chicks (post-hatch days 1-11) exhibited high probability of firing a well timed postsynaptic action potential during high-frequency stimulation of the auditory nerve. Improvements in reliability and timing of postsynaptic spikes were accompanied by a developmental increase in steady-state EPSCs during stimulus trains and a decline in the extent of synaptic depression. Synchrony of EPSCs during stimulus trains improved with age. An increased pool of synaptic vesicles, lower release probability, larger and faster transmitter quanta, and reduced AMPA receptor desensitization contributed to these changes. Together, these factors improve the ability of cochlear nucleus magnocellularis neurons to faithfully transmit timing information encoded by the auditory nerve.

Temporal processing from the auditory nerve to the medial nucleus of the trapezoid body in the rat

This investigation examines temporal processing through successive sites in the rat auditory pathway: auditory nerve (AN), anteroventral cochlear nucleus (AVCN) and the medial nucleus of the trapezoid body (MNTB). The degree of phase-locking, measured as vector strength, varied with intensity relative to the cell’s threshold, and saturated at a value that depended upon stimulus frequency. A typical pattern showed decline in the saturated vector strength from approximately 0.8 at 400 Hz to about 0.3 at 2000 Hz, with similar profiles in units with a range of characteristic frequencies (480–32 000 Hz). A new expression for temporal dispersion indicates that this variation corresponds to a limiting degree of temporal imprecision, which is relatively consistent between different cells. From AN to AVCN, an increase in vector strength was seen for frequencies below 1000 Hz. At higher frequencies, a decrease in vector strength was observed. From AVCN to MNTB a tendency for temporal coding to be improved below 800 Hz and degraded further above 1500 Hz was seen. This change in temporal processing ability could be attributed to units classified as primary-like with notch (PL N). PL N MNTB units showed a similar vector strength distribution to PL N AVCN units. Our results suggest that AVCN PL N units, representing globular bushy cells, are specialised for enhancing the temporal code at low frequencies and relaying this information to principal cells of the MNTB.

Modulation of a presynaptic hyperpolarization-activated cationic current (I(h)) at an excitatory synaptic terminal in the rat auditory brainstem.

1. A hyperpolarization-activated non-specific cation current, I(h), was examined in bushy cell bodies and their giant presynaptic terminals (calyx of Held). Whole-cell patch clamp recordings were made using an in vitro brain slice preparation of the cochlear nucleus and the superior olivary complex. The aim was to characterise I(h) in identified cell bodies and synaptic terminals, to examine modulation by presynaptic cAMP and to test for modulatory effects of I(h) activation on synaptic transmission. 2. Presynaptic I(h) was activated by hyperpolarizing voltage-steps, with half-activation (V(1/2)) at -94 mV. Activation time constants were voltage dependent, showing an e-fold acceleration for hyperpolarizations of -32 mV (time constant of 78 ms at -130 mV). The reversal potential of I(h) was -29 mV. It was blocked by external perfusion of 1 mM CsCl but was unaffected by BaCl(2). 3. Application of internal cAMP shifted the activation curve to more positive potentials, giving a V(1/2) of -74 mV; hence around half of the current was activated at resting membrane potentials. This shift in half-activation was mimicked by external perfusion of a membrane-permeant analogue, 8-bromo-cAMP. 4. The bushy cell body I(h) showed similar properties to those of the synaptic terminal; V(1/2) was -94 mV and the reversal potential was -33 mV. Somatic I(h) was blocked by CsCl (1 mM) and was partially sensitive to BaCl(2). Somatic I(h) current density increased with postnatal age from 5 to 16 days old, suggesting that I(h) is functionally relevant during maturation of the auditory pathway. 5. The function of I(h) in regulating presynaptic excitability is subtle. I(h) had little influence on EPSC amplitude at the calyx of Held, but may be associated with propagation of the action potential at branch points. Presynaptic I(h) shares properties with both HCN1 and HCN2 recombinant channel subunits, in that it gates relatively rapidly and is modulated by internal cAMP.

Morphology and physiology of neurons in the ventral nucleus of the lateral lemniscus in rat brain slices.

