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The auditory nerve of birds and mammals exhibits differences and similarities, but given the millions of years since the two classes diverged from a common ancestor, the similarities are much more impressive than the differences. The avian nerve is simpler than that of mammals, but share many fundamental features including principles of development, structure, and physiological properties. Moreover, the available evidence shows that the human auditory nerve follows this same general organizational plan. Equally impressive are reports that homologous genes in worms, flies, and mice exert the same heredity influences in man. The clear implication is that animal studies will produce knowledge that has a direct bearing on the human condition.

At maturity, the AMPA receptors of auditory neurons exhibit very rapid desensitization kinetics and high permeability to calcium, reflecting the predominance of GluR3 flop and GluR4 flop subunits and the paucity of GluR2. We used mRNA analysis and immunoblotting to contrast the development of AMPA receptor structure in the chick cochlear nucleus [nucleus magnocellularis (NM)] with that of the slowly desensitizing and calcium-impermeable AMPA receptors of brainstem motor neurons in the nucleus of the glossopharyngeal/vagal nerves. The relative abundance of transcripts for GluRs 1-4 changes substantially in auditory (but not motor) neurons after embryonic day (E)10, with large decreases in GluR2 and increases in GluR3 and GluR4. Relative to the motor neurons, NM neurons show a higher abundance of flop isoforms of GluRs 2-4 at E10, suggesting that auditory neurons are already biased toward expression of flop isoforms before the onset of synaptic function at E11. Immunoreactivities in NM show very distinct developmental patterns from E13 onward: GluR2 declines by >90%, GluR3 increases threefold, and GluR4 remains relatively constant. Our results show that there are a series of critical points during normal development, most occurring after the onset of function, when rapid changes in receptor structure (occurring via both transcriptional and post-transcriptional control mechanisms) produce the specialized AMPA receptor functions that enable auditory neurons to accurately encode acoustic information., (Copyright 2002 Wiley Periodicals, Inc.)

Zinc is widely distributed in the central nervous system (CNS), it functions normally as a synaptic modulator, and it contributes to neuronal death under pathologic conditions. Zinc colocalizes with glutamate in excitatory synapses, and the presence of zinc is well characterized in the synapses of the auditory system. Since chick cochlear nucleus neurons depend upon synaptic activation of metabotropic glutamate receptors (mGluRs) for maintenance and survival, the goal of this study was to determine (1) if zinc is released from the eighth nerve calyces onto nucleus magnocellularis (NM) neurons in the chick cochlear nucleus, and, if so, (2) what effect it has on group I mGluR-mediated calcium homeostasis of these neurons. Using in vitro slices and a fluorescent dye relatively specific to vesicularized zinc, we show that zinc is indeed localized to the presynaptic calyces and is released upon nerve stimulation or KCl depolarization. Experiments employing fura-2 calcium imaging show that zinc inhibits group I mGluR release of calcium from internal stores of NM neurons and disrupts activity-dependent calcium homeostasis in a manner identical to the mGluR5-specific antagonist 2-methyl-6-(phenylethynyl)pyridine. The mGluR1-specific antagonist 7-hydroxyiminocyclopropan-[b]chromen-la-carboxylic acid ethyl ester did not affect release of calcium from stores by the nonspecific mGluR agonist aminocyclopentane dicarboxylic acid, nor did it affect activity-dependent calcium homeostasis. We conclude that zinc is present in and released from the glutamatergic eighth nerve calcyes. The presence of zinc inhibits mGluR5, a major component of calcium homeostasis of NM neurons, and plays a modulatory role in the activity-dependent, mGluR-mediated calcium homeostasis of auditory neurons.

The ionotropic glutamate receptor (GluR) subtype known as the AMPA receptor, which mediates rapid excitatory synaptic transmission in many regions of the nervous system, is composed of four different protein subunits, termed GluRs 1-4. The functional properties of each AMPA receptor are determined by the relative levels of GluRs 1-4 and by post-transcriptional modifications of these proteins through mRNA editing and alternative exon splicing. The present paper reviews the published evidence for (1) localization of mRNAs and immunoreactivity for GluRs 1-4 in the cochlea and subcortical central nervous system auditory pathways of mammals and birds, and (2) involvement of AMPA receptors in synaptic transmission in the auditory system. Recent biochemical and electrophysiological evidence concerning the specialized properties of AMPA receptors on brainstem auditory neurons is also reviewed, along with data concerning how these properties emerge during normal development.

Although dependence on afferent synaptic activity has been shown for central neurons in every sensory system, the mechanisms of afferent maintenance of target sensory neurons are not understood. Neurons in the cochlear nucleus (CN) require afferent activity for maintenance and survival. One of the earliest changes seen after activity deprivation is an increase in intracellular calcium that leads to the death of 30% of the neuronal population. Sixty minutes after deafferentation, the surviving neurons show increased phosphorylation of the transcription factor calcium/cAMP response element-binding protein (CREB). CREB phosphorylation in activity-deprived CN neurons is dependent on increased intracellular calcium resulting from influx through AMPA receptors and is mediated by calcium/calmodulin-dependent kinases and protein kinase A. We conclude that in CN neurons, the deafferentation-induced increase in calcium activates at least two kinase pathways that phosphorylate CREB in surviving neurons. We hypothesize that this phosphorylation results in the transcription of genes containing the calcium/cAMP response element within their promoter regions, and these genes code for proteins that allow the neurons to compensate for their hypercalcemic, activity-deprived state.

Neurons of the chick cochlear nucleus, nucleus magnocellularis (NM), require eighth nerve activation of metabotropic glutamate receptors (mGluRs) for maintenance of intracellular calcium homeostasis. Interrupting this activation results in an increase in intracellular calcium concentration ([Ca(2+)](i)) followed by cell atrophy, degeneration, and death of many neurons. Although these phenomena are well characterized in late embryonic and posthatch chicks, little is known about the role of mGluRs and calcium homeostasis during the development of synaptic activity in NM. Using Fura-2 imaging, fluorescent immunohistochemistry, and Western immunoblotting, we investigated (1) the expression and function of group I mGluRs and their role in calcium regulation during development of NM, and (2) the expression of two other key molecules involved in regulating neuronal [Ca(2+)](i) : inositol trisphosphate receptors (IP(3)Rs) and sarcoplasmic/endoplasmic reticulum calcium ATPases (SERCAs). Confocal imaging of Fluo-3-labeled NM was used to investigate the kinetics of global NM neuron calcium signals. Measurements were made at four ages that extend from before synaptic function begins in NM, through functional onset, to mature patterns of spontaneous activity, namely, embryonic days (E) 10, 13, 15, and 18. mGluR5, mGluR1, and SERCA expression peaked at E13 and then decreased with age. IP(3)R expression increased to peak at E18. [Ca(2+)](i) response to mGluR activation increased with age. The rise time of [Ca(2+)](i) signals in NM neurons did not change with development, but E13 neurons were slower to reestablish baseline [Ca(2+)](i). These results suggest that the mGluR-mediated calcium homeostasis of NM neurons develops in parallel with synaptic activity and appears to be refined with increasing synaptic activity., (Copyright 2000 Wiley-Liss, Inc.)

