Afferent regulation of cytochrome-c and active caspase-9 in the avian cochlear nucleus

During development, a subpopulation (approximately 30%) of neurons in the avian cochlear nucleus, nucleus magnocellularis (NM), dies following removal of the cochlea. It is clear that neuronal activity coming from the auditory nerve provides trophic support critical for cell survival in the NM. Several aspects of the intracellular signaling cascades that regulate apoptosis have been defined for naturally occurring, or programmed cell death, in neurons. These intracellular cascades involve the extrusion of cytochrome-c from the mitochondria into the cytosol and the subsequent activation of proteolytic caspase cascades, which ultimately act on substrates that lead to the death of the cell. In contrast, the intracellular signaling cascades responsible for deafferentation-induced cell death are not fully understood. In the present series of experiments, the potential extrusion of cytochrome-c from the mitochondria into the cytosol, and the activation of caspases were examined in the NM following deafferentation. Cytochrome-c immunoreactivity increased within 6 h following deafferentation and persisted for at least 3–5 days following surgery. However, cytochrome-c was not detectable within immunoprecipitates obtained from cytosolic fractions of deafferented NM neurons. This suggests that the increased immunoreactivity of cytochrome-c is related to mitochondrial proliferation. As a positive control, cytochrome-c was detected in cytosolic fractions of deafferented NM neurons treated with kainic acid, a substance known to cause cytochrome-c release into the cytosol. In addition, immunoreactivity for downstream active caspase-9 did increase following cochlea ablation. This increase was observed within 3 h following cochlea removal, but was not observed 4 days following surgery, a time point after the dying population of NM neurons have already degenerated. Together, these findings suggest that deafferentation of NM neurons results in caspase activation, but this activation may be cytochrome-c independent.     

The influence of chronic lithium administration on deafferentation-induced cellular changes in the chick cochlear nucleus

The avian brainstem serves as a useful model system to address the question of how afferent activity influences viability of target neurons. Approximately 20-30% of neurons in the chick cochlear nucleus, nucleus magnocellularis (NM) die following deafferentation (i.e. deafness produced by cochlea removal). Previous studies have identified cellular events that occur within hours following cochlea removal, which are thought to lead to the ultimate death of NM neurons. We have recently shown that chronic lithium treatment increases neuronal survival following deafferentation. To assess where in the cell death cascade lithium is having its effect, we evaluated some of the early deafferentation-induced cellular changes in NM neurons. Lithium did not affect deafferentation-induced changes that occur across the entire population of NM neurons. There were still deafferentation-induced increases in intracellular calcium concentrations and early changes in the ribosomes, as indicated by Y10b immunolabeling. Lithium did, however, affect changes that are believed to be indicative of the subpopulation of NM neurons that will eventually die. Ribosomes recovered in all of the deafferented NM neurons (as assessed by Y10b labeling) by 10 h following cochlea removal in subjects pretreated with lithium, while a subpopulation of the NM neurons in saline-treated subjects showed dramatic reduction in Y10b labeling at that time. Lithium treatment also prevented the robust upregulation of b cell leukemia/lymphoma-2 (Bcl-2) mRNA that is observed in a subpopulation of deafferented NM neurons 6 h following cochlea removal.     

Deafferentation-induced caspase-3 activation and DNA fragmentation in chick cochlear nucleus neurons

