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.     

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.     

Effects of unilateral cochlea removal on anteroventral cochlear nucleus neurons in developing gerbils

Afferent regulation of neurons in the cochlear nucleus as a function of age was investigated at the light microscope level. Unilateral cochlea removal was performed on Mongolian gerbils of three age groups: 1, 8, and 20 weeks postnatal. Animals survived for either 2 days or 2 weeks. An additional group of neonatally operated animals had a prolonged survival of 9 weeks. The number of neurons in anteroventral cochlear nucleus (AVCN) was counted, and cross-sectional area measurements of large spherical cells in AVCN were made. In animals 1 week old at the time of surgery, there was a 35% reduction in neuron number in AVCN after 2 days, a 58% reduction after 2 weeks, and a 59% reduction 9 weeks after inner ear destruction. However, in animals 20 weeks old at the time of surgery, there was no cell loss in AVCN either 2 days or 2 weeks after surgery. Animals in each age group showed a reduction in cross-sectional area of large spherical cells in AVCN after cochlea ablation. The gerbils that underwent cochlea removal at 8 and 20 weeks showed an average decrease of 14-18%. This effect was seen as early as 2 days after cochlea removal. Animals that underwent cochlea removal at 1 week exhibited the greatest change; a 25% decrease at 2 days progressed to 38% at 2 weeks following cochlea removal. No appreciable further changes were seen at 9 weeks after neonatal cochlea removal. The results indicate greater susceptibility of 1-week-old gerbil cochlear nucleus neurons to peripheral loss than found in older animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphometric changes in the chick nucleus magnocellularis following acoustic overstimulation

The present investigation considered the effects of cochlear damage caused by exposure to intense sound on the nucleus magnocellularis of the chick. Neonatal chicks exposed to intense sound were separated into four groups with post-exposure recovery durations of 0, 15, 27, and 43 days. Four age-matched, non-exposed control groups were also formed. At each recovery interval, the control and exposed birds were sacrificed and their brains prepared for paraffin embedding. The brain stem region containing the nucleus magnocellularis (NM) was serially sectioned in the coronal plane. All sections containing NM cells were identified and then coded in terms of their percentile distance from the most caudolateral section. Sections along the nucleus at the 15th, 30th, 50th, 65th, 80th, and 95th percentile positions were selected for evaluation, and the cross-sectional areas of individual NM cells in these sections were then measured. Cell areas were corrected for the bias introduced by eccentricity of the nucleus. The number of NM cells per 1,000 μm² was also calculated at the 50th and 65th percentile positions. These procedures were repeated for the age-matched, non-exposed control animals. The cross-sectional cell area in exposed animals, immediately after the exposure, was reduced significantly at all positions, but returned to near normal by 43 days of recovery. However, the coronal area of NM in the sections at the 50th and 65th percentile position, as well as NM cell density, were unaffected by the exposure at all recovery intervals. The observation of structural recovery in NM cells at 43 days post-exposure was remarkable because it occurred at least 4 weeks after complete functional restoration of single-cell activity in the NM. The shrinkage in NM cell size throughout the nucleus may be due to a general reduction in spontaneous activity in the cochlear nerve fibers caused by the acoustic injury to the chick basilar papilla. J. Comp. Neurol. 390:412–426, 1998. © 1998 Wiley-Liss, Inc.     

Effects of unilateral cochlear removal on dendrites in the gerbil medial superior olivary nucleus

