Afferent influences on brainstem auditory nuclei of the chicken: Regulation of transcriptional activity followiqg cochlea removal

Neuronal survival in the cochlear nucleus of young animals is regulated by afferent activity. Removal or blockade of nerve VIII input results in the death of 20–40% of neurons in the cochlear nucleus, nucleus magnocellularis (NM), of the 10–14 days posthatch chick. Neuronal death in NM is preceded by complete failure of protein synthesis and degradation of ribosomes. In addition, there is a biphasic change in the immunoreactivity of ribosomes for a monoclonal antiribosomal RNA antibody, Y10B. Initially, the entire population of afferentdeprived NM neurons loses Y10B immunoreactivity, but, after 6 or 12 hours of afferent deprivation, lack of Y10B immunoreactivity specifically marks dying NM neurons. Whether RNA synthesis is also altered in afferent-deprived NM neurons has not previously been studied. To determine whether RNA synthesis in NM neurons is regulated by loss of afferent activity, we injected chicks with ³H-uridine following unilateral cochlea removal and measured the incorporation of RNA precursor with tissue autoradiography. As early as 1 hour after cochlea removal, there was a significant decrease in ³H-uridine incorporation by afferent-deprived NM neurons. After longer periods of afferent deprivation (6 or 12 hours), the majority of dying NM neurons (marked by loss of Y10B immunoreactivity) fail to incorporate RNA precursor. Six or 12 hours following cochlea removal, the subpopulation of surviving NM neurons incorporates ³H-uridine at increased levels over those observed 1 or 3 hours after cochlea removal. These findings suggest that nuclear function is regulated by afferent synaptic activity and that failure of RNA synthesis occurs early in the cell death process. © 1995 Wiley-Liss, Inc.     

Afferent influences on brain stem auditory nuclei of the chicken: changes in succinate dehydrogenase activity following cochlea removal

We have examined one of the metabolic consequences of unilateral cochlea (basilar papilla) removal in the chick brain stem auditory system. We assessed changes in succinate dehydrogenase (SDH), a mitochondrial enzyme involved in energy metabolism, in neurons of second-order n. magnocellularis (NM) and third-order n. laminaris (NL). Chickens undergoing surgery at 10 days of age were perfused 4 hours to 35 days postlesion. Chickens 6 or 66 weeks of age at cochlea removal were examined 1 or 8 days after surgery. In all groups, cryostat sections were prepared for SDH histochemistry or Nissl staining. In normal chickens, NM cell bodies and NL neuropil contain SDH reaction product. In young birds, the density of SDH reaction product in NM shows a rapid biphasic response to cochlea removal. From 8 to 60 hours postlesion, density increases ipsilateral to cochlea removal; for survival times of 3-35 days, SDH density decreases in ipsilateral NM. In NL, no changes were observed until 3 days after cochlea removal. Then we observed a long-lasting decrease in density of SDH reaction product in the neuropil regions receiving input from the deafferented NM. All of these changes are age-dependent in that they were observed only following cochlea removal on or before 6 weeks of age.     

Astrocyte proliferation in the chick auditory brainstem following cochlea removal

Astrocytes in the central nervous system (CNS) respond to injury and disease by proliferating and extending processes. The intermediate filament protein of astrocytes, glial fibrillary acidic protein (GFAP) also increases in astrocytes. These cells are called “reactive astrocytes” and are thought to play a role in CNS repair. We have previously demonstrated rapid increases (< 6 hours) in GFAP-immunoreactive and silver-impregnated glial processes in the chick cochlear nucleus, nucleus magnocellularis (NM), following cochlea removal or activity blockade of the eighth nerve. It was not known whether these changes were the result of glial proliferation, glial hypertrophy, or both. The present study examined the time course of astrocyte proliferation in NM following cochlea removal. Postnatal chicks received unilateral cochlea removal and survived for 6, 12, 18, 24, 36, 48, and 72 hours. Bromodeoxyuridine was used to label proliferating cells. The volume and number of labeled cells in NM was calculated for both the experimental and control sides of the brains for experimental animals was well as for unoperated control animals. A subset of astrocytes continuously divide in the normal posthatch chick brainstem. The percentage of labeled nuclei increases within NM 36 hours following cochlea removal and is robust by 48 hours. This increase is due to astrocyte proliferation within, rather than migration to, NM. These resulis indicate that rapid increases in GFAP following reduced activity are independent of cell proliferation. The time course of astrocyte proliferation suggests that cellular degeneration within the nucleus may play a role in upregulating astrocyte proliferation. © 1994 Wiley-Liss, Inc.     

