Neurotrimin expression during cerebellar development suggests roles in axon fasciculation and synaptogenesis.

We investigated the temporal expression of the neural cell adhesion molecule, neurotrimin, in the rat cerebellum and the brainstem from birth to adulthood using immunoreactive labeling. A wave of expression accompanied the development of projection pathways extending from brainstem nuclei (pons/inferior olive) through the cerebellar peduncles into the arbor vitae and disappeared with myelination by P14. Immuno-EM revealed expression of neurotrimin on the surface of unmyelinated axons but not on astrocytes or oligodendroglia. With the development of the molecular and internal granular layers, intense labeling occurred on the surface of parallel fiber bundles, granule cells and mossy fibers. With synaptogenesis, each excitatory junction was labeled by the immunoreaction. By P21, neurotrimin reactivity decreased on the surfaces of neuronal somata, dendrites and axons but remained at excitatory synaptic contact sites in both the molecular and granular layers. The spatial-temporal expression pattern of neurotrimin suggests that this adhesion molecule plays a role in axonal fasciculation of specific cerebellar systems and may also be involved in the formation of excitatory synapses and their stabilization into adulthood.

Alpha 1E subunit of the R-type calcium channel is associated with myelinogenesis.

During myelinogenesis, we found an exceedingly strong, transient expression of the alpha(1E) gene for the R-type voltage-gated calcium channel in CNS white matter. This immunoreactivity appeared in glial cells along specific pathways of the brainstem, cerebellum, and telencephalon. The reactivity followed a wave that progressed from the brainstem at P5, to the cerebellar peduncles by P8, the arbor vitae by P14, and the granular layer by P17. The reactivity-peaked about 3-4 days later and decreased gradually to become negligible in all areas before adulthood. Ultrastructural analysis confirmed that alpha(1E) immunoreactivity was located in oligodendroglial somata, their projections, paranodal wraps and loose myelin sheaths. There was a distinct association of the channel protein reactivity on oligodendroglial membranes in contact with the axon. We propose that glial projections, contacting axons, sense axonal firing through small K(+) currents and open the high voltage R-type calcium channels to signal myelination.

c-fos gene expression parallels auditory adaptation in the adult rat.

This study investigated the pattern of c-fos gene expression corresponding with auditory adaptation to novel sound. Using six groups of adult rats (naive control, 1 h, and 1, 2, 3, and 4 days of continuous stimulation), we quantified c-fos expressing cells in the dorsal and ventral cochlear nuclei and found a 54 fold increase in 1 h following novel sound stimuli. The number of reactive cells decreased sharply within 24 h and nearly disappeared by 96 h. Our results reveal that c-fos gene expression in the adult rat is attenuated in parallel with the expected auditory adaptation to novel sounds indicating an association with auditory learning and memory.

Dying-back of Purkinje cell dendrites with synapse loss in aging rats.

Qualitative and quantitative changes were found in the cerebellar circuitry of old as compared to young rats. The old group had a reduced number of synapses (at least 30%), however, there was an increase in the size of remaining synaptic components (13.5% for spine head volume, 66% for bouton volume, and 17% for the area of synaptic contact zones). Furthermore, there were pronounced morphological changes in the older group appearing as: 1) prominent lipofuscin bodies in Purkinje cell somata, 2) numerous myelinated fibers in the lower part of the molecular layer, 3) tortuous Purkinje cell dendrites in a thinned molecular layer, and 4) abundant vacuolar profiles and membrane swirls in small and intermediate-sized dendrites. Our findings suggest that Purkinje cell dendrites are dying-back reducing the target field for granule cells and that remaining synaptic sites compensate by increasing synaptic contact area as well as the size of pre- and postsynaptic structures.

Transient c-fos gene expression in cerebellar development and functional stimulation.