Cochlea removal severs peripheral processes of cochlear ganglion cells and permanently abolishes afferent input to nucleus magnocellularis (NM) neurons. Deafferented chick NM neurons undergo a series of morphologic and metabolic changes, which ultimately trigger the death of 20%–40% of neurons. Previous studies suggested that this cell specific death involves activation of the intrinsic apoptotic pathway, including increased presence of cytochrome c and active caspase-9 in the cytoplasm of deafferented NM neurons. Interestingly, however, both markers were detected pan-neuronally, in both degenerating and surviving NM neurons [Wilkinson BL, Elam JS, Fadool DA, Hyson RL (2003) Afferent regulation of cytochrome-c and active caspase-9 in the avian cochlear nucleus. Neuroscience 120:1071–1079]. Here, we provide evidence for the increased appearance of late apoptotic indicators and describe novel characteristics of cell death in deafferented auditory neurons. Young broiler chickens were subjected to unilateral cochlea removal, and brainstem sections through NM were reacted for active caspase-3 and terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL). Caspase-3 activation is observed in the cytoplasm of both dying and surviving deafferented NM neurons 24 h to 7 days following cochlea removal, suggesting that caspase-3, usually considered an “executioner” of apoptotic death, may also function as a “modulator” of death. In addition, we find that TUNEL labeling of degraded DNA is observed in deafferented NM. In contrast to upstream apoptotic markers, however, TUNEL labeling is restricted to a subpopulation of deafferented neurons. Twelve hours following cochlea removal, TUNEL labeling is observed as punctate accumulations within nuclei. Twenty-four hours following cochlea removal, TUNEL accumulates diffusely throughout neuronal cytoplasm in those neurons likely to die. This cytoplasmic TUNEL labeling may implicate mitochondrial nucleic acid degradation in the selective death of some deafferented NM neurons. Our study examines the subcellular distributions of two prominent apoptotic mediators, active caspase-3 and TUNEL, relative to known histochemical markers, in deafferented NM; provides new insight into the apoptotic mechanism of cell death; and proposes a role for mitochondrial DNA in deafferentation-induced cell death.     

Immunocytochemical localization of neuronal calcium sensor-1 in the hippocampus and cerebellum of the mouse,with special reference to presynaptic terminals

Neuronal calcium sensor-1 (NCS-1) is a member of the EF-hand calcium-binding protein superfamily, which is considered to modulate synaptic transmission and plasticity. In this work, we first examined the distribution patterns of NCS-1 in the hippocampus and cerebellum. The intense NCS-1-immunoreactive (IR) elements in the hippocampus were restricted to dendritic layers, while those in the cerebellum occurred in both dendritic and cellular layers. Then, we examined the exact localization of NCS-1 using immunofluorescent double labeling for NCS-1 and synaptophysin, a marker of presynaptic terminals. In the hippocampus, the mossy fiber systems (terminals and bundles) exhibited intense NCS-1 immunoreactivity. On the other hand, the presumed principal cell dendrites were also NCS-1-IR in the stratum lacunosum-moleculare of Ammon's horn and molecular layer of the dentate gyrus, where NCS-1-IR elements and synaptophysin-IR presynaptic terminals showed characteristic complementary distribution patterns. In the cerebellum, some of the basket cell axon terminals surrounding the somata of Purkinje cells exhibited NCS-1 immunoreactivity, while the pinceau showed consistent labeling for NCS-1. Higher magnification observations revealed that the NCS-1-IR presumed granule cell dendrites and synaptophysin-IR mossy fiber terminals in the glomeruli of the cerebellum showed characteristic complementary distribution patterns. Furthermore, we estimated quantitatively the relative amount of NCS-1 in the presynaptic terminals in individual layers, and confirmed that the mossy fiber terminals in the hippocampus contained comparatively high amounts of NCS-1. These results showed the diverse localization of NCS-1 in pre- and/or postsynaptic elements of the hippocampus and cerebellum, and suggest potential roles in specific synaptic transmission.     

Neuronal calcium sensor-1 is expressed by dorsal root ganglion cells, is axonally transported to central and peripheral terminals, and is concentrated at nodes

Neuronal calcium sensor-1 (NCS-1) is a member of the EF-hand calcium-binding protein superfamily which has been implicated in the modulation of a number of neuronal functions. In this study we have examined the expression of NCS-1 in adult rat dorsal root ganglion (DRG) neurons. NCS-1 immunoreactivity was present in most DRG neurons, including many calcitonin gene-related peptide (CGRP) expressing ones. NCS-1 showed some colocalization with the synaptic vesicle protein synaptophysin and underwent both anterograde and retrograde axonal transport. NCS-1 immunoreactivity was also present in the dorsal horn of the spinal cord, and in peripheral cutaneous terminals innervating blood vessels, where it was coexpressed with CGRP. In addition, NCS-1 in peripheral nerves was concentrated at nodes and adjoining paranodes. These results suggest novel roles for NCS-1, particularly in relation to channel function at nodes and to the peripheral release of vasoactive peptides.     