Afferent influences on brain stem auditory nuclei of the chicken: neuron number and size following cochlea removal

The consequences of cochlea removal on neuron number and soma cross-sectional area were examined in the second order auditory nucleus (n. magnocellularis) of chickens. Both the age of the subjects at the time of cochlea (basilar papilla) removal (1-66 weeks) and the survival period (1-45 days) were varied. Neuron number and soma cross-sectional area were determined from Nissl stained sections. Additional material was processed to examine the relationship of ganglion cell loss to changes in n. magnocellularis. Neuron number decreased by 25-30% and soma cross-sectional area decreased by 10-20% ipsilateral to the cochlea removal in chickens operated on during the first 6 weeks after hatching. In contrast, in chickens operated on at 66 weeks posthatch neuron number decreased less than 10% and there was no change in soma area. The changes were rapid, being nearly complete 2 days after cochlea removal. An initial change (1 and 2 days after surgery) observed in animals operated on up to 6 weeks posthatch was the presence of a large number of neurons in which no Nissl substance could be detected. These results demonstrate an age-dependent change in the susceptibility of NM neurons to deafferentation. This change is not temporally related to other measures of functional maturation of the auditory system.     

Afferent influences on brain stem auditory nuclei of the chicken: presynaptic action potentials regulate protein synthesis in nucleus magnocellularis neurons

Studies of the avian auditory system indicate that neurons in nucleus magnocellularis (NM) and nucleus laminaris of young animals are dramatically altered by changes in the auditory receptor. We examined the role of presynaptic activity on these transneuronal regulatory events. TTX was used to block action potentials in the auditory nerve. TTX injections into the perilymph reliably blocked all neuronal activity in the cochlear nerve and NM. Far-field recordings of sound-evoked potentials revealed that responses returned within 6-12 hr after a single TTX injection. Changes in protein synthesis by NM neurons were measured by determining the incorporation of 3H-leucine using autoradiography. NM neurons on the side of the brain ipsilateral to the TTX injection were compared to normally active cells on the other side of the same tissue section. Grain counts over individual neurons revealed that a single injection of TTX produced a 40% decrease in grain density in ipsilateral NM neurons within 1.5 hr after the TTX injection. However, by 24 hr after a single TTX injection, grain densities were not different on the 2 sides of the brain. Continuous activity blockade for 6 hr caused the cessation of amino acid incorporation in a portion of NM neurons and a 15-20% decrease in the remaining neurons. These changes in amino acid incorporation are comparable to those following complete removal of the cochlea (Steward and Rubel, 1985). We also examined NM for neuron loss and soma shrinkage after blocking eighth nerve action potentials. TTX injected every 12 hr for 48 hr caused a 20% neuron loss and an 8% shrinkage of the remaining neurons. Similar reductions were found following cochlea removal (Born and Rubel, 1985). It is concluded that neuronal activity plays a major role in the maintenance of normal NM neurons. Furthermore, these results suggest that transneuronal morphological changes seen in neurons following deafferentation or alterations of sensory experience are a result of changes in the level of presynaptic activity.     

Afferent influences on brain stem auditory nuclei of the chicken: effects of conductive and sensorineural hearing loss on n. magnocellularis

Nucleus magnocellularis is the avian homologue of the spherical cell region of the mammalian anteroventral cochlear nucleus. Its primary excitatory synaptic input is from large end bulbs of Held from the eighth nerve ganglion cells. We have examined the effects of three peripheral manipulations--middle ear ossicle (columella) removal (monaural and binaural), columella removal and oval window puncture (monaural), and monaural earplug--on cross-sectional cell area ("cell size") of second-order auditory neurons in n. magnocellularis of the chicken. Manipulations were performed between embryonic day 19 and posthatch day 4. Survival time was varied from 2 to 60 days. Air conduction and bone conduction thresholds were determined to assess for conductive and sensorineural hearing loss associated with each of these manipulations. Hair cell counts were made from basilar papillae of each experimental group. We found that a columella removal alone, which produced a 50-55-dB purely conductive hearing loss, was not associated with changes in cell size of n. magnocellularis neurons. Similarly, chronic monaural earplugging did not affect the cross-sectional area of these second-order auditory neurons. Conversely, a combined columella removal and oval window puncture, which produced a mixed hearing loss with a 15-40-dB sensorineural component was associated with an 18-20% reduction in n. magnocellularis cell area. Hair cell counts for experimental ears were not significantly different from control ears. These results, in conjunction with measurements of multiunit activity recorded in n. magnocellularis, suggest that manipulations which markedly attenuate extrinsic auditory stimulation, but do not result in chronic change in the average activity levels, also do not influence the size of n. magnocellularis cell bodies. On the other hand, a manipulation which influences overall activity levels, but does not result in degeneration of receptor cells, resulted in marked changes in n. magnocellularis cell size.     

