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Abstract Background Borrelia burgdorferi contains one 16S and two tandem sets of 23S-5S ribosomal (r) RNA genes whose patterns of transcription and regulation are unknown but are likely to be critical for survival and persistence in its hosts. Results RT-PCR of B. burgdorferi N40 and B31 revealed three rRNA region transcripts: 16S rRNA-alanine transfer RNA (tRNAAla); tRNAIle; and both sets of 23S-5S rRNA. At 34°C, there were no differences in growth rate or in accumulation of total protein, DNA and RNA in B31 cultured in Barbour-Stoenner-Kelly (BSK)-H whether rabbit serum was present or not. At 23°C, B31 grew more slowly in serum-containing BSK-H than at 34°C. DNA per cell was higher in cells in exponential as compared to stationary phase at either temperature; protein per cell was similar at both temperatures in both phases. Similar amounts of rRNA were produced in exponential phase at both temperatures, and rRNA was down-regulated in stationary phase at either temperature. Interestingly, a relBbu deletion mutant unable to generate (p)ppGpp did not down-regulate rRNA at transition to stationary phase in serum-containing BSK-H at 34°C, similar to the relaxed phenotype of E. coli relA mutants. Conclusions We conclude that rRNA transcription in B. burgdorferi is complex and regulated both by growth phase and by the stringent response but not by temperature-modulated growth rate.

The C57BL6 mouse (B6) is homozygous for the gene for age-related hearing loss (ahl/ahl) and shows normal adult-like hearing before subtle changes in hearing begin at about 30 days of age. The B6Cast mouse is congenic to B6, having the wild type allele for normal hearing from Castaneous Ei on a B6 background. It has normal hearing throughout most of its lifespan. This study characterized the morphology of octopus cell (OC) somata in the posterior-ventral cochlear nucleus and of synaptic terminals on the OC somata in 8-week-old B6 and B6Cast mice, and the immunolocalization of antibodies to GluR1 (glutamate receptor subunit 1) and GRIP-C (glutamate receptor interacting protein-C terminus). By 8 weeks of age there are significant changes in the morphology of OCs and synaptic terminals around their somata in B6 mice compared to B6Cast mice. The distribution of immunoreactivity for the proteins GluR1 and GRIP is also significantly different in B6 mice from that in B6Cast mice. The modest degenerative changes reported in some B6 outer hair cells of the basal turn at this age do not seem adequate to explain the major changes observed in most OCs at a time when physiological studies show that many measures of the animals' hearing are still near normal. The findings suggest that changes in the alpha-amino-3-hydroxy-5-methyl-4-isoxazole glutamate receptor subunits and/or their binding proteins are part of the phenotype of ahl, and may reflect a role of the glutamate receptor pathway in the mechanism of ahl.

Differences were demonstrated in the distribution of glutamate receptors (GluR) 1, 2, 2/3 and 4 postsynaptic immunoreactivity (PSIR) on the somata of cartwheel and octopus cells in the adult gerbil cochlear nucleus (CN). Montages of electron micrographs of cartwheel and octopus cells immunoreacted with antibodies to GluR 1, 2, 2/3 and 4 were prepared. The number of synaptic terminals with PSIR were counted on all cells for each antibody, normalized to the total length of somatic surface analyzed. The density of terminals apposed to PSIR on octopus cells was similar for the antibodies GluR1, 2/3 and 4, but significantly less for GluR2. On cartwheel somata the numbers of terminals apposed to immunoreactive postsynaptic specializations with GluR1, 2, 2/3 or 4 were not significantly different from each other. The density of terminals apposed to GluR2/3 and 4 positive postsynaptic specializations was significantly less on cartwheel cells than on octopus somata. The data suggest that the decreased presence of the GluR2 subunit, which confers calcium impermeability to the assembled receptor and slower gating kinetics to receptors with a high GluR4 content, is the major difference in the AMPA receptors on the somata of these cell types. The presence on cartwheel cells of a majority of AMPA receptors which contain GluR2 may account for the fact that cartwheel cells respond to shocks to the auditory nerve with 100 ms excitatory postsynaptic potentials (EPSPs), while octopus cells, most of whose AMPA receptors lack GluR2, respond with 1 ms EPSPs.

The alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system. The GluR2 subunit confers calcium impermeability to AMPA receptors. Various calcium binding proteins play a role in calcium regulation within the neurons. This study sought to identify possible relationships between calcium binding proteins and glutamate receptor subunits, especially GluR2, in gerbil cochlear nucleus neurons. Our immunohistochemical observations reveal no particular correlation between GluR2 and calbindin; all the cell types show labeling for all the antibodies studied except calretinin. There was coincidence of strong GluR4 and strong parvalbumin staining in octopus cells, although calbindin was also present in these cells. This study suggests a possible relationship between parvalbumin and predominantly GluR4 containing receptors, even when calbindin is present. The absence of a strong inverse correlation between the presence of ionotropic AMPA receptor subunit GluR2 and calbindin suggests a more significant role of non-AMPA ionotropic glutamate receptors or other voltage-gated channels in the regulation of calcium in the neurons of cochlear nucleus. Alternatively, more detailed analysis of receptor composition at particular synapses and the subcellular localization of specific calcium binding proteins may be required.