For most neurons, dendrites serve as the major pathway for incoming activity from other neurons. It might therefore be expected that dendrites are particularly sensitive to variations in the level of afferent input they receive. In the auditory brainstem, this expectation has been confirmed in neurons of the medial superior olivary nucleus (MSO). The MSO is uniquely suited to studies of afferent influences on dendrites, as lateral and medial dendrites of MSO neurons receive inputs almost exclusively from the ipsilateral and contralateral ears, respectively. Thus, the effects of unilateral afferent manipulations may be compared between defined dendrites on the same neurons. We have used unilateral deafening (by surgical destruction of the cochlea) in immature [postnatal day 18 (P18)] and adult gerbils to study the late maturation and effect of peripheral deafferentation on the dendrites of MSO neurons. In semi-thin, frontal sections from unoperated animals, we found a change between P18 and adulthood from a lateral to a medial bias in the symmetry of MSO dendrites. Cochlear removal in adulthood led to a reduction in the density of dendritic profiles on the side of the ablation in both MSOs. Cochlear removal at P18 led to a rapid (< 3 days) and sustained dendritic atrophy that was most marked in the caudal part of the nucleus. Electron microscope (EM) measurements in the sagittal plane on MSO dendrite profiles of animals unilaterally deafened at P18 showed a reduction in the number, but not in the area, of profiles on the side of the deafened ear. These results demonstrate a developmental change in the symmetry of MSO medial and lateral dendrites, and a rapid and long-lasting reduction in the number of distal dendrites produced by unilateral deafening either in infancy or adulthood.     

Cytochrome oxidase response to cochlea removal in chicken auditory brainstem neurons

Changes in cytochrome oxidase (CO) activity were studied in the chick brainstem auditory nuclei, n. magnocellularis (NM) and n. laminaris (NL), following unilateral cochlea removal. Chickens aged 10 days or 56 weeks underwent unilateral cochlea removal. Following survival periods of 30 minutes to 14 days for the 10-day-old birds and 6 hours or 14 days for the 56-week-old birds, the animals were perfused with paraformaldehyde/glutaraldehyde fixative. Cryostat sections of the brainstem were then prepared for CO histochemistry. Microdensitometry was used to quantify the difference in CO staining in NM and NL ipsilateral and contralateral to the cochlea removal. Since the cochlea projects to the ipsilateral NM, the contralateral NM was used as a within-animal control. In normal chickens, NM cell bodies and the cell bodies and dendrites of NL neurons stain darkly for CO in both young and adult birds. In 10-day-old birds, there is no significant change in CO staining in NM from 30 minutes to 3 hours after cochlea removal. Then, a rapid biphasic change in CO staining was found in the ipsilateral NM. An increase in staining was observed 6 to 24 hours postoperatively, followed by a decrease in CO staining at 3- to 14-day survival times. In the 56-week-old birds, no increases in CO staining were observed 6 hours after cochlea removal, but a decrease in CO staining was found 14 days postoperatively. In NL, no changes were observed until 3 days (10-day-old birds) or 14 days (56-week-old birds) after cochlea removal. Then a decrease in CO staining was observed in the dendritic and glial/fiber regions of NL containing axons from the deafferented NM. Thus it appears that afferent input has a regulatory effect on the oxidative metabolism of neurons in the chicken auditory brainstem nuclei, an effect that differs with the age of the animal at the time of afferent manipulation.     

Auditory brainstem of the ferret: early cessation of developmental sensitivity of neurons in the cochlear nucleus to removal of the cochlea

The role of primary afferent innervation in the maintenance of neurons in the mammalian auditory system was assessed by performing unilateral removals of the cochlea in neonatal and mature ferrets of known birth dates. Removals were performed under steroid anesthesia and resulted in the complete destruction of the organ of Corti and the loss of at least 80% of type I spiral ganglion neurons. Four main age groups [postnatal days (P)5, P24, P90, and P180] were used. Additional animals received no surgery, partial removals, or complete removals at older ages. Three months after the cochlear removal the animals were reanesthetized and perfused. The brainstem and the temporal bones were wax-embedded, frontally sectioned, and Nissl-stained. Sections of the right and left cochlear nuclei were compared quantitatively. Removal of the cochlea at P5 resulted in the loss of more than 50% of large (nongranular) neurons throughout the ipsilateral cochlear nucleus. Lesions at older ages did not produce any neuron loss. The size of the remaining neurons was reduced by 10-15% in all age groups. Partial lesions at P5 produced a graded response in the cochlear nucleus that was related to the extent of the lesion. The developmental sensitive period for the effects of cochlear removal on the ferret cochlear nucleus is therefore over before the age (P28-P30) at which the animal begins to hear. The present result differs markedly from the chicken, in which the sensitive period for removal of the cochlea persists for at least 2 months after the onset of hearing.     