Brainstem auditory neurons in the chick nucleus magnocellularis (NM) express high levels of the neuron-specific calcium-binding protein calretinin (CR). CR has heretofore been considered a diffusible calcium buffer that is dispersed uniformly throughout the cytosol. Using high-resolution confocal microscopy and complementary biochemical analyses, we have found that during the development of NM neurons, CR changes from being expressed diffusely at low concentrations to being highly concentrated beneath the plasma membrane. This shift in CR localization occurs at the same time as the onset of spontaneous activity, synaptic transmission, and synapse refinement in NM. In the chick brainstem auditory pathway, this subcellular localization appears to occur only in NM neurons and only with respect to CR, because calmodulin remains diffusely expressed in NM. Biochemical analyses show the association of calretinin with the membrane is detergent-soluble and calcium-independent. Because these are highly active neurons with a large number of Ca2+-permeable synaptic AMPA receptors, we hypothesize that localization of CR beneath the plasma membrane is an adaptation to spatially restrict the calcium influxes.

1. Neurons in the brainstem auditory pathway exhibit a number of specializations for transmitting signals reliably at high rates, notably synaptic AMPA receptors with very rapid kinetics. Previous work has not revealed a common structural pattern shared by the AMPA receptors of auditory neurons that could account for their distinct functional properties. 2. We have used whole-cell patch-clamp recordings, mRNA analysis, immunofluorescence, Western blots and agonist-evoked cobalt uptake to compare AMPA receptors on the first-, second- and third-order neurons in the chick ascending auditory pathway with those on brainstem motor neurons of the glossopharyngeal/vagal nucleus, which have been shown to have very slow desensitization kinetics. 3. The results indicate that the AMPA receptors of the cochlear ganglion, nucleus magnocellularis and nucleus laminaris share a number of structural and functional properties that distinguish them from the AMPA receptors of brainstem motor neurons, namely a lower relative abundance of glutamate receptor (GluR)2 transcript and much lower levels of GluR2 immunoreactivity, higher relative levels of GluR3 flop and GluR4 flop, lower relative abundance of the C-terminal splice variants GluR4c and 4d, less R/G editing of GluR2 and 3, greater permeability to calcium, predominantly inwardly rectifying I-V relationships, and greater susceptibility to block by Joro spider toxin. 4. We conclude that the AMPA receptors of auditory neurons acquire rapid kinetics from their high content of GluR3 flop and GluR4 flop subunits and their high permeability to Ca2+ from selective post-transcriptional suppression of GluR2 expression.

Manipulating the expression of a protein can provide a powerful tool for understanding its function, provided that the protein is expressed at physiologically-significant concentrations. We have developed a simple method to measure (1) the concentration of an overexpressed protein in single cells and (2) the covariation of particular physiological properties with a protein's expression. As an example of how this method can be used, teratocarcinoma cells were transfected with the neuron-specific calcium binding protein calretinin (CR) tagged with green fluorescent protein (GFP). By measuring GFP fluorescence in microcapillaries, we created a standard curve for GFP fluorescence that permitted quantification of CR concentrations in individual cells. Fura-2 measurements in the same cells showed a strong positive correlation between CR-GFP fusion protein expression levels and calcium clearance capacity. This method should allow reliable quantitative analysis of GFP fusion protein expression.

Motoneurons have been shown to be particularly sensitive to Ca2+-dependent glutamate excitotoxicity, mediated via AMPA receptors (AMPARs). To determine the molecular basis for this susceptibility we have used immunocytochemistry, RT-PCR, and electrophysiology to profile AMPARs on embryonic day 14.5 rat motoneurons. Motoneurons show detectable AMPAR-mediated calcium permeability in vitro and in vivo as determined by cobalt uptake and electrophysiology. Motoneurons express all four AMPAR subunit mRNAs, with glutamate receptor (GluR) 2 being the most abundant (63.9+/-4.8%). GluR2 is present almost exclusively in the edited form, and electrophysiology confirms that most AMPARs present are calcium-impermeant. However, the kainate current in motoneurons was blocked an average of 32.0% by Joro spider toxin, indicating that a subset of the AM PARs is Ca2+-permeable. Therefore, heterogeneity of AMPARs, rather than the absence of GluR2 or the presence of unedited GluR2, explains AMPAR-mediated Ca2+ permeability. The relative levels of flip/flop isoforms of each subunit were also examined by semiquantitative PCR. Both isoforms were present, but the relative proportion varied for each subunit, and the flip isoform predominated. Thus, our data show that despite high levels of edited GluR2 mRNA, some AMPARs are Ca2+-permeable, and this subset of AMPARs can account for the AMPAR-mediated Ca2+ inflow inferred from cobalt uptake and electrophysiology studies.

Glutamate activates three distinct classes of ionotropic receptors: AMPA, kainate and NMDA. AMPA receptors (AMPARs) are of particular importance as they mediate the majority of fast excitatory synaptic transmission and are implicated in a variety of neurological disorders [B. Bettler, C. Mulle, AMPA and kainate receptors, Neuropharmacology 34 (1995) 123-139]. Functional AMPARs are believed to be a heteromer comprising a combination of four closely related subunits, GluRs1-4 [B. Bettler, C. Mulle, AMPA and kainate receptors, Neuropharmacology 34 (1995) 123-139]. Diversity of AMPARs is obtained through multiple combinations of AMPAR subunits, by alternative splicing of subunits at the flip/flop and/or C-terminal sites, and by mRNA editing of a single amino acid at multiple sites [M. Hollmann, M. Hartley, S. Heinemann, Ca2+ permeability of KA-AMPA-gated glutamate receptor channel depends on subunit composition, Science 252 (1991) 851-853; B. Sommer, K. Keinanen, T.A. Verdoorn, W. Wisden, N. Burhashev, A. Herb, M. Kohler, T. Takagi, B. Sakmann, P.H. Seeburg, Flip and flop: a cell-specific functional switch in glutamate-operated channels in the CNS, Science 249 (1990) 1580-1585; B. Sommer, M. Kohler, R. Sprengel, P.H. Seeburg, RNA editing in brain controls a determinant of ion flow in glutamate-gated channels, Cell 67 (1991)]. The subunit combination, editing status, and splice variant expression have profound effects on channel kinetics and can serve as predictors of the channel's properties [M. Hollmann, M. Hartley, S. Heinemann, Ca2+ permeability of KA-AMPA-gated glutamate receptor channel depends on subunit composition, Science 252 (1991) 851-853; B. Sommer, K. Keinanen, T.A. Verdoorn, W. Wisden, N. Burhashev, A. Herb, M. Kohler, T. Takagi, B. Sakmann, P.H. Seeburg, Flip and flop: a cell-specific functional switch in glutamate-operated channels in the CNS, Science 249 (1990) 1580-1585; B. Sommer, M. Kohler, R. Sprengel, P.H. Seeburg, RNA editing in brain controls a determinant of ion flow in glutamate-gated channels, Cell 67 (1991)]. In this manuscript, we detail procedures for profiling AMPAR composition, namely: relative subunit ratios, expression of flip/flop isoforms, Q/R and R/G editing status, and Ca2+ permeability using small amounts of cDNA from identified cell populations., (Copyright 1998 Elsevier Science B. V.)