Cochlear ablation in adult ferrets results in changes in insulin-like growth factor-1 and synaptophysin immunostaining in the cochlear nucleus

Afferent activity modulates synaptic plasticity as well as the levels of activity-dependent molecules such as growth factors. Disruption of this activity due to deafferentation has been shown to result in an altered trophic support and consequently in changes in neuronal excitability and synaptic transmission. In the present study, to test whether lack of cochlear integrity results in changes in insulin-growth factor-1 (IGF-1) and synaptophysin immunostaining in the cochlear nucleus, the first relay structure in the auditory pathway, unilateral cochlear ablations were performed in adult ferrets. Changes in IGF-1 and synaptophysin immunostaining were assessed in the anteroventral (AVCN), posteroventral (PVCN) and dorsal cochlear nucleus (DCN) at 1, 20 and 90 days after deafferentation. An increase in IGF-1 immunostaining within AVCN, PVCN and DCN was observed ipsilaterally at all survival times after cochlear ablation when compared with the contralateral side and unoperated animals. This increase was accompanied by a significant ipsilateral increase in the mean gray level of synaptophysin immunostaining as well as a decrease in the area of synaptophysin immunostaining at 1 and 20 days after the ablation in AVCN, PVCN and DCN compared with the contralateral side and control animals. These changes in synaptophysin immunostaining were no longer present 90 days after cochlear ablation. The present results provide evidence of a persistent upregulation in IGF-1 and a transitory upregulation in synaptophysin levels in the cochlear nucleus that may reflect neuroprotective mechanisms following the loss of trophic support from spiral ganglion neurons.     

黄雀(carduelis spinus)耳蜗神经元至延髓耳蜗主核的投射——HRP法研究

将HRP溶液注入鸣禽黄雀的耳蜗内,顺行追踪了耳蜗神经元至延髓听觉核团的上行投射。①在同侧的听神经(NⅧ)有密集标记的神经纤维束并分别投射至延髓的巨细胞杉和角状核;②在巨细胞核和角状核出现大量密集的标记终末,表明:黄雀的耳蜗神经元发出的纤维组成听神经后分别投射至同侧的巨细胞核和角状核做为听觉通路的第一级换元站的延髓耳蜗主核只由此二亚核组成,延髓的层状核并不接受耳蜗纤维的直接投射。     

Neuroprotection and neurogenesis: modulation of cornus ammonis 1 neuronal survival after transient forebrain ischemia by prior fimbria-fornix deafferentation

Severe transient forebrain ischemia causes selective neuronal death in the hippocampal cornus ammonis 1 region. We tested the hypothesis that fimbria-fornix deafferentation can provide long-term protection to cornus ammonis 1 neurons and modulate neurogenesis following ischemia. Fimbria-fornix lesion or sham-fimbria-fornix lesion was performed on Wistar rats 13 days prior to 10 min forebrain ischemia or sham ischemia. Temperature was regulated and rats survived for 7, 14 or 28 days. Immunofluorescent bromodeoxyuridine and neuron specific nuclear protein staining and immunochemistry terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining were performed. At 7 days after ischemia, 73%+/-14% of cornus ammonis 1 neurons were damaged, while deafferentation reduced the injury to 36%+/-17% of cornus ammonis 1 neurons. This protection persisted for at least 28 days. Ischemia significantly increased the number of bromodeoxyuridine-positive cells (85-90 cells/section in stroke group vs. 6 to 11 cells/section in normal or sham stroke group), with very few terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-stained cells adjacent to the hippocampal cornus ammonis 1. Fimbria-fornix lesioning followed by ischemia increased the percentage of new neurons 13-fold over ischemia alone and 6.5-fold over sham lesion plus ischemia. The results indicate that fimbria-fornix deafferentation provides long-term neuroprotection in cornus ammonis 1 following forebrain ischemia and promotes neurogenesis after ischemic insults.     