Rapid changes in protein synthesis and cell size in the cochlear nucleus following eighth nerve activity blockade or cochlea ablation.

Destruction of the cochlea causes secondary changes in the central auditory pathway through transynaptic regulation. These changes appear to be mediated by an activity-dependent process and can be detected in the avian auditory system as early as 30 minutes after deafferentation. We compared the early changes in cochlear nucleus neurons following deafferentation by cochlea ablation with those seen following activity deprivation by perilymphatic tetrodotoxin (TTX) exposure. Protein synthesis and size of large spherical cells in the anteroventral cochlear nucleus (AVCN) of 14-day-old gerbils were measured during the first 48 hours after the manipulations. Both cochlea ablation and TTX produced a reliable decrease in protein synthesis by AVCN neurons (30-40%) by 1 hour. The magnitude of change in tritiated leucine incorporation was similar at all survival times, in both experimental groups. In contrast to the rapid changes in protein synthesis, the decrease in cell size was first evident 18 hours after TTX exposure and 48 hours after cochlea ablation. There was no significant change in protein synthesis or cell size in control groups at any of the survival times. These findings are consistent with changes in the avian auditory system in response to deafferentation and TTX exposure. Cochlea ablation and TTX exposure induced similar transneuronal changes, supporting the hypotheses that activity of auditory afferents in young mammals plays a regulatory role in the metabolism and morphology of their target neurons in the central auditory pathway, and that early changes following destruction of the peripheral receptor are due to reduction of activity-dependent interactions of presynaptic and postsynaptic cells.     

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.     

Influence of mitochondrial protein synthesis inhibition on deafferentation—induced ultrastructural changes in nucleus magnocellularis of developing chicks

Following cochlea removal in developing chicks, about 30% of the neurons in the ipsilateral second-order auditory nucleus, nucleus magnocellularis, undergo cell death. Administration of chloramphenicol, a mitochondrial protein synthesis inhibitor, results in a pronounced increase in deafferentation-induced cell death. In this study, we examined whether the chloramphenicol enhancement of deafferentation-induced cell death reveals the same ultrastructural characteristics that are seen in degenerating nucleus magnocellularis neurons after cochlea removal alone. Unilateral cochlea removal was performed on anaesthetized posthatch chicks. One group of animals was simultaneously treated with chloramphenicol. Six, twelve, or twenty-four hours following cochlea removal, n. magnocellularis neurons were studied by routine transmission electron microscopy. Particular attention was paid to the integrity of the polyribosomes and rough endoplasmic reticulum. Two ultrastructurally different types of neuronal degeneration were observed in the deafferented nucleus magnocellularis neurons: an early onset electron-lucent type that always involved ribosomal dissociation and a late-onset electron-dense type displaying nuclear pyknosis and severely damaged mitochondria. The percentage of nucleus magnocellularis neurons displaying ribosomal disintegration following cochlea removal was found to be markedly increased after chloramphenicol treatment. This finding suggests that mitochondrial function is important for the maintenance of a functional protein synthesis apparatus following deafferentation. © 1996 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.     

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.     

Rapid changes in cochlear nucleus cell size following blockade of auditory nerve electrical activity in gerbils