To identify chick GluR4 isoforms, we used PCR to amplify a C-terminal region that is the site of alternative splicing in rat. We report here the cloning of three novel chick GluR4 isoforms. GluR4c has a 113-bp insert in the C-terminus, is expressed in flip and flop isoforms, is most strongly expressed in the cerebellum, midbrain and forebrain, and appears from embryonic day (E) 2.5 through at least post-hatching day (P) 2, with a peak of expression at E17. GluR4d has a 184-bp segment inserted at the 4c splice site, occurs as flip and flop isoforms, is expressed most strongly in cerebellum, hindbrain and forebrain, and is present from E11 through P2, with peak expression at E17. GluR4s is a shortened form that lacks the nominal 4th transmembrane and flip/flop domains and shares a common C-terminal region with GluR4. GluR4s is expressed most strongly in the hindbrain and cerebellum and its expression increases from E11 through P2. Experiments on purified cerebellar cells show that glia express GluR4c and GluR4d at combined levels nearly twice that of GluR4 and that flip isoforms predominate. In contrast, granule cells express GluR4c and GluR4d at a level comparable to GluR4 and express GluR4s at a level less than half that in cerebellar glia. Thus, the independence of alternative splicing at the flip/flop and C-terminal splice sites allows seven alternatively spliced forms of GluR4 to exist in chick CNS. This structural diversity increases the potential for functional diversity in neuronal and glial GluRs incorporating GluR4.

The expression of the calcium-binding protein calretinin (CR) in the chick brainstem auditory nuclei angularis (NA), laminaris (NL), and magnocelularis (NM) was studied during normal development and after deafening by surgical removal of the otocyst (embryonic precursor of the inner ear) or columella (middle ear ossicle). CR mRNA was localized by in situ hybridization by using a radiolabeled oligonucleotide chick CR probe. CR immunoreactivity (CR-IR) was localized on adjacent tissue sections. CR mRNA signal in the auditory nuclei was expressed at comparable levels at embryonic day (E)9 and E11 and increased thereafter to reach the highest levels in posthatch chicks. CR-IR neurons were apparent in NM and NA at E11 and in NL by E13, and CR-IR increased in all three auditory nuclei thereafter. Neither unilateral nor bilateral otocyst removal caused detectable changes in the intensity of CR mRNA expression or CR-IR in the auditory nuclei at any of the several ages examined. Similarly, columella removal at posthatching day 2 or 3 failed to significantly affect CR mRNA or CR-IR levels at 3 hours, 1 day, or 3-4 days survival times. We conclude that cochlear nerve input is not necessary for expression of either calretinin mRNA or protein and that the profound decrease in sound-evoked activity caused by columella removal does not affect the maintenance of CR expression after hatching.

The role of glutamate receptors in regulating programmed neuronal death and deafferentation-induced neuronal death in the brainstem auditory nuclei was studied by in ovo drug administration to chick embryos. The nucleus laminaris (NL) undergoes programmed developmental cell death of 19% between embryonic day 9 (E9) and E17. The AMPA/kainate receptor antagonist CNQX, when administered at doses of 200-300 microg/d from E8 to E15, prevented programmed neuronal death in NL through at least posthatching day 8, without producing anatomical or behavioral abnormalities. 3-((RS)-2-Carboxypiperazin-4-yl)-propyl-1-phos-phonic acid, an antagonist of NMDA receptors, had no effect on normal cell death in the NL. CNQX, given from E8 to E15 or only from E8 to E10, also blocked the 33% neuronal loss in the nucleus magnocellularis (NM) that follows surgical destruction of the otocyst on E3, a procedure that deafferents NM neurons by preventing formation of the cochlear nerve. Treatment either with CNQX or the more highly selective NBQX from E8 to E10, before the onset of synaptic transmission in NM and NL, was also effective in preventing normal neuronal death in NL. Analysis of the effects of CNQX or NBQX on spontaneous embryonic motility at E10 showed that the doses effective in preventing neuronal death suppressed motility for <8 hr. We conclude that periodic blockade of AMPA/kainate receptors can protect CNS neurons against subsequent programmed cell death or deafferentation-induced death.

Early destruction of the otocyst (embryonic precursor of the inner ear) in chick embryos results in complex changes in developing central auditory pathways. In the cochlear nucleus angularis (NA) and nucleus magnocellularis (NM), 30% to 40% of the neurons die after otocyst removal, the survivors are shrunken, and some neurons in the NA migrate to an abnormal position in the brain stem. The characteristic forms of cochlear nucleus neurons develop normally in the absence of cochlear nerve input, however. In the nucleus laminaris (NL), development of normal dendritic size is dependent on a normal inner ear, but most of the highly specialized dendritic organization of this nucleus, which is important for low-frequency sound localization, can develop normally in the absence of cochlear influences. Otocyst removal induces formation of a permanent functional aberrant axonal projection to the ipsilateral NM from the contralateral NM. Although these aberrant axons form functional glutamatergic synapses, these show immature functional properties, suggesting that cochlear nerve inputs are necessary for normal maturation of glutamate receptors on auditory neurons. Treatment of chick embryo for a brief period in ovo with 6-cyano-7-nitroquinoxaline-2,3-dione, a quinoxalinedione antagonist of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-kainate subtype of glutamate receptor, completely and permanently prevents the neuronal loss in the NM produced by otocyst removal. The work reviewed has 1) identified aspects of development in central auditory neurons that are perturbed by profound early sensorineural loss, 2) identified aspects of development that appear independent of cochlear influences, and 3) suggested a potential chemotherapeutic approach to prevention of central neuronal death after early damage to the cochlea.

