Effects of ketamine on response properties of neurons in the superior paraolivary nucleus of the mouse.

The superior paraolivary nucleus (SPON; alternative abbreviation: SPN for the same nucleus in certain species) is a prominent brainstem structure that provides strong inhibitory input to the auditory midbrain. Previous studies established that SPON neurons encode temporal sound features with high precision. These earlier characterizations of SPON responses were recorded under the influence of ketamine, a dissociative anesthetic agent and known antagonist of N-methyl-d-aspartate glutamate (NMDA) receptors. Because NMDA alters neural responses from the auditory brainstem, single unit extracellular recordings of SPON neurons were performed in the presence and absence of ketamine. In doing so, this study represents the first in vivo examination of the SPON of the mouse. Herein, independent data sets of SPON neurons are characterized that did or did not receive ketamine, as well as neurons that were recorded both prior to and following ketamine administration. In all conditions, SPON neurons exhibited contralaterally driven spikes triggered by the offset of pure tone stimuli. Ketamine lowered both evoked and spontaneous spiking, decreased the sharpness of frequency tuning, and increased auditory thresholds and first-spike latencies. In addition, ketamine limited the range of modulation frequencies to which neurons phase-locked to sinusoidally amplitude-modulated tones.

Connections of the superior paraolivary nucleus of the rat: projections to the inferior colliculus.

GABAergic neurotransmission contributes to shaping the response properties of inferior colliculus (IC) neurons. In rodents, the superior paraolivary nucleus (SPON) is a prominent and well-defined cell group of the superior olivary complex that sends significant but often neglected GABAergic projections to the IC. To investigate the trajectory, distribution and morphology of these projections, we injected the neuroanatomical tracer biotinylated dextran amine into the SPON of albino rats. Our results demonstrate that: (1) the SPON innervates densely all three subdivisions of the ipsilateral IC: central nucleus (CNIC), dorsal cortex (DCIC) and external cortex (ECIC). The SPON also sends a sparse projection to the contralateral DCIC via the commissure of the IC. (2) SPON axons are relatively thick (diameter >1.2 microm), ascend to the midbrain tectum in the medial aspect of the lateral lemniscus, and, for the most part, do not innervate the nuclei of the lateral lemniscus. (3) SPON fibers ramify profusely within the IC and bear abundant en passant and terminal boutons. (4) The axons of neurons in discrete regions of the SPON form two laminar terminal plexuses in the ipsilateral IC: a medial plexus that spans the CNIC and DCIC parallel to the known fibrodendritic laminae of the CNIC, and a lateral plexus located in the ECIC and oriented more or less parallel to the surface of the IC. (5) The projection from SPON to the ipsilateral IC is topographic: medial SPON neurons innervate the ventromedial region of the CNIC and DCIC and the ventrolateral region of the ECIC, whereas more laterally situated SPON neurons innervate more dorsolateral regions of the CNIC and DCIC and more dorsomedial regions of the ECIC. Thus, SPON fibers follow a pattern of distribution within the IC similar to that previously reported for intracollicular and corticocollicular projections.

Encoding of temporal features of auditory stimuli in the medial nucleus of the trapezoid body and superior paraolivary nucleus of the rat.

