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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.

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.

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.

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.

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 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.

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)