Using oligonucleotide primers, we have amplified and sequenced the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors from the brain of 17-day-old chick embryos. Both flip and flop isoforms of each of these glutamate receptors (GluR) were identified and cloned. Nucleotide comparisons showed that the two isoforms for each chick receptor subtype were 71-78% identical, whereas homologous chick and rat isoforms were 94-98% identical. Reverse transcriptase-polymerase chain reaction and restriction enzyme analysis were employed to identify regional variation in flip and flop levels of each AMPA receptor. Flip isoforms of GluR 1-3 predominated in forebrain, while flop variants of GluR 1-4 were more prevalent in the cerebellum. This differential regional expression suggests that alternative splicing of AMPA receptor subunits contributes importantly to synaptic diversity in chick central nervous systems.

Kainate, an agonist at non-NMDA glutamate receptors, evoked strong concentration-dependent cobalt accumulation in the chick brain stem auditory nuclei angularis, laminaris (NL) and magnocellularis (NM). This effect could be blocked completely by the AMPA/kainate receptor antagonist CNQX and the AMPA antagonist GYKI 53655 but not by antagonists of NMDA receptors or voltage-sensitive calcium channels. Kainate (30 microM) evoked cobalt uptake from embryonic day (E)9 through E21, with a peak intensity at E15. Before E13, uptake occurred mainly in NL but declined markedly in NM after E15 and ceased in NL by E21. We conclude that calcium-permeable non-NMDA receptors are transiently expressed at the time when mature neuronal number, form and synaptic connectivity are established.

Conantokins are peptide antagonists of the N-methyl-D-aspartate (NMDA) subclass of excitatory amino acid receptors. We compared conantokin-G and AP5 antagonism of NMDA receptor activity expressed in cultures of neonatal rat cerebellar granule cells, using the fluorescent calcium indicator dye fura-2. The results were consistent with the binding of two molecules of agonist (NMDA) for channel activation and one antagonist molecule (AP5) for inhibition. However, conantokin-G antagonism was more complex: the peptide inhibited only approximately 70% of the elevation of intracellular free calcium produced by NMDA. These results, when combined with previous ones [8], suggest that conantokin-G may have different affinities for, and functional effects on, different subtypes of NMDA receptor complexes expressed in the mammalian CNS.

Surgical destruction of the otocyst in chick embryos prevents formation of the *** ear, abolishes normal cochlear input to the cochlear nucleus (nucleus magnocellularis, NM) and results in axons from the contralateral NM forming (in addition to their normal bilateral endings in nucleus laminaris, NL) a novel and functional aberrant projection to the deafferented NM. We studied the pharmacology of synaptic transmission at aberrant synapses in an in vitro preparation of the brainstem in chick embryos and hatchlings. Transmission at the aberrant synapses (as with cochlear nerve synapses in NM and NM synapses in NL) is blocked by the quinoxalinedione antagonists CNQX and NBQX, confirming the presence of excitatory amino acid receptors of the non-NMDA subtype. At cochlear nerve synapses in NM, the antagonist potency of NBQX normally decreases rapidly after embryonic day (E)18 (IC50 = 0.69 +/- 0.06 microM, mean +/- S.E.M.), reaching an asymptotic value by E21 (IC50 = 2.7 +/- 0.4 microM) that is maintained at least through posthatching day (P)14 (IC50 = 3.6 +/- 0.3 microM). In the case of the aberrant endings, the potency of NBQX remained (from E21 [IC50 = 0.6 +/- 0.1 microM] through at least P14[IC50 = 0.5 +/- 0.1 microM]) at levels that are statistically indistinguishable from the E18 value for normal cochlear nerve synapses.(ABSTRACT TRUNCATED AT 250 WORDS)

We reacted dispersed cultures of newborn rat cerebellar granule cells with serum, purified IgG, and CSF from patients with type IIa ("anti-Hu") antibody response accompanying paraneoplastic neurologic syndromes. All type IIa sera, IgGs, and CSFs, but not those of normal or cancer controls, produced bright nuclear immunofluorescence of cultured granule neurons. Type IIa serum and CSF labeled proteins of 35-42 kd in rat granule cell blots, identical in molecular weight to proteins labeled by type IIa antibodies in blots of human granule cells. IgGs eluted from the 35-42 kd band in blots of rat granule cells labeled proteins of similar molecular weights in blots of human granule cells and produced typical type IIa immunostaining of human cerebellar sections. Human IgG could be identified in nuclei and cytoplasm of neurons incubated for 72 hours with 2/4 type IIa sera tested, but not with normal sera. Type IIa sera or IgGs from 4/7 patients produced specific lysis of rat granule cells in the presence of complement, as compared with controls using normal serum or heat-inactivated complement. Prolonged (7-day) incubation of cultures with type IIa antibody without complement also resulted in specific lysis, whereas incubation with normal serum or serum from neurologically normal patients with small-cell carcinoma of the lung did not. Rat granule cell cultures provide a valuable in vitro system with which to study the interaction of type IIa antibody with neurons. The present study provides the first reported evidence that type IIa antibodies may cause cell injury directly, in the absence of lymphocyte-mediated immune response.

The effects of the glutamate receptor antagonist gamma-D-glutamylaminomethyl sulfonic acid (GAMS) on inward currents induced by bath application of kainic acid (KA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) were studied with single-electrode voltage clamp methods in Xenopus oocytes injected 3-5 days previously with mRNA from the brain of E16-17 chick embryos. Both AMPA and KA induced smooth inward currents, with Hill coefficients of 1.5 (AMPA) and 2.1 (KA). GAMS, at concentrations up to 1 mM, produced no reliable antagonism of AMPA-induced currents but showed a consistent, dose-dependent and reversible antagonism of KA-induced responses; the slope of the Schild plot was 0.76 and the pA2 value 4.32. In the presence of GAMS, however, the Hill coefficient for AMPA is reduced significantly and approaches unity, suggesting that AMPA interacts with both KA and AMPA binding sites on chick brain glutamate receptors. The selectivities of three quinoxalinedione antagonists (6,7-dinitroquinoxaline-2,3-dione [DNQX], 6-cyano-7- nitroquinoxaline-2,3-dione [CNQX] and 6-nitro-7-sulfamoyl-benzo(F)quinoxaline-2,3-dione [NBQX]) were then compared with that shown by GAMS. DNQX, CNQX and NBQX all blocked the effects of both KA and AMPA completely, competitively, reversibly and dose-dependently, with Schild-plot slopes very close to 1.0. Against AMPA, observed pA2 values were 6.58 for DNQX, 6.43 for CNQX and 6.77 for NBQX. Against KA, pA2 values were 6.42 for DNQX, 6.56 for CNQX and 7.21 for NBQX.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of axon-target cell interactions in shaping the prevalence and distribution of synaptic densities was studied in an experimentally-induced aberrant functional projection from the chick cochlear nucleus (nuc. magnocellularis, NM) to the contralateral NM. Contact with an abnormal target appears to induce in the aberrant axons a pattern of presynaptic densities resembling that in normal cochlear nerve endings in NM. NM axon terminals induced similar numbers of postsynaptic densities (PSDs) per unit length of membrane apposition in both their normal and abnormal targets but the longer membrane apposition in the highly invaginated aberrant terminal in NM results in a significantly greater amount of postsynaptic density per ending. These auditory neurons thus appear able to adjust a variety of features to permit assembly and maintenance of a novel functional synapse.