Neurons in the superior paraolivary nucleus (SPON) of the rat respond to the offset of pure tones with a brief burst of spikes. Medial nucleus of the trapezoid body (MNTB) neurons, which inhibit the SPON, produce a sustained pure tone response followed by an offset response characterized by a period of suppressed spontaneous activity. This MNTB offset response is duration dependent and critical to the formation of SPON offset spikes [Kadner A, Kulesza RJ Jr, Berrebi AS (2006) Neurons in the medial nucleus of the trapezoid body and superior paraolivary nucleus of the rat may play a role in sound duration coding. J Neurophysiol. 95:1499-1508; Kulesza RJ Jr, Kadner A, Berrebi AS (2007) Distinct roles for glycine and GABA in shaping the response properties of neurons in the superior paraolivary nucleus of the rat. J Neurophysiol 97:1610-1620]. Here we examine the temporal resolution of the rat's MNTB/SPON circuit by assessing its capability to i) detect gaps in tones, and ii) synchronize to sinusoidally amplitude modulated (SAM) tones. Gap detection was tested by presenting two identical pure tone markers interrupted by gaps ranging from 0 to 25 ms duration. SPON neurons responded to the offset of the leading marker even when the two markers were separated only by their ramps (i.e. a 0 ms gap); longer gap durations elicited progressively larger responses. MNTB neurons produced an offset response at gap durations of 2 ms or longer, with a subset of neurons responding to 0 ms gaps. SAM tone stimuli used the unit's characteristic frequency as a carrier, and modulation rates ranged from 40 to 1160 Hz. MNTB neurons synchronized to modulation rates up to approximately 1 kHz, whereas spiking of SPON neurons decreased sharply at modulation rates >or=400 Hz. Modulation transfer functions based on spike count were all-pass for MNTB neurons and low-pass for SPON neurons; the modulation transfer functions based on vector strength were low-pass for both nuclei, with a steeper cutoff for SPON neurons. Thus, the MNTB/SPON circuit encodes episodes of low stimulus energy, such as gaps in pure tones and troughs in amplitude modulated tones. The output of this circuit consists of brief SPON spiking episodes; their potential effects on the auditory midbrain and forebrain are discussed.

Characterization of an alternatively spliced AATYK mRNA: expression pattern of AATYK in the brain and neuronal cells.

The AATYK gene encodes a tyrosine kinase whose expression is up-regulated during the apoptosis and differentiation of 32Dcl3 myeloblastic cells. Because high levels of AATYK mRNA have also been detected in the brain, and because these transcripts differ in size from that observed in the 32Dcl3 cell line, it was of interest to determine whether this gene encodes mRNAs that are alternatively spliced and whether these mRNAs are expressed in a tissue-specific manner. We have isolated a novel, alternatively spliced AATYK mRNA using cDNA library screening and RT-PCR, whose expression is readily detected in the brain but not myeloid cells. Western blot analysis revealed that the AATYK protein was expressed in virtually all regions of the adult rat brain in which neurons are present, including olfactory bulb, forebrain, cortex, midbrain, cerebellum and pons. Immunohistochemical labeling of adult brain sections showed the highest levels of AATYK expression in the cerebellum and olfactory bulb. Expression of AATYK was also up-regulated as a function of RA-induced neuronal differentiation of p19 embryonal carcinoma cells, supporting a role for this protein in mature neurons and neuronal differentiation.

Anisotropic organization of the rat superior paraolivary nucleus.

In rodents, the superior paraolivary nucleus (SPON) is one of the major nuclei of the superior olivary complex that innervate the inferior colliculus. To analyze the intrinsic organization of the SPON and to gain further insight into its relationship with the inferior colliculus, the neuroanatomical tracers biotinylated dextran and horseradish peroxidase were unilaterally injected into different regions of the central nucleus of the inferior colliculus of adult albino rats. Both tracers resulted in retrograde labeling of SPON cell bodies. In addition, biotinylated dextran rendered excellent filling of dendritic and axonal processes within the nucleus. Our results confirm that the projection from the SPON to the central nucleus of the inferior colliculus is nearly exclusively ipsilateral and strictly topographic. Furthermore, our data show that virtually all SPON neurons participate in this projection. The labeling with biotinylated dextran reveals that typical SPON neurons are medium to large multipolar cells with four to seven thick, long, scarcely branched and smooth dendrites that extend over long distances within a nearly parasagittal plane and intermingle with similarly oriented axonal plexuses. Some of the neurons located ventrally within the nucleus possess dendrites that extend ventrally beyond the limits of the SPON to penetrate into the underlying ventral nucleus of the trapezoid body. The parallel arrangement of flattened dendritic and axonal fields within the SPON is reminiscent of the fibrodendritic laminae found in other mammalian auditory nuclei. This fact and the available data about the connectivity of the nucleus stress the similarities between the SPON and the principal nuclei of the superior olivary complex.