Patterns of functional innervation in the auditory nuclei of the chick brainstem following early unilateral removal of the otocyst

Neurons in the third-order auditory nuclei in the brainstem of chicks (nuclei laminares, NL) receive functional innervation from the ipsilateral and contralateral second-order nuclei (nuclei magnocellulares, NM) which is restricted to the dorsal and ventral dendrites respectively. This pattern of innervation in NL is established by embryonic stage 40 (day 15 of incubation). We have examined the distribution of this innervation in both NL at this age or older in embryos from which one otocyst had been removed or damaged on day 3 of incubation. The distribution of functional synapses was determined by analysis of the changes in polarity of field potentials evoked by electrical stimulation of either the ipsilateral or contralateral NM. The distribution of field potential polarity in NL of 40% of recordings in operated embryos and in all sham-operated embryos was the same as that observed in unoperated embryos. However, in the remaining operated embryos, the time course of the field potentials and the changes in the polarity of the responses as the recording electrode penetrated NL were abnormal. The abnormal complexity of responses and the abnormal distribution of field potential amplitude in NL in the operated embryos suggests that loss or damage to the first-order auditory innervation can result in (a) the formation of novel, functional synapses between second-order auditory neurons, and/or (b) disruption of processes that produce segregated innervation of the dendrites of the third-order auditory neurons.     

Afferent influences on brain stem auditory nuclei of the chicken: time course and specificity of dendritic atrophy following deafferentation

The time course and specificity of the changes in dendritic morphology following deafferentation were examined in nucleus laminaris of young chickens. The dendrites of nucleus laminaris neurons are segregated into dorsal and ventral domains, which are innervated separately from the ipsilateral and contralateral nucleus magnocellularis, respectively. Transection of the crossed dorsal cochlear tract deafferents the ventral dendrites of nucleus laminaris bilaterally without interrupting the matching input to the dorsal dendrites. In 10-day-old chicks, atrophy of the ventral dendrites began immediately after transecting the tract; the ventral dendrites were 10% shorter by 1 hour and 16% shorter by 2 hours after deafferentation. The length of the ventral dendrites progressively decreased over the next 2 weeks, resulting in at least a 60% loss of ventral dendrite 16 days after surgery. The dorsal dendrites of the same cells, whose afferents remained intact, did not change in length during the time course of this study. However, 16 days after the lesion, spines appeared on the normally smooth dorsal and ventral dendrites. The time course of dendritic atrophy and its restriction to the deafferented postsynaptic surface are related to possible mechanisms by which afferents regulate and maintain their target neurons.     

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 perated 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 embronic day 11. The results indecate 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 promary 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.     

Rapid growth of astrocytic processes in N. magnocellularis following cochlea removal.

Removal of the cochlea or pharmacological blockade of eighth nerve activity in young postnatal chickens results in rapid transneuronal cell death and atrophy in neurons of n. magnocellularis. The present experiments were designed to examine the influence of afferent input on astrocyte structure in n. magnocellularis. Young chickens were subjected to unilateral cochlea removal. At times ranging from 5 minutes to 72 hours later, the brainstems were histologically processed with a polyclonal antibody against glial fibrillary acidic protein (GFAP). A second group of chick brainstems was impregnated by a Golgi method 6 hours after unilateral cochlea removal and impregnated three-dimensional reconstructions were made of glial cells in n. magnocellularis (NM). Analyses of GFAP positive processes in NM revealed an observable increase in the number of astrocytic processes at the borders of the nucleus within 30 minutes of cochlea removal and a twofold increase in GFAP + glial processes by 6 hours. A secondary increase in the number and density of GFAP + processes occurred between 24 and 72 hours following cochlea removal, during the period of axonal degeneration, and transneuronal cell atrophy and death. Analyses of astrocytes impregnated by the Golgi method revealed that individual glial cells had increased their total process length and the number of processes by approximately twofold by 6 hours after cochlea removal. These results suggest that the structure of astrocytes is rapidly and dramatically influenced by the level of excitatory activity in a neuronal system. Furthermore, the similarity of results obtained with GFAP immunohistochemistry and three-dimensional reconstruction of astrocytes provides evidence that the short-term changes observed following cochlea removal represent the actual growth of glial processes. We speculate that modulations in glial processes as a function of afferent activity may act to influence synaptic efficacy.     

