Activity-dependent regulation of [Ca2+]i in avian cochlear nucleus neurons: roles of protein kinases A and C and relation to cell death
Neurons in the cochlear nucleus, specifically the nucleus magnocellularis (NM) of young chicks, depend on excitatory afferent input from the eighth nerve for their survival and maintenance. One of the earliest alterations observed in NM neurons following deafferentation is an increase in intracellular calcium concentration ([Ca²⁺]ₖᵢ). This elevation in [Ca²⁺]ₖᵢ results from the loss of metabotropic glutamate receptor (mGluR) activation, which normally initiates second-messenger cascades involved in regulating [Ca²⁺]ₖᵢ. Since mGluRs are known to operate through phospholipase C and adenylate cyclase signaling pathways, the objective of this study was to investigate the roles of protein kinases A (PKA) and C (PKC) in regulating [Ca²⁺]ₖᵢ in NM neurons during eighth nerve stimulation. Additionally, we aimed to explore the relationship between increased [Ca²⁺]ₖᵢ and cell death, as assessed by propidium iodide incorporation.
To monitor [Ca²⁺]ₖᵢ levels in individual NM neurons, we used fura-2 ratiometric fluorescence imaging in brainstem slices. NM field potentials were recorded during eighth nerve stimulation. Continuous orthodromic stimulation at 5 Hz maintained [Ca²⁺]ₖᵢ levels in NM neurons at approximately 110 nM for 180 minutes. In the absence of stimulation, [Ca²⁺]ₖᵢ levels gradually increased, reaching a mean of 265 nM by 120 minutes. This Adenosine Cyclophosphate increase was partially prevented by superfusing with PKC activators (phorbol-12,13-myristate acetate, 100 nM or dioctanoylglycerol, 50 µM) or PKA activators (8-bromoadenosine-3′,5′-cyclic monophosphate sodium [8-Br-cAMP], 1 mM; forskolin, 50 µM; Sp-adenosine 3′,5′-cyclic monophosphothioate triethylamine, 100 µM).
Inhibition of PKA (100 µM Rp-cAMPS) or PKC (50 nM bisindolymaleimide or 10 µM U73122) during continuous orthodromic stimulation caused [Ca²⁺]ₖᵢ levels to rise above 170 nM and 180 nM, respectively, by 120 minutes. Nonspecific kinase inhibition with 1 µM staurosporine during stimulation resulted in a similar [Ca²⁺]ₖᵢ increase, matching that observed in the absence of stimulation, but smaller than the response seen with mGluR inhibition. Furthermore, any manipulation that resulted in a [Ca²⁺]ₖᵢ increase ≥250 nM also led to a corresponding rise in the number and percentage of propidium iodide-labeled NM neurons.
These findings suggest that eighth nerve activity helps maintain [Ca²⁺]ₖᵢ in NM neurons at physiological levels through mGluR-mediated activation of PKA and PKC. Disruption of these signaling pathways, or activity deprivation, leads to an increase in [Ca²⁺]ₖᵢ that predicts subsequent cell death.