The development of gamma-aminobutyric acid-immunoreactivity (GABA-I) in nucleus magnocellularis (NM) and nucleus laminaris (NL) of the chick was studied by using an antiserum to GABA. In posthatch chicks, GABA-I is localized to small, round punctate structures in the neuropil and surrounding nerve cell bodies. Electron microscopic immunocytochemistry demonstrates that these puncta make synaptic contact with neuronal cell bodies in NM; thus, they are believed to be axon terminals. GABAergic terminals are distributed in a gradient of increasing density from the rostromedial to the caudolateral regions of NM. The distribution of GABA-I was studied during embryonic development. At embryonic days (E) 9-11, there is little GABA-I staining in either NM or NL. Around E12-14, a few fibers are immunopositive but no gradient is seen. More GABA-I structures are present at E14-15. They are reminiscent of axons with varicosities along their length, preterminal axonal thickenings and fiber plexuses. At E15, terminals become apparent circumscribing neuronal somata and are also discernible in the neuropil of both nuclei. In E16-17 embryos, terminals are the predominantly labeled GABA-I structures and they are uniformly distributed throughout NM. The density of GABAergic terminals increases in caudolateral regions of NM such that by E17-19, there is a gradient of increasing density of GABA-I terminals from the rostromedial to caudolateral regions of NM. The steepness of this gradient increases during development and is the greatest in posthatch (P) chicks. Cell bodies labeled with the GABA antiserum are located around the borders of both NM and NL and in the neuropil between these two nuclei. Occasionally, GABA-I neurons can be found within these auditory brainstem nuclei in both embryonic and posthatch chicks. Nucleus angularis (NA) contains some GABAergic cells. The appearance of GABA-I terminals around E15 is correlated in time with the formation of end-bulbs of Held on NM neurons. Thus, the ontogeny of presumed inhibitory inputs to chick auditory brainstem nuclei temporally correlates with, and could modulate the development of, excitatory auditory afferent structure and function.

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The second- and third-order auditory nuclei in the brainstem of the chicken, nucleus magnocellularis (NM) and nucleus laminaris (NL), receive afferents that are immunoreactive to gamma-aminobutyric acid (GABA). In order to investigate the source(s) of these GABAergic afferents, we examined the distribution, morphology, and connectivity of GABAergic neurons in and adjacent to NM and NL in chicks from 7 days of incubation to 12 days posthatch. Immunocytochemical techniques revealed the presence of approximately 150 GABA-labeled neurons within the neuropil surrounding NM and NL on each side of the brainstem. Most of these neurons are located between NM and NL and along the lateral border of NM. GABAergic neurons are multipolar; their thick dendritic processes branch extensively and give rise to several thin, secondary processes. Frequently, the GABA-labeled processes arborize within NM or NL. The morphology of these non-NM/NL neurons was investigated further with Golgi impregnation and specific neuronal markers (antisera to microtubule-associated protein). Our observations suggest that a considerable portion of GABAergic input to NM and NL originates from local GABAergic neurons. In order to determine other possible sources of GABAergic input to NM and NL, we injected tracers unilaterally into NM/NL. A small number (20-30) of neurons were retrogradely labeled in the trapezoid body, almost exclusively ipsilaterally. No labeled cells were found in other regions of the brainstem, except for the contralateral NM. Unilateral injections of horseradish-peroxidase-labeled wheat germ agglutinin into the paraflocculus revealed only minor terminal labeling in the lateral region of NL bilaterally. The number and distribution of GABAergic terminals in NM and NL appeared normal after transection of the crossed dorsal cochlear tract.

The types of layer III neurons in cat primary auditory cortex (AI) projecting to the contralateral AI were studied with horseradish peroxidase or horseradish peroxidase conjugated to wheat germ agglutinin. Injections between the anterior and posterior ectosylvian sulci retrogradely labeled both pyramidal and non-pyramidal somata in contralateral cortical layers III, V, and VI in AI, and in the ventral nucleus of the ipsilateral medial geniculate body. Three-quarters (72%) of the retrogradely labeled cells were found in layer III and one-quarter (28%) lay in layers V and VI. Every part of AI was innervated by commissural neurons. The topographical distribution of the labeled cells varied systematically. Injections in the caudal part of AI labeled cells in the caudal part of the opposite AI, while more rostral injections labeled cells in the contralateral, rostral AI. Injections covering the rostro-caudal extent of AI labeled cells throughout the opposite AI. Each part of AI thus projects most strongly to a contralateral, homotypic area, and less strongly to other, adjacent sectors of AI. The types of labeled cells were distinguished from one another on the basis of size, somatic and dendritic morphology, laminar distribution, and nuclear membrane morphology. Their somatodendritic profiles were compared to, and correlated with, those in Golgi-impregnated material from adult animals. Among the pyramidal cells of origin were small, medium-sized, and large neurons, and star pyramidal cells. The non-pyramidal cells of origin included bipolar and multipolar cells. Thus, at least six of the 12 kinds of neurons, as defined by morphological methods, participate in the interhemispheric pathway. Pyramidal cells comprised 65% of the cells of origin, 14% of the labeled cells in layer III were non-pyramidal, and 21% of the neurons could not be classified. It is unknown if these different types of commissural neurons have the same laminar or cytological targets in AI, or if they represent more than one functional or parallel pathway within AI. In any case, cytologically diverse layer III neurons contribute to the commissural system.

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.