The ventral nucleus of the lateral lemniscus (VNLL) is a prominent neuronal group that lies within the auditory pathway connecting the auditory lower brainstem and midbrain. Previous physiologic studies showed that VNLL neurons respond mainly to contralaterally presented sounds and display various firing patterns. To understand better the role that VNLL neurons play in transmitting and processing of auditory information, we examined the morphology of VNLL neurons and their cellular physiology in young rat brain slices. We made whole-cell patch-clamp recordings and labeled cells intracellularly with neurobiotin to investigate the relation between morphologic neuronal types, intrinsic membrane properties, and postsynaptic responses. VNLL neurons fell into two distinct morphologic groups, i.e., bushy cells and stellate cells, based on their dendritic patterns. Stellate cells were grouped further into stellate I, II, and elongate cells according to soma shape, dendritic branches, and orientation. Bushy cells showed an onset firing pattern and a nonlinear current-voltage relationship. All three subtypes of stellate cells had a linear current-voltage relationship, but exhibited different firing patterns. Stellate I cells showed regular and onset-pause firing patterns, whereas stellate II cells showed adapting and elongate cells showed burst firing patterns. Bushy cells and stellate cells responded to stimulation of the lateral lemniscus with excitatory and/or inhibitory synaptic potentials. These results suggest that the VNLL is a heterogeneous neuronal group and that it contains many channels for processing different kinds of auditory information. Neuronal morphology and intrinsic membrane properties contribute to the behavior of individual neurons.

Role of Biophysical Specialization in Cholinergic Modulation in Neurons of the Ventral Cochlear Nuclei

In contacting arrays of different types of neurons whose axons have differing targets in the brain stem, the auditory pathway is subdivided into parallel ascending pathways, each of which carries a different type of information. Several distinct arrays of neurons in the ventral cochlear nuclei have anatomical and biophysical specializations which enable them to extract differing facets of acoustic information and to convey it up the auditory pathway. T stellate cells have higher input resistances and have lower firing thresholds than bushy or octopus cells, enabling their firing to be modulated by small currents. Cholinergic currents, driven by neurons in the ventral nucleus of the trapezoid body that are likely to include medial olivocochlear efferents, excite T stellate cells, but have subtle effects on the firing of bushy cells, and have no detectable influence on octopus cells and D stellate cells. We suggest that cholinergic excitation of T stellate cells contributes toward shifting their acoustic dynamic ranges and increasing the encoding of spectral peaks in noisy conditions and in awake animals.

An immunogold investigation of the relationship between the amino acids GABA and glycine and their transporters in terminals in the guinea-pig anteroventral cochlear nucleus

The majority of terminals contacting spherical bushy cell bodies in the guinea-pig anteroventral cochlear nucleus contain GABA, glycine or both (colocalizing). Double labeling with antibodies to each amino acid and the plasma membrane transporter for the other was performed using different sizes of gold particles. The transporter for GABA occurs in the plasma membranes of some terminals containing glycine and vice versa suggesting that colocalizing terminals can retrieve both amino acids.

The effects of congenital deafness on auditory nerve synapses and globular bushy cells in cats

It is well known that auditory deprivation affects the structure and function of the central nervous system. Congenital deafness represents one form of deprivation, and in the adult white cat, it has been shown to have a clear effect upon the synaptic interface between endbulbs of Held and spherical bushy cells. It is not known, however, whether all primary synapses are affected and/or whether they are affected in the same way and to the same extent. Thus, we studied a second neuronal circuit in the deaf white cat involving modified (small) endbulbs and globular bushy cells. Compared to normal hearing cats, modified endbulbs of congenitally deaf cats were 52.2% smaller but unchanged in structural complexity. There was also a striking loss of extracellular space between ending and cell body. The somata of postsynaptic globular bushy cells were 13.4% smaller and had enlarged postsynaptic densities. These data reveal that axosomatic synapses demonstrate abnormal structure as a consequence of deafness and that the extent of the abnormalities can vary with respect to the circuits involved. The implication of these observations is that synaptic anomalies would introduce differential delays within separate circuits, thereby desynchronizing neural activity from sound stimuli. This loss of synchronization could in turn disrupt temporal processing and compromise a host of related functions, including language comprehension.

Diversity and plasticity in amino acid receptor subunits in the rat auditory brain stem

Glutamate, gamma aminobutyric acid (GABA) and glycine receptors have different properties depending on the specific subunit combination utilized. The subunit composition of amino acid receptors may help to shape the responses of neurons and can provide a diversity of response properties in different neuronal types and regions. This allows a synaptic fine tuning for an optimization of processing requirements and may also allow for changes in response to changes in input. This article reviews the diversity that has been found in the subunit composition of GABA, glycine, alpha amino-3-hydroxy-5-methyl-4 isoxazole propionic acid and N-Methyl, D-aspartate (NMDA) receptors in the mammalian auditory brain stem and provides new data on how the NMDAR1 glutamate receptor subunit changes as a consequence of deafness. In the latter study, quantitative in situ hybridization was used to assess NMDAR1 mRNA expression in six cell types of the rat cochlear nucleus. A unilateral cochlear ablation was performed and expression determined in the ipsilateral and contralateral cochlear nucleus 5 and 20 days later. Significantly decreased expression, compared to normal, was found 5 days following deafness, in ipsilateral spherical bushy cells, octopus cells and shell neurons, but not in fusiform cells, corn cells or granule cells. At 20 days the expression was not significantly different from normal in any of the six cell types.