The distribution of AMPA-preferring ionotropic glutamate receptors (GluR) within the gerbil lateral superior olive (LSO) was investigated immunocytochemically using antibodies to GluR1, 2, 2/3 and 4. Light microscopy showed GluR1 antibody preferentially labeling a population of small neurons located in the dorsal hilus and a population mainly at or near the margins of the LSO. GluR4 antibody strongly stained most large LSO neuronal somata and proximal dendrites including all principal cells. GluR2/3 antibody showed very modest staining and appeared in most cell types. GluR2 showed less intense neuronal staining than GluR2/3 and was observed as a punctate accumulation at the surface of some neuronal profiles. GluR1, 2, 2/3 and 4 immunoreactivity was found along dendrites of most large LSO neurons and in their somata. Postsynaptic specializations positive for GluR2 were rare on LSO somata compared to the high frequency of GluR4 and 1 specializations. Double labeling studies showed that different portions of the distal dendrites showed a preponderance of GluR1 or GluR4 subunits. Electron microscopic observations confirm similarities in the localization of immunoreactivity for the antibodies tested in the cytoplasm of somata and dendrites, but reveal differences at the plasmalemma, at synaptic appositions and appositions with glial processes. Receptor composition varied with cell type and location on cells.

Inhibitory synaptic transmission is known to play an important role during the maturation of central auditory pathways. While there is a lot of information on the modulatory role of glycine (Gly) on the postsynaptic target nuclei in the developing auditory brain stem, such a role for gamma-aminobutyric acid (GABA) in the lateral superior olive (LSO) of neonatal gerbil has been only recently reported (Kotak and Sanes [1997] Soc Neurosci Abst 23:1549; Kotak et al. [1998] J Neurosci 18:4646-4655). Here we present further immunohistochemical findings and the first ultrastructural evidence documenting a significant decrease in the postsynaptic localization of the beta2,3 subunit of the GABA(A) receptor from postnatal day (P)4 to P14 in the LSO of gerbil and the shift in the location of most of the staining from dendritic to astroglial over the same time course. There was a concomitant increase in staining for the Gly receptor (GlyR) anchoring protein, gephyrin. At the same time, GABA and Gly did not show a significant change in their staining pattern, suggesting that the transmitter levels are not particularly indicative of the inhibitory function in the neonatal gerbil LSO, but their receptors on the postsynaptic cells are. The observations of the present study suggest that the early GABAergic inhibition may be important in establishing appropriate synaptic contacts in the LSO of gerbil.

GABAergic and glycinergic circuits are found throughout the auditory brainstem, and it is generally assumed that transmitter phenotype is established early in development. The present study documents a profound transition from GABAergic to glycinergic transmission in the gerbil lateral superior olive (LSO) during the first 2 postnatal weeks. Whole-cell voltage-clamp recordings were obtained from LSO neurons in a brain slice preparation, and IPSCs were evoked by electrical stimulation of the medial nucleus of the trapezoid body (MNTB), a known glycinergic projection in adult animals. GABAergic and glycinergic components were identified by blocking transmission with bicuculline and strychnine (SN), respectively. In the medial limb of LSO, there was a dramatic change in the GABAergic IPSC component, decreasing from 78% at postnatal day 3 (P3)-P5 to 12% at P12-P16. There was an equal and opposite increase in the glycinergic component during this same period. Direct application of GABA also elicited significantly larger amplitude and longer duration responses in P3-P5 neurons compared with glycine-evoked responses. In contrast, MNTB-evoked IPSCs in lateral limb neurons were more sensitive to SN throughout development. Consistent with the electrophysiological observations, there was a reduction in staining for the beta2,3-GABAA receptor subunit from P4 to P14, whereas staining for the glycine receptor-associated protein gephyrin increased. Brief exposure to baclofen depressed transmission at excitatory and inhibitory synapses for approximately 15 min, suggesting a GABAB-mediated metabotropic signal. Collectively, these data demonstrate a striking switch from GABAergic to glycinergic transmission during postnatal development. Although GABA and glycine elicit similar postsynaptic ionotropic responses, our results raise the possibility that GABAergic transmission in neonates may play a developmental role distinct from that of glycine.

The distribution of immunoreactivity for the ionotropic N-methyl-D-aspartate (NMDA) receptor subunits was mapped in the cochlear nucleus of postnatal day (P) 7, P14, P21, and P28 gerbils. Frozen sections and serial plastic sections of tissue were incubated with antibodies to NMDAR1 (NR1), NMDAR2A (NR2A), NMDAR2A/B (NR2A/B), and NMDAR2B (NR2B). An overall diffuse stain was noted at P7 for NR1 and NR2A/B. Staining of neuronal somata in the dorsal cochlear nucleus molecular layer and fusiform cell layer, the posteroventral cochlear nucleus octopus cell area, and the anteroventral cochlear nucleus increased from P7 to P28. Staining of the neuropil (the unresolved mass of processes and axons, excluding only neuronal somata and distinctly stained proximal dendrites) of the deep dorsal cochlear nucleus and posteroventral cochlear nucleus showed a steady decrease, while molecular layer neuropil remained moderately stained. The NR2A antibody produced a distinctive staining of dendrites in the dorsal cochlear nucleus deep and fusiform cell layers seen first at P14 with increasing dendritic lengths stained at P21 and P28. Giant neurons of the deep dorsal cochlear nucleus were the most conspicuous somata stained by the NR2A. Their stained dendrites spanned much of the dorsal cochlear nucleus deep and fusiform cell layers and even extended into the octopus cell area of the posteroventral cochlear nucleus. Dendritic staining was also present in caudal and rostral posteroventral cochlear nucleus, first distinguishable at P14 and becoming increasingly strong. The Chemicon polyclonal NR2B antibody produced glial staining especially prominent in the caudal posteroventral cochlear nucleus and the dorsal cochlear nucleus fusiform cell layer, most intense at P7 and subsequently decreasing, although not disappearing, in all areas through P28. The Molecular Probes (Eugene, OR) polyclonal NR2B produced a light granular staining pattern over a number of somata but no glial staining. Neuropil staining was not prominent with either NR2B antibody. Differences in changes of neonatal immunoreactivity patterns in different populations of cochlear nucleus neuronal somata and dendrites for NR1, NR2A, NR2A/B, and NR2B suggest that alterations in some receptor composition is occurring over the period spanning the onset of hearing.

