Postnatal development of spiral ganglion cells in the rat.

The postnatal development of the spiral ganglion in the albino rat was studied using light and electron microscopy. The morphological characteristics distinguishing type 1 from type 2 spiral ganglion cells were defined, and the critical period for distinguishing the two types of neurons was identified. At birth, the spiral ganglion consists of a homogeneous population of small, densely packed, spherical cells that have large cytoplasmic-to-nuclear ratios. During the first postnatal week, the cells mature slowly. At this period the myelin sheath around the cell bodies generally consists of only a few layers of loose myelin. Long glial fingers extend around the cell processes and soma, particularly the filopodial extensions of the somatic membrane. Type 2 spiral ganglion cells can be distinguished at postnatal day 8. Viewed with the phase-contrast microscope these cells are smaller and have more darkly staining nuclei and more lightly staining cytoplasm than the type 1 cells. The most characteristic ultrastructural features of the type 2 neurons are the densely packed neurofilaments in the cytoplasm and lack of compact myelin around the cell soma. By day 14, spiral ganglion cells are morphologically mature, although the myelin sheath continues to thicken. The results are discussed in relation to the electrophysiological development of the auditory system and the morphological maturation of the organ of Corti.

Species differences in the synaptic membranes of the end bulb of held revealed with the freeze-fracture technique.

Two morphological differences distinguish the membranes of the end bulb-spherical cell synapse in rats and mice from those in guinea pigs and chinchillas. First, in freeze-fracture replicas, the membranes of rat and mouse spherical cells lack perisynaptic aggregates which are present in the other species. Second, small gap junctions are present between the end bulb and spherical cell soma of rats and mice. These interspecies differences are not reflected in thin-sectioned material. This observation points out the difficulty in attempting to generalize about the significance of intramembrane specializations in synaptic membranes.

The maturation of the end bulb of Held in the rat anteroventral cochlear nucleus.

The maturation of the end bulb of Held was studied in serial thin sections through the rostral pole of the rat anteroventral cochlear nucleus (AVCN) at 2-day intervals from birth through 16 days of age. In the neuropil of newborn rats primary auditory terminals occasionally synapse with large dendritic processes of spherical cells. Between 6 and 10 days of age, the spherical cell has numerous, long, fingerlike appendages at the base and proximal part of its primary dendrite which extend into the neuropil and envelope a single primary terminal. Small processes from the terminal extend along the appendages toward the cell body. At 10 days, these processes reach the dendritic pole of the soma and fill the interstices between the appendages. Synaptic contacts are present between the end bulb processes and the cell body. At 12 days, the end bulb covers most of the dendritic pole of the spherical cell. The remaining somatic appendages are small and are clustered beneath the end bulb. Synapses between the end bulb and soma are frequent. At 16 days, the end bulb resembles that seen in the mature rat. Nonprimary terminals appear on the soma at 10 days. These terminals are randomly distributed, with fine processes which extend from the end bulb over the remaining regions of the cell body.

A freeze-fracture study of the maturation of synapses in the anteroventral cochlear nucleus of the developing rat.

The freeze-fracture technique was used to study the maturation of the postsynaptic membrane of spherical cells of the anteroventral cochlear nucleus (AVCN) of the developing rat brain. Observations were made at 2-day intervals from birth to 16 days of age. At all ages, the external leaflet (E-face) of the spherical cell membrane has aggregates of particles opposite presynaptic active zones. From birth to 6 days of age, these particle aggregates were found only on dendritic processes, not on the cell body. At 8 days of age, the aggregates were found on small somatic appendages and on the cell body adjacent to somatic appendages. In animals from 10 to 12 days of age, particle aggregates became increasingly common on the cell body. Morphometric analysis demonstrated a decrease in size but not packing density of the particles in the aggregates. These changes in the location and size of the aggregates correspond to the location of asymmetrical synapses seen in thin section material at these same time intervals (Neises et al., 1982). At 14 days the fracture plane no longer followed the postsynaptic membrane at the active zone; instead it shifted to the presynaptic membrane and the postsynaptic specializations were rarely seen. This fracture pattern is typical of adult animals.

