Calcium-Dependent Regulation of Cholinergic Cell Phenotype in the Hypothalamus In Vitro

Abstract

Glutamate is a major fast excitatory neurotransmitter in the CNS including the hypothalamus. Our previous experiments in hypothalamic neuronal cultures showed that a long-term decrease in glutamate excitation upregulates ACh excitatory transmission. Data suggested that in the absence of glutamate activity in the hypothalamus in vitro, ACh becomes the major excitatory neurotransmitter and supports the excitation/inhibition balance. Here, using neuronal cultures, fura-2 Ca2+ digital imaging, and immunocytochemistry, we studied the mechanisms of regulation of cholinergic properties in hypothalamic neurons. No ACh-dependent activity and a low number (0.5%) of cholinergic neurons were detected in control hypothalamic cultures. A chronic (2 wk) inactivation of N-methyl-d-aspartate (NMDA) ionotropic glutamate receptors, L-type voltage-gated Ca2+ channels, calmodulin, Ca2+/calmodulin-dependent protein kinases II/IV (CaMK II/IV), or protein kinase C (PKC) increased the number of cholinergic neurons (to 15–24%) and induced ACh activity (in 40–60% of cells). Additionally, ACh activity and an increased number of cholinergic neurons were detected in hypothalamic cultures 2 wk after a short-term (30 min) pretreatment with bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid tetrakis(acetoxy-methyl) ester (BAPTA AM; 2.5 μM), a membrane permeable Ca2+-chelating agent that blocks cytoplasmic Ca2+ fluctuations. An increase in the number of cholinergic neurons following a chronic NMDA receptor blockade was likely due to the induction of cholinergic phenotypic properties in postmitotic noncholinergic neurons, as determined using 5-bromo-2′-deoxyuridine (BrdU) labeling. In contrast, a chronic inactivation of non-NMDA glutamate receptors or cGMP-dependent protein kinase had little effect on the expression of ACh properties. The data suggest that Ca2+, at normal intracellular concentrations, tonically suppresses the development of cholinergic properties in hypothalamic neurons. However, a decrease in Ca2+ influx into cells (through NMDA receptors or L-type Ca2+ channels), inactivation of intracellular Ca2+ fluctuations, or downregulation of Ca2+-dependent signal transduction pathways (CaMK II/IV and PKC) remove the tonic inhibition and trigger the development of cholinergic phenotype in some hypothalamic neurons. An increase in excitatory ACh transmission may represent a novel form of neuronal plasticity that regulates the activity and excitability of neurons during a decrease in glutamate excitation. This type of plasticity has apparent region-specific character and is not expressed in the cortex in vitro; neither increase in ACh activity nor change in the number of cholinergic neurons were detected in cortical cultures under all experimental conditions.