Histochemical and Fluorescent Analyses of Mitochondrial Integrity in Chick Auditory Neurons following Deafferentation
Authors: Karnes Hope Elizabeth , Scaletty Peter Nicholas , Durham Dianne 
Published: April 2011

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.