The developmental pharmacology of excitatory amino acid (EAA) receptors in the chick cochlear nucleus (nucleus magnocellularis, NM) was studied by means of bath application of drugs and recording of synaptically-evoked field potentials in brain slices taken from chicks aged embryonic day (E) 14 through hatching (E21). The abilities of various EAA agonists (N-methyl-D-aspartate [NMDA], kainic acid, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA]) to suppress postsynaptic responses by depolarization block and of EAA antagonists ((3-[RS]-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid [CCP], dizocilpine [MK-801], 6-nitro-7-sulfamoyl-benzo(F)quinoxaline-2,3 dione [NBQX], 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX] and 6,7-dinitroquinoxaline-2,3-dione [DNQX]) to suppress these responses directly were assessed quantitatively. The results support the existence of NMDA receptors in NM and suggest that the ability of these receptors to influence synaptically-evoked responses declines dramatically during the last week of embryonic life. The results similarly suggest that the non-NMDA receptors in NM undergo changes in density and/or function during a period of development when the cochlear nucleus is undergoing a variety of morphological and functional transformations.

Because kainic acid (KA) is more potent than other excitatory amino acids (EAAs) in affecting synaptic transmission in the cochlear nucleus, previous reports have concluded that primary afferent neurotransmission to the cochlear nucleus in birds and mammals is mediated by KA-preferring non-N-methyl-D-aspartate (non-NMDA) EAA receptors. Since this conclusion is at odds with a number of studies suggesting that rapid excitatory neurotransmission in the CNS is mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring non-NMDA receptors, we re-examined the pharmacology of synaptic transmission between the cochlear nerve and nucleus magnocellularis (NM) in chickens, using bath application of drugs and recording of field potentials evoked in NM by electrical stimulation of the cochlear nerve in vitro. A series of EAA agonists produced complete, concentration-dependent and reversible suppression of postsynaptic responses: the order of potency was domoic acid (DO) greater than KA greater than AMPA much greater than quisqualic acid much greater than L-glutamic acid (Glu). Three quinoxalinedione antagonists of non-6-nitro-7-sulphamobenzo[f]quinoxaline-2,3-dione NMDA receptors also produced complete, concentration-dependent and reversible suppression of postsynaptic responses in NM without affecting the presynaptic action potential; the half-maximal inhibitory concentrations (IC50's) were 2.7 +/- 0.4 microM for 6-nitro-7-sulphamobenzo[f]quinoxaline-2,3-dione (NBQX), 5.3 +/- 0.1 microM for 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and 10.6 +/- 1.2 microM for 6,7-dinitroquinoxaline-2,3-dione (DNQX).(ABSTRACT TRUNCATED AT 250 WORDS)

The venom of the North American funnel-web spider Agelenopsis aperta contains a variety of arylamine toxins (the alpha-agatoxins) that paralyze insects by blocking glutamatergic neuromuscular transmission. We have tested six synthetic alpha-agatoxins for their ability to antagonize glutamate receptor function in mammalian brain. These compounds produce, at submicromolar concentrations, noncompetitive inhibition of N-methyl-D-aspartate (NMDA) receptor-mediated elevations in the concentration of cytosolic free calcium in cultured rat cerebellar granule neurons. In contrast, the alpha-agatoxins are relatively weak antagonists of elevations in the cytosolic free calcium concentration induced by non-NMDA receptor agonists. The alpha-agatoxins also produce reversible suppression of the NMDA receptor-mediated excitatory postsynaptic potential in rat hippocampal slices at concentrations that have little effect on the non-NMDA receptor-mediated population spike. We conclude that the alpha-agatoxins are selective and reversible noncompetitive antagonists at NMDA receptors in mammalian brain.

The aims of the present study were to determine which structures in the stria vascularis (SV) may depend upon the presence of pigmented melanocytes both for normal morphology and for the expression of gentamicin ototoxicity in the inner ear. These pigment-dependent influences were inferred through comparisons of the SV in pigmented guinea pigs and in albinos containing nonpigmented melanocytes. Results were obtained from 6 albino and 8 pigmented guinea pigs given gentamicin, and from 3 albino and 3 pigmented control animals not receiving the drug. One-month old animals received gentamicin daily (100 mg/kg) for 14 days and recovered for an additional 14 days before being prepared for electron microscopy. The SV from each of the 4 cochlear turns was analyzed using stereological point counting procedures. In control animals, differences were found in the higher cochlear turns, where volume density for the marginal cells in albinos was abnormally large (turns 3 and 4), while the volume density for intermediate cells (melanocytes) was abnormally small (turn 3). Cell volume estimates for the intermediate cells were significantly smaller in the albino than pigmented control animals in the higher cochlear turns, indicating that functional abnormalities may be found in the albino cochlea. In animals exposed to gentamicin, marginal cell volume density was reduced significantly in turn 4 of albinos, but not in any region of the pigmented inner ears. Radial area of SV and estimates of the absolute volumes for marginal cells in albinos given gentamicin also were significantly reduced in turn 1 compared to their controls; such differences were not observed in the pigmented animals. The results indicate that marginal cell size is significantly reduced in albino but not pigmented animals 14 days after gentamicin exposure, and further suggest a role of pigmented melanocytes in ameliorating gentamicin-induced cochlear damage.