Phosphorylation regulates spontaneous and evoked transmitter release at a giant terminal in the rat auditory brainstem.

1. The role of phosphorylation in synaptic transmission was investigated at a large glutamatergic terminal, the endbulb of Held, on bushy cells in the rat anteroventral cochlear nucleus (AVCN). 2. Whole-cell recordings of excitatory postsynaptic currents (EPSCs) were used to examine the effects of kinase inhibitors and activators on low-frequency (baseline) evoked release, spontaneous release, paired-pulse facilitation (PPF) or depression (PPD), repetitive stimuli and recovery from depression. 3. Application of the kinase inhibitor H7 (100 microM) reduced low-frequency evoked EPSC amplitude (by 15 %) and simultaneously increased PPF (or reduced PPD), with no significant change in other aspects of transmission. H7 did not affect the amplitude or frequency of spontaneous miniature EPSCs. 4. Phorbol esters increased EPSC amplitude (by 50 %) with a concomitant decrease in PPF (or increase in PPD), and reduced the final EPSC amplitude during repetitive stimuli. The effect of phorbol esters was due exclusively to protein kinase C (PKC) activation, as the specific PKC inhibitor bis-indolylmaleimide (Bis) completely blocked the potentiating effect of phorbol esters on EPSC amplitude. 5. Significantly, phorbol esters did not increase the evoked EPSC amplitude at connections in which release was maximized using high extracellular calcium concentrations (4-6 mM). 6. Phorbol esters increased the frequency of spontaneous miniature EPSCs in physiological calcium (by 275 %), and in high extracellular calcium (by 210 %) when phorbol esters did not increase the evoked EPSC amplitude. 7. Our results are most consistent with the actions of H7 to decrease low-frequency release probability and phorbol esters to increase low-frequency release probability at the endbulb-bushy cell synaptic connection in the AVCN. The effects of H7 and phorbol esters on paired-pulse responses and tetanic depression appear to be largely consequential to these changes in low-frequency release probability.

Identification of tuberculo-ventral neurons in the polymorphic layer of the rat dorsal cochlear nucleus.

The tuberculo-ventral tract represents a short nervous circuit within the auditory cochlear nuclei. Tuberculo-ventral neurons of the dorsal cochlear nucleus send isofrequency inhibitory inputs to bushy cells of the ventral cochlear nucleus. Injection of wheat germ agglutinin conjugated to horseradish peroxidase into the rat ventral cochlear nucleus, labelled tuberculo-ventral neurons retrogradely in the deep polymorphic layer of the ipsilateral dorsal cochlear nucleus. Five to 20% of the perimeter of these cells was covered by synaptic boutons, most of which contained flat and pleomorphic vesicles. These boutons contained glycine and sometimes GABA. Occasional small axo-somatic boutons contained round vesicles and were immunonegative for both glycine and GABA. This study shows that the synaptic profile of tuberculo-ventral neurons is different from that of other medium-size glycinergic neurons within the polymorphic layer or more superficial regions of the dorsal cochlear nucleus like cartwheel neurons. In fact the latter mostly receive boutons that contain pleomorphic vesicles.

Computational model for the bushy cell of the cochlear nucleus

A model of the cochlear nucleus bushy cell, adapted from Rothman, Young and Manis (J. Neurophysiol. 70 (1993) 2562–2583), was implemented using GENESIS. The model had one compartment that included Na+(GNa) and low- and high-threshold K+ channels (GKLT and GKHT). When GKLT was set to zero, the model cell's response to a current step changed from onset to regular sustained. When GKHT (representing Kv3.1) was reduced, the action potential became wider. The present model results support the view that the specialized functional properties of the bushy cell, i.e., fast membrane time constant, onset discharges and entrainment to high-rate pulse train, are produced by combined actions of the GKLT,GKHT and GNa channels, and that these channels adapted to optimize the bushy cell's precise temporal-encoding ability.

GABA mediates presynaptic inhibition at glycinergic synapses in a rat auditory brainstem nucleus.