Superior paraolivary nucleus of the rat is a GABAergic nucleus.

The presence of the inhibitory neurotransmitters glycine and GABA (gamma-amino butyric acid) and GAD (glutamic acid decarboxylase), the synthesizing enzyme for GABA, was examined by immunocytochemistry in the superior paraolivary nucleus (SPON) of the rat. Only rarely were SPON neurons observed to be glycine-immunoreactive, but the majority were GABA-immunoreactive. Using unbiased stereological counting methods, we estimated that this nucleus contains approximately 2500 neurons. Moreover, 90% of SPON neurons were immunolabeled by antisera directed against either the 65- or 67-kD isoform of GAD, or a third antiserum that recognizes both GAD isoforms. Morphometric analysis of GAD-immunolabeled neurons indicated that SPON neurons possess cell bodies and dendritic arbors that are elongated rostrocaudally and relatively flattened parasagittally. Abundant glycine-, GABA-, and GAD-immunoreactive punctate profiles-presumed to represent, for the most part, presynaptic axon terminals-were observed in apposition to SPON neurons. We conclude that the rat SPON contains a homogeneous population of multipolar GABAergic neurons that receive abundant GABAergic and glycinergic innervation. The vast majority of glycinergic inputs to SPON are presumed to originate in the ipsilateral medial nucleus of the trapezoid body, but the source(s) of its GABAergic innervation remains to be determined.

Immunologic and histologic observations in reovirus-induced otitis media in the mouse.

The goals of this study were to develop a mouse model for virally induced otitis media, and to study the immune response to infection. Intranasal inoculation of mice by reovirus was used to induce otitis media. Immunohistochemical evidence for the presence of reovirus in the nasopharynx, eustachian tubes, and middle ears and the amount of infiltrating B-cells and T-cells in those sites were serially evaluated by painlessly sacrificing animals over a 21 -day period. Reovirus antigen was detected in the middle ear mucosa by day 4 in 75% of infected animals, and histologic evidence for otitis media was found in 54% of all infected animals. A significant increase in B-cells in the nasopharynx and eustachian tubes was noted 7 to 10 days following infection. The number of infiltrating T-cells did not vary significantly from that in the control animals at any of the sites. These results provide a basis for further investigations of the immune response in otitis media.

Ultrastructure of neurons and large synaptic terminals in the lateral nucleus of the trapezoid body of the cat.

Neurons of the lateral nucleus of the trapezoid body (LNTB), the most prominent periolivary nucleus of the cat superior olivary complex, form an important component of the descending auditory pathways and also innervate the medial superior olive. Cells forming the posteroventral subnucleus (pvLNTB), when investigated by light microscopy, exhibit morphological similarities with globular bushy cells of the cochlear nucleus and principal cells of the medial nucleus of the trapezoid body. These latter two cell types are integral components of brainstem circuitry mediating the early stages of sound localization. In this report, ultrastructural features of LNTB neurons are described. pvLNTB cell bodies are characterized by a round to oval shape, smooth nuclear membrane, and the relative paucity of stacks of rough endoplasmic reticulum. In addition, pvLNTB cell bodies and proximal dendrites are contacted by large synaptic terminals which contain round synaptic vesicles and form multiple asymmetric synaptic junctions. These ultrastructural characteristics are similar to those previously described for globular and principal cells and distinguish pvLNTB cells from cells of the main subnucleus. Large terminals contacting pvLNTB cells contain a specialized organelle assembly, including an adherens plaque associated by filamentous strands with a mitochondrion. We name this organelle assembly the mitochondria-associated adherens complex (MAC) and note its proximity to synaptic junctions. Because high activity rates are characteristic of large terminals in the lower auditory system, the MAC may play a specialized role in membrane stabilization at synapses which generate high rates of vesicle membrane turnover.