To investigate the etiology of hole formation in the gerbil and rat central auditory system, organotypic cultures were grown in control and veratridine-containing media. The latter condition is known to increase neuronal activity. Tissue was obtained at postnatal day 6 and grown for 6-9 days in vitro, a period prior to the formation of holes in vivo. In both rats and gerbils, veratridine led to the appearance of large numbers of holes, and these were phenotypically similar to those found in vivo. These results support the idea that hole formation is an activity-dependent phenomenon, and suggest that it is not restricted to the mature gerbil auditory system.

This study used immunocytochemical markers for various classes of glial cells to investigate the relationship between glial elements and microcysts in the gerbil auditory system at the light and electron microscopic level. Monoclonal antibodies S-100, GFAP and Rip were used on tissue from 3- and 12-month old animals and acutely deafened 12 month old animals to localize astrocytes and oligodendrocytes and their processes around microcysts. No differences in the number and distribution of astrocytes were found in the PVCN as a result of aging or deafening. S-100 and GFAP labeling showed a high correlation between astrocytic processes and microcysts. The results indicate that up to 80% of microcysts are either contacted by astrocytic profiles over much of their perimeter or are labeled internally by the astrocytic markers S-100 or GFAP. Some microcysts appear to originate in neuronal dendrites or in axons.

Perfusion of the gerbil cochlea with micromolar quantities of 3H-gamma-aminobutyric acid (GABA) results in rapid, selective labeling of 50-60% of the olivocochlear (OC) efferent terminals on afferent dendrites beneath the inner hair cells, and all of the efferent terminals beneath the outer hair cells. In order to identify the neurons from which these GABA-accumulating terminals originate, the cell bodies were localized by using retrograde transport of 3H-nipecotic acid, a metabolically inert GABA analog. With survival times of 6-30 hours after cochlear injection, myelinated OC efferent fibers and cell bodies were well labeled, with the greatest number being labeled at 12-18 hours. All of the labeled neurons belonged to the medial OC system, and no lateral OC neurons were labeled. It is concluded that the GABA-accumulating endings in the gerbil cochlea arise from medial OC neurons, and therefore that medial OC efferent neurons in this species project to both inner and outer hair cell regions.

This study was designed to test the hypothesis that microcysts in the gerbil auditory system are formed from neuronal somata. Six neuronal types (octopus, multipolar, bushy, elongate, miscellaneous and small) were distinguished, counted and measured along with the microcysts in the posteroventral cochlear nuclei (PVCNs) of 3, 12 and 36 month old gerbils. No decrease was observed in the numbers of neurons in any neuronal class, or in the neuronal population as a whole, in the PVCN of the gerbil as a function of age. Neither was any change observed in the PVCN area occupied by non-neuronal, non-microcyst elements. Neuronal sizes were unchanged between 3 and 12 months, but multipolar and bushy cells, as well as the total neuronal population decreased significantly in size between 12 and 36 months. The number and size of microcysts increased significantly between 3 and 12 months of age and accounts for increases in PVCN volume. The number and size of microcysts decreased significantly between 12 and 36 months. Thus, the appearance of microcysts can not result from the selective loss of any single class of neurons. Hearing was assessed in five 36 month old animals with auditory brainstem responses (ABR) and number and size of microcysts were found to correlate with hearing status, being largest and most numerous in animals with the best hearing, and smallest and fewest in the deaf animal. It is concluded that microcysts cannot represent a neurodegenerative disease of neuronal somata. Microcyst formation appears to be a dynamic process related to the degree of auditory stimulation.

Two populations of superior olivary neurons which project to different sensory cell regions in the cochlea also give off collateral projections to the ventral cochlear nucleus (VCN) and adjacent brainstem. To determine whether these VCN projections also have different targets they were characterized by selective retrograde amino acid transport. Retrograde transport of 3H-d-aspartate (D-ASP) selectively labeled the unmyelinated fibers and neurons of the lateral olivocochlear (OC) system including a dense collateral projection to the central VCN. Retrograde transport of 3H-nipecotic acid (NIP) labeled the myelinated fibers and neurons of the medial OC system, including collateral projections to the peripheral VCN, subpeduncular granule cells, and nucleus Y. Medial and lateral OC efferent collaterals thus innervate different regions of the CN. Lateral system collaterals overlap extensively with Type I spiral ganglion cell afferent input. They are well positioned to play a role in modulating afferent input to the central auditory system, as is the primary projection of these efferents to the cochlea. The medial system collaterals project near the recently described afferent projections of Type II spiral ganglion cells. The medial system collaterals may therefore be related to the function of outer hair cells, as the medial system primary axons appear to be in the cochlea.

The cochlea is well suited for studies of the uptake properties of auditory neurons and nonneuronal supporting cells. Probe concentrations of radioisotopically labeled amino acids, including putative neurotransmitters and their precursors, breakdown products, and blockers, can be introduced via the natural, fluid-filled channels of the inner ear. Uptake patterns can be mapped at cellular and intracellular levels using light and electron microscopic autoradiographic methods. The procedures for introduction of label, fixation, plastic embedment, and light and electron microscopic autoradiography are described with special reference to the cochlea. Labeling patterns observed with over 20 amino acids are summarized for hair cells, spiral ganglion neurons, efferents, and nonneural elements of the stria vascularis, limbus, and modiolus. Limitations on the interpretation of results and their implications for the general usefulness of the methods are discussed.