Many inhibitory nerve terminals in the mammalian anteroventral cochlear nucleus (AVCN) contain both glycine and GABA, but the reason for the co-localization of these two inhibitory neurotransmitters in the AVCN is unknown. We have investigated the roles of glycine and GABA at synapses on bushy cells in the rat AVCN, using receptor immunohistochemistry and electrophysiology. Our immunohistochemical results show prominent punctate labelling of postsynaptic clusters of glycine receptors and of the receptor clustering protein gephyrin over the surface of bushy cells. In contrast, weak diffuse membrane immunolabelling of GABAA receptors was observed. Whole-cell recordings from bushy cells in AVCN slices demonstrated that evoked inhibitory postsynaptic currents (IPSCs) were predominantly (81 %) glycinergic, based on the decrease in amplitude of the IPSCs in bicuculline (10 microM). This observation was supported by the effect of strychnine (1 microM), which was to decrease the evoked IPSC (to 10 % of control IPSC amplitude) and to produce a greater than 90 % block of spontaneous miniature IPSCs. These results suggest a minor role for postsynaptic GABAA receptors in bushy cells, despite a high proportion of GABA-containing terminals on these cells. Therefore, a role for metabotropic GABAB receptors was investigated. Activation of GABAB receptors with baclofen revealed a significant attenuation of evoked glycinergic IPSCs. The effect of baclofen was presynaptic, as indicated by a lack of change in the mean amplitude of spontaneous IPSCs. Significantly, the decrease in the amplitude of evoked glycinergic IPSCs observed following repetitive nerve stimulation was reduced in the presence of the GABAB antagonist, CGP 35348. This indicates that synaptically released GABA can activate presynaptic GABAB receptors to reduce transmitter release at glycinergic synapses. Our results suggest specific pre- versus postsynaptic physiological roles for GABA and glycine in the AVCN.

Microglial and astrocytic reactions prior to onset of thalamic cell death after traumatic lesion of the rat sensorimotor cortex

The temporospatial relationship between microglial and astrocytic reactions and delayed thalamic cell death was examined 1-7 days following a traumatic cold lesion of the rat sensorimotor cortex using immunocytochemistry in combination with terminal deoxynucleotidyltransferase-mediated biotinylated dUTP nick end labeling (TUNEL) of nuclear DNA fragmentation. No or only occasional TUNEL-positive cells were found in the thalamic relay nuclei up to 3 days after trauma. After 7 days, on the other hand, a considerable number of TUNEL-positive cells were seen in the ventrobasal, the ventrolateral and posterior thalamic nuclei. Already 3 days after trauma, i.e., before cell injury was detectable, many protoplasmic astrocytes, which were reactive for glial fibrillary acidic protein, and ramified microglia, which were positive for complement receptor type 3b (CR3b) but negative for major histocompatibility complex (MHC) class II antigen, were noticed in the thalamus. The number of labeled astro- and microglia further increased after 7 days, when DNA fragmentation became evident. At this time, the morphology of microglia shifted towards bushy and rod-like cells, and microglia became also reactive for MHC class II antigen. Clusters of CR3b- and MHC class II-positive microglia were found in the ventrobasal thalamus. The present findings demonstrate that trauma-induced microglial and astrocytic reactions appear in the thalamus prior the onset of cell damage.

Contrasting Ca2+ channel subtypes at cell bodies and synaptic terminals of rat anterioventral cochlear bushy neurones

1. Whole-cell patch clamp recordings were made from bushy cells of the anterioventral cochlear nucleus (aVCN) and their synaptic terminals (calyx of Held) in the medial nucleus of the trapezoid body (MNTB). 2. Both high voltage-activated (HVA) and low voltage-activated (LVA) calcium currents were present in acutely dissociated aVCN neurones and in identified bushy neurones from a cochlear nucleus slice. 3. The transient LVA calcium current activated rapidly on depolarization (half-activation, -59 mV) and inactivated during maintained depolarization (half-inactivation, -89 mV). This T-type current was observed in somatic recordings but was absent from presynaptic terminals. 4. On the basis of their pharmacological sensitivity, P/Q-type Ca2+ channels accounted for only 6 % of the somatic HVA, while L-, N- and R-type Ca2+ channels each accounted for around one-third of the somatic calcium current. 5. The divalent permeabilities of these native calcium channels were compared. The Ba2+/Ca2+ conductance ratios of the somatic HVA and LVA channels were 1.4 and 0.7, respectively. The conductance ratio of the presynaptic HVA current was 0.9, significantly lower that that of the somatic HVA current. 6. We conclude that LVA currents are expressed in the bushy cell body, but are not localized to the excitatory synaptic terminal. All of the HVA current subtypes are expressed in bushy cells, but there is a strong polarity to their localization; P-type contribute little to somatic currents but predominate at the synaptic terminal; L-, N- and R-types dominate at the soma, but contribute negligibly to calcium currents in the terminal.