N-methyl-D-aspartate receptors are present in vagal afferents and their dendritic targets in the nucleus tractus solitarius.

N-Methyl-D-aspartate receptors are present in the nodose ganglion, which contains the cell bodies of vagal afferents, and in the nucleus tractus solitarius, where these afferent fibers terminate. This suggests that N-methyl-D-aspartate receptors are located presynaptically on visceral vagal afferents and/or their target neurons in the nucleus tractus solitarius. To test this hypothesis, we combined anterograde transport of biotinylated dextran amine, following injections into the left nodose ganglion, with electron microscopic immunogold labeling of antipeptide antiserum against the R1 subunit of the N-methyl-D-aspartate receptor in the nucleus tractus solitarius of rat brain. Within the medial nucleus tractus solitarius, the N-methyl-D-aspartate receptor R1 immunoreactivity was seen in dendrites (39% of 639 profiles), axons and axon terminals (41%), and a few neuronal perikarya and glia. Many vagal afferent axons and terminals (40% of 468 profiles) contained N-methyl-D-aspartate receptor R1 immunogold labeling. In addition, 42% of the dendrites contacted by vagal afferent terminals (n = 206) contained N-methyl-D-aspartate receptor R1 immunoreactivity. In axons and dendrites, the gold particles were occasionally seen within asymmetric postsynaptic junctions or at non-synaptic sites on the plasma membrane. More commonly, however, N-methyl-D-aspartate receptor R1 labeling was seen on membranes of vesicular cytoplasmic organelles, suggesting that there is abundant N-methyl-D-aspartate receptor protein available for activity-dependent mobilization to the plasmalemma. Since many vagal afferents are glutamatergic, our results implicate N-methyl-D-aspartate receptors in autoregulation of the presynaptic release and postsynaptic responses to glutamate at the level of the first central synapse in the nucleus tractus solitarius.     

Calcium-sensing mechanism in TRPC5 channels contributing to retardation of neurite outgrowth

The calcium- and sodium-permeable transient receptor potential channel TRPC5 has an inhibitory role in neuronal outgrowth but the mechanisms governing its activity are poorly understood. Here we propose a mechanism involving the neuronal calcium sensor-1 (NCS-1) protein. Inhibitory mutants of TRPC5 and NCS-1 enhance neurite outgrowth similarly. Mutant NCS-1 does not inhibit surface-expression of TRPC5 but generally suppresses channel activity, irrespective of whether it is evoked by carbachol, store depletion, lanthanides or elevated intracellular calcium. NCS-1 and TRPC5 are in the same protein complex in rat brain and NCS-1 directly binds to the TRPC5 C-terminus. The data suggest protein–protein interaction between NCS-1 and TRPC5, and involvement of this protein complex in retardation of neurite outgrowth.     

Endocannabinoid-mediated synaptically evoked suppression of GABAergic transmission in the cerebellar cortex