The option of predeposit autologous blood donation (PABD) before elective surgery has been gaining popularity as a means of eliminating the transmission of the acquired immune deficiency syndrome and hepatitis. It also prevents potential antigen sensitization and transfusion reactions. The use of PABD in pregnant women has been described, but its safety for both mother and fetus, especially in the first and third trimester, has not been established. After studying 16 third-trimester pregnant women with antenatal surveillance techniques and continuous fetal monitoring, we concluded that PABD is a safe procedure for both mother and fetus.

A fresh brain-slice preparation that allows simultaneous characterization of the anatomic localization of radioactively labeled substances in a variety of different auditory brain stem areas has been used to examine localization patterns for a number of putative neurotransmitter amino acids in the anteroventral and dorsal cochlear nuclei of the cat. Following incubation of fresh brain sections in oxygenated salt solutions containing micromolar amounts of the tritiated amino acids, differences in the pattern of label localization are revealed by light microscopic autoradiography. Electron microscopic autoradiography of the same material demonstrates that the label is restricted to different synaptic terminal populations. In the dorsal cochlear nucleus, gamma aminobutyric acid (GABA) and glutamic acid incubations label two different terminal populations in the molecular layer. Glycine incubation labels a population more uniformly distributed among the molecular, fusiform cell, and deep layers. In the anteroventral cochlear nucleus the pattern after GABA and glycine incubations is similar, with multiple endings on all spherical cells labeled. Studies are underway to characterize fully the different labeling patterns and the conditions under which they occur.

Light and electron microscope autoradiography were performed on goldfish (Carassius auratus) retinas incubated in [125I]labelled alpha-bungarotoxin. The toxin was bound preferentially to membrane receptors in the inner and outer plexiform layers. Binding was suppressed by 10(-5) M nicotine or 10(-5) M native alpha-bungarotoxin. Electron microscopic analysis of the outer plexiform layer (OPL) strongly suggested that alpha-bungarotoxin binding sites were located on small bipolar cell dendritic processes that invaginated rod and cone synaptic terminals, and on large bipolar cell dendritic processes more proximally situated in the OPL. Large horizontal cell processes in the OPL and horizontal cell processes that invaginated rod and cone synaptic terminals did not appear to be labelled.

In lizards (Sceloporus undulatus), long term (13 or 19 weeks) acclimation to an environment of 6 degrees C produces a striking increase in the argyrophilic neurofibrillar network in most large perikarya of the trochlear nucleus. In electron micrographs the cells contain numerous bundles of 10-30 regularly-spaced 90 A neurofilaments. In the cells from warm acclimated animals, a plexus of neurofibrils is seen by light microscopy. The electron micrographs show scattered neurofilaments and fewer, thinner bundles than in the cold. Within the cell bodies of the cold animals, glycogen particles are organized in regional accumulations from which other organelles are excluded except for the bundles of neurofilaments which are distributed throughout the cytoplasm. The aggregations of rough endoplasmic reticulum (RER) are also penetrated by the neurofilament bundles. The increased neurofilamentous network in the cold is not accompanied by obvious changes in the amount or distribution of RER or of microtubules which are present in limited numbers in both conditions. The dendrites of trochlear cells and axon terminals within the nucleus also show a cold induced increase in neurofilaments, as well as in the distinctive accumulations of glycogen particles.

The cochleas and brain stems of normal-hearing CBA/CaJ and CaH mice ranging in age from 1 to 18 months were examined by light microscopy to document normal age-related changes. At all ages examined, the cochlear morphologic structure appeared normal with no obvious loss of hair cells or spiral ganglion cells. In the cochlear nucleus, qualitative and quantitative changes were observed in the total nuclear volume, in globular cell size, and in neuronal packing density.

Correlated studies of the ear and brainstem in deaf and hearing white cats have demonstrated early and progressive changes both peripherally and centrally, including organ of Corti degeneration, loss of spiral ganglion cells and auditory nerve fibers and decrease of neuronal size in the medial superior olivary nucleus (MSO). Loss of synaptic appositions on MSO neuronal perikarya is already pronounced in the youngest deaf animal, a time before the spiral ganglion cell population has decreased significantly. Thus, spiral ganglion cell populations alone cannot be used as a reliable indicator of the integrity of the central auditory pathways.

Study was made of the dimensions of the nerves to the receptor organs in the anterior branch of the bullfrog vestibular nerve. The number of fibers and their diameters and trajectories in the nerve and Scarpa's ganglion were studied. A correlation was made between the anatomical and physiological properties of neurons identified with intracellular injections of horseradish peroxidase. The data suggest a specific pattern of innervation of the organs, organization of fibers and cells in the vestibular nerve, and significant correlation between the anatomical and physiological properties of individual neurons. A comparison was made between the information obtained from the bullfrog and that from other animals, which suggests a similarity between species and supports the hypothesis of a differential physiological role of the neurons in each vestibular organ according to anatomical characteristics.