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in rat cerebellar granule cells using the fluorescent indicator fura-2. Culturing the cells as monolayers on plastic squares which could be placed into cuvettes allowed measurements of [Ca2+]i to be performed on large and homogeneous populations of CNS neurons. Granule cells so cultured maintained low levels of [Ca2+]i (around 90 nM) which increased promptly upon the addition of various excitatory amino acids including N-methyl-D-aspartate (NMDA). Increases in [Ca2+]i elicited by NMDA were inhibited by Mg2+ (1 mM) and often potentiated by glycine (1 microM). The addition of TTX or strychnine (5 microM each) did not alter responses to NMDA or NMDA plus glycine. Cytosolic Ca2+ responses to NMDA/glycine were dependent on the presence of extracellular Ca2+ and were unaffected by concentrations of nifedipine or verapamil that blocked increases in [Ca2+]i elicited by K+ depolarization. Responses elicited by NMDA/glycine were inhibited competitively by 2-amino-5-phosphonovalerate or 3-((+-)-2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid and non-competitively by MK-801 or Mg2+. HA-966 and 7-chlorokynurenate inhibited responses to NMDA alone and blocked competitively the potentiating effects of glycine. The results demonstrate NMDA-mediated increases in [Ca2+]i in cerebellar granule cells that arise solely from influx of extracellular Ca2+ through dihydropyridine-insensitive channels. The strict dependence of the NMDA-evoked response on extracellular Ca2+ provides little evidence for a coupling of NMDA receptors to inositol phosphate metabolism and mobilization of intracellular Ca2+. The effect of various agents on NMDA/glycine-induced increases in [Ca2+]i parallels their effects on ligand binding to or current flow through the NMDA receptor-channel complex. The measurement of cytosolic Ca2+ in this preparation of neuronal cells thus appears especially well suited for assessing, on a functional level, the regulation of NMDA receptors in the CNS.

The receptors mediating excitatory neurotransmission from the cochlear nucleus (nuc. magnocellularis, NM) to third-order auditory neurons in nucleus laminaris (NL) of the chicken were studied using in vitro brain slices, bath application of drugs, and electrophysiological recording of postsynaptic field potentials. Postsynaptic responses in NL were blocked completely, in a concentration-dependent and reversible fashion, by bath application of the broad-spectrum excitatory amino acid (EAA) antagonist kynurenic acid, the 'non-NMDA' EAA receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX), and the EAA agonists domoic acid, kainic acid, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and quisqualic acid. The selective NMDA receptor antagonists 3-[-)-2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP) and dibenzocycloheptenimine (MK-801) had no effect. The results demonstrate that excitatory input from the cochlear nucleus to NL is mediated by non-NMDA (G2) EAA receptors which exhibit some of the pharmacologic features typical of the AMPA receptors defined by binding studies.

A low molecular weight fraction (AG2) containing arylamine compounds has been isolated from venom of the spider Agelenopsis aperta. When administered intravenously or intracerebroventricularly, AG2 produces dose-dependent suppression of behavioral convulsions induced in rats by kainic acid, picrotoxin, or bicuculline. The low molecular weight compounds in spider venoms may provide novel tools for anticonvulsant research and therapy.

Conantokin-T, a 21-amino acid peptide which induces sleep-like symptoms in young mice was purified from the venom of the fish-hunting cone snail, Conus tulipa. The amino acid sequence of the peptide was determined and verified by chemical synthesis. The peptide has 4 residues of the modified amino acid, gamma-carboxyglutamate (Gla). The sequence of the peptide is: Gly-Glu-Gla-Gla-Tyr-Gln-Lys-Met-Leu-Gla-Asn-Leu-Arg-Gla-Ala-Glu-Val-Lys- Lys-Asn-Ala-NH2. Conantokin-T inhibits N-methyl-D-aspartate (NMDA) receptor-mediated calcium influx in central nervous system neurons. This observation suggests that like conantokin-G (a homologous Conus peptide with recently identified NMDA antagonist activity) conantokin-T has NMDA antagonist activity. A sequence comparison of conantokins-T and -G identifies the 4 Gla residues and the N-terminal dipeptide sequence as potential key elements for the biological activity of this peptide.

Cochlear nerve axons and their target neurons in nucleus magnocellularis (NM) of the chicken undergo extensive parallel structural transformations during development. Between embryonic days 12 and 17 (E12-E17), each immature highly branched axon condenses into a mature calyxlike ending applied to a single NM neuron. Simultaneously, NM neurons are transformed from multipolar cells with many long dendrites into spherical unipolar neurons with only an axon. We tested the hypothesis that cochlear nerve input is necessary for the transformation of NM cells by surgically destroying one otocyst on E3, thereby preventing formation of the nerve. Nucleus magnocellularis neurons from embryos at E11-E12, E13-14, and E17-18 were stained by horseradish peroxidase injected into their axons or by a Golgi-Hortega method. In camera lucida drawings, the number of dendrites on each cell was counted and the cell's position along the posterior-to-anterior and lateral-to-medial axes of the nucleus quantified. At E11-12, neurons throughout NM on both the deafferented and normally innervated sides of the brain have about ten dendrites. At E13-14, there is a steep spatial gradient in dendritic number bilaterally; cells at anteromedial positions have about two dendrites, while cells in posterolateral positions have an average of nine dendrites. By E17-18, only 14% of the neurons on either side have a dendrite, and these cells are evenly distributed throughout the nucleus. We conclude that cochlear nerve axons are not required for normal spatio-temporal gradients of dendritic loss, even though the absence of these axons causes severe atrophic changes in NM.(ABSTRACT TRUNCATED AT 250 WORDS)

The axonal endings on the somata and dendrites of third-order auditory neurons in nucleus laminaris (NL) were measured and classified in thin-sectioned material from adult chickens. Two methods were used to determine which ending types arise from second-order auditory neurons in nucleus magnocellularis (NM): 1) degeneration of axonal endings in NL after transection of the crossed dorsal cochlear tract (CTrX) carrying the axons of each NM to the contralateral NL, and 2) injection of horseradish peroxidase (HRP) into NM or the CTrX to label endings in NL by anterograde transport. About 42% of the perikaryl surface of NL neurons and 63% of the dendritic surface are apposed by axon terminals arising from NM; these endings are also prevalent on the axon hillock and initial segment. The NM endings are characterized by round, clear synaptic vesicles distributed at an average density of 76/microns2 behind small punctate synaptic junctions with thick synaptic densities. These endings degenerate preferentially after transection of the CTrX and are the only endings consistently labeled after HRP injections. About 31% of the perikaryl surface of NL and 10% of the distal dendritic surface are apposed by a prominent non-NM axonal ending type. This ending is characterized by a synaptic vesicle density of 135/microns2 and a single large area of synaptic contact which bears very slight densities on both pre- and postsynaptic membranes. On the perikaryon, about 80% of these non-NM terminals end on the cell body proper, with the rest found on proximal dendrites or capping the short somatic processes or spines occasionally seen in NL. The distribution of the two ending types does not differ significantly along the posterior-to-anterior axis of NL.