This study examined the temporal appearance of c-fos expression in rat cerebella from birth to postnatal day 21 (P21) and following systemic kainic acid administration in adults. During development, Fos positive granule cells appeared first at P3 in caudal lobules and extended to all folia by P9. These reactive cells occurred in clusters throughout the granular layer and reached their highest reactivity by P15. In addition, Fos positive basket and stellate cells were seen in the molecular layer at this time. A steep decline in Fos protein labeling of the cerebellum began by P18 and was barely detectable in adulthood. In adult rats, systemic injection of kainic acid induced c-fos expression in granule cells and stellate/basket interneurons within 1 h of treatment. Fos reactive granule cells appeared in clusters with some forming distinct parasagittal bands in caudal folia. One day after unilateral cerebellar lesioning, a limited number of reactive cells were found on the lesion margins. A combination of lesioning and systemic kainic acid produced a strong, c-fos expression throughout the ipsilateral granular layer as well as in Purkinje cell nuclei. Contralateral to the lesion, the pattern of granule cell reactivity appeared the same but slightly stronger than those with kainate alone. We conclude that c-fos gene expression occurs transiently in granule cells during postnatal development and can be rapidly re-induced in the adult following systemic injection of glutamate agonists. The c-fos expression patterns suggest that subpopulations of granule cells are clustered and related to the parasagittal compartments of Purkinje cells.

Distribution of glutamate receptors GluR 2/3 and NR1 in the developing rat cerebellum.

The distribution of glutamate receptors GluR2/3 and NR1 was analysed immunohistochemically during development of the rat cerebellum. GluR2/3 immunoreactivity appeared by postnatal day P0 in somata of Purkinje cells. Throughout P7, P15, P20 and adulthood, GluR2/3 immunoreactivity was found in the entire Purkinje cell dendritic arbor reaching to the external granular layer and, by P15, the surface of the cerebellum. By P7, the granular layer revealed scattered, mildly reactive, cells. NR-1 immunoreactivity first gained prominence about P7 in the region of the multi-layered Purkinje cell somata. By P15, NR1 was prominent in Purkinje cell somata and Golgi cells. The reaction product extended into the primary main dendrite of Purkinje cells. By P21, stellate and basket cells had intense reactivity throughout the molecular layer and reactive large-diameter dendrites of Golgi cells projected toward the molecular layer. Granule cells remained very weak among strongly reactive Golgi cell somata and dendrites. Ultrastructural immunohistochemistry revealed NR1 reaction product in Purkinje cell somata, in stellate cell somata and dendrites and on postsynaptic membranes of scattered spines throughout the molecular layer. The later appearance and restricted location of NR1 in somata and proximal dendrites of Purkinje cells contrasted markedly with GluR2/3 which appeared before birth and remained prominent throughout Purkinje cell dendritic arbors of adults. The time of NR1 expression correlated with the generation of granule cells, their synaptogenesis on Purkinje cells, the formation of stellate/baske cells and the shift of climbing fibre synapses from distal to proximal dendrites. The developmental appearance of stellate/basket cells and Golgi cells as well as their high reactivity remaining into adulthood suggest that these inhibitory molecular and granular layer interneurons are the principal targets of glutamate axons serving NR1 synaptic properties while Purkinje cells and brush type granule cells are targets for glutamate connections with GluR2/3 characteristics.

Plasmalemmal ATPase calcium pump localizes to inner and outer hair bundles.

Recent studies demonstrate calcium ion influx at the tips of hair cell stereocilia during mechano-transduction. These ions must be either pumped from the cytosol into the extracellular space or endoplasmic envelope, or else sequestered by binding to specific proteins. A plasma membrane calcium pump (ATPase-type) was analysed in whole-mounts of rat organ of Corti using a monoclonal antibody to a large cytoplasmic loop of this protein. The reactivity was particularly high on the tips of longer stereocilia and was found along the shafts. Inner hair cell stereocilia had much less reactivity than outer hair cells. The reactivity lined the plasma membrane of inner hair cell bodies while a higher reactivity appeared in the cytoplasm of outer hair cells. Supporting cells were unreactive. Ultrastructural examination confirmed the plasma membrane calcium pump location on stereocilia and along the endolymph surface of receptor cells. Reaction product lined the plasma membrane of stereocilia as intense puncta. More reactive puncta occurred near the distal ends of stereocilia and the number decreased toward the ciliary base. The endolymph plasma membrane over the cuticular notch was especially reactive. The finding of more intense pump reactivity at the tips of stereocilia than the base is consistent with the hypothesis that during transduction, calcium ions enter stereocilia, distally, and the ATPase plasma membrane calcium pump rapidly extrudes these ions to the extracellular space.

Effect of unilateral tympanotomy on auditory induced c-fos expression in cochlear nuclei.