This study characterizes morphologically the neurons residing within the matrix of the cat lateral superior olive (LSO), excluding the hili and myelinated axon envelope. Several light microscopic techniques including Golgi impregnations, Nissl stains, and acetylcholinesterase histochemistry were used, as well as electron microscopy. Five distinct classes of neurons have been identified: principal neurons, multiplanar neurons, marginal neurons, small neurons, and class 5 neurons. These neuronal classes differ in regard to their size and shape, dendritic organization, perikaryal synaptic density, and their relative numbers. Principal neurons compose approximately three-quarters of the LSO neurons. They are multipolar and uniplanar in their dendritic arborization, radiating from the hili in rostrocaudal planes perpendicular to the curvatures of the LSO. In transverse sections the principal cell perikarya are fusiform and bipolar, with mean dimensions of 23 X 11 microns. More than 60% of the surface of these cells is contacted by synaptic terminals. Multiplanar neurons (averaging 23 X 19 microns) compose only 11% of the LSO neuronal population. Their dendritic arborization is not restricted to any particular plane, and their somal surface receives synaptic contacts similar, in number and type, to principal cells. Marginal neurons, although they are similar to principal neurons in shape and dendritic arborization, differ in that they are generally smaller (averaging 20 X 10.5 microns). They also possess fewer axosomatic synaptic contacts (approximately 33%), are oriented perpendicularly to principal neurons, are limited in distribution to the contours of the LSO immediately beneath the myelinated axon envelope, and constitute only 4% of the neuronal population. Small neurons (mean dimensions = 9 X 8 micron) compose 8% of the LSO neurons. They possess a multiplanar array of primary dendrites and have nuclei with multiple deep infoldings. Small neurons have the fewest axosomatic synaptic contacts of all classes of LSO neurons (approximately 10%). Additionally, there are neurons that are similar to principal neurons, but receive fewer axosomatic contacts (approximately 33%). These cells have been tentatively identified as class 5 neurons until more information on this type allows for the assignment of a more descriptive name. A number of acetylcholinesterase-positive neurons are also found within the LSO, whose relationship to the other classes of neurons is presently unresolved. Possible functions of the multiple neuronal types are discussed.

Label localization was compared by light and electron microscopic autoradiography in the organ of Corti of the gerbil, following in vivo incubation with one of eight 3H-labeled amino acids. Following incubation with GABA, efferent endings underneath the outer hair cells were heavily labeled, as were all tunnel crossing fibers, many small fibers in the inner spiral bundle and some efferents beneath the inner hair cells. This pattern was absent following incubations with the GABA analog muscimol. Following L- and especially D-aspartic acid incubations, efferent endings under the inner hair cells and small fibers of the inner spiral bundle were preferentially labeled. It is concluded that these differential labeling patterns after GABA and aspartic acid incubations reflect two populations of efferent fibers in the organ of Corti: one terminating primarily upon the outer hair cells and one underneath the inner hair cells. Cochlear hair cells showed the highest level of labeling following incubation with alanine. Inner hair cells were more heavily labeled than outer hair cells following incubations with alanine and glycine. Hair cell labeling was comparable to that of supporting cells for most amino acids. However, for alanine and glycine the labeling of inner hair cells, and for L-aspartic and glutamic acid the labeling of all hair cells, was higher than that of structural cells. Labeling of hair cells was substantially lower than that of structural cells following taurine incubations.

Immunocytochemical staining has been widely used for localizing various hormonal antigens, protein markers and putative neurotransmitters in tissues. Immunostained sections can be examined light microscopically and specific areas selected for electron microscopic study.

1. The correlations between anatomic and physiological characteristics of primary afferent neurons innervating the anterior semicircular canal in the bullfrog were investigated. These characteristics were examined separately in large groups of neurons, and the direct correlations between them were established in a subset of neurons by means of intraaxonal recording and labeling. 2. Anatomic features of the anterior canalicular nerve that were related with fiber diameter were studied. This nerve was composed of an average of 1,142 fibers (standard deviation of 171 in 5 samples), of which 42% were less than 2 microns in diameter and 8% were greater than 7 microns. The nerve branched into 6 clearly defined bundles, whose fiber diameter-dependent composition could be determined in 5 samples. In the 2 center bundles, 32% of the fibers had diameters greater than 7 microns. In contrast, these thick fibers comprised only 4% of the fiber population in the 2 lateralmost bundles, in which 44% of the fibers had diameters less than 2 microns. The projections of labeled afferent fibers were traced into the neuroepithelium, and it was demonstrated that all thick fibers, even those of the lateral bundles, turned toward more central regions of the crista. Consequently, in the bullfrog, there is a clear predominance of thick afferent fibers innervating the anterior crista's central region and thin fibers in the peripheral region. 3. The dendritic morphology of the broad classes of afferent fibers (i.e., thick and thin) was elucidated. Individually labeled thick afferents possessed dendrites forming short, thick, clawlike extensions to contact a few hair cells. The thinnest afferents were labeled through extracellular horseradish peroxidase (HRP) injections. In contrast to the thick fibers, thin afferents were characterized by an unbranched trajectory with serially located bouton-like structures that were apposed to successive hair cells. 4. The characteristics of spontaneous firing and the responses to rotational stimuli were determined for 138 anterior canalicular neurons. Spontaneous firing rates ranged from 0 to 95 spikes.s-1. The coefficient of variation (CV) of spontaneous firing ranged from 0.12 to 2.5. Response gains to high- (0.5 and 0.4 Hz) and medium- (0.05 Hz) frequency sinusoidal acceleration stimuli were positively correlated with CV (P less than 0.001) for neurons with a CV value less than or equal to 0.5. The gain of neurons characterized by more irregular spontaneous firing (CV values greater than 0.5) was uncorrelated with CV.(ABSTRACT TRUNCATED AT 400 WORDS)