Descending projections from auditory brainstem nuclei to the cochlea and cochlear nucleus of the guinea pig

Projections from auditory brainstem nuclei to the cochlea and cochlear nuclei in the guinea pig were studied by injection of two retrograde fluorescent neuronal tracers. For seven experiments fast blue was injected into the scala tympani of one cochlea and diamidino yellow was injected into dorsal or anteroventral cochlear nucleus of the same side. The results show that the efferent projections to the cochlea and cochlear nucleus generally form two separate neuronal systems even though they share many common nuclei of origin. The largest projections to the cochlear nucleus come bilaterally from the lateral and ventral nuclei of the trapezoid body. Other nuclei, the lateral superior olive, the ventral nucleus of the lateral lemniscus, the dorsomedial periolivary nuclei, and the medial nucleus of the trapezoid body showed an ipsilateral bias in their projections to the cochlear nucleus. An upper limit of 3.5% of the medial system olivocochlear efferent neurones projecting to the cochlea were labelled with both diamidino yellow and fast blue, suggesting that few efferent neurones projecting to the cochlea send collaterals to the cochlear nucleus in this species. However, the site of medial system olivocochlear efferent collateral terminations is the granule cell area for the cat, mouse, and gerbil. When diamidino yellow was injected in the superficial layers of the cochlear nucleus, including the superficial granule cell layer of the ventral cochlear nucleus, approximately 3.6% of medial system olivocochlear efferents projecting to the cochlea sent collaterals to the cochlear nucleus. In three animals fast blue was injected into the cochlear nucleus and diamidino yellow into the cochlea. These experiments revealed a greater proportion of the medial system olivocochlear efferents projecting to the cochlea sending collaterals to the cochlear nucleus, but this proportion was still less than 10%. These results were confirmed by the extracellular injection of horseradish peroxidase into the intraganglionic spiral bundle. Only three medial system olivocochlear efferents were observed to send collaterals to the cochlear nucleus. This number was less than 10% of all labelled medial system fibres. Although these experiments suggest that in the guinea pig the number of olivocochlear efferents sending collaterals to the cochlear nucleus is considerably smaller than is found for the cat, mouse, and gerbil, it is not possible with the current experimental procedures to conclude whether the results are due to species or methodological differences.     

GABAergic neurons in brainstem auditory nuclei of the chick: distribution, morphology, and connectivity

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.     

Specific alterations in cardiovascular function and in glucose utilisation within lower brainstem nuclei following intracisternal neuropeptide Y in the conscious rat

In the conscious normotensive rat, intracisternal neuropeptide Y (NPY) (1.25 nmol i.c.) gave rise to alterations in peripheral haemodynamic variables and glucose use within discrete areas of the CNS as measured by [14C]2-deoxyglucose autoradiography. The haemodynamic response to i.c. NPY comprised a transient hypertension followed by a prolonged hypotension and bradycardia. These cardiovascular responses to NPY were accompanied by a significant reduction in function related glucose use in the area postrema (-29% from vehicle-injected controls), nucleus tractus solitarius (caudal portion -24%, rostral portion -19%), Kolliker-Fuse nucleus (-14%), inferior colliculus (-18%) and subfornical organ (-19%). It is proposed that the area postrema, nucleus tractus solitarius and Kolliker-Fuse nucleus in the brainstem are involved functionally in the haemodynamic response to i.c. NPY.     

