Development of the place principle: tonotopic organization.

The tonotopic organization of brainstem auditory nuclei was compared in embryonic and hatchling chickens. In embryos, neurons at any given position in these nuclei were maximally sensitive to lower frequency sounds than the best frequency after hatching. This finding indicates that neurons are maximally stimulated by sounds of different frequencies as development proceeds and supports the hypothesis that during development there is a change in the spatial encoding of frequency along the cochlea.

Hair cell regeneration after acoustic trauma in adult Coturnix quail.

Recovery of hair cells was studied at various times after acoustic trauma in adult quail. An initial loss of hair cells recovered to within 5 percent of the original number of cells. Tritium-labeled thymidine was injected after this acoustic trauma to determine if mitosis played a role in recovery of hair cells. Within 10 days of acoustic trauma, incorporation of [3H]thymidine was seen over the nuclei of hair cells and supporting cells in the region of initial hair cell loss. Thus, hair cell regeneration can occur after embryonic terminal mitosis.

Development of the place principle: acoustic trauma.

Developmental changes in the site of receptor damage following pure-tone acoustic overstimulation were examined in the basilar papillae of embryonic and hatchling chickens. During development, a systematic shift in the position of damage toward the apex of the cochlea was produced by each of three frequencies, suggesting that the transduction properties of the sensory epithelium systematically shift with age. These results imply that neurons in the central nervous system may be maximally stimulated by different sounds during development.

Development of polysensory responses in association cortex of kitten.

Sensory responsiveness of single neurons in posterior association cortex of kittens that were 7 to 50 days old was investigated. The percentage of trimodal cells (that is, cells that respond to visual, auditory, and somesthetic stimulation) increased gradually until day 50, when percentages of trimodally responsive cells approached the adult level. In the youngest kittens, cells were predominantly responsive to only visual stimulation. With maturation, responsiveness to auditory and then to somesthetic stimulation was observed in increasing percentages of cells.

Rapid regulation of microtubule-associated protein 2 in dendrites of nucleus laminaris of the chick following deprivation of afferent activity.

Differential innervation of segregated dendritic domains in the chick nucleus laminaris (NL), composed of third-order auditory neurons, provides a unique model to study synaptic regulation of dendritic structure. Altering the synaptic input to one dendritic domain affects the structure and length of the manipulated dendrites while leaving the other set of unmanipulated dendrites largely unchanged. Little is known about the effects of neuronal input on the cytoskeletal structure of NL dendrites and whether changes in the cytoskeleton are responsible for dendritic remodeling following manipulations of synaptic input. In this study, we investigate changes in the immunoreactivity of high-molecular weight microtubule associated protein 2 (MAP2) in NL dendrites following two different manipulations of their afferent input. Unilateral cochlea removal eliminates excitatory synaptic input to the ventral dendrites of the contralateral NL and the dorsal dendrites of the ipsilateral NL. This manipulation produced a dramatic decrease in MAP2 immunoreactivity in the deafferented dendrites. This decrease was detected as early as 3 h following the surgery, well before any degeneration of afferent axons. A similar decrease in MAP2 immunoreactivity in deafferented NL dendrites was detected following a midline transection that silences the excitatory synaptic input to the ventral dendrites on both sides of the brain. These changes were most distinct in the caudal portion of the nucleus where individual deafferented dendritic branches contained less immunoreactivity than intact dendrites. Our results suggest that the cytoskeletal protein MAP2, which is distributed in dendrites, perikarya, and postsynaptic densities, may play a role in deafferentation-induced dendritic remodeling.

Accommodation enhances depolarizing inhibition in central neurons.

Neurons in the avian cochlear nucleus are depolarized by GABAergic synaptic input. We recorded GABAergic synaptic currents using the gramicidin-perforated-patch method and found their reversal potential (V(rev)) to be depolarized relative to spike threshold, which is surprising given that these inputs are inhibitory. Depolarizing IPSPs (dIPSPs) are kept below spike generation threshold by the activation of a dendrotoxin-I-sensitive, voltage-gated K(+) conductance. We show experimentally that the polarity of IPSPs contributes to their efficacy; dIPSPs induce accommodation, the positive shift in spike threshold, and are therefore more strongly inhibitory than conventional, hyperpolarizing IPSPs in the same neurons. A similar inhibitory mechanism has been described in invertebrate sensory fibers and axons of dorsal root ganglion cells and may be a general means of amplifying the strength of inhibition in cases where the size of excitatory conductances greatly exceeds that of inhibitory conductances.

GABAergic inhibition in nucleus magnocellularis: implications for phase locking in the avian auditory brainstem.

