Background: Neurons rely exclusively on mitochondrial oxidative phosphorylation to meet cellular
energy demands, and disruption of mitochondrial function often precipitates neuronal
cell death. Auditory neurons in the chick brain stem (n. magnocellularis [NM]) receive
glutamatergic innervation exclusively from ipsilateral eighth nerve afferents. Cochlea
removal permanently disrupts afferent support and ultimately triggers apoptotic cell
death in 30–50% of ipsilateral, deafferented neurons. Here, we evaluated whether disruption
of mitochondrial function occurs during deafferentation-induced neuronal cell death.
Purpose: To determine whether mitochondrial dysfunction occurs preferentially within dying
NM neurons.
Research Design: An experimental study. All birds underwent unilateral cochlea removal. Normally innervated
neurons contralateral to surgery served as within-animal controls.
Study Sample: Hatchling broiler chickens between 8 and 12 days of age served as subjects. A total
of 62 birds were included in the study.
Intervention: Cochlea removal was performed to deafferent ipsilateral NM neurons and trigger neuronal
cell death.
Data Collection and Analysis: Following unilateral cochlea removal, birds were sacrificed 12, 24, 48, or 168 hours
later, and brain tissue was harvested. Brainstems were sectioned through NM and evaluated
histochemically for oxidative enzyme reaction product accumulation or reacted for
Mitotracker Red, an indicator of mitochondrial membrane potential (m) and cytoplasmic
TdT-mediated dUTP Nick-End Labeling (TUNEL), an indicator of cell death. Histochemical
staining intensities for three mitochondrial enzymes, succinate dehydrogenase (SDH),
cytochrome c oxidase (CO), and ATP synthase (ATPase) were measured in individual neurons and compared
in ipsilateral and contralateral NM. Comparisons were made using unpaired t-tests
(CO) or Kruskal Wallis one way ANOVA followed by Dunn's post hoc pairwise comparisons
(ATPase, SDH). Mitotracker Red tissue was examined qualitatively for the presence
of and extent of colocalization between Mitotracker Red and TUNEL label in NM.
Results: Results showed global upregulation of all three oxidative enzymes within deafferented
NM neurons compared to contralateral, unperturbed NM neurons. In addition, differential
SDH and ATPase staining intensities were detected across neurons within the ipsilateral
nucleus, suggesting functional differences in mitochondrial metabolism across deafferented
NM. Quantitative analyses revealed that deafferented neurons with preferentially elevated
SDH and ATPase activities represent the subpopulation destined to die following cochlea
removal. In addition, Mitotracker Red accumulated intensely within the subset of deafferented
NM neurons that also exhibited cytoplasmic TdT-mediated dUTP Nick-End Labeling (TUNEL)
and subsequently died.
Conclusions: Taken together, our results demonstrate that a subset of deafferented NM neurons,
presumably those that die, preferentially upregulates SDH, perhaps via the tricarboxylic
acid (TCA) cycle. These same neurons undergo ATPase uncoupling and an eventual loss
of Δψm.
Key Words
ATPase - auditory system - cytochrome c oxidase - deafferentation - Mitotracker Red
- succinate dehydrogenase - TUNEL