Ultrastructural and immunocytochemical characterization of neurons in the rat ventral cochlear nucleus projecting to the inferior colliculus

Neurons in the rat ventral cochlear nucleus which project to the inferior colliculus were identified after retrograde labelling of the neural tracer wheat germ agglutinin conjugated to horse radish peroxidase. After tracer injection into the Inferior Colliculus, electron microscopy and immunocytochemical localization of glycine, GABA and glutamate in retrograde labelled neurons were employed. In the acoustic root area and posterior ventral cochlear nucleus, most of the body surface of the neurons projecting to the inferior colliculus was 10-30% covered by axo-somatic boutons and appeared as multipolar cells of type I. These large to medium size cells with sparse stacks of ergastoplasmic cisternae organized in Nissl bodies, were heavily labelled and were the main projecting neurons to the inferior colliculus. Most of these cells were glycine and GABA negative but variably glutamate positive, suggesting that they are excitatory neurons. This result suggests the absence of an inhibitory innervation of the inferior colliculus from these cells. A few retrograde labelled, large to giant neurons showed an irregular surface, sparse short stacks of ergastoplasmic reticulum, numerous microtubules, cell bodies 60-80% covered by synaptic boutons, and they appeared as multipolar cells of type II. These cells were less strongly retrograde labelled than multipolar type I, and were occasionally glycine positive, presumably inhibitory. This suggests that at least a small caliber collateral axon stemming from these neurons can reach the inferior colliculus. Occasional glycine positive octopus cells not labelled with the tracer were also observed. The contribution of glycinergic axons to the innervation of the inferior colliculus appears therefore to by very limited. Occasional labelled cells were represented by apparently globular bushy neurons, but the weak labelling suggests that the tracer was taken up by collateral axons reaching the inferior colliculus and not by the main axon. Globular bushy cells were consistently negative for both glycine and GABA, and variably positive for glutamate. In the anteroventral cochlear nucleus, labelled multipolar type I and II showed similar immunocytological and ultrastructural characteristics to those in the posteroventral cochlear nucleus but their dimension was smaller. Cells identified as spherical bushy neurons were never retrograde labelled.

Ultrastructural and immunocytochemical characterization of commissural neurons in the ventral cochlear nucleus of the rat

Medium to large-giant multipolar neurons in the rat ventral cochlear nucleus were retrograde labelled after injection of the tracer Wheat Germ Agglutinin conjugated to Horse Radish Peroxidase into the contralateral cochlear nucleus. Light microscopy immunocytochemistry showed that 42.45% of these retrograde labelled neurons, generally strongly labelled with the tracer, were markedly glycine immunopositive, and that 57.55%, usually weakly retrograde labelled neurons, were immunonegative or weakly positive for glycine. These commissural neurons were generally GABA negative and variably immunopositive for glutamate. About 1/3rd of the commissural neurons had variably developed a rough endoplasmic reticulum whilst axo-somatic boutons covered 20-40% of the cell body. These cells were recognized as multipolar neurons of type I. Most of them were weakly glycine positive or even negative and a few appeared glycinergic. A little less than the remaining 2/3rds of the whole commissural population in the postero-ventral cochlear nucleus presented a surface which was 65-85% covered with synaptic boutons, among which some also appeared labelled. These cells were recognized as multipolar neurons of type II. Many microtubules and neurofilaments were present, free ribosomes being more numerous around Nissl bodies with short cisternae. A few low retrograde labelled type II were weakly or non glycinergic. A small number of large to giant neurons type II, strongly retrograde labelled, appeared to be glycine positive, consistently GABA negative and variably glutamate positive. A very small proportion of retrograde labelled neurons appeared having the characteristics of globular bushy neurons. Their weak labelling, however, suggests that they project by collaterals or thin axons to the contralateral cochlear nucleus. Spherical bushy cells in the rat anteroventral cochlear nucleus lack the nuclear capping of rough endoplasmic reticulum observed in the cat, and none was labelled after injection into the contralateral cochlear nucleus. Globular and spherical neurons were variably glutamate positive but glycine and GABA negative. In conclusion, the present study suggests that commissural neurons include a small number of strongly labelled large to giant glycinergic and presumably inhibitory type II and, less frequently type I. A large group of less heavily labelled commissural neurons of type I and II contain low levels or no glycine, which is probably used for metabolic purposes rather than as a neurotransmitter. This suggests that these neurons are presumably excitatory.