Ectopic overexpression of engrailed-2 in cerebellar Purkinje cells causes restricted cell loss and retarded external germinal layer development at lobule junctions.

Members of the En and Wnt gene families seem to play a key role in the early specification of the brain territory that gives rise to the cerebellum, the midhindbrain junction. To analyze the possible continuous role of the En and Wnt signaling pathway in later cerebellar patterning and function, we expressed En-2 ectopically in Purkinje cells during late embryonic and postnatal cerebellar development. As a result of this expression, the cerebellum is greatly reduced in size, and Purkinje cell numbers throughout the cerebellum are reduced by more than one-third relative to normal animals. Detailed analysis of both adult and developing cerebella reveals a pattern of selectivity to the loss of Purkinje cells and other cerebellar neurons. This is observed as a general loss of prominence of cerebellar fissures that is highlighted by a total loss of sublobular fissures. In contrast, mediolateral patterning is generally only subtly affected. That En-2 overexpression selectively affects Purkinje cells in the transition zone between lobules is evidenced by direct observation of selective Purkinje cell loss in certain fissures and by the observation that growth and migration of the external germinal layer (EGL) is selectively retarded in the deep fissures during early postnatal development. Thus, in addition to demonstrating the critical role of Purkinje cells in the generation and migration of granule cells, the heterogeneous distribution of cellular effects induced by ectopic En expression suggests a relatively late morphogenetic role for this and other segment polarity proteins, mainly oriented at lobule junctions.

PEP-19 immunoreactivity in the cochlear nucleus and superior olive of the cat.

We applied antiserum to PEP-19, a presumptive calcium-binding polypeptide, to the auditory brainstem of cats to determine whether this antiserum would selectively reveal cochlear nucleus neurons and their projections. We report that the entire populations of ventral cochlear nucleus bushy and multipolar cells, but not octopus cells, express this peptide in their somata and dendrites. Presumed axons of spherical bushy cells located dorsally and thicker globular bushy cell fibers located ventrally in the trapezoid body are immunostained, as are thin fibers presumed to represent the axons of multipolar cells. Large calyceal endings in the medial nucleus of the trapezoid body are densely immunoreactive as are smaller punctate profiles that outline immunonegative neuronal profiles in the medial and lateral superior olives. These features of immunolabeling indicate that PEP-19 is expressed in all neuronal compartments. Within the entire superior olivary complex, relatively few neurons are immunolabeled, and the vast majority of these are found in the periolivary nuclei. There are many more immunostained neurons in lateral than in medial periolivary cell groups, but their combined numbers are dwarfed by the numbers of immunolabeled cells in the ventral cochlear nucleus. The borders of the principal nuclei and some of the periolivary cell groups are well defined by the distribution of PEP-19-immunoreactive fibers and puncta. Since ventral cochlear nucleus bushy cells comprise the predominant input to principal nuclei of the superior olive, and the entire bushy cell population is immunolabeled by PEP-19 antiserum, the numbers and distribution of their inputs can be quantified. In this study we report that immunoreactive puncta apposed to the cell bodies and proximal dendrites of neurons in the medial superior olive occur at a density of 20/100 microns2. Moreover, we demonstrate by pre-embedding immunoelectron microscopy that the PEP-19-immunoreactive punctate profiles observed in the medial superior olive by light microscopy represent presynaptic terminal boutons that contain round synaptic vesicles and form asymmetric synaptic junctions, features traditionally associated with excitatory synapses. Thus, this antiserum represents a useful tool for investigating the distribution of ventral cochlear nucleus fibers and synaptic terminals within their target nuclei in the superior olive.

Glycine immunoreactivity in the lateral nucleus of the trapezoid body of the cat.

