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.

Second-order auditory neurons in nucleus magnocellularis (NM) of the chick brainstem undergo a series of rapid metabolic changes following unilateral cochlea removal, culminating in the death of 25% of NM neurons. Within hours of cochlea removal, ipsilateral NM neurons show marked increases in histochemical staining for the mitochondrial enzymes succinate dehydrogenase and cytochrome oxidase. We investigated corresponding ultrastructural changes in NM neurons by preparing animals undergoing unilateral cochlea removal for transmission electron microscopy. We quantified changes in NM mitochondrial volume by stereological methods and qualitatively compared mitochondrial morphology between NM neurons destined to survive and those destined to die after cochlea removal. Within hours of cochlea removal, ipsilateral NM neurons show striking increases in mitochondrial volume (84% at 6 hours and 236% at 12 hours after cochlea removal compared to unoperated, control animals). At 2 week survival times, ipsilateral NM neurons contain fewer mitochondria than contralateral neurons. Surprisingly, anesthesia alone causes short-term increases in NM mitochondrial volume. Animals anesthetized with pentobarbital and ketamine and sacrificed 6 or 12 hours later showed a 45% increase in mitochondrial volume compared to previously unanesthetized animals. NM neurons destined to die within days of cochlea removal can be identified within several hours after deafferentation by the appearance of their ribosomes. We observed qualitative differences in mitochondrial morphology in dying neurons. Mitochondria in neurons destined to die consistently showed mitochondrial swelling and vacuolization indicative of metabolic dysfunction. Similar mitochondrial changes have been reported when mitochondria take up excess calcium. Ultrastructural changes in NM after cochlea removal display features of both programmed and pathological cell death, in which increased intracellular calcium is thought to play a role.

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The role of mitochondrial biogenesis in hair cell survival after injury was evaluated by inhibiting mitochondrial protein synthesis with chloramphenicol and then studying the effects on hair cell survival after exposure to two different types of ototoxins, gentamicin and acoustic trauma. Seven- to 10-day-old chicks were treated with either a single injection of gentamicin (250 mg/kg) or noise (1500 Hz at 120 dB sound pressure level for 14 hours). A subset of the gentamicin- and noise-treated animals also received chloramphenicol (1200 mg/kg during a 24-hour period) through a subcutaneous osmotic pump. A control group received chloramphenicol alone (1200 mg/kg during a 24-hour period). All animals were sacrificed after 5 days, and their basilar papillae (cochleas) were prepared for scanning electron microscopy. Hair cell loss was quantified with stereologic techniques. Animals treated with chloramphenicol alone did not have any evidence of hair cell loss. Gentamicin-treated animals had characteristic hair cell loss beginning at the basal tip and tapering out along the inferior edge more distally. The addition of chloramphenicol to gentamicin treatment significantly increased hair cell loss by 30%, extending the area of hair cell loss into the superior hair cell region at the distal boundary of the lesion. Pure-tone noise exposure characteristically produced hair cell loss along the inferior edge and occasionally included hair cells along the most superior edge. Addition of chloramphenicol to noise exposure significantly increased hair cell loss by 80%, with extension of the lesion across the full width of the sensory epithelium and basally. These results demonstrate that mitochondrial biogenesis is involved in cellular responses to injury. They suggest that mitochondrial function may regulate the probability of survival after metabolic challenges to hair cell integrity.

NADH: nitrate reductase (EC 1.6.6.1) (NR) is present in small amounts in plant tissues and its polypeptide in inherently labile. Consequently, NR is difficult to purify. We have generated 20 monoclonal antibodies (McAb) for corn and squash NR and selected two for use in immunoaffinity chromatography. Squash McAb CM 15(11) and corn McAb ZM 2(69)9, which both bind corn and squash NR, were covalently coupled to Sepharose and used for purification of NR with elution of the purified enzyme by a pH 11 buffer. Although this procedure yielded highly purified NR, its activity was diminished by the pH 11 treatment. When corn leaf crude extract was applied to McAb CM 15(11)-Sepharose, NR bound and could be eluted in homogeneous form by its substrate, NADH. Corn leaf NR prepared by substrate elution retained a high level of NADH: NR activity. Immunoaffinity-purified corn and squash NR were shown to have an interchain disulfide bond as well as a reactive thiol group. These results are discussed in relation to the recently obtained sequences of NR clones and suggestions made for site-directed mutagenesis experiments to aid in identifying the cysteine residues of NR associated with these features of the enzyme.