Effects of unilateral and bilateral cochlea removal on 2-deoxyglucose patterns in the chick auditory system

The 2-deoxyglucose (2DG) method was used to map functional activity in the auditory system of chicks that had been subjected to unilateral or bilateral cochlea removal. Following survival times of 1 day to 4 weeks, chicks were exposed to continuous white noise in the 2DG experiments. In monaural subjects nucleus angularis and nucleus magnocellularis showed faint 2DG uptake on the side contralateral to the intact ear. In the binaural nucleus laminaris, the asymmetrical and almost mirror-imaged labeling pattern (Lippe, Stewart, and Rubel: Brain Res. 196:43-58, '80) was produced. The superior olive (OS) was strongly labeled on the ipsilateral side, whereas the contralateral OS showed only a slight 2DG uptake at its medial border. The lateral lemniscus and nucleus lemnisci lateralis, pars ventralis (LLv) showed stronger activation on the contralateral side. Both Nissl stains and 2DG patterns provide evidence that nucleus ventralis lemnisci lateralis (VLV) can be subdivided into an anterior (VLVa) and a posterior (VLVp) part. Whereas VLVp is labeled only contralaterally, VLVa is labeled on both sides with similar intensity. Nucleus mesencephalicus lateralis, pars dorsalis (MLD) is strongly labeled throughout contralaterally. The ipsilateral MLD shows a defined ventral portion of high 2DG uptake. Intensity of labeling here is symmetrical to the corresponding area of the contralateral MLD. These symmetrical patterns were related to the tonotopic organization of MLD, which was mapped in intact animals by using tone stimuli. Assuming that symmetrical 2DG uptake in monaural animals indicates excitatory input from both ears (EE-cells), it appears that these EE-cells occupy a sector of each isofrequency plane in MLD. Nucleus ovoidalis (Ov) generally was stronger labeled on the contralateral side. The columnar organization of field L as seen in monaural chicks has already been described (Scheich, Exp. Brain Res. 51:199-205, '83). In bilaterally deafened chicks, MLD, Ov, and layer L2 of field L showed strong but spatially restricted 2DG accumulation in contrast to absence of labeling in peripheral nuclei. The 2DG patterns in monaural chicks are likely to reflect excitatory input within the auditory system. In addition they reveal new insights into the functional organization of some of its nuclei. In particular, they support the notion that MLD contains maps of several interaural integration mechanisms similar to field L. Labeling in the auditory system of bilaterally deafened chicks may result from descending projections or from other than auditory inputs.     

Effects of cochlea removal on GABAergic terminals in nucleus magnocellularis of the chicken

The effects of unilateral cochlea removal on GABA-immunoreactive (GABA-I) terminals in nucleus magnocellularis (NM) of the chick were assessed by immunocytochemical (ICC) techniques. Posthatch chicks (5-8 days old) survived from 1-37 days following unilateral cochlea removal. In the ipsilateral NM, the density of GABA-I terminals appeared to increase relative to normal controls 10-37 days after cochlea removal. However, most of that increase could be attributed to a decrease in cell size, cell number, and volume of the nucleus as a result of deafferentation. In the contralateral NM, the density of GABA-I terminals decreased relative to the ipsilateral NM and to normal animals 1-21 days after cochlea removal. The number of GABA-I terminals per NM neuron also decreased in the contralateral NM while that in the ipsilateral NM was comparable to normal controls. To ascertain whether these changes represented changes in the number of terminals or in the amount of GABA contained within the terminals, we also examined these terminals using an antibody to glutamic acid decarboxylase (GAD), the biosynthetic enzyme for GABA. Following unilateral cochlea removal, there was no difference in the density of GAD-I terminals in NM between the two sides of the brain for any of the survival times. Similarly, bilateral cochlea removal had no discernible effect on the density of GABA-I terminals in NM. These data suggest that unilateral deafferentation may temporarily downregulate the biosynthesis of GABA in the contralateral NM.     

Development of GABA immunoreactivity in brainstem auditory nuclei of the chick: ontogeny of gradients in terminal staining

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.     

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. © 1997 Wiley-Liss, Inc.     