In the avian auditory brainstem, nucleus magnocellularis (NM) functions to relay phase-locked signals to nucleus laminaris for binaural coincidence detection. Although many studies have revealed that NM neurons exhibit intrinsic physiological and anatomical specializations for this purpose, the role of inhibition has not been fully explored. The present study characterizes the organization of GABAergic feedback to NM. Anterograde and retrograde labeling methods showed that NM receives a prominent projection from the ipsilateral superior olivary nucleus (SON). The functional features of this projection were explored in a brain slice preparation. Stimulating fibers from the SON evoked long-lasting, depolarizing responses in NM neurons that were blockable by bicuculline, a GABA(A) receptor antagonist. The slow time course of these responses allowed them to undergo temporal summation during repetitive stimulation. The summed GABAergic response was capable of blocking spikes generated in NM neurons by suprathreshold current injection. This inhibitory effect was attributable to a large reduction in input resistance caused by a combination of the opening of a GABAergic Cl(-) conductance and the recruitment of a low-voltage activated K(+) conductance. This large reduction of input resistance increased the amount of current necessary to drive NM neurons to threshold. The results lead us to propose that GABAergic inhibition enhances phase-locking fidelity of NM neurons, which is essential to binaural coincidence detection in nucleus laminaris.

Rapid changes in ultrastructure during deafferentation-induced dendritic atrophy.

This study describes qualitative and quantitative changes in dendritic ultrastructure during the rapid atrophy of nucleus laminaris (NL) dendrites following deafferentation. The dendrites of n. laminaris neurons in the chick auditory system are segregated into dorsal and ventral dendritic tufts, which receive spatially separated innervation from the ipsilateral and contralateral nucleus magnocellularis, respectively. We have previously shown that removing the input to the ventral side of NL results in the rapid atrophy of the ventral dendrites, whereas the nondeafferented dorsal dendrites of the same cells do not change in length. The ultrastructure of NL was examined in normal animals and after deafferentation. Changes in dendritic ultrastructure were not qualitatively apparent 4 hours after deafferentation. Between 12 and 48 hours the cytoplasm of the ventral dendrites became progressively more lucent, and a gap formed in the transition between the soma and ventral dendritic cytoplasm. Many of the dendrite tips, however, appeared normal even 2 days after deafferentation. Degeneration of dendrite plasma membrane was not visible until 2 days after deafferentation. On the other hand, quantitative measurements revealed a 30% decrease in microtubule density in the initial portion of the ventral dendrite by 4 hours, and a 50-60% decrease from 12 to 48 hours after deafferentation. Neurofilament density in the initial ventral dendrites decreased 50% by 12 hours, and 70% by 2 days after deafferentation. Many of the terminals of the severed afferents remained attached to the atrophying dendrite until 2 days after surgery, when they were in advanced stages of degeneration. Glia apparently were not involved in dendrite loss. The implications of these results on the role of cytoskeleton in the production and maintenance of dendritic shape are discussed.

Changes in neuronal cell bodies in N. laminaris during deafferentation-induced dendritic atrophy.

N. laminaris dendrites begin to atrophy almost immediately after they are deafferented. Accompanying this rapid change in shape is a loss of microtubules and neurofilaments at the base of the dendrite, and a decrease in the density of the dendritic cytoplasm. However, degenerative changes in the dendritic plasma membrane were not evident until 2 days after deafferentation. Thus it was unknown what happened to the volume and membrane lost from the atrophying dendrites before this time. The soma was investigated in this study as a possible recipient of the volume of the atrophying dendrite. Soma size increased significantly by 2 hours after deafferentation and continued to increase for 1-8 days after deafferentation. The nucleus, which is normally concentric with the soma, moved continuously to the dorsal pole of the soma, toward the innervated side of the cell. The cytoplasm on the ventral side of the soma showed a decrease in density and loss of cytoskeleton similar to what was found in the initial portion of the ventral primary dendrites in the accompanying paper. These changes are interpreted as indicative of a rapid resorption of the ventral dendrite back into the soma following deafferentation.

Temporal, spatial, and morphologic features of hair cell regeneration in the avian basilar papilla.

Hair cell-selective antibodies were used in combination with the nucleotide bromode-oxyuridine (BrdU) to examine the temporal, spatial, and morphologic progression of auditory hair cell regeneration in chicks after a single gentamicin injection. New hair cells are first identifiable with an antibody to class III beta (beta) tubulin (TuJ1) by 14 hours after BrdU incorporation, but progenitor cells in S phase and M phase are TuJ1-negative. TuJ1 labeling reveals that new hair cells are first detected at 3 days after gentamicin, in the base, and the emergence and maturation of regenerating hair cells spreads apically over time. Differentiation of regenerating hair cells consists of a progressive series of morphologic changes. During early differentiation (14 hours to 1 day after BrdU), regenerating hair cells are round or fusiform and remain near the lumen, where they are generated. During intermediate differentiation (2-4 days after BrdU), regenerating hair cells resemble support cells; their somata are elongated, their nuclei are in the support cell layer, and they appear to contact both the lumenal surface and the basal lamina. The 275-kDa hair cell antigen is first expressed in regenerating hair cells during this period. During late differentiation (7 days after BrdU and later), TuJ1-positive cells acquire the globose shape of mature hair cells. Labeling with antibodies to hair cell antigen, calmodulin, and ribosomal RNA confirms this morphologic progression. Examination of sister cells born at 3 days post-gentamicin reveals that there is equal likelihood that they will assume the hair cell or support cell fate (i.e., both asymmetric and symmetric differentiation occur).