The immediate early gene, c-fos, signals expression of target genes. Three natural occurring physiological entities: (1) learning, (2) plasticity, and (3) stress are proposed to use c-fos gene expression to signal molecular changes in neurons. The objective of this study was to determine whether c-fos expression is predominately activated by stress or by novel events associated with learning and plasticity. The approach was to quantitate the number of neurons in cochlear nuclei which express Fos protein following short-term novel sound stimuli together with either uni- or bilateral tympanotomy so as to differentiate novel sound stimuli from stress activation. The results show that routinely experienced sounds do not elicit c-fos expression in medullary cochlear nuclei, but novel sounds produced a 25-fold increase in the number of active cells. Following unilateral tympanotomy with novel sound stimulation, only a small number of cells were activated, ipsilaterally, (partially deafened side) while contralaterally, there was a 30-fold increase. After normalization of the data for control values, the data clearly indicate that novelty of sound stimuli induce c-fos gene expression. Furthermore, bilateral tympanotomy (bilateral partial deafening) with sound stimulation activated both sides by 20-fold, indicating that the c-fos response followed the sound stimulation. The data allow us to conclude that stress generates only a small contribution to c-fos gene expression while novel stimuli are potent signals, strongly implicating c-fos in novelty induced adaptation processes involved in learning and plasticity.

Activation of fos in mouse amygdala by local infusion of norepinephrine or atipamezole.

Norepinephrine (NE) is known to activate a number of immediate-early genes (IEGs) in the brain which may be involved in prolonged changes in neuronal function. To investigate the function of these genes it would be useful to have a model system in which they are induced in specific populations of cells in specific brain regions without systemic drug administration which can affect multiple sites. In the present paper we have shown that local infusions of NE or of the alpha2-adrenoceptor antagonist, atipamezole, in the mouse amygdala produces localized expression of fos. The expression of fos was blocked by a cocktail of an alpha1-(prazosin) and beta1-adrenoceptor (betaxolol) blocker but not by a selective 5-HT1A blocker (WAY100135). Prazosin and betaxolol did not have a nonspecific reducing action on fos expression. It is concluded that localized expression of fos after NE infusion in the mouse amygdala represents a model system for further studies of the role of IEG expression in central noradrenergic function.

Ultrastructural localization of the plasmalemmal calcium pump in cerebellar neurons.

In a previous study, fluorescence labeling of a plasmalemmal ATPase protein with the 5F10 monoclonal antibody revealed prominent antigen in the cerebellar molecular layer surrounding the somata and dendrites of Purkinje cells. In the present study, this antibody labeled with silver enhanced nano-sized gold particles on semithin plastic sections revealed a clearly demarcated plasma membrane outlining the somata and entire dendritic arbors of Purkinje cells including their spines. Ultrastructural analysis of horseradish peroxidase preparations showed reaction product along the plasmalemma and extending on to the sub-plasmalemmal endoplasmic reticulum. In the granular layer, somata of granule cells were reactive, as were their dendritic extensions into glomeruli where reactive claws surrounded voids formed by mossy fiber rosettes. Somata and dendrites of cerebellar nuclear cells also had reactive zones that were limited to the plasma membrane and a narrow zone of the sub-plasmalemmal endoplasmic reticulum. Comparative labeling of this protein and P channel protein revealed similar plasmalemmal locations. This study shows that a specific calcium ATPase pump protein is located on the plasmalemma of certain types of cerebellar neurons. The ultrastructural distribution of calcium pump and P channel antibodies occurred in punctate sites along the plasma membrane of dendrites and spines of Purkinje cells. The close association between P-type calcium channels and the plasma membrane calcium pump is consistent with rapid extrusion of intracellular calcium from neurons endowed with large numbers of voltage-gated calcium channels.

Transient expression of lyn gene in Purkinje cells during cerebellar development.