Afferent influences on brain stem auditory nuclei of the chicken: cessation of amino acid incorporation as an antecedent to age-dependent transneuronal degeneration

Previous studies of the avian auditory system have revealed that removal of the peripheral receptor (the cochlea) leads to a transneuronal degeneration of auditory relay neurons in nucleus magnocellularis (NM) of the brain stem. An early manifestation of the degeneration which can be observed within 12 hours is a decrease of histochemical staining for RNA (Nissl staining); such a decrease could reflect an alteration in protein synthetic activity within the NM neurons. The present study evaluates this possibility by determining whether the cochlea removal led to an alteration incorporation of protein precursors in the target neurons which exhibit transneuronal degeneration and if so, how early the changes appeared. The cochlea was removed unilaterally in seventeen 10-day-old chicks and two 66-week-old mature chickens, and incorporation of protein precursors was evaluated in the neurons of NM at 0.5, 1.5, 3, 6, 12, and 24 hours following the cochlea removal. Each chick received an intravenous injection of 3H leucine, and was allowed to survive for 30 minutes after the injection of precursor. The brains were then prepared for autoradiography. The extent of incorporation by neurons in NM was determined by counting grains overlying each cell body and determining grain density/micrometers2 of neuron cross-sectional area. We found that auditory relay neurons whose synaptic inputs have been silenced exhibit dramatic decreases in protein synthesis within 30 minutes after removal of the cochlea; leucine incorporation was reduced by about 50%. In chicks sacrificed 3 to 24 hours after removal of the cochlea, some neurons (about 1/3) were entirely unlabeled despite heavy labeling of their neighbors and heavy labeling of all NM neurons on the opposite side of the brain. The remaining neurons exhibited about a 15% reduction in incorporation in comparison with the cells in the contralateral (control) NM. While the decreases in incorporation were apparent at all survival intervals, there was no consistent decrease in Nissl staining until 6 hours after cochlea removal. There were no changes in protein precursor incorporation following removal of the cochlea in adult birds, a result which is in keeping with the relative absence of transneuronal degeneration following removal of the cochlea at maturity. The results suggest a very rapid transneuronal regulation of protein metabolism within target neurons in young animals, perhaps by activity-related events.     

Central migration of neuronal tissue and embryonic stem cells following transplantation along the adult auditory nerve

The regeneration of the auditory nerve remains a challenge in restoring hearing. An interesting approach would be to use a cell replacement therapy with the potential to establish connections from the inner ear to the central auditory system. This hypothesis was tested by xenografted (mouse to rat) implantation of embryonic dorsal root ganglion (DRG) neurons and embryonic stem (ES) cells along the auditory nerve in the adult host. DRG neurons were obtained at embryonic day 13-14 in transgenic animals expressing enhanced green fluorescence protein (EGFP). For embryonic stem cells, a tau-GFP ES cell line was used as a donor. The fibers of the auditory nerve in the adult rat were transected through the modiolus at the first cochlear turn, and the biological implants were transplanted into the transection. The transplanted DRG neurons and ES cells survived for a postoperative survival time ranging from 3 to 9 weeks, verified by EGFP/GFP fluorescence, and neurofilament or TUJ1 immunostaining. At 9 weeks following implantation, the implanted DRG neurons were found to have migrated along the auditory nerve in the internal meatus. At the same postoperative time, the ES cells had migrated into the brain stem close to the ventral cochlear nucleus. The results demonstrate not only the survival and migration of xenografted DRG neurons and stem cells along the adult auditory nerve but also the feasibility of a cell replacement therapy in the degenerated auditory system.     

Temporal changes in neuronal dropout following inductions of lithium/pilocarpine seizures in the rat

Estimates of neuronal dropout for approximately 100 structures as defined by Paxinos-Watson were completed for brains of male Wistar albino rats between 1 and 50 days after status epilepticus was evoked by a single systemic injection of lithium and pilocarpine. Sample estimates of neuronal loss were strongly correlated with direct measures of cell density. The most extensive immediate damage occurred within the substantia nigra reticulata, CA1 field of the hippocampus, the piriform cortex and the reuniens and paratenial nuclei of the thalamus. Neuronal dropout continued in many other structures over a 50-day period. Structures that showed the greatest 2-deoxyglucose (2-DG) uptake during discrete seizures and waxing and waning seizures within the early stages of status epilepticus but the least 2-DG uptake at the time of late continuous spiking and fast spiking with pauses [Neuroscience 64 (1995) 1057, 1075] exhibited the most neuronal dropout. Relationships between the delay of injection of acepromazine (which facilitated survival) and the amount of damage suggested that the source of the process that results in permanent brain damage may originate within the region of the piriform cortices and its subcortices.