The central auditory system contains several predominantly glycine-immunoreactive nuclei, and one of these, the lateral nucleus of the trapezoid body, contains cell bodies exhibiting a spectrum of labeling intensity. By using post-embedding glycine immunocytochemistry on thin sections, and toluidine blue staining of adjacent sections, we established that darkly glycine-immunoreactive neurons constituted a distinct morphological class and form one of three subnuclei of the lateral nucleus of the trapezoid body, called the posteroventral subnucleus. These neurons resemble, in both labeling intensity and cell body morphology, the principal cells of the medial nucleus of the trapezoid body. The other two subnuclei of the lateral nucleus of the trapezoid body, its main and hilus subnuclei, contained predominantly glycine-immunoreactive and glycine-immunonegative neurons, respectively. Glycine immunoreactivity was compared with gamma-aminobutyric acid (GABA) immunoreactivity in order to identify other organizational features of the lateral nucleus of the trapezoid body. Cell bodies that displayed either dark glycine-immunoreactivity or which were glycine-immunonegative were GABA-immunonegative. Cell bodies that displayed GABA immunoreactivity were preferentially located in the main subnucleus. Patterns of distribution of axosomatic innervation in the lateral nucleus of the trapezoid body were revealed in which glycine-immunoreactive puncta were (1) more numerous than GABA-immunoreactive puncta on glycine-immunonegative cell bodies and (2) equal to or less numerous than GABA-immunoreactive puncta on glycine-immunoreactive cell bodies. The characteristics of neural circuitry revealed by glycine and GABA immunoreactivity in the lateral nucleus of the trapezoid body may be generalizable to other populations of neurons of the superior olivary complex and to other regions of the central nervous system containing glycinergic neurons, such as the retina.

Organization of ventrolateral periolivary cells of the cat superior olive as revealed by PEP-19 immunocytochemistry and Nissl stain.

Ventrolateral periolivary cell groups, through their descending projections to the cochlear nucleus (CN) and local projections to principal nuclei of the superior olive, may participate in brainstem mechanisms mediating such tasks as signal detection in noisy environments and sound localization. Understanding the function of these cell groups can be improved by increased knowledge of the organization of their synaptic inputs in relation to their cellular characteristics. Immunocytochemistry for PEP-19 (a putative calcium binding protein) reveals four patterns of immunolabeling within the ventrolateral periolivary region. Three of the patterns, which have distinct fiber and punctate labeling characteristics, help to define three subdivisions of the lateral nucleus of the trapezoid body (LNTB). The fourth pattern defines two other nuclei, the anterolateral periolivary nucleus (rostral) and the posterior periolivary nucleus (caudal), which display many immunoreactive cell bodies but little fiber and punctate labeling. One of the subdivisions of the LNTB contains large PEP-19 immunolabeled puncta arranged in pericellular nests. Analysis of Nissl-stained sections reveals a neuronal population that resembles globular cells of the ventral cochlear nucleus (VCN) and which colocalizes with pericellular nests of large immunolabeled puncta. Cell counts reveal that roughly 10,000 neurons constitute the cat ventrolateral periolivary region, 9,000 of which are found in the LNTB. Three-dimensional reconstructions of auditory brainstem nuclei clarify the complex spatial relationships among these structures.

Postsynaptic targets of Purkinje cell terminals in the cerebellar and vestibular nuclei of the rat.

