Activity-dependent regulation of the subcellular localization of neuronal calcium sensor-1 in the avian cochlear nucleus

Abstract

Neurons in the avian cochlear nucleus, nucleus magnocellularis (NM), are highly sensitive to manipulations of afferent input, and removal of afferent activity through cochlear ablation results in the death of approximately 20-40% of ipsilateral NM neurons. The intracellular cascades that determine whether an individual NM neuron will die or survive are not fully understood. One early event observed in NM following deafferentation is a rapid rise in intracellular calcium concentration. In most cellular systems, the activity of calcium-binding proteins is believed to accommodate calcium influx. The calcium-binding protein, neuronal calcium sensor-1 (NCS-1), is an intracellular neuronal calcium sensor belonging to the EF-hand superfamily. NCS-1 has been implicated in calcium-dependent regulation of signaling cascades. To evaluate NCS-1 action in NM neurons, the localization of NCS-1 protein was examined. Double-label immunofluorescence experiments revealed that NCS-1 expression is evident in both the presynaptic nerve terminal and postsynaptic NM neuron. The postsynaptic expression of NCS-1 typically appears to be closely associated with the cell membrane. This close proximity of NCS-1 to the postsynaptic membrane could allow NCS-1 to function as a modulator of postsynaptic signaling events. Following deafferentation, NM neurons were more likely to show diffuse cytoplasmic NCS-1 labeling. This increase in the number of cells showing diffuse cytoplasmic labeling was observed 12 and 24 h following cochlea ablation, but was not observed 4 days following surgery. This activity-dependent regulation of NCS-1 subcellular localization suggests it may be associated with, or influenced by, processes important for the survival of NM neurons.

Fig. 1

Pre-and Post-synaptic localization of neuronal calcium sensor (NCS)-1. Laser confocal photomicrographs from sections processed for double-label immunofluorescence showing: (A) immunofluorescent labeling for NCS-1 (Cy3/red), (B) immunofluorescent labeling of the presynaptic terminal marker synaptic vesicle protein 2 (SV2) (FITC/green) immunofluorescent labeling, and (C) both channels superimposed. The yellow coloring in the superimposed image is indicative of some co-localization of NCS-1 with the presynaptic marker SV2, which is present in auditory nerve fibers synapsing on NM neurons. As can be seen in both the single channel and the combined image, NCS-1 is also clearly present in postsynaptic nucleus magnocellularis (NM).

Fig. 2

Deafferentation induced change in neuronal calcium sensor (NCS)-1 labeling. (A) Low-power photomicrographs of anti-NCS-1 peroxidase-stained tissue sections showing a “ringing” of nucleus magnocellularis (NM) cell bodies on the intact side of the brain. By 24 h following cochlea removal, an increase in the number cells with diffuse cytoplasmic NCS-1 immunolabeling can be observed in deafferented NM neurons when compared with intact NM neurons on the contralateral side of the brainstem. (B) High-power photomicrographs where white arrows point to NCS-1 diffuse labeling in the cytoplasm of deafferented NM neurons, and black arrows point to NCS-1 labeling in close proximity to the membrane.

Fig. 3

Time course of deafferentation-induced change in neuronal calcium sensor (NCS)-1 labeling. A difference between the two sides in the number of nucleus magnocellularis (NM) neurons showing diffuse cytoplasmic labeling is evident by 12 h following cochlea removal; this difference is not evident by 6 h following surgery. The increased labeling in the deafferented NM persists for at least 24 h following cochlea removal (CR), but by 96 h following surgery the difference between sides is no longer evident. Bars = S.E.M. Statistically reliable differences in the number of labeled neurons are only observed at 12, 18 and 24 h following cochlea removal.