Cytoskeletal organization at the postsynaptic complex.

Postsynaptic densities and the adjacent cytoskeleton were examined in deep-etched, unfixed slices of guinea pig anteroventral cochlear nucleus. The postsynaptic density seen in conventional thin sections corresponds to a meshwork of 4-nm filaments associated with intramembrane particles at the postsynaptic active zone of inhibitory as well as excitatory synapses. These filaments intermesh with a lattice of 8- to 9-nm microfilaments, tentatively identified as F-actin, that is concentrated under the postsynaptic density. We postulate that the meshwork of 4-nm filaments anchors receptors to the adjacent microfilament lattice; this extended postsynaptic complex may limit the mobility of receptors and help maintain the curvature of the postsynaptic membrane.

Effects of neuronal activity on kainic acid neurotoxicity in the ventral cochlear nucleus.

Kainic acid was injected into the brain stem of adult guinea pigs, and the animals were either placed in a sound reducing-chamber or stimulated with 90 dB noise. The pattern and rate of kainic acid-induced degeneration in the anteroventral cochlear nucleus (AVCN) of sound-deprived animals was similar to that in animals exposed to ambient noise [2]. The amount of degeneration was greatly increased in animals stimulated with 90 dB noise. Therefore, although decreased activity in primary auditory fibers does not protect neurons in the AVCN from kainate-induced neurotoxicity, increased auditory stimulation augments the effects of kainic acid in the cochlear nucleus.

Fast axonal transport in auditory neurons of the guinea pig: a rapidly turned-over glycoprotein.

Proteins of the fast component of axonal transport were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis in the guinea pig spiral ganglion, which has its cell bodies in the cochlea and its axons in the eighth cranial nerve projecting to the ipsilateral cochlear nucleus. We found that we could easily identify the proteins of the fast component even though these axons are only about 3 mm long because the cochlea minimized diffusion of labeled precursor into the cochlear nucleus. The composition of the fast component of the spiral ganglion cells was similar, but not identical, to the fast component of guinea pig retinal ganglion cells. One difference was the predominance in the spiral ganglion cell fast component of a rapidly turned-over glycoprotein (RTGP) with a molecular weight of 110,000-140,000 and an isoelectric point of 5.0 RTGP accumulated in the cochlear nucleus for just the first 3 hr after the application of the labeled precursor and then rapidly disappeared, whereas the other major fast component polypeptides continued to accumulate for 12-24 hr. RTGP was also tentatively identified in the fast component of retinal ganglion cells, but was not as prominently labeled relative to the other fast-component proteins in those cells. The rapid disappearance of RTGP from spiral ganglion cell terminals in the cochlear nucleus may be a result of secretion, perhaps as part of a synaptic vesicle, or retrograde transport as a feedback signal. The difference in the relative amounts of RTGP found in spiral ganglion and retinal ganglion cell terminals may reflect differences in the fundamental properties of the two groups of neurons.

Freeze-fracture studies on the synapse between the type I hair cell and the calyceal terminal in the guinea-pig vestibular system.

The apposition between type I hair cells and the calyceal terminals of vestibular ganglion cell peripheral processes was studied in the vestibular epithelium of the guinea-pig, using thin-sectioned and freeze-fractured specimens. Chemical synaptic junctions were exceedingly rare in thin-sectioned specimens, and were not seen in freeze-fracture replicas. Furthermore, no gap junctions were present between the hair cell and the calyx. There were, however, regions along the apposition where the membranes were closely apposed. At these regions, the hair cell was invaginated by cytoplasmic protrusions of the calyx and the plasmalemmata of the two cells were separated by only 6-7 nm. The number and conformation of the close appositions varied between different cells. In freeze-fracture replicas, the closely-apposed plasmalemmata of the hair cell and the calyx had no special distribution of intramembrane particles on either membrane leaflet. However, on the external membrane leaflet of the hair cell, a large patch of widely-spaced, large particles surrounded the regions of close apposition. The corresponding region of the plasmalemma of the calyx had no special distribution of particles on either membrane leaflet. The scarcity of chemical synaptic junctions, the absence of gap junctions between the cells and the unique arrangement of particles in the hair cell plasmalemma surrounding regions of close membrane apposition may indicate an unusual mode of synaptic transmission between the type I hair cell and the calyx.

