Thomas N. Parks

The AMPA receptors 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.

Primary innervation of the avian and mammalian cochlear nucleus

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.

Origin of ascending auditory projections to the nucleus mesencephalicus lateralis pars dorsalis in the chicken

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.

Characterization of the AMPA‐Activated Receptors Present on Motoneurons

Abstract: 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 AMPARs 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.

Contrasting molecular composition and channel properties of AMPA receptors on chick auditory and brainstem motor 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.

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)

Developmental changes in the effects of drugs acting at NMDA or non-NMDA receptors on synaptic transmission in the chick cochlear nucleus (nuc. magnocellularis)

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.

Pharmacological evidence for synaptic transmission mediated by non-n-methyl-d-aspartate receptors in the avian cochlear nucleus ☆

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.

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. J. Comp. Neurol. 383:112–121, 1997.

Cobalt-permeable non-NMDA receptors in developing chick brainstem auditory nuclei

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.