The cerebellar and vestibular nuclei consist of a heterogeneous group of inhibitory and excitatory neurons. A major proportion of the inhibitory neurons provides a GABAergic feedback to the inferior olive, while the excitatory neurons exert more direct effects on motor control via non-olivary structures. At present is is not clear whether Purkinje cells innervate all types of neurons in the cerebellar and vestibular nuclei or whether an individual Purkinje cell axon can innervate different types of neurons. In the present study, we studied the postsynaptic targets of Purkinje cell axons in the rat using a combination of pre-embedding immunolabelling of the Purkinje cell terminals by L7, a Purkinje cell-specific marker, and postembedding GABA and glycine immunocytochemistry. In the cerebellar nuclei, vestibular nuclei and nucleus prepositus hypoglossi Purkinje cell terminals were found apposed to GABAergic and glycinergic neurons as well as to larger non-GABAergic, non-glycinergic neurons. In the cerebellar and vestibular nuclei individual Purkinje cell terminals innervated both the inhibitory and excitatory neurons. Both types of neurons were contacted no only by non-GABAergic Purkinje cell terminals but also by GABA-containing terminals that were not labelled for L7 and by non-GABAergic, non-glycinergic terminals that formed excitatory synapses. Glycine-containing terminals were relatively scarce ( < 2% of the GABA-containing terminals) and frequently contacted the larger non-GABAergic, non-glycinergic neurons. To summarize, Purkinje cell axons evoke their effects through different types of neurons present in the cerebellar and vestibular nuclear complex. The observation that individual Purkinje cells can innervate both excitatory and inhibitory neurons suggests that the excitatory cerebellar output system and the inhibitory feedback to the inferior olive are controlled simultaneously.

Sex differences in the distribution of axon types within the genu of the rat corpus callosum.

Neuroanatomical sex differences have been documented in the rat neocortex, including dimorphism of its predominant commissure, the corpus callosum (CC). In particular, CC sex differences have been reported in the ultrastructure of the posterior callosal region, the splenium. Since the CC is a heterogeneous fiber tract with its axons arising from distinct cortical areas and passing through restricted regions along its length, it became of interest to ascertain whether cellular sexual dimorphisms may also be present in another division of the CC. To test this hypothesis, electron microscopy was used to examine axon composition in adult male and female rats in the anterior portion, the genu. The number and size of axons, the thickness of the myelin sheath, and the area within the genu occupied by these constituents, were quantified. Results showed a significant sex difference in the ratio of unmyelinated to myelinated axons, with females having a larger proportion of unmyelinated fibers. This effect was present for both (1) the number of axons, and (2) the area taken up by axonal fibers. No differences were found in the size of either axon type, or for myelin thickness. Comparison of these results with those from the splenium and possible mechanisms underlying this dimorphism are discussed.

Evidence for GABAergic interneurons in the red nucleus of the painted turtle.

Immunocytochemical and electrophysiological evidence supporting the presence of GABAergic interneurons in the turtle red nucleus is presented. Injections of HRP into the spinal cord produced labeling of large neurons in the contralateral red nucleus. The peroxidase-antiperoxidase (PAP) method revealed smaller cells immunoreactive to an antibody against glutamate decarboxylase (GAD), the synthetic enzyme for the inhibitory neurotransmitter GABA, that were interspersed among larger immunonegative neurons. Similar small neurons were densely immunostained by antibodies to GABA-glutaraldehyde conjugates obtained from different sources and applied according to pre-embedding and postembedding protocols. Rubrospinal neurons retrogradely labeled with HRP measured 16 and 27 microns in mean minor and major cell body diameters, while GABA-like immunopositive neurons situated within the red nucleus measured 7 and 13 microns. There was very little overlap in soma size between the two cell populations. Therefore, we suggest that the GAD- and GABA-positive neurons may be local inhibitory interneurons. This notion is further supported by observations of pre-embedding immunostaining for GAD and postembedding immunostaining for GABA showing that the turtle red nucleus is amply innervated by immunoreactive axon terminals. These puncta are closely apposed to cell bodies and dendrites of both immunonegative large neurons and immunopositive small neurons. Moreover, immunogold staining at the electron microscopic level demonstrated that GABA-like immunoreactive axon terminals with pleomorphic synaptic vesicles formed symmetric synapses with cell bodies and dendrites of the two types of red nucleus cells. These ultrastructural features are commonly assumed to indicate inhibitory synapses. A moderately labeled bouton with round vesicles and asymmetric synapses was also observed. In addition, the two types of red nucleus neurons received asymmetric axosomatic and axodendritic synapses with GABA-negative boutons provided with round vesicles, features usually associated with excitatory functions. To obtain electrophysiological evidence for inhibition, intracellular recordings from red nucleus neurons were conducted using an in vitro brainstem-cerebellum preparation from the turtle. Small, spontaneous IPSPs were recorded from 7 out of 14 red nucleus cells studied. These morphological and physiological results provide strong support for concluding that the turtle red nucleus, like its mammalian counterpart, contains GABAergic inhibitory interneurons. While we have not identified the main source of input to these interneurons, in view of the scarce development of the reptilian cerebral cortex, this input is unlikely to come from the motor cortex as it does in mammals.(ABSTRACT TRUNCATED AT 400 WORDS)