Surgical extirpation of the otocyst on embryonic day (E) 3 in chick embryos prevents formation of the cochlear nerve and results in development of an aberrant axonal projection from the contralateral cochlear nucleus (nucleus magnocellularis, NM) to the deafferented NM. We have studied the morphology of this projection using horseradish peroxidase injections in NM axons and light and electron microscopy. The ability of the projection to activate its target neurons synaptically was assessed by means of extracellular microelectrode recording from in vitro preparations of the chick brainstem. The aberrant projection arises as a vertically directed branch from the contralaterally traveling NM axon at the medial border of nucleus laminaris (NL). This axonal branch forms boutonal endings that may terminate anywhere in NM but are most common in its ventral and medial regions. In our experiments, this projection is not seen on the unoperated side of experimental animals or in normal controls from E11 onward but is found on the operated sides of all experimental animals, including those with bilateral removal of the otocysts. The aberrant projection persists at least from E11 through hatching and has essentially identical features in unilaterally and bilaterally lesioned animals. The endings of the aberrant projection are boutonal in form and, in the electron microscope, exhibit all of the elements associated with normal synapses. Electrophysiological studies confirm that stimulation of the aberrant axons can elicit postsynaptic responses in NM and suggest that these synapses use an excitatory amino acid neurotransmitter.

The tonotopic and topographic organization of the bilateral projection from second-order auditory neurons of nucleus magnocellularis (NM) to nucleus laminaris (NL) was examined in young chickens. In one group of birds, the NM axons which innvervate the contralateral NL were severed by cutting the crossed dorsal cochlear tract at the midline. Heavy terminal degeneration in NL was confined to the neuropil area immediately ventral to the perikaryl lamina. Very little degeneration was seen in the dorsal neuropil region. In a second series of animals, the charactertistic frequency (CF) of cells in an area of NM was first determined by microelectrode recording techniques and then a small electrolytic lesion was made through the recording electrode. Following survival periods of 24-48 hours, the distribution of projections from the lesioned area to the ipsilateral and contralateral NL was examined using the Fink-Heimer method. As previously described in the pigeon, projections from NM terminate densely in the neuropil region immediately dorsal to the ipsilateral NL cell bodies and ventral to the perikaryl layer on the contralateral side, providing each NL neuron with segregated binaural innervation. Lesions in any area of the NM produced degeneration confined to a limited caudo-rostral and medio-lateral portion of both laminar nuclei. To investigate this topographic relationship, the cuado-rostral extents of the lesion in NM and of the resulting degeneration in both NL were determined. Linear regression and correlation analyses then related these positional values to each other and to the CF found at the center of each lesion. All correlations were highly significant and ranged from 0.78 between the position of the lesion in NM and CF to 0.91 between the caudo-rostral position of degeneration in the NL ipsilateral and contralateral to the lesion. It is concluded that neurons in NM project in a very discrete topographic, tonotopic and symmetrical fashion to NL on both sides of the brain, contributing to the binaural response properties and tonotopic organization of neurons in NL. The results also suggest that the organization of projections from NM to NL could provide a mechanism for the differential transmission delay required by a "place" model of low-frequency sound localization.

The pattern of primary auditory projections to the brain stem of young chickens was investigated using terminal degeneration methods and orthograde transport of horseradish peroxidase (HRP) or tritiated amino acid. Of particular interest was the question of whether nucleus laminaris (NL) receives primary afferents. A study of silver-stained degeneration pattersn in nucleus magnocellularis (NM) and NL at three intervals following unilateral interruption of the cochlear nerve revealed that by 48 hours after the lesion, degenerating terminals were found only in the ipsilateral nucleus angularis (NA), NM and lagenar projection areas but not in NL. Five- and eight-day survival times, however, also revealed degeneration bilaterally in NL. The appearance of terminal degeneration in NL at the longer survival times is attributed to the previously-reported severe and rapid transneuronal degeneration of neurons in NM following deafferentation and not to the presence of cochlear nerve terminals in NL. Injection of HRP or tritiated proline into the basilar papilla produced patterns of labeling similar to that seen in the 2-day degeneration material; HRP reaction product or autoradiographic label were seen only in the ipsilateral NA and NM and in the ipsilateral projection areas of the macula lagena but not in either NL. The patterns of primary auditory projections revealed by the three methods were quite similar to each other and to that previously reported for the pigeon and confirm the conslucion that the laminar nucleus of chickens does not receive primary afferents.

Previous studies using various excitatory amino acid antagonists have shown that synaptic transmission between the auditory nerve and the cochlear nucleus of chickens (nuc. magnocellularis; NM) is mediated by non-N-methyl-D-aspartate (non-NMDA) receptors. In the present study we have attempted to define the subclass of non-NMDA receptor in the NM by examining the effects of various excitatory amino acid agonists on synaptically evoked field potentials in an in vitro preparation of the chicken brain stem. Both quisqualate and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), whose actions operationally define the quisqualate receptor class, caused variable and weak depression of evoked responses in the NM, as did L-glutamate. Kainic acid, on the other hand, completely blocked postsynaptic responses at micromolar concentrations. We conclude that kainate-preferring non-NMDA receptors play a predominant role in mediating transmission in the NM.

The hypothesis that synaptic transmission between the auditory nerve and the cochlear nucleus is mediated by an excitatory amino acid acting through N-methyl-D-aspartate (NMDA) receptors was examined in an in vitro preparation of the chicken brainstem. The ability of various bath-applied excitatory amino acid receptor antagonists to inhibit synaptically-evoked responses was assessed by recording field potentials from nucleus magnocellularis (NM) following electrical stimulation of the cochlear nerve. Antagonists that selectively block responses mediated by NMDA receptors, such as D-alpha-aminoadipate and 2-amino-5-phosphonovalerate, were without effect on evoked transmission in NM. In contrast, antagonists that additionally act on non-NMDA receptors, such as cis-2,3-piperidine dicarboxylate and gamma-D-glutamylglycine, reversibly suppressed transmission. The results indicate that (1) transmission in the chicken auditory system is mediated by non-NMDA receptors, and (2) a substance(s) chemically akin to aspartate and glutamate may be the transmitter used by the auditory nerve in NM.

In a previous paper (Brain Res., 260:1-9, 1983) we reported that albino cats show abnormal auditory brainstem evoked responses that appear to arise from structural defects in or near the superior olivary complex. In the present study, neuronal cross-sectional area in brainstem nuclei was compared in albino and normally pigmented adult cats. The albinos were true tyrosinase-negative (cc) and should not be confused with the deaf white cat (W); the albinos are not deaf. Neurons in the medial superior olivary nucleus (MSO) of albinos were, on average, 41% smaller than in pigmented animals; there was no overlap in the neuronal size distributions for the two groups of animals. Cell size in the lateral superior olive, medial nucleus of the trapezoid body, ventral nucleus of the lateral lemniscus, anteroventral cochlear nucleus, dorsal cochlear nucleus, and facial nucleus was also smaller (by 9-21%) in albinos than in pigmented animals but none of these differences was statistically reliable. In the abducens nucleus, neurons were 12% larger in albinos than in pigmented animals, demonstrating that neuronal size in the albinos is not uniformly smaller. Several lines of evidence suggest that the auditory system defects in albinos are related to abnormal pigmentation rather than to other gene effects. It is possible that a subtle pigment-related disruption of inner ear development in albinos results in a central cascade of atrophic changes along the auditory pathway.