Uptake of putative neurotransmitters in the organ of Corti.

In vitro uptake of putative neurotransmitters into the organ of Corti of the guinea pig was studied by autoradiography. After incubation in 3H-glycine the label was heaviest over the inner hair cell, but was not confined to the synaptic region of the cell. After incubation in 3H-GABA, 3H-glutamate and 3H-aspartate, heavy labeling was seen over the fibers and terminals of the efferent olivocochlear bundle. Leucine, an amino acid not thought to be a neurotransmitter, was uniformly taken up by all cochlear structures. The fact that GABA, glutamate and aspartate are taken up into efferents, which are almost certainly cholinergic, suggests that high affinity uptake of these substances is not restricted to terminals in which these substances are released as neurotransmitters.

Synaptic structures in the type II hair cell in the vestibular system of the guinea pig. A freeze-fracture and TEM study.

The synaptic contacts of the type II hair cell in the vestibular system of the guinea pig was described in thin-sectioned and freeze-fractured specimens. Synaptic bodies were present at the apposition with both large and small afferent terminals. About 20% of the synaptic bodies observed consisted of complexes of two or more adjacent synaptic discs. In freeze-fracture replicas, the cytoplasmic leaflet of the hair cell plamalemma beneath the synaptic body had a bar-shaped aggregate of large particles. The size and shape of the particle aggregate was the same as that of the synaptic body. Small plasmalemmal deformations, interpreted as sites of synaptic vesicle exocytosis, were found immediately adjacent to the particle aggregate. On the postsynaptic membrane, an aggregate of intramembrane particles was present at the synaptic junction. The type II hair cell had no gap junctions or close membrane appositions between it an the apposed afferent fiber. Efferent boutons ending on the type II hair cell had no intramembrane particle specialization on the postsynaptic membrane; however those efferent boutons ending on large and small afferent fibers had an aggregate of medium-sized particles on the external leaflet of the postsynaptic bouton beneath the presynaptic active zone.

Kainic acid injections result in degeneration of cochlear nucleus cells innervated by the auditory nerve.

When kainic acid, a putative neurotoxin for neurons with glutamatergic input, is injected into the brainstem, it produces a selective pattern of degeneration in the cochlear nucleus. The rate and extent of degeneration is correlated with the distribution of the primary auditory fibers. This evidence supports the hypothesis that glutamate is the neurotransmitter for primary auditory fibers.

Changes in the synapses of spiral ganglion cells in the rostral anteroventral cochlear nucleus of the waltzing guinea pig following hair cell loss.

Between 10 and 60 days of age in the waltzing guinea pig, there is a genetically induced loss of all hair cells in the organ of Corti. About 43% of the spiral ganglion cells degenerate between 30 and 60 days of age. After 90 days of age, there is no further loss of spiral ganglion cells. Both Type I and II ganglion cells remain and are without afferent input. The terminals of these ganglion cells in the rostral AVCN, the end bulbs of Held, are normal until 30 days of age. During the period of ganglion cell loss degenerating end bulbs are seen. After 60 days of age, when most ganglion cell degeneration is complete, the remaining end bulbs have fewer synaptic vesicles and their synaptic junctions are flattened. The channels of enlarged extracellular space, which normally surround each synaptic junction or small groups of junctions, are only infrequently present. In freeze-fracture replicas of the rostral AVCN of waltzing guinea pigs after hair cell loss, the number of large, non-aggregate particles on the external leaflet of the principal cell opposite the end bulb is increased, and the number of perisynaptic aggregates is decreased compared to waltzing guinea pigs 10 days of age. The junctional aggregates are unaltered. These changes in the presynaptic terminal and postsynaptic membrane may be related to the loss of afferent input to the spiral ganglion cells, suggesting that activity is important for maintaining the synapse.