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 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.

Ontogeny of tonotopic organization of brain stem auditory nuclei in the chicken: implications for development of the place principle.

The morphological development of the cochlea begins in the base or midbasal region and spreads toward the apex. In adults, the base responds maximally to high-frequency sounds and lower frequencies are represented progressively toward the apex. This predicts that responses to sound should occur initially to high frequencies and gradually change to include lower frequencies. Paradoxically, animals respond first to relatively low frequencies and last to high frequencies. We have previously proposed that this discrepancy results from an ontogenetic change in spatial coding of frequency along the cochlea (Rubel et al., '76). According to this model, only the basal end of the cochlea transduces sound early in development but it responds to low frequencies. During maturation the representation of low and midrange frequencies shifts apically and the base becomes responsive to high frequencies. This hypothesis predicts that the tonotopic organization within the central nervous system should change during development; neurons at any given location within an auditory nucleus should become maximally responsive to successively higher frequency sounds during development. In the present study this prediction was tested by using microelectrode recording procedures to map the tonotopic organization of nucleus magnocellullaris (NM) and nucleus laminaris (NL), first- and second-order auditory nuclei, in chickens at three ages: embryonic day 17, 1 day posthatch, and 2-4 weeks posthatch. The characteristic frequencies of neurons having the same anatomical location were quantitatively compared across ages. The tonotopic order in NM and NL was similar at all ages; responses to high-frequency sounds were recorded anteromedially and lower frequencies were located progressively more caudolaterally. However, there was a striking quantitative change in tonotopic organization. Neurons at a given location in both nuclei became maximally responsive to progressively higher frequencies during development. The characteristic frequencies of neurons in embryos and newly hatched chicks averaged, respectively, 1.00 (+/- 0.06, S.E.M.) and 0.34 (+/- 0.04) octaves lower than their predicted adult values. All regions in both nuclei showed a statistically significant increase in characteristic frequency during development except the most posterolateral (low-frequency) sector. Too few neurons were recorded from this region to be able to reliably estimate characteristic frequency. These results support the hypothesis that the spatial coding of frequency along the cochlea shifts during development.(ABSTRACT TRUNCATED AT 400 WORDS)

Organization and development of brain stem auditory nuclei of the chicken: dendritic gradients in nucleus laminaris.

Nucleus laminaris (NL) is a third-order auditory nucleus in the avian brain stem which receives spatially-segregated binaural inputs from the second-order magnocellular nuclei. The organization of dendritic structure in NL was examined in Golgi-impregnated brains from hatchling chickens. Quantitative analyses of dendritic size and number were made from camera lucida drawings of 135 neurons sampled from throughout the nucleus. The most significant results of this study may be summarized as follows: (1) The preponderant neuron in n. laminaris may be characterized as having a cylindrical-to-ovoid cell body, about 20 micrometer in diameter. The neurons comprising NL were found to be nearly completely homogeneous in issuing their dendrites in a bipolar fashion: one group of dendrites is clustered on the dorsal surface of the cells, the other group on the ventral. The dendrites of NL are contained within the glia-free neuropil surrounding the nucleus. From the rostromedial to the caudolateral poles of NL there is a gradient of increasing extension of the dendrites, increasing number of tertiary and higher-order dendrites, and increasing distance from the somata of the occurrence of branching. (2) The total dendritic size (sum of the dorsal) and ventral dendritic lengths of the cells) increases 3-fold from the rostromedial to the caudolateral poles of NL. About 50% of the variance in dendritic size is accounted for by the position of the cells in NL, and the gradient of dendritic size increase has the same orientation across NL as the tonotopic gradient of decreasing characteristic frequency in NL. (3) From the rostromedial pole to the caudolateral pole of NL there is an 11-fold decrease in the number of primary dendrites along a gradient coinciding with the length and frequency gradients. Sixty-six percent of the variance in dendrite number is accounted for by position in the nucleus. (4) The correlation of dorsal and ventral dendritic size on a cell-by-cell basis is not high (r = 0.47), indicating a fair amount of variability on the single-cell level. On the other hand, the average dorsal dendritic length within an isofrequency band in NL correlates very highly with the average ventral dendritic length. Thus, on an areal basis, the amount of dendritic surface area offered to the dorsal and ventral afferents is tightly regulated. (5) The dorsal and ventral dendrites have separate gradients of increasing length and number across NL. The dorsal gradients are skewed toward the rostrocaudal axis, while the ventral dendritic gradients are skewed mediolaterally. (6) There was no correlation between either dendritic size or number of primary dendrites and the size of the somata in NL, which remains relatively constant throughout the nucleus. Several hypotheses about the ontogenetic control of dendritic structure are examined in light of the above data. Of these, the hypotheses that the ontogeny of dendritic size and number is largely under afferent control receives a great deal of circumstantial support.

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.

Organization and development of the brain stem auditory nuclei of the chicken: primary afferent projections.

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.

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.

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: 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.