Expression of the lyn gene, a member of sarcoma proto-oncogene family, was analyzed immunohistochemically during cerebellar development in the rat. Lyn immunoreactivity was intense in axons, dendrites and somata of Purkinje cells from gestational day 18 to postnatal day 15 and then decreased. lyn gene expression clearly followed the appearance and the maturation of dendritic arbors. A rapid decrease in Lyn protein, after the 18th postnatal day, left only a few scattered positive Purkinje cell somata in the adult. External and undifferentiated internal granule cells were weak in Lyn immunoreactivity but gradually increased during development. Clusters of positive granule cells were found along the Purkinje cell layer with parasagittal bands crossing the granular layer by 21 days. These bands persisted into adulthood. Cerebellar nuclei lacked immunoreactivity in early development but only fastigial neurons began to acquire lyn gene expression by the 15th postnatal day. The corresponding appearance of the lyn gene expression and the formation of Purkinje cell dendritic arbors suggests that Lyn protein is involved in dendrogenesis. On the other hand, the late onset of immunoreactivity in fastigial neurons and granule cells implies a role in cell maintenance.

Immunohistochemical localization of Lyn (p56) protein in the adult rat brain.

Expression of a sarcoma proto-oncogene, c-lyn, was mapped in the adult rat brain using immunohistochemistry. Lyn protein was prevalent in restricted cell populations of the olfactory bulb and the basal forebrain which included nuclei of accumbens, fundal striatum, bed stria, ventral pallidum and central amygdala as well as deep entorhinal and pyriform cortices. Tightly packed Lyn-positive cells formed discrete multiple stripes crossing perpendicular to the rostral limb of the anterior commissure, and intense masses surrounding the caudal limb. In the thalamus, the habenula, anterodorsal nucleus and medial geniculate body, together with the paraventricular hypothalamic nuclei, had prominent reactive neuronal somata and dendrites in the neuropil. The lateral septal nucleus also had intense Lyn-positive neurons with overlapping dendritic fields. In addition, scattered neurons were evenly distributed throughout the striatum. The red, interpeduncular, auditory and trigeminal tract nuclei were intensely reactive. The cerebellar molecular layer was uniformly labeled except for a few isolated fiber bundles in the lowest part of this layer. The granule cells adjacent to the Purkinje cell layer appeared in reactive patches. In the spinal cord, the posteromarginal nucleus had intense labeling. The significance of this highly localized distribution pattern of Lyn protein may be related to connections forming functional compartments serving signal transduction within specific central nervous system circuitry.

Effects of glycerin, hyaluronic acid, and hydroxypropyl methylcellulose on the spiral ganglion of the guinea pig cochlea.

A cochlear lubricant may facilitate the surgeon's ability to place the electrode array deep within the cochlea. Patient performance with the multichannel cochlear implant may be enhanced with a deeper electrode insertion. Theoretically, deeper insertion and stimulation will recruit and activate more surviving spiral ganglion neurons. Several studies have shown that neuron survival is a factor for cochlear implant success, especially in the postmeningitis patient. We studied the histologic and electrophysiologic effects of the intracochlear injection of three potential lubricants in the guinea pig: glycerin, hyaluronic acid, and hydroxypropyl methylcellulose. All three have approved medical uses, reduce friction, and are readily available. Results show that when compared to surgical controls (cochleostomy without injection), there is no significant reduction in the spiral ganglion neuronal count at 2 and 8 weeks postinjection, and the dendrite and axon histology is well preserved. Injection of any of the substances within the cochlea causes severe hearing loss (detected by direct round window electrocochleographic responses to auditory stimuli) that only partially recovers with time. These findings suggest that any of the three tested substances could be considered as lubricants for intracochlear electrode insertion.

Ultrastructural findings in the vestibular end-organs of AIDS cases.

Neurotologic manifestations are apparent in human immunodeficiency virus (HIV) infection, but are poorly understood. Symptoms related to the vestibular system include episodes of vertigo, imbalance, ataxia, and nausea. Although patients present more often with hearing impairment, vestibular complaints are described and electrophysiologic studies indicate vestibular dysfunction in HIV-infected patients. Whether the disease involvement includes the central, or the peripheral nervous system has not been established. Ultrastructural analysis of vestibular end-organs obtained from HIV autopsy cases revealed pathologic changes in the labyrinth wall, the epithelial lining, and the receptor maculae and cristae. Cytologic changes in hair cells included inclusion bodies, viral-like particles, and hair bundle malformations. Epithelial lining cells, supporting cells, and connective tissue cells had inclusions and viral-like particles. These findings are consistent with those of a previous cochlear study demonstrating intracellular viral-like particles with the morphologic characteristics of HIV. Further cytologic evaluation of decalcified temporal bones and immunohistochemical analysis of freshly harvested HIV-infected temporal bones may provide further insight into the pathogenesis of viral-induced hearing loss and vestibular impairment.