Large spherical cells of the mammalian anteroventral cochlear nucleus (AVCN) receive direct excitatory input from auditory nerve axons. Trans-synaptic regulation of neuronal cell size and cell number after cochlear ablation has been previously demonstrated in neonates of several vertebrate species, including the gerbil. Such changes may be related to loss of spontaneous or evoked auditory nerve electrical activity or to loss of activity-independent factors. We have developed a method to chronically, yet reversibly, block auditory nerve electrical activity without violating the integrity of the inner ear. Tetrodotoxin (TTX) was embedded in an ethylene-vinyl acetate copolymer resin (Elvax). A small piece of Elvax containing TTX was placed next to the round window membrane, which allowed TTX to diffuse into the inner ear. As a measure of the effectiveness of manipulation, the onset, duration, and magnitude of the auditory threshold shift were measured by the auditory brainstem response. The sound-evoked response was abolished within 10 minutes of placement of TTX on the round window membrane. The duration of threshold shift was dose-dependent and lasted 24-46 hours. Implants of Elvax without TTX did not produce a significant threshold shift. TTX, which blocks voltage-gated sodium channels, did not abolish the potassium-based cochlear microphonic response. The consequence of blocking afferent electrical activity on gerbil AVCN large spherical cells was examined by measuring their cross-sectional area after each of four manipulations: unilateral auditory nerve action potential blockade with TTX; unilateral surgical cochlear ablation; ipsilateral TTX exposure/contralateral cochlear ablation; and unilateral sham operation (Elvax without TTX).(ABSTRACT TRUNCATED AT 250 WORDS)     

Projections from the cochlear nucleus to the inferior colliculus in normal and neonatally cochlea-ablated gerbils

The distribution of the projection from one cochlear nucleus (CN) within each inferior colliculus (IC) was studied in adult, normal gerbils and adult gerbils subjected to unilateral ablation of the contralateral cochlea at 2 days of age. The projection was studied by using the Fink-Heimer technique for impregnating degenerating axons and their terminal processes with silver. Following an extensive, unilateral lesion of the CN, degeneration was seen in both ICs of all animals. In normal animals, degeneration was both more widespread and heavier in the contralateral than in the ipsilateral central nucleus of IC (ICC). Degeneration was most widespread in the rostral and lateral parts of both ICCs and in the ventral part of the contralateral ICC. Degeneration was observed in 26% of the area examined in ipsilateral ICC and in 73% of the area examined in contralateral ICC. In cochlea-ablated animals there was a much greater similarity in the area of degeneration in the ICC ipsilateral (57%) and contralateral (67%) to the CN lesion. The same regional distributions of degeneration were observed as in the normal animals except that the distribution of degeneration in the ipsilateral ICC more closely resembled the normal contralateral than the normal ipsilateral profile. We conclude that the normal distribution of projections from the CN within the ipsilateral ICC is substantially modified by neonatal ablation of the contralateral cochlea.     

Experience-independent development of dendritic organization in the avian nucleus laminaris

The third-order auditory neurons of the avian nucleus laminaris (NL) have distinct dorsal and ventral dendritic tufts that receive their predominant synaptic input from, respectively, the ipsilateral and contralateral cochlear nucleus. Beginning about embryonic day (E) 14 in the chick and continuing for some weeks after hatching, NL neurons undergo a complex series of morphological transformations that result in the formation of a steep anteromedial-to-posterolateral gradient of increasing total dendritic length across the nucleus. This gradient perfectly parallels the tonotopic axis of NL. It has been proposed that acoustically evoked activity in the auditory pathway contributes importantly to formation of the gradient of dendritic length in NL and to several other features of dendritic development. The present experiment tested this hypothesis by surgically removing both otocysts (embryonic precursors of the inner ear) and studying the developing NL in the absence of peripheral input. The results of a quantitative study of Golgi-impregnated material show that at E17 both the steepness and predictability of the spatial gradient of dendritic length in operated animals are indistinguishable from normal. Similarly, the correlation of dorsal and ventral dendritic lengths on individual cells in operated animals is not significantly different from normal. The absolute length of both dendritic fields is reduced below normal, although only dorsal dendrites show a statistically reliable (14%) decrease. This is a significantly smaller effect than the 44% length reduction seen previously in animals with unilateral otocyst removal (T.N. Parks: J. Comp. Neurol. 202:47-57, '81); symmetrical afferent input appears more important to the regulation of NL dendritic length than the absolute level of this input.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective changes in hippocampal neuropeptide Y neurons following removal of the cholinergic septal inputs

