1. Neurons in the cochlear nucleus, nucleus magnocellularis (NM), of embryonic and neonatal chicks are dependent on eighth nerve activity for their maintenance and survival. Removing this input results in the death of 20-40% of the NM neurons and profound changes in the morphology and metabolism of surviving neurons. 2. One of the first changes in NM neurons after an in vivo cochlea removal is an increase in intracellular calcium concentration ([Ca2+]i). Increased [Ca2+]i has been implicated in a number of neuropathologic conditions. 3. In this study, we orthodromically and antidromically stimulated NM neurons in an in vitro brain stem slice preparation and monitored NM field potentials while simultaneously assessing the [Ca2+]i of NM neurons using fura-2. 4. During continuous orthodromic stimulation, [Ca2+]i of NM neurons remained constant at 80 nM. In the absence of stimulation, NM neuron [Ca2+]i increased steadily to 230 nM by 90 min. Antidromic and contralateral stimulation produced a [Ca2+]i increase in NM neurons that was similar in magnitude but slightly more rapid than that observed in the absence of stimulation. 5. Addition of the metabotropic glutamate receptor (mGluR) antagonists (R,S)-alpha-methyl-4-carboxyphenylglycine or 2-amino-3-phosphonopropionic acid to the superfusate during continued orthodromic stimulation resulted in a dose-dependent, rapid, and dramatic increase in NM neuron [Ca2+]i without affecting the postsynaptic field potentials recorded from NM. 6. The ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione and 2-amino-5-phosphonovalerate eliminated NM field potentials during continued orthodromic stimulation but did not result in an increase in [Ca2+]i. 7. Continuous superfusion of trans-(+/-)-aminocyclopentane dicarboxylate, but not glutamate, prevented the increase in [Ca2+]i in the absence of stimulation. 8. These results suggest that NM neurons rely on eighth nerve activity-dependent activation of a mGluR to maintain physiological [Ca2+]i. Removal of this mGluR activation results in an increase in [Ca2+]i that may contribute to the early stages of degeneration and eventual death of these neurons.

Neurons of the cochlear nucleus, nucleus magnocellularis (NM), of young chicks require excitatory afferent input from the eighth nerve for maintenance and survival. One of the earliest changes seen in NM neurons after deafferentation is an increase in intracellular calcium concentration ([Ca2+]i). This increase in [Ca2+]i is due to loss of activation of metabotropic glutamate receptors (mGluR) that activate second-messenger cascades involved in [Ca2+]i regulation. Because mGluRs are known to act via the phospholipase C and adenylate cyclase signal transduction pathways, the goal of this study was to determine the roles of protein kinases A (PKA) and C (PKC) activities in the regulation of NM neuron [Ca2+]i by eighth nerve stimulation. Additionally, we sought to determine the relationship between increased [Ca2+]i and cell death as measured by propidium iodide incorporation. [Ca2+]i of individual NM neurons in brain stem slices was monitored using fura-2 ratiometric fluorescence imaging. NM field potentials were monitored in experiments in which the eighth nerve was stimulated. Five hertz orthodromic stimulation maintained NM neuron [Ca2+]i at approximately 110 nM for 180 min. In the absence of stimulation, NM neuron [Ca2+]i increased steadily to a mean of 265 nM by 120 min. This increase was attenuated by superfusion of PKC activators phorbol-12,13-myristate acetate (100 nM) or dioctanoylglycerol (50 microM) and by activators of PKA: 1 mM 8-bromoadenosine-3',5'-cyclophosphate sodium (8-Br-cAMP), 50 microM forskolin or 100 microM Sp-adenosine 3',5'-cyclic monophosphothioate triethylamine. Inhibition of PKA (100 microM Rp-cAMPS) or PKC (50 nM bisindolymaleimide or 10 microM U73122) during continuous orthodromic stimulation resulted in an increase in NM neuron [Ca2+]i that exceeded 170 and 180 nM, respectively, by 120 min. Nonspecific kinase inhibition with 1 microM staurosporine during stimulation resulted in an [Ca2+]i increase that was greater in magnitude than that seen with either PKA or PKC inhibition alone, equal to that seen in the absence of stimulation, but much smaller than that seen with inhibition of mGluRs. In addition, manipulations that resulted in a [Ca2+]i increase >/=250 nM resulted in an increase in number and percentage of propidium iodide-labeled NM neurons. These results suggest that eighth nerve activity maintains [Ca2+]i of NM neurons at physiological levels in part via mGluR-mediated activation of PKA and PKC and that increases in [Ca2+]i due to activity deprivation or interruption of the PKA and PKC [Ca2+]i regulatory mechanisms are predictive of subsequent cell death.