The cellular and cytoarchitectural features of the lateral superior olive, the medial superior olive, the superior paraolivary nucleus and the medial, lateral and ventral nuclei of the trapezoid body are described in C57BL/6 mice using Nissl, Bodian and Golgi techniques. Principal, spindle and marginal cells are present in a well-defined lateral superior olive. The dendrites of these cells run primarily within rostrocaudal sheets as in the cat. The principal cells of the medial nucleus of the trapezoid body are similar to the principal cells in the cat. Large multipolar cells characterize the lateral nucleus of the trapezoid body and bipolar cells with a medial-lateral orientation are found in the medial superior olive. The largest neurons are found in the superior paraolivary nucleus and the lateral superior olive, and the medial and ventral nuclei of the trapezoid body. While brain weight and neuronal packing density change with development, the characteristic location of cell groups and the shape and Nissl-staining pattern of neurons in the youngest brains examined were essentially unchanged in the adult mice, although dendritic maturation had occurred. The homologies of the C57BL/6 superior olivary complex nuclei with the same areas described in other mouse strains, rat and cat are discussed. This study expands our understanding of the organization of the superior olivary complex in an inbred strain of Mus musculus and relates it to other species. The data about changes occurring during postnatal maturation may aid in the interpretation of behavioral and physiological studies of neonatal plasticity of the auditory system.

Rosettes of platelets around granulocytes-platelet satellitism-previously described in Behcet's syndrome led to the discovery of a case that may have been 80 years in duration. A strong relationship between calcium ions and the phenomenon was suggested by its specificity in edetic-acidanticoagulated blood, and the subsequent migration of platelets on supravital preparation from around neutrophils upon addition of 0.2 M calcium chloride to heparinized EDTA-treated blood. Plasma from the patient was able to cause the phenomenon with donor granulocytes and platelets. Platelet agglutinins were also demonstrable. Specificity in Behcet's syndrome is possible, but remains unproved.

Electron microscopic analysis of the perikaryal surfaces of central and marginal cells in the cat medial superior olivary nucleus has revealed three differences in their relationships with synaptic terminals. Marginal cells have a smaller percentage of their surface covered by synaptic terminals; synaptic endings on marginal cells tend to be smaller in size; and a higher proportion of synaptic terminals on marginal cells contain small vesicles. These differences suggest that central and marginal cells differ in the information which they receive, as well as in the cells to which they pass their information.

The fibers from the nerves innervating each of the three semicircular canals and the saccule were labeled by injecting horseradish peroxidase extracellularly into these nerves. The projections into the various vestibular nuclei of each receptor were studied in transverse sections of the brain stem throughout the vestibular nuclear area. All five vestibular nuclei receive primary afferents throughout their areas. There are differences in the projection patterns of the canals. In the superior and ventral vestibular nuclei, the location of the projections depends on the crista injected. The anterior canal projects ventrally, the horizontal canal centrally, and the posterior canal more dorsally. Each canal, however, sends fibers to all areas, with overlap of fibers from the different cristae. The cerebellar nucleus receives uniform innervation from the three canals. The medial vestibular nucleus in the rostral and caudal areas receives only thin fibers from each canal, with considerable overlap. The descending nucleus in the rostral and caudal areas receives innervation from the cristae, also with considerable overlap, but with greater intensity in the ventral part of the caudal portion of the nucleus. Each crista sends fibers to the cerebellar granular layer and to the base of the cerebellar Purkinje cell layer. These fibers also innervate the reticular formation below the entry zone of the eighth nerve. The saccule innervates both the dorsal (acoustic) and the ventral nuclei, the latter in the most dorsal position. The innervation of the utricle could be ascertained only in the middle section of the descending and the medial nuclei, an area which does not receive significant innervation from the cristae. Primary afferent fibers course in the vestibular tract, forming a longitudinal bundle lateral to the vestibular nuclei. In the bundle the larger fibers are medially situated.

Contraction of the cricothyroideus muscles (CTMs), innervated by the superior laryngeal nerves (SLNs), modulates the voice by tilting the thyroid cartilage anteriorly onto the top of the cricoid and tensing the vocal cords. Either unilateral or bilateral paralysis of the SLNs is disabling for individuals with above-average voice demands. Some patients never compensate for this paralysis; there is no surgical procedure recognized to correct it. This study tested the hypothesis that surgical fusion of the thyroid and cricoid cartilages anteriorly can correct the problems of SLN injury by duplicating the mechanical tilt of the thyroid onto the cricoid cartilage normally produced by the CTMs. The SLNs were cut in 12 dogs. In six the cricoid and thyroid cartilages were fused anteriorly. Vocal cord and airway function was assessed preoperatively, immediately postoperatively, and 6 to 10 weeks after surgery. Following surgery there was no airway compromise and there appeared to be a more satisfactory compensation for the SLN paralysis in the fused larynges as compared with the unfused controls as determined by cinelaryngoscopic analysis.

The localization of 3H-labeled amino acids was compared by light and electron microscopic autoradiography in the spiral ganglion of the gerbil, following in vivo intracochlear incubations. Following incubations with taurine, a population of heavily labeled neurons could be distinguished from lightly labeled neurons. The heavily labeled population comprised 5-6% of spiral ganglion neurons, and included the least myelinated cells. Ultrastructurally, the heavily labeled neurons were characterized by a loosely coiled Schwann cell sheath covering the cell body, and the presence of abundant cytoplasmic microfilaments. The unlabeled cells showed a typical perikaryal myelin sheath and cytoplasm rich in rough endoplasmic reticulum. It is concluded that the spiral ganglion of the gerbil contains both Type I and Type II neurons, and that Type II neurons preferentially incorporate the amino acid taurine. Type II neurons are therefore biochemically as well as morphologically distinct from Type I neurons.