The number and distribution of subpopulations of hilar interneurons containing neuropeptide Y (NPY), somatostatin (SOM), or γ-aminobutyric acid (GABA) immunoreactivities were examined in the hilus of the dentate gyrus following removal of the cholinergic septal inputs. One, 2, 4, 8, 12, and 24 weeks after intracerebroventricular injections of immunotoxin, consisting of antibody to the low-affinity nerve growth factor receptor conjugated to saporin (192 IgG-saporin), lesioned rats were processed simultaneously with controls for NPY, SOM, or GABA immunolabeling. Across all time points, the number of NPY-labeled neurons was reduced to a statistically significant level (paired t-test, P = 0.001) in the injected rats (73% of control values, on average). The decrease in the number of NPY-labeled neurons was not limited to any particular subregion rostrally but appeared greater in the central region caudally. The size of NPY-labeled neurons did not differ statistically between control and immunolesioned rats examined at 1, 2, and 24 week time points. In contrast, the number of both SOM- and GABA-immunoreactive neurons in injected rats did not appear to be affected in any consistent manner. Examination of the hilus in adjacent Nissl-stained sections with the optical dissector revealed that although the total number of small nonprincipal cells (5–15 μm in diameter) did not appear affected at the 4-week time point, there was a statistically significant (P = 0.03) reduction across the 8–24-week time points (to 80% of control values, on average). Dual-labeling studies on separate rats showed that a small subpopulation of the NPY- and SOM-labeled neurons, primarily in the infragranular hilus, were colocalized with neurons containing GABA immunoreactivity (18% and 5%, respectively). These studies demonstrate that removal of the cholinergic septal inputs (1) can cause relatively rapid, selective decreases in the number of NPY-immunoreactive hippocampal interneurons and (2) appears to lead to the death of hippocampal interneurons over a longer time course. The changes in NPY immunoreactivity seem to occur in the portion of interneurons that probably does not contain either SOM or GABA immunoreactivity. J. Comp. Neurol. 386:46–59, 1997. © 1997 Wiley-Liss, Inc.     

The effect of unilateral basilar papilla removal upon nuclei laminaris and magnocellularis of the chick examined with [H]2-deoxy-d-glucose autoradiography

The effect of unilateral basilar papilla removal on glucose uptake in the 2nd and 3rd order auditory nuclei in the chick's brain stem, nucleus magnocellularis and nucleus laminaris, respectively, was examined with [³H]2-deoxy-D-glucose (2-DG) autoradiography. The tissue was processed according to a thaw-mount technique, and the number of grains in the resulting autoradiographs was counted to assess changes in glucose uptake. It was observed that there is a greater density of grains over the neuropil regions of nucleus laminaris which receive input from the normal ear than over the corresponding regions which receive input from the operated ear. Similarly, differences in grain density are found between the normally innervated and deafferented magnocellular nuclei although these differences are not as great as those in nucleus laminaris. Differences in grain density were also apparent between the glial/fiber regions which bound the neuropil areas of nucleus laminaris; there is a greater density of grains overlying those glial/fiber regions through which fibers receiving input from the normal ear course than over those regions through which fibers which normally carry input from the operated ear travel. It is likely that this difference mainly reflects glucose uptake in the fibers although a possible contribution of glial tissue cannot be excluded. All these effects of basilar papilla removal are seen with survival times as short as 70 min and thus likely reflect the reduction of neural activity rather than the degeneration of pre- or postsynaptic elements. Finally, the same pattern of results as described above was found when using the more common [¹⁴C]2-DG procedure or when using [³H]2-DG but processing the tissue using the freeze-dried technique.The present results thus show the neuropil regions of nucleus laminaris and the adjacent glial/fiber areas to be areas of high glucose utilization. Unilateral basilar papilla removal results in the removal of an excitatory input to these regions, and this results in a reduction of glucose utilization that is specific to those neuropil regions and glial/fiber areas that receive input from the operated ear. These findings are contrasted with another study in which removal of a major excitatory input to the dentate gyrus of the rat results in a reduced glucose utilization which is not specific to the deafferented region and which largely reflects post- rather than presynaptic events.     