Hearing loss induced by ototoxicity is a worldwide problem despite the development of newer antibiotics and chemotherapy agents. The cellular mechanisms responsible for aminoglycoside-induced hearing loss are still poorly understood. We have developed two different methods of studying the dynamic cellular and subcellular changes in the chick auditory sensory epithelium that occur during hair cell death. The first study was performed in mature chicks after a single, high dose injection of gentamicin, which results in the rapid loss of all hair cells in the basal third of the cochlea. Chicks were sacrificed at discrete time points after drug treatment, and transmission electron microscopy was performed to study the ultrastructural changes in basal hair cells during the course of cell death. We noted various changes in the cell morphology including accumulation of cytoplasmic inclusion bodies, dispersion of the cytoplasmic polyribosomes, mitochondrial swelling, and cellular extrusion by 24 h after injection. The next two studies were performed using tissue cultures from mature avian auditory sensory epithelium. Cultured cells were labeled using vital fluorescent markers, and levels of intracellular calcium and reactive oxygen species within hair cells were studied following aminoglycoside exposure. We identified a dose-dependent increase in the levels of intracellular calcium, which was blocked by an inhibitor of voltage-gated calcium channels. We also found that levels of reactive oxygen species in hair cells greatly increased after exposure to gentamicin, and this response was blocked by two different antioxidants. These studies serve to identify key cellular and molecular changes in hair cells in response to ototoxic drugs. Further study of these processes may lead to a better understanding of how ototoxicity is induced and to potential preventative interventions. 389d To whom correspondence may be addressed.

1. Ratiometric fura-2 imaging was used to measure the intracellular calcium concentration ([Ca2+]i) of neurons in the embryonic avian cochlear nucleus, nucleus magnocellularis (NM), after an in ovo unilateral cochlea removal (deafferentation). 2. The mean [Ca2+]i of NM neurons receiving normal input was 113 nM. 3. Deafferentation increased the mean [Ca2+]i of NM neurons to 247, 311, 339, and 314 nM at 1, 3, 6, and 12 h after cochlear removal, respectively. These values did not differ significantly. 4. The percent frequency distribution of deafferented NM neuron [Ca2+]i shifts away from normative levels toward higher [Ca2+]i at 1 and 3 h after cochlear removal, but shifts back toward normative levels at 6 and 12 h after cochlear removal. 5. This increased [Ca2+]i following cochlear removal temporally coincides with well-characterized changes in NM neurons following activity deprivation. 6. These data suggest that deregulation of [Ca2+]i homeostasis plays a key role in NM neuron degeneration and death following activity deprivation.

The auditory nerve serves as the only excitatory input to neurons in the avian cochlear nucleus, nucleus magnocellularis (NM). NM neurons in immature animals are dependent upon auditory nerve signals; when deprived of them, many NM neurons die, and the rest atrophy. Auditory nerve terminals release glutamate, which can stimulate second messenger systems by activating a metabotropic glutamate receptor (mGluR). Therefore, it is possible that the effecters of mGluRstimulated signal transduction systems are needed for NM neuronal survival. This study shows that mGluR activation in NM neurons attenuates voltage-dependent changes in [Ca2+],. Voltage-dependent Ca2+ influx was also attenuated by increasing CAMP with forskolin, VIP, or 8-bromo-CAMP, indicating that mGluR activation may stimulate adenylate cyclase. The main results may be summarized as follows. NM neurons possess high voltage-activated Ca*+ channels that were modulated by quisqualate, glutamate, and (?)transACPD, in that order of potency. Glutamatergic inhibition of Ca2+ influx was not blocked by L-AP3 or L-AP4, which antagonize the actions of mGluRs in other neural systems; it was blocked by serine-Ophosphate. Finally, the attenuation of voltage-dependent Ca*+ influx was duplicated by CAMP accumulators. Since NM neurons have high rates of spontaneous activity and higher rates of driven activity, the expression of this mGluR turns out to be very valuable: without it, [Ca2+], could reach lethal concentrations. These results provide an important clue as to the identity of an intracellular signal that may play an important role in NM neuronal survival.