Developmental changes in the subcellular localization of calretinin.

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.

Afferent influences on the development of the brain stem auditory nuclei of the chicken: otocyst ablation.

The effects of embryonic deafferentation on the morphological development of the avian cochlear nuclei, n. angularis (NA) and n. magnocellularis (NM), were investigated. The right otocyst was surgically removed from chick embryos at 55 to 60 hours of incubation and the subsequent development of total volume, neuron number, and neuron cross-sectional area were studied with quantitative methods in animals sacrificed at 2-day intervals between embryonic days 9 and 19 and at 28 days posthatching. The development of NA and NM is severely affected by otocyst ablation. Between embryonic days 9 and 19, a large group of NA neurons in the medioventral portion of the nucleus on the operated side moves to an ectopic ventromedial position, while the remainder of this nucleus stays in its normal dorsolateral position. Beginning about day 13 of incubation, the normal increase in the volume of NA and the size of its neurons becomes progressively retarded and 40% of its neurons are lost. The growth of NM is also retarded after day 11 of incubation and the growth of mean neuron size is retarded after day 15. There is a 30% loss of neurons in NM which begins after embryonic day 11. The results indicate the primary cochlear fibers make a critical contribution to the growth and maintenance of their target neurons. The absence of this facilitative influence following otocyst ablation becomes apparent just at the time synapses would normally be formed between the the primary auditory afferents and the brain stem auditory neurons. The abnormal movement of neurons in nucleus angularis to an ectopic position after otocyst ablation suggests that primary auditory afferents may serve to stabilize the position of their target cells within the developing brain.

Morphology of axosomatic endings in an avian cochlear nucleus: nucleus magnocellularis of the chicken.

The axonal endings formed on the somata of neurons in the brainstem auditory nucleus magnocellularis (NM) were measured and classified in thin-sectioned material from adult chickens. Degeneration of primary endings after destruction of the basilar papilla and labeling of cochlear nerve fibers by injection of horseradish peroxidase (HRP) into the inner ear were used to determine which ending types arise from the cochlear ganglion. About 60% of the perikaryal surface is apposed by primary type terminals. These primary endbulbs are characterized by round clear synaptic vesicles distributed at an average density of 63 vesicles/micrometers 2 and a number of small, punctate, highly asymmetrical synaptic contacts. The primary type is the only class of endings which disappears after destruction of the basilar papilla and which is consistently labeled after HRP injections into the ear. These endings probably account for the "fast" EPSP seen in NM during stimulation of the cochlear nerve. NM neurons receive two types of nonprimary ending. About 13% of the perikaryal surface is apposed by a morphologically homogeneous class of small "symmetrical" endings; these are characterized by a flattened rhomboidal shape, numerous mitochondria, frequent coated vesicles, and small round or ovoid synaptic vesicles at an average density of 165 vesicles/micrometers 2. Most of the length of the apposition between ending and cell body is occupied by a synaptic complex with thin symmetrical presynaptic and postsynaptic densities. These endings were frequently found on short somatic processes. The second nonprimary axosomatic ending type in NM is most easily identified in experimental material; these endings occupy about 5% of the cell surface area and have a distinctly rounded profile in cross section. These endings typically exhibit clear round synaptic vesicles at a density of 111 vesicles/micrometers 2 arrayed before synaptic contacts which occupy a substantially larger fraction of the total apposition length than in the endbulbs. Many of these synaptic contacts show well-defined presynaptic grids and have postsynaptic densities intermediate in width between the endbulbs and the symmetrical endings. This second type of nonprimary ending may be responsible for the long-latency excitatory post-synaptic potentials seen in intracellular recordings from NM during electrical stimulation of the cochlear nerve. The morphology and distribution of the three ending types does not differ significantly along the posterior-to-anterior axis of NM.

Organization and development of the brain stem auditory nuclei of the chicken: primary afferent projections.

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.

Age- and position-dependent effects of monaural acoustic deprivation in nucleus magnocellularis of the chicken.

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.

Organization and development of brain stem auditory nuclei of the chicken: tonotopic organization of n. magnocellularis and n. laminaris.

Extracellular recordings of responses to tone-burst stimulation were used to determine the tonotopic organization of n. magnocellularis (NM) and n. laminaris (NL) in hatching chickens. NM cells show "primary-like" response patterns to ipsilateral stimulation, and are arranged in dorso-ventral isofrequency columns. Units responding to the highest frequency tones (about 4,100 Hz) are situated at the rostromedial pole of the medial division. Units with lower characteristic frequencies (CF's) are found at successively caudal and lateral sites, until extremely low CF's ( less than 500 Hz) are represented dorsoventrally in the daudolateral tail of the lateral division. No evidence was found of auditory input to the region which receives projections from the macula lagena. NL receives polarized, binaural, excitatory input. Units have similar CF's and thresholds to tones presented to either ear. The tonotopic organization in NL matches that found in NM--high CF's rostromedially and low CF's caudal and lateral. Quantitative procedures were developed for relating CF to the position of a unit within either nucleus. These analyses account for 79% and 89% of the frequency variance found within NM and NL, respectively, and predict the CF of a neuron by its position within each nucleus.

Organization and development of brain stem auditory nuclei of the chicken: organization of projections from n. magnocellularis to n. laminaris.

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.

Induction of aberrant functional afferents to the chick cochlear nucleus.

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.

A developmental gradient of dendritic loss in the avian cochlear nucleus occurring independently of primary afferents.

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)

Morphology and origin of axonal endings in nucleus laminaris of the chicken.

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.

Experience-independent development of dendritic organization in the avian nucleus laminaris.

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)

Developmental changes in metabotropic glutamate receptor-mediated calcium homeostasis.

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.

Auditory brainstem anomalies in albino cats: II. Neuronal atrophy in the superior olive.

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.

Calretinin expression in the chick brainstem auditory nuclei develops and is maintained independently of cochlear nerve input.

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.

Type IIa ('anti-Hu') antineuronal antibodies produce destruction of rat cerebellar granule neurons in vitro.

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.

Gamma-D-glutamylaminomethyl sulfonic acid (GAMS) distinguishes kainic acid- from AMPA-induced responses in Xenopus oocytes expressing chick brain glutamate receptors.

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)

Non-N-methyl-D-aspartate receptors mediating synaptic transmission in the avian cochlear nucleus: effects of kynurenic acid, dipicolinic acid and streptomycin.

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)

New isoforms of the chick glutamate receptor subunit GluR4: molecular cloning, regional expression and developmental analysis.

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.

Zinc inhibition of group I mGluR-mediated calcium homeostasis in auditory neurons.

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.

Flip and flop isoforms of chick brain AMPA receptor subunits: cloning and analysis of expression patterns.

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.