Afferent influences on brainstem auditory nuclei of the chick: Nucleus magnocellularis neuronal activity following cochlea removal

Elimination of presynaptic elements often results in marked changes, such as atrophy and death, in postsynaptic neurons in the central nervous system. These transneuronal changes are particularly rapid and profound in young animals. In order to understand the cellular events underlying transneuronal regulation it is necessary to explore changes in the local environment of neurons following manipulations of their afferents. In previous investigations we have documented a variety of rapid and marked cellular changes in neurons of the cochlear nucleus of neonatal chicks (n. magnocellularis) following cochlea removal. In adult chickens, however, these transneuronal changes are either absent or minor. The goals of the studies presented here were to examine changes in the electrical activity of nucleus magnocellularis cells and their afferents following removal of the cochlea and to determine if these changes were similar in adult and neonatal animals. Two measures of electrical activity were used; multiunit recording with microelectrodes and incorporation of radiolabeled 2-deoxyglucose (2-DG). Microelectrode recordings revealed high levels of spontaneous activity in n. magnocellularis and n. laminaris, the binaural target of n. magnocellularis neurons. Neither puncturing of the tympanic membrane nor removal of the columella causes significant changes in spontaneous activity, although the latter results in a profound hearing loss (40-50 dB). Removal of the cochlea, on the other hand, results in immediate cessation of all extracellular electrical activity in the ipsilateral n. magnocellularis. Recordings from the same location for up to 6 h failed to reveal any return of spontaneous activity. When the electrode tip was placed in n. laminaris, unilateral cochlea removal had no discernible effect on extracellularly recorded spontaneous activity, probably due to the high levels of excitatory input from the intact ear. Bilateral cochlea removal, however, completely eliminated activity in n. laminaris. 2-DG studies conducted 1 h to 8 days following unilateral cochlea removal revealed marked decreases in 2-DG incorporation in the ipsilateral n. magnocellularis and bilaterally in the n. laminaris target of the ablated cochlea. No compensatory return of 2-DG incorporation was observed for up to 8 days. Comparisons of adult and neonatal chicks failed to reveal significant differences in the effects of cochlea removal on multiunit activity or 2-DG incorporation, suggesting that age differences in transneuronal regulation are due to intrinsic biochemical differences in young and adult neurons rather than differences in the proportion of synaptic input that has been abolished.     

Patterns of cell death in mouse anteroventral cochlear nucleus neurons after unilateral cochlea removal

Developmental changes that influence the results of removal of afferent input on the survival of neurons of the anteroventral cochlear nucleus (AVCN) of mice were examined with the hope of providing a suitable model for understanding the cellular and molecular basis for these developmental changes in susceptibility. We performed unilateral cochlear ablation on wild-type mice at a variety of ages around the time of hearing onset to determine developmental changes in the sensitivity of AVCN neurons to afferent deprivation. In postnatal day 5 (P5) mice, cochlea removal resulted in 61% neuronal loss in the AVCN. By age P14, fewer than 1% of AVCN neurons were lost after this manipulation. This reveals a rather abrupt change in the sensitivity to disruption of afferent input, a critical period. We next investigated the temporal events associated with neuron loss after cochlea removal in susceptible animals. We demonstrate that significant cell loss occurs within 48 hours of cochlea removal in P7 animals. Furthermore, evidence of apoptosis was observed within 12 hours of cochlea removal, suggesting that the molecular events leading to cell loss after afferent deprivation begin to occur within hours of cochlea removal. Finally, we began to examine the role of the bcl-2 gene family in regulating afferent deprivation-induced cell death in the mouse AVCN. AVCN neurons in mature bcl-2 knockout mice demonstrate susceptibility to removal of afferent input comparable to neonatal sensitivity of wild-type controls. These data suggest that bcl-2 is one effector of cell survival as these cells switch from afferent-dependent to -independent survival mechanisms.