Presynaptic CB₁ cannabinoid receptors are frequently targets of endogenous cannabinoids (endocannabinoids) released from postsynaptic neurons. It is known that the glutamatergic afferent input to a neuron can trigger endocannabinoid production and that the released endocannabinoid can suppress the glutamatergic input. We tested the hypothesis that activation of the glutamatergic input to a neuron leads to an endocannabinoid-mediated suppression of the GABAergic afferent input to the same neuron. Spontaneous postsynaptic currents (sPSCs) were recorded with patch-clamp techniques in Purkinje cells in mouse cerebellar brain slices. Activation of the climbing fiber-mediated glutamatergic input to Purkinje cells led to a suppression of the sPSCs by 34±3%. This suppression was mostly due to suppression of GABAergic spontaneous inhibitory postsynaptic current (sIPSCs), because 93% of the sPSCs recorded in Purkinje cells were GABAergic sIPSCs. Blockade of ionotropic, but not metabotropic glutamate receptors, prevented the suppression. The climbing fiber activation led to an increase in calcium concentration in the Purkinje cells, and this increase was necessary for the suppression of sPSCs, because the suppression did not occur when the calcium increase was prevented by BAPTA. No sPSC suppression was observed in the presence of the CB₁ antagonist rimonabant or the diacylglycerol lipase inhibitor orlistat. In a further series of experiments GABAergic sIPSCs were recorded: these sIPSCs were also suppressed after climbing fiber activation, and the suppression was sensitive to the CB₁ antagonist SLV319. Finally, the GABAergic synaptic transmission between molecular layer interneurons and Purkinje cells was directly studied on simultaneously patch-clamped neuron pairs. Climbing fiber activation led to suppression of the interneuron → Purkinje cell synaptic transmission. The results point to a novel form of endocannabinoid-mediated heterosynaptic plasticity. The endocannabinoid production in a neuron is triggered by its glutamatergic synaptic input and is dependent on an increase in intracellular calcium concentration. The produced endocannabinoid, in turn, suppresses the GABAergic synaptic input to the neuron by activating CB₁ cannabinoid receptors.     

Complex response to afferent excitatory bursts by nucleus accumbens medium spiny projection neurons

The nucleus accumbens (NAc) of the ventral striatum is involved in attention, motivation, movement, learning, reward, and addiction. GABAergic medium spiny projection neurons that make up approximately 90% of the neuronal population are commonly driven by convergent bursts of afferent excitation. We monitored spiny projection neurons in mouse striatal slices while applying stimulus trains to mimic bursts of excitation. A stimulus train evoked a simple, short-lived postsynaptic response from CA1 hippocampal pyramidal neurons, but the train evoked a complex series of excitatory postsynaptic potentials (EPSPs) or currents (EPSCs) from the NAc spiny projection neurons. As is commonly seen with projection neurons, the EPSC amplitudes initially displayed facilitation followed by depression, and that pattern was sensitive to the extracellular calcium concentration. In addition, there were two other novel observations. The spiny projection neurons responded to the stimulus train with a prolonged depolarization that was accompanied by a posttrain increase of spontaneous glutamatergic synaptic activity. Blocking AMPA/kainate glutamate receptors strongly inhibited the evoked EPSP/EPSCs, the posttrain spontaneous synaptic activity, and the prolonged depolarization. A potassium channel inhibitor increased and extended the prolonged postsynaptic depolarization, causing a long-lasting depolarized plateau potential. Our results indicate that burst-like activity along ventral striatal afferents is extended in time by additional spontaneous glutamate release that is integrated by the postsynaptic spiny projection neurons into a prolonged depolarization. The results suggest that the posttrain quantal glutamate release helps to blend and maintain multiple afferent inputs. That convergent excitation is further integrated by the postsynaptic neuron into a prolonged depolarization that may contribute to the depolarized "up state" observed in vivo.     

Neuron morphology and synaptic architecture in the medial superior olivary nucleus

Summary Dendritic arborization pattern, spatial and synaptic relations of various neuron types and the terminal distribution of afferent axons of various origin were studied in the medial superior olivary nucleus of the cat using Golgi, degeneration, electron microscope and horseradish peroxidase techniques. Three types of neurons clearly different in morphological features, distribution, neighbourhood relations, input and output characteristics were distinguished: (1) fusiform cells having specific dendritic orientations and arborization patterns and synaptic relations to various types of terminal axon arborizations (2) multipolar neurons with wavy dendrites bearing spine-like appendages, receiving relatively few synaptic contacts and having a locally arborizing axon, and (3) elongated marginal cells, largely restricted to the fibrous capsule of the nucleus. The fusiform and marginal neurons were identified by retrograde peroxidase labeling as the olivo-collicular projection cells.Ultrastructural analysis of normal and experimental material revealed the presence of four distinct kinds of axon terminals differing in size, synaptic vesicles type, relation to postsynaptic targets and in origin: (i) large terminals with multiple extended asymmetric synaptic membrane specializations and containing round, clear vesicles arise from the spherical cells of the ipsilateral anteroventral cochlear nucleus, (ii) most of the small axon terminal profiles — engaged in asymmetric synaptic contacts — originated from the trapezoid nucleus, (iii) terminal boutons containing pleomorphic vesicles belong to fibers descending from the ipsilateral multipolar neurons in the central nucleus of the inferior colliculus and from the nuclei of the lateral lemniscus while (iv) boutons containing exclusively ovoid vesicles and remaining intact after complete deafferentation of the nucleus were considered to be of local origin.     