Regeneration of the auditory midbrain intercommissural projection in organotypic culture.

The aim of this study was to determine whether postnatal mammalian central neurons retain the capacity for axonal regeneration across a lesion site in organotypic cultures of the auditory midbrain. Brain slices from the gerbil inferior colliculus (IC) were obtained from postnatal 6-8 d old animal and cultured for 6-15 d in vitro. IC explants containing an intact commissural projection exhibited robust axonal and dendritic morphologies as assessed with biocytin labelling. In transected explants, the two lobes of the inferior colliculus were cut at the midline and then reapposed to one another in vitro. There was a robust regeneration of commissural fibers across the lesion site in 78% of the biocytin-labeled explants. Massive axonal regeneration was also revealed by immunostaining explants for Tau (100% of sections), an axon-specific microtubule-associated protein. Ultrastructural analyses demonstrated that biocytin-labeled regenerating fibers established de novo synaptic profiles in the contralateral lobe of the inferior colliculus. Finally, the distribution of astrocytes and oligodendrocytes were assessed by staining for glial fibrillary acidic protein (GFAP) and myelin-associated glycoprotein (MAG), respectively. GFAP-positive astrocytes were more widely distributed than in vivo, and oligodendrocytes remained immature, and evenly distributed in all explants. Taken together, these data demonstrate that the postnatal mammalian auditory midbrain can be maintained in vitro, and that central axons are capable of regenerating across the site of injury without the aid of an artificial substrate.

The antinociceptive effect of S-(+)-ibuprofen in rabbits: epidural versus intravenous administration.

This study was designed to determine whether systemic absorption plays any role in the antinociceptive effect of epidural (EP) sodium S(+)-ibuprofen (IB). One week after surgical implantation of EP catheters, six rabbits were given EP injections with either normal saline (NS) 0.4 mL or IB 10 mg in 0.4 mL NS (Group 1) on separate days. Each animal was injected with IB 10 mg intravenously (i.v.) on another day. Six control rabbits (Group 2) had neither surgery nor any injection. Analgesic testing was performed using electric stimulation through two electrocardiogram (ECG) skin electrodes with built-in adhesive, attached to shaved hip areas using 50 V, 1 Hz, 3 ms, before and 0.5,1,2 and 3 h after injection in Group 1, and in similar times in controls. The 95% confidence intervals (CI) of the mean difference between baseline and maximal nociceptive response latency of all groups were compared using analysis of covariance (ANCOVA) adjusted for baseline measurements. This comparison covered all possible pairs among all groups. Significant antinociceptive effects were seen after EP IB but not after control or i.v. IB. Neither motor dysfunction nor evidence of systemic toxicity or neurotoxicity was observed in any animal.

Immunohistochemical localization of protein kinase C delta during postnatal development of the cerebellum.

In cerebella of neonatal rats, immunoreactivity of protein kinase C delta (PKC delta) was moderate to strong in radial glia, Bergmann fibers and astrocytes but was absent in neurons. Beginning on the 7th day, a few small clusters of Purkinje cells expressed weak PKC delta reactions in caudal lobules of the vermis. From 10 to 20 days, clusters of labeled Purkinje cells increased in number, size and intensity while astrocytes and palisades of aligned Bergmann glia gradually diminished in intensity. At 21 days, glial cells had weak reactions while Purkinje and stellate-basket cells had intensity and distribution patterns found in adults. A transient occurrence of PKC delta in glia and later appearance in selective groups of neurons strongly support a significant role for this enzyme in signal transduction.

Ultrastructural findings in the cochlea of AIDS cases.

