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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.

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 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.

Surgical removal of the otocyst in chick embryos induces axons from the contralateral cochlear nucleus (nucleus magnocellularis, NM) to form, in addition to their normal endings in nucleus laminaris (NL), anomalous and persistent functional contacts in the ipsilateral NM (Jackson and Parks, 1988). We have examined how interaction between the abnormal synaptic partners during development influences the form of the axon terminal and its relation to the target neuron. In the light microscope, aberrant axon terminals labeled in vitro with HRP appear to form boutons quite unlike the large calycine endbulbs made by the normal cochlear nerve (CN) endings in NM. In the electron microscope, however, the anomalous endings appear embedded in the NM cells, something never seen normally in NM or NL. Morphometric analyses were performed on electron micrographs from NM and NL in animals aged embryonic day (E) 19 to posthatching day (P) 2 from which the right otocyst had been removed on E3 and in normal control animals. Aberrant endings appose 18% of the circumference of operated NM cells, versus 45% for CN axons in the normal NM at this age. The mean length of membrane apposition for the anomalous NM-to-NM endings was 215% greater than for normal NM-to-NL endings but 54% smaller than that in normal CN endings. These results support the idea that developmental interactions between synaptic partners can influence the form of the contact between the 2 neurons. The results also demonstrate, however, that formation of persistent and functional synapses with NM neurons throughout development is not sufficient to induce any axon to assume the calycine form of a cochlear nerve endbulb.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

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.

We studied the chick auditory system to determine whether there is a normal developmental reduction in the number of cochlear nerve axons innervating individual cochlear nucleus (nucleus magnocellularis, NM) neurons. We also examined the preterminal branching patterns of cochlear nerve axons during development. The number of cochlear nerve axons innervating individual NM neurons was estimated by counting the increments in the postsynaptic response as the intensity of cochlear nerve electrical stimulation was varied gradually; this number fell from a mean of 4.0 on embryonic day 13 (E13) to a mean of 2.2 on E17 and E18 and the 4th day after hatching. This highly reliable decline in functional convergence was accompanied by a decrease in the number of preterminal branches of cochlear nerve fibers innervating the NM. On E13 and E14, most axons stained by iontophoretic injections of horseradish peroxidase showed two distinct preterminal branches in the NM. By E17 and E18 and thereafter, cochlear nerve axons were unbranched and terminated with a characteristic single large calycine ending in the NM. There are about twice as many cochlear nerve axons as neurons in the NM and the number of fibers in the nerve appears to decline only slightly between E13 and E17. The 50% decline in the number of cochlear nerve axons making functional synapses on individual NM neurons therefore is associated principally with the concurrent elimination of cochlear nerve axon branching in the NM.

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.

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.

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)

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.

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.