Lithium increases bcl-2 expression in chick cochlear nucleus and protects against deafferentation-induced cell death

Approximately 20-30% of neurons in the avian cochlear nucleus (nucleus magnocellularis) die following deafferentation (i.e. deafness produced by cochlea removal) and the remaining neurons show a decrease in soma size. Cell death is generally accepted to be a highly regulated process involving various pro-survival and pro-death molecules. One treatment that has been shown to modify the expression of these molecules is chronic administration of lithium. The present experiments examined whether lithium treatment can protect neurons from deafferentation-induced cell death. Post-hatch chicks were treated with LiCl or saline for 17 consecutive days, beginning on the day of hatching. On the 17th day, a unilateral cochlea ablation was performed. Five days following surgery, the nucleus magnocellularis neurons were counted stereologically on opposite sides of the same brains. Lithium reduced deafferentation-induced cell death by more than 50% (9.8% cell death as compared with 22.4% in saline-treated subjects). Lithium did not affect cell number on the intact side of the brain. Lithium also did not prevent the deafferentation-induced decrease in soma size, suggesting a dissociation between the mechanisms involved in the afferent control of soma size and those involved in the afferent control of cell viability. A possible mechanism for lithium's neuroprotective influence was examined in a second set of subjects. Previous studies suggest that the pro-survival molecule, bcl-2, may play a role in regulating cell death following deafferentation. Tissues from lithium- and saline-treated subjects were examined using immunocytochemistry. Chronic administration of lithium dramatically increased the expression of bcl-2 protein in nucleus magnocellularis neurons. These data suggest that lithium may impart its neuroprotective effect by altering the expression of molecules that regulate cell death.     

Morphology and electrophysiology of superior laryngeal nerve afferents and postsynaptic neurons in the medulla oblongata of the cat.

Intra-axonal recordings were made from 24 afferent fibres of the superior laryngeal nerve in and around the nucleus tractus solitarius, in 26 pentobarbitone-anaesthetized cats. Conduction velocity ranged from 15 to 38 m/s. Four afferents were injected with horseradish peroxidase. They showed dense terminal arborization in the region of the ventral and ventrolateral subnuclei of the nucleus tractus solitarius, both rostral and caudal to the obex. Six other intra-axonal recordings were thought to originate from axons of neurons postsynaptic to superior laryngeal afferents; one of these was injected with horseradish peroxidase and showed a similar arborization pattern to that of the afferent axons. In the same region, intracellular recordings were made from 124 neurons which responded to superior laryngeal nerve stimulation with excitatory postsynaptic potentials (mean latency 2.7 +/- 1.0 ms). Ninety-nine of these neurons were thought to receive a monosynaptic input. The stimulation threshold evoking these responses was similar to that which inhibited phrenic nerve discharge. Eleven of the monosynaptically excited neurons were injected with horseradish peroxidase. They had fusiform or stellate somata and simple dendritic trees, radiating mainly in the transverse plane. In one experiment, in which both a superior laryngeal nerve afferent fibre and a neuron were labelled, afferent terminal varicosities were found in close apposition with the postsynaptic membrane of the injected neuron. Four of 14 (29%) tested neurons could be antidromically activated from the C3 spinal segment. The stimulus thresholds and onset latencies of the responses of superior laryngeal nerve afferents and medullary neurons to stimulation of the superior laryngeal nerve are consistent with their involvement in the reflex inhibition of respiratory neurons evoked by superior laryngeal nerve stimulation.     