The cochlear uptake of amino acids which are putative neurotransmitters, or closely-related compounds, was examined autoradiographically in the gerbil. Hair cells showed no preferential uptake of most compounds tested. However, preferential accumulation of glutamine by cochlear hair cells was striking. Vestibular hair cells showed no affinity for this amino acid. Glutamine uptake by cochlear hair cells may play an important role in afferent synaptic transmission, by providing transmitter precursor and/or by clearing the synaptic cleft.

[3H]Nipecotic acid was shown to be preferentially accumulated by the same cochlear structures which selectively accumulate [3H]gamma-aminobutyric acid ([3H]-GABA), including the terminals of a subset of olivocochlear neurons. With both amino acids, olivocochlear fibers selectively transported label in a retrograde direction, from cochlea to brainstem. However, only [3H]nipecotic acid produced dense labeling, and labeling of cell bodies in the superior olive, presumably because it is metabolized very slowly. Nipecotic acid appears to provide a selective retrograde tracer, specific to neurons whose terminals exhibit preferential GABA uptake.

Light microscopic autoradiography and electron microscopic autoradiography were used to study the distribution of label in the cochlear efferents following in vivo incubation with tritiated amino acids. Two basic patterns of labeling were observed. These patterns correspond closely to the lateral and medial superior olivary complex (SOC) olivocochlear systems identified by Warr and Guinan ('79, Brain Res. 173:152-155). Our electron microscopic observations suggest that, at least in the gerbil, the complete separation of outer hair cell (OHC) versus inner hair cell (IHC) efferent innervation proposed by these investigators based upon light microscopic data does not occur. Rather, our data suggest that while the lateral SOC system supplies endings only to the region under the IHC, the medial SOC system may supply endings beneath both the IHCs and OHCs.

Although Corti in 1851 first described the presence of cochlear pigmentation in the stria vascularis (SV) of "very old" cats, modern studies have failed to find pigment consistently in the feline stria. While the variable presence of pigment in the feline SV would appear to contrast with this structure's uniform pigmentation in other mammalian species, variability in both the distribution and abundance of inner ear pigment has rarely been studied in any species. In the present study, the SV was examined light microscopically in sectioned material or whole-mounts from pigmented and albino animals of 5 species, including the cat, guinea pig, rabbit, ferret and mouse. In these species, the SV of each pigmented animal contained varying amounts of melanin pigment and none was found in the albino inner ear. Pigmented guinea pigs contained the most uniformly dense and least variable distribution of strial melanin, followed by the rabbit, mouse, ferret and cat. Several species also displayed more strial pigment apically and less basally. In cats, pigmented cells were principally located adjacent to the strial capillaries. Ultrastructural studies of the stria in pigmented cats revealed that these perivascular cells frequently contained an abundance of pigmented organelles and other structural features which allowed them to be distinguished from intermediate cells.

Two populations of olivocochlear (OC) neurons have been identified in the gerbil brain stem on the basis of differential labeling patterns of 3H-D-aspartic acid (D-ASP) and wheatgerm agglutinin-horseradish peroxidase conjugate (WGA/HRP) from the cochlear perilymph. While both populations are capable of uptake and retrograde uptake of WGA/HRP, one population accumulates and retrogradely transports D-ASP (D-ASP OC neurons) and the other does not (non-D-ASP OC neurons). D-ASP OC neurons are found in or near the lateral superior olive, are small in size, and receive very few synaptic contacts. The vast majority of these synapses contain small, mildly pleomorphic vesicles with scattered dense core vesicles. Synapses with distinctly larger pleomorphic vesicles have also been observed. These neurons possess all of the features common to neurons of the lateral olivocochlear system. Non-D-ASP OC neurons are found primarily in the ventral nucleus of the trapezoid body, as well as in the area between the medial superior olive and the medial nucleus of the trapezoid body. These neurons are larger and receive greater numbers and types of synaptic contacts than those found on D-ASP OC neurons. The 2 most common synapses found on non-D-ASP OC neurons are axosomatic ones containing small, mildly pleomorphic vesicles and scattered dense core vesicles similar to those seen on the D-ASP OC neurons, and axodendritic synapses containing large, round vesicles. Much less frequently observed are synapses containing small, round vesicles or ones containing predominantly flat vesicles. The ultrastructural features of the non-D-ASP OC neurons correspond to those described for neurons of the medial olivocochlear system.

The biochemical parameters of bone mineral metabolism in patients with nonhypercalcemic malignancy have not been extensively investigated. Therefore, a group of 29 such patients with different types of malignancy was studied. Ten patients received corticosteroids. In the entire group, serum ionized calcium (Ca2+), bone gla protein (BGP), 25-hydroxyvitamin D (25OHD), and 1,25-dihydroxyvitamin D (1,25(OH)2D) were all lower than in age-matched controls, and carboxy-terminal parathyroid hormone (CPTH) was higher. Although both corticosteroid- and noncorticosteroid-treated patients had decreased BGP values, the corticosteroid-treated patients had lower BGP levels than those not on steroids (4.24 +/- 0.70 SE vs. 11.50 +/- 2.20 ng/ml; P less than 0.005). Patients on corticosteroids had lower 1,25(OH)2D values than controls (18.81 +/- 2.71 vs. 27.83 +/- 1.17 pg/ml; P less than 0.01), whereas those not on corticosteroids had normal 1,25(OH)2D values. These results suggest that patients with nonhypercalcemic malignancy have nutritional vitamin-D deficiency and secondary hyperparathyroidism with perhaps corticosteroid-induced suppression of serum 1,25(OH)2D and BGP. The decreased levels of serum BGP in the nonsteroid-treated patients suggest, in addition, a defect in osteoblast function.

