Activity-Dependent Regulation of [Ca2+]iin Avian Cochlear Nucleus Neurons: Roles of Protein Kinases A and C and Relation to Cell Death

Abstract

Zirpel, Lance, William R. Lippe, and Edwin W Rubel. Activity-dependent regulation of [Ca²⁺]_i in avian cochlear nucleus neurons: roles of protein kinases A and C and relation to cell death. J. Neurophysiol. 79: 2288–2302, 1998. Neurons of the cochlear nucleus, nucleus magnocellularis (NM), of young chicks require excitatory afferent input from the eighth nerve for maintenance and survival. One of the earliest changes seen in NM neurons after deafferentation is an increase in intracellular calcium concentration ([Ca²⁺]_i). This increase in [Ca²⁺]_i is due to loss of activation of metabotropic glutamate receptors (mGluR) that activate second-messenger cascades involved in [Ca²⁺]_i regulation. Because mGluRs are known to act via the phospholipase C and adenylate cyclase signal transduction pathways, the goal of this study was to determine the roles of protein kinases A (PKA) and C (PKC) activities in the regulation of NM neuron [Ca²⁺]_i by eighth nerve stimulation. Additionally, we sought to determine the relationship between increased [Ca²⁺]_i and cell death as measured by propidium iodide incorporation. [Ca²⁺]_i of individual NM neurons in brain stem slices was monitored using fura-2 ratiometric fluorescence imaging. NM field potentials were monitored in experiments in which the eighth nerve was stimulated. Five hertz orthodromic stimulation maintained NM neuron [Ca²⁺]_i at ∼110 nM for 180 min. In the absence of stimulation, NM neuron [Ca²⁺]_i increased steadily to a mean of 265 nM by 120 min. This increase was attenuated by superfusion of PKC activators phorbol-12,13-myristate acetate (100 nM) or dioctanoylglycerol (50 μM) and by activators of PKA: 1 mM 8-bromoadenosine-3′,5′-cyclophosphate sodium (8-Br-cAMP), 50 μM forskolin or 100 μM Sp-adenosine 3′,5′-cyclic monophosphothioate triethylamine. Inhibition of PKA (100 μM Rp-cAMPS) or PKC (50 nM bisindolymaleimide or 10 μM U73122) during continuous orthodromic stimulation resulted in an increase in NM neuron [Ca²⁺]_i that exceeded 170 and 180 nM, respectively, by 120 min. Nonspecific kinase inhibition with 1 μM staurosporine during stimulation resulted in an [Ca²⁺]_i increase that was greater in magnitude than that seen with either PKA or PKC inhibition alone, equal to that seen in the absence of stimulation, but much smaller than that seen with inhibition of mGluRs. In addition, manipulations that resulted in a [Ca²⁺]_i increase ≥250 nM resulted in an increase in number and percentage of propidium iodide-labeled NM neurons. These results suggest that eighth nerve activity maintains [Ca²⁺]_i of NM neurons at physiological levels in part via mGluR-mediated activation of PKA and PKC and that increases in [Ca²⁺]_i due to activity deprivation or interruption of the PKA and PKC [Ca²⁺]_i regulatory mechanisms are predictive of subsequent cell death.