Ascending auditory projections to the nucleus mesencephalicus lateralis pars dorsalis (MLd) were studied in white Leghorn chickens by means of unilateral injections of horseradish peroxidase into the MLd and by injections of tritiated leucine into nucleus angularis or the combined nucleus magnocellularis and nucleus laminaris. The experiments showed that nucleus angularis sends an extensive projection to the contralateral MLd and a smaller projection to the rostral pole of the ipsilateral MLd; the lagenar region contributes to these bilateral connections. Nucleus angularis also projects bilaterally to the superior olive and nucleus ventralis lemnisci lateralis and to the contralateral nucleus lemnisci lateralis pars ventralis and dorsal nucleus of the lateral lemniscus. Projections from nucleus laminaris were demonstrated to the ipsilateral superior olive, to the contralateral lemniscal nuclei and a small medial region in MLd bilaterally; the contralateral projection is much denser than the ipsilateral one. Other nuclei having ascending connections with MLd include the contralateral superior olive, the ipsilateral nucleus lemnisci lateralis pars ventralis, the contralateral nucleus ventralis lemnisci lateralis and the contralateral MLd. The ipsilateral superior olive and nucleus ventralis lemnisci lateralis also project to MLd but much more sparsely than in their contralateral projection. Although several of these findings correspond with auditory connections previously shown in the pigeon brainstem, they differ fundamentally in that we find both nucleus angularis and nucleus laminaris projecting to different areas of the MLd on both sides of the brain. In particular, our observation that the cochlear nucleus has bilateral connections with MLd demonstrates an important avian similarity with the brainstem auditory pathways of other terrestrial vertebrates.

The third-order auditory neurons of the avian nucleus laminaris (NL) have distinct dorsal and ventral dendritic tufts that receive their predominant synaptic input from, respectively, the ipsilateral and contralateral cochlear nucleus. Beginning about embryonic day (E) 14 in the chick and continuing for some weeks after hatching, NL neurons undergo a complex series of morphological transformations that result in the formation of a steep anteromedial-to-posterolateral gradient of increasing total dendritic length across the nucleus. This gradient perfectly parallels the tonotopic axis of NL. It has been proposed that acoustically evoked activity in the auditory pathway contributes importantly to formation of the gradient of dendritic length in NL and to several other features of dendritic development. The present experiment tested this hypothesis by surgically removing both otocysts (embryonic precursors of the inner ear) and studying the developing NL in the absence of peripheral input. The results of a quantitative study of Golgi-impregnated material show that at E17 both the steepness and predictability of the spatial gradient of dendritic length in operated animals are indistinguishable from normal. Similarly, the correlation of dorsal and ventral dendritic lengths on individual cells in operated animals is not significantly different from normal. The absolute length of both dendritic fields is reduced below normal, although only dorsal dendrites show a statistically reliable (14%) decrease. This is a significantly smaller effect than the 44% length reduction seen previously in animals with unilateral otocyst removal (T.N. Parks: J. Comp. Neurol. 202:47-57, '81); symmetrical afferent input appears more important to the regulation of NL dendritic length than the absolute level of this input.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of a moderately severe monaural conductive hearing loss on the development of neuronal size in the avian nucleus magnocellularis (NM) was investigated. NM is considered to be the homologue of the mammalian anteroventral cochlear nucleus and receives large calyceal synaptic endings from the cochlear nerve. Silicone plastic earplugs which produce a 40 dB broadband conductive hearing loss were placed in one external auditory canal of chick embryos on the 18th day of incubation. After hatching, all animals were housed in communal brooders and sacrificed at 4, 10, 25, and 60 days of age. Nissl-stained sections from paraffin- and plastic-embedded brains were used to sample neuronal cross-sectional areas in NM on the deprived and nondeprived sides of the brain. These samples were obtained separately in each brain from three posterior-to-anterior percentage quartiles within NM. Statistical analyses of these data showed that the severity of deprivation-induced cell size changes in NM varied as a function of both age and position. In the 4- and 10-day groups, no significant deprived-versus-nondeprived differences in neuronal size were seen in any area of the nucleus. At 25 days after hatching, only the third posterior-to-anterior quartile (i.e., 50-74%) showed a significant difference; this difference was also significantly greater than those in the second and fourth quartiles at this age. By 60 days, all three sampled areas in NM showed highly significant differences (averaging 12%) in mean neuronal cross-sectional area. Cell size values from the deprived and nondeprived sides of 60-day-old experimental animals were also compared with values from 60-day-old control subjects. Whereas the deprived NM cells were significantly smaller than controls, there was no evidence for a reliable hypertrophy in the nondeprived cells. Thus, the principal effect of the acoustic deprivation produced was to retard continued growth of the deprived neurons after 4 days of age.

We have examined the effects of a number of excitatory amino acid antagonists on transmission at the cochlear nerve-nucleus magnocellularis synapse in the chicken. Using an in vitro preparation and bath application of drugs, we studied the effects of kynurenic acid and several related substances, streptomycin and a selective N-methyl-D-aspartate receptor antagonist, DL-alpha-aminosuberate. The last compound had no effect on evoked transmission. Of the various kynurenic acid-related compounds tested, only kynurenic and dipicolinic acid selectively altered responses in nucleus magnocellularis. Quinolinic acid, a kynurenic acid analogue that is structurally akin to dipicolinic acid but which acts selectively at N-methyl-D-aspartate receptors, was without effect. The effect of kynurenic acid was solely inhibitory, completely blocking postsynaptic responses with a potency dependent on the frequency of nerve stimulation. No such frequency dependence was seen with dipicolinic acid although this compound also completely suppressed evoked responses. In addition dipicolinic acid potentiated postsynaptic responses at concentrations only slightly lower than those causing inhibition. Streptomycin inhibited responses in nucleus magnocellularis but this effect seems to result partially from the ability of the drug to inhibit presynaptic calcium influx. Our finding that selective antagonists of N-methyl-D-aspartate receptors were ineffective while antagonists of both receptor types, such as kynurenic and dipicolinic acids, inhibited evoked responses reinforces the conclusion that postsynaptic receptors mediating transmission at this synapse are of the non-N-methyl-D-aspartate type [Nemeth et al. (1983) Neurosci. Lett. 40, 39-44].(ABSTRACT TRUNCATED AT 250 WORDS)

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.