We have previously shown that perfusion of the gerbil cochlea with probe concentrations of 3H-D-aspartic acid (D-ASP) results in immediate, selective labeling of 50-60% of the efferent terminals under the inner hair cells, presumably by high-affinity uptake. The present study was undertaken to determine the origin of these endings. Twenty-four hours after cochlear perfusion with D-ASP, labeled neurons were observed in the ipsilateral, and to a much lesser extent in the contralateral, lateral superior olivary nucleus (LSO). The cells were small, primarily fusiform, and showed fewer synaptic contacts than other LSO cells. Combined transport of D-ASP and horseradish peroxidase indicated that all olivocochlear neurons within the LSO that projected to the injected cochlea were labeled by D-ASP. Labeled fibers coursed dorsally from the LSO, joined contralateral fibers that had passed under the floor of the fourth ventricle, and entered the VIIIth nerve root at its ventromedial edge. Adjacent to the ventral cochlear nucleus (VCN), densely labeled collateral fibers crossed the nerve root to enter the VCN. Labeled fibers and terminals were prominent in the central VCN. Neither retrograde transport of D-ASP by medial olivocochlear and vestibular efferents nor anterograde transport by VIIIth nerve afferents was observed. The D-ASP-labeled cells and fibers are clearly lateral olivocochlear efferents. Retrograde transport of D-ASP thus allows the cells, axons, and collaterals of the lateral olivocochlear system to be studied, morphologically, in isolation from other cells that project to the cochlea. Since the olivocochlear neurons are almost certainly cholinergic, retrograde amino acid transport does not necessarily identify the primary neurotransmitter of a neuron. Rather, it indicates the presence of selective uptake by the processes of that neuron at the site of amino acid injection. Retrograde labeling appears to be markedly enhanced by the use of metabolically inert compounds such as d-isomer amino acids.

Five morphologically distinct classes of neurons can be identified within the neuropil of the gerbil lateral superior olivary nucleus (LSO) by using a variety of histological techniques and electron microscopy. The physical features of these five classes resemble those found in the cat LSO and are identified, by using criteria and nomenclature established for the cat, as principal neurons, multiplanar neurons, marginal neurons, small neurons, and class 5 neurons. Principal cells compose approximately 75% of the total LSO neuronal population. They possess a discoid dendritic organization and are oriented rostrocaudally, perpendicular to the transverse curvatures of the LSO. Roughly 8% of the LSO population is composed of multiplanar neurons, whose dendritic fields are not restricted to any single plane of section. Both principal and multiplanar neurons share similar cytoplasmic features, and greater than 65% of their perikaryal surface is in contact with synaptic terminals. Small neurons compose approximately 11% of the LSO neurons, have the lowest percentage of their somal surface contacted by synaptic terminals (approximately 8%), and are found mostly in the middle/medial portions of the LSO. Marginal neurons, which compose approximately 6% of the LSO population, appear similar to principal neurons at the light microscopic level except that they are found along the contours of the LSO, oriented orthogonal to principal neurons. Approximately 28% of the somal surface of marginal neurons is in contact with synaptic terminals. The class 5 neuronal somata receive a similar number of axosomatic synaptic contacts as marginal neurons (approximately 31%) but are found well within the matrix of the LSO, aligned parallel to principal neurons. Class 5 neurons share the same light microscopic features as principal neurons and can be identified electron microscopically based only on the reduced percentage of somal surface occupied by synaptic terminals.

The central projections of the vestibular end organs in the bullfrog Rana calesbeiana were analyzed by using horseradish peroxidase labeling of the primary vestibular afferents. Separate extracellular injections were made of the anterior branch, the posterior branch, the ampullary nerve of each of the three semicircular canals, and the branch to the saccule. The anterior and posterior branches of the bullfrog eighth nerve, each containing both vestibular and auditory fibers, merge and enter the brain stem as a single nerve root. The thin caliber fibers of the anterior branch enter the brain stem on the ventral-posterior aspect of the nerve and immediately divide dichotomously into ascending (rostral) and descending (caudal) branches. The thick caliber fibers of the anterior branch enter the brain stem on the ventral-anterior aspect of the eighth nerve and traverse medially into the alar plate before dividing into ascending and descending branches. The primary afferent fibers of the vestibular nerve innervate an ipsilateral area of the brain stem which extends caudally from the rhombencephalon at the level of the twelfth nerve nucleus and rostrally up to and including the cerebellar nucleus and the cerebellum. The following vestibular nuclei can be identified by the fact that they receive primary vestibular afferents: the ventral vestibular nucleus, medial vestibular nucleus, descending vestibular nucleus, superior vestibular nucleus, and cerebellar nucleus. The dorsal (acoustic) nucleus, which receives primary auditory input, also receives afferents from the saccule. In addition to these nuclei, the cerebellum and the reticular formation receive significant primary input from the various vestibular receptors. The primary vestibulo-cerebellar fibers terminate mainly among the granular cells of the lobus auricularis and of the corpus cerebelli on the ipsilateral side. Each of the three semicircular canals projects into the cerebellum, while no such projection was observed in the saccular nerve preparations. Fibers from each of the three semicircular canals project to all of the vestibular nuclei. Heavily labeled large neurons, presumably vestibular efferents, are seen in the ipsilateral reticular formation, adjacent to the seventh motor nucleus.