Internal organization of membranes at end bulbs of Held in the anteroventral cochlear nucleus.

The end of bulb of Held in the rostral ventral cochlear nucleus of the chinchilla and guinea pig was studied with the freeze-fracture technique. The end bulb has multiple, small active zones which are uniformly distributed within the calyceal portion of this terminal. Single or small groups of active zones are surrounded by enlarged channels of extracellular space often containing processes of astrocytes. Small plasmalemmal deformations occur at these active zones. The number of these deformations is thought to be indicative of exocytotic transmitter release because they are more frequent in animals fixed in a noisy environment compared to animals fixed in a quiet environment. Thus, our study provides a basis for the quantitative study of changes in transmitter secretion at a central nervous system synapse driven by a controllable natural stimulus. The postsynaptic active zone at end bulbs resembles other excitatory synapses in the central nervous system in having an aggregate of large particles on the external membrane leaflet. This junctional aggregate of particles is coextensive with the presynaptic active zone and with the postsynaptic density seen in thin sections. Several perisynaptic aggregates of particles are deployed around each active zone on the external membrane leaflet. These irregularly-shaped aggregates occur preferentially opposite the channels of enlarged extracellular space and along the edge of the end bulb and are not components of intercellular junctions or plasmalemmal contacts with cytoplasmic organelles. Although the function of the different particle aggregates on the postsynaptic membrane is not clear, our findings provide a basis for studying the factors controlling and maintaining their structure as well as more evidence that a consistent relationship exists between types of synaptic action and structure of the postsynaptic membrane.

Aspartic acid and glutamic acid levels in the cochlear nucleus after auditory nerve lesion.

Aspartic acid, glutamic acid and alanine were measured in the cochlear nucleus after lesioning the auditory nerve by cochlear ablation. Ultrastructural analysis of the cochlear nucleus showed that most primary auditory terminals were degenerating one day after cochlear ablation; the terminals were enlarged and the number of synaptic vesicles was reduced. Primary auditory terminals were virtually gone three days after cochlear ablation. Aspartic acid decreased after cochlear ablation in parallel with the morphological degeneration of the primary auditory terminals. The level of total aspartic acid in the cochlear nucleus had decreased more than 8% one day after cochlear ablation and more than 30% after two days, and remained at this level up to 28 days. Glutamic acid also decreased in the cochlear nucleus after cochlear ablation but not in parallel with the morphological degeneration of the primary auditory terminals. Following a slight increase one day after cochlear ablation, total glutamic acid decreased about 10% after two days and continued to decrease slowly through to day 28. Alanine dropped slowly after cochlear ablation and not in parallel with the degeneration of the primary terminals. Levels of other amino acids measured were unchanged or had increased two days after cochlear ablation. Aspartic acid and glutamic acid did not decrease in the superficial layers of the dorsal cochlear nucleus, an area receiving little or no primary innervation.

Remodeling of neuronal membranes as an early response to deafferentation. A freeze-fracture study.

The early effects of deafferentation on the postsynaptic membrane beneath the end bulb of Held in the anteroventral cochlear nucleus (AVCN) were studied with the freeze-fracture technique. Three distinct responses were seen on the external membrane leaflet after cochlear ablation. Within 12 h the number of nonaggregate particles increased 147% by the addition of new particles to the membrane. The increase in number of nonaggregate particles continued until 4 days after cochlear ablation. The other responses occurred later, after degenerative changes were present in the end bulb. Between 1 and 2 days after cochlear ablation, the number of perisynaptic aggregates surrounding the postsynaptic active zone decreased to 10% of normal numbers. By 4 days, all perisynaptic aggregates had disappeared from the membrane. Coated vesicles may be involved in removing these aggregates. Between 1 and 3 days, the number of junctional aggregates decreased, but the size of the aggregates increased, apparently as a result of coalescence of nearby junctional aggregates. The total number of particles in junctional aggregates in the membrane was not altered during the first 6 days after cochlear ablation. The three separate responses suggest the existence of at least three different types of intramembranous particles on the external leaflet of the principal cell membrane, with each type dependent upon different cues for its maintenance in the membrane.