Characteristics of labeling of the cerebellar Purkinje neuron by L7 antiserum.

Previously, it has been shown by light microscopy that antiserum to the L7 protein labels cerebellar Purkinje neurons. Herein we show by light and electron microscopic immunocytochemistry that all cerebellar Purkinje cells express L7 and that the gene product is distributed to all neuronal compartments, including the nucleus. Possible functional roles for L7, based on its subcellular localization, are discussed. L7 is proposed as an excellent marker molecule for future studies of normal and aberrant cerebellar development.

Dynamic organization of developing Purkinje cells revealed by transgene expression.

The cerebellum has many properties that make it a useful model for investigating neural development. Purkinje cells, the major output neurons of the cerebellar cortex, have drawn special attention because of the availability of biochemical markers and mutants that affect their development. The spatial expression of L7, a protein specific for Purkinje cells, and L7 beta Gal, a gene expressed in transgenic mice that was constructed from the L7 promoter and the marker beta-galactosidase, delineated bands of Purkinje cells that increased in number during early postnatal development. Expression of the transgene in adult reeler mutant mice, which show inverted cortical lamination, and in primary culture showed that the initial expression of L7 is intrinsic to Purkinje cells and does not depend on extracellular signals. This may reflect an underlying developmental map in cerebellum.

Cerebellar Purkinje cell markers are expressed in retinal bipolar neurons.

Previous studies have been directed at the elucidation of neuron-specific gene expression in the mammalian central nervous system. In particular, we have identified a series of marker molecules that are expressed in cerebellar Purkinje cells with varying degrees of specificity. Here, we show by light microscopic immunocytochemistry and Northern transfer and hybridization that two of these markers, namely, L7 and PEP19, are expressed in the retina of mouse and rabbit, while a third marker, cerebellin, is absent. Light and electron microscopic immunocytochemistry proves that L7-like immunoreactivity is restricted to rod bipolar cells, while PEP 19-like immunoreactivity is distributed in both rod and cone bipolars. PEP19 is also expressed by subsets of amacrine and ganglion cells. The density of PEP19-positive bipolar cells is greater than that of L7-positive bipolar cells, although the density of each is approximately equal in central and peripheral portions of the retina. An antiserum to a fourth Purkinje cell marker, vitamin D-dependent calcium-binding protein-28 kD (CaBP), reveals primarily axonless horizontal cells, but also subsets of rod bipolar, amacrine, and, in the mouse but not in the rabbit, ganglion cells. The processes of immunoreactive cell bodies form discrete bands in the internal plexiform layer, and mixtures of the antisera help distinguish their identity. Thus, these Purkinje cell markers can be used at the electron microscopic level to unravel the extremely complex neuropil of this retinal layer. Furthermore, knowledge of the retinal distribution of this panel of molecules is of general value for future studies of retinal neuronal typology and can serve to map the densities of subsets of bipolar cells throughout the retina. The expression of L7 and PEP19 in bipolar cells and in Purkinje cells suggests a biochemical relationship between these two spatially distant neuronal populations.

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

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 hind-brain 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 sheet-like 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.