Neurotologic manifestations associated with the human immunodeficiency virus (HIV-1) are poorly understood. Eight temporal bones of acquired immunodeficiency syndrome (AIDS) cases were dissected and investigated ultrastructurally to determine if pathogens and pathologic changes were present within the cochlea. Extracellular viral-like particles with morphologic characteristics of HIV-1 were identified on the tectorial membrane in three cases. Numerous viral-like particles with cores appeared trapped within lacunae along the tectorial membrane in one specimen. Intracellular viral-like particles, appearing essentially similar to identified HIV-1 particles of infected lymphocyte cultures, were found within the cytoplasm of connective tissue cells. These same cells had gradients of enlarged viral envelopes forming cisterns that contained cores and varying amounts of cistern particulate. Some sectioned profiles of enlarged cytoplasmic cisterns revealed the attachment and inward protrusion of the core from the cistern wall. Other ultrastructural findings included cytoplasmic globular-dense particulate bodies, aggregates of smaller viral-like particles, and mitochondrial changes. This demonstration of prominent viral-like particles and cochlear pathology may help to explain the neurotologic manifestations associated with HIV-1 infection.

Structural and molecular heterogeneity of astrocytes and oligodendrocytes in the gerbil lateral superior olive.

The goal of this study was to determine the distribution and diversity of astrocytes and oligodendrocytes within the lateral superior olive of the gerbil. We used morphometric analyses and several immunocytochemical markers to assess differences in glial cell composition between the lateral (low-frequency projection) and the medial (high-frequency projection) limb of the lateral superior olive. Cell counts from Toluidine-stained semithin sections revealed a similar density of total astrocytes in both the lateral and the medial limbs. However, based on cytologic features, there was a prevalence of fibrous-like astrocytes in the lateral limb and protoplasmic-like astrocytes in the medial limb. In a similar manner, glial fibrillary acidic protein staining of astrocytes was intense in the lateral limb, but was largely restricted to the nucleus borders in the medial limb of the lateral superior olive. While glial fibrillary acidic protein was largely restricted to astrocytic processes, glutamine synthetase and S100 protein staining occurred, for the most part, in glial cell bodies. The density of glutamine synthetase positive cell bodies was homogeneous between the two limbs, while the density of S100-positive somata was significantly greater in the lateral limb. Cell counts obtained from semithin sections demonstrated a greater density of oligodendrocytes in the lateral limb than in the medial limb of the lateral superior olive. In a similar manner, there was a 40% greater density of carbonic anhydrase-positive somata in the lateral limb compared to the medial limb. Transferrin immunostaining was restricted to oligodendrocytes, but the density of labeled somata was identical in the lateral and medial limbs. 2',3'-Cyclic nucleotide 3'-phosphodiesterase and myelin-associated glycoprotein were also localized to the somata of oligodendrocytes, labeling both perisomatic and interfascicular cells. At the ultrastructural level, specialized contacts were found between pairs or clusters of oligodendrocytes. These results suggest that more than one type of astrocyte and oligodendrocyte is present within the gerbil lateral superior olive. Furthermore, glial cells were unevenly distributed, such that a greater density of oligodendrocytes and fibrous-like astrocytes were found in the low-frequency projection region. This heterogeneity is well correlated with known differences in the neuronal morphology within the lateral superior olive.

Antinociception without motor blockade after subarachnoid administration of S-(+)-ibuprofen in rats.

This study was designed to determine whether the nonsteroidal anti-inflammatory drug (NSAID) sodium S-(+)-ibuprofen (IB), can be used intrathecally as a substitute analgesic for opiates to avoid the side effects of intrathecal narcotics. One week or more after surgical implantation of subarachnoid catheters, four groups of Sprague-Dawley rats were given 0.05 ml subarachnoid injections containing one of the following: Group A, normal saline (NS); Group B, IB 0.25 mg, 0.5 mg and 1.5 mg; Group C, morphine (M) 0.05 mg and 0.025 mg; Group D received NS or IB 1.5 mg. Animals were sacrificed for spinal cord examination one week after injection. Tail flick response latency (TFL) was determined before and 15, 30, 60, 120 and 180 minutes after each injection. TFL differences were compared. IB 1.5 mg vs NS, IB 0.5 mg vs NS, IB 0.25 mg vs M 0.05 mg, IB 0.25 mg vs M 0.025 mg, M 0.05 mg vs NS, and M 0.025 vs NS showed p < 0.05. IB 1.5 mg vs M 0.05 mg and M 0.025 mg, IB 0.5 mg vs M 0.05 mg and M 0.025 mg revealed no significant difference. No motor impairment was observed in any animal. Light microscopy of the spinal cord revealed no evidence of pathological changes in any animal (group D).