Rapid effects of neurotrophic factors on calcium homeostasis in rat visceral afferent neurons

Neurotrophic factors maintain and modulate neuron function in adults. We tested the hypothesis that neurotrophic factors rapidly alter intracellular calcium concentrations, thereby affecting neuron excitability. The majority of rat nodose neurons express TrkA, TrkB and TrkC receptor after 1 day in culture. Addition of nerve growth factor, brain derived neurotrophic factor or glial derived neurotrophic factor increased cytosolic calcium in about one third of the neurons within less than 10 min. This increase was due to calcium release from intracellular stores and could be blocked by the tyrosine kinase inhibitor K252a. The rapid effect of neurotrophic factors suggests a role of these molecules in the early response after inflammation as potential mediators for sensitization of afferent neurons.     

Apoptosis of inner hair cells caused by laser ablation of their spiral ganglion neurons in cultures of the mouse organ of Corti

Laser beam ablation of spiral ganglion neurons was performed in seven organotypic cultures of the newborn mouse cochlea between 5 and 8 days in vitro, with a recovery period of from 18 hours to 3 days. Direct somatic injury (laser or mechanical) inflicted on hair cells does not necessarily cause their death; many of them survive, repair damage and re-establish their neurosensory connections. By contrast, laser irradiation and ablation of their afferent spiral ganglion neurons causes a most spectacular degeneration of sensory cells within 18-48 hours after the insult. Ultrastructurally, the degenerated hair cells-characteristically the inner hair cells-display "dark-cell vacuolar degeneration" that combines the signs of apoptotic death (the peripheral condensation of nuclear chromatin and nuclear pyknosis) with signs of cell edema, vacuolization and necrosis. The ultimate condensation of the cytoplasm gives the dead cells a jet black appearance. The irradiated spiral ganglion neurons die displaying similar pathological characteristics. The extent and locus of inner hair cell degeneration correspond to that of ablated spiral ganglion neurons: ultimately the ablation of one neuron causes degeneration of a single inner hair cell within the closest radial segment of the afferent innervation. The elimination of spiral ganglion neurons by mechanical means does not affect hair cell survival.It is inferred that the laser pulse acts as a stimulus depolarizing the neuronal membrane of the spiral ganglion neurons and their radial fibers and causing the excitotoxic death of their synaptic sensory cells through excessive stimulation of the glutamatergic receptors. Reciprocal pre-and postsynaptic synapses between the afferent dendrites and inner hair cells in culture could possibly serve as entryways of the stimulus. The pathogenesis of this apparent transsynaptically-induced apoptotic death of inner hair cells will be further examined in culture.     

Glutamatergic calcium responses in the developing lateral superior olive: receptor types and their specific activation by synaptic activity patterns

The lateral superior olive (LSO) is a binaural auditory brain stem nucleus that plays a central role in sound localization. Survival and maturation of developing LSO neurons critically depend on intracellular calcium signaling. Here we investigated the mechanisms by which glutamatergic afferents from the cochlear nucleus increase intracellular calcium concentration in LSO neurons. Using fura-2 calcium imaging in slices prepared from neonatal mice, we found that cochlear nucleus afferents can activate all major classes of ionotropic and metabotropic glutamate receptors, each of which contributes to an increase in intracellular calcium. The specific activation of different glutamate receptor classes was dependent on response amplitudes and afferent stimulus patterns. Low-amplitude responses elicited by single stimuli were entirely mediated by calcium-impermeable AMPA/kainate receptors that activated voltage-gated calcium channels. Larger-amplitude responses elicited by either single stimuli or stimulus trains resulted in additional calcium influx through N-methyl-d-aspartate receptors. Finally, high-frequency stimulation also recruited group I and group II metabotropic glutamate receptors, both of which mobilized intracellular calcium. This calcium release in turn activated a strong influx of extracellular calcium through a membrane calcium channel that is distinct from voltage-gated calcium channels. Together, these results indicate that before hearing onset, distinct patterns of afferent activity generate qualitatively distinct types of calcium responses, which likely serve in guiding different aspects of LSO development.