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

Abstract

Denisova. Calcium-dependent regulation of cholinergic cell phenotype in the hypothalamus in vitro. J Neurophysiol 88: 1352-1362, 2002; 10.1152/jn.00827.2001. 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 Ca 2ϩ 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, Ltype voltage-gated Ca 2ϩ channels, calmodulin, Ca 2ϩ /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,NЈ,NЈ-tetraacetic acid tetrakis(acetoxy-methyl) ester (BAPTA AM; 2.5 M), a membrane permeable Ca 2ϩ -chelating agent that blocks cytoplasmic Ca 2ϩ 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-2Ј-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 Ca 2ϩ , at normal intracellular concentrations, tonically suppresses the development of cholinergic properties in hypothalamic neurons. However, a decrease in Ca 2ϩ influx into cells (through NMDA receptors or L-type Ca 2ϩ channels), inactivation of intracellular Ca 2ϩ fluctuations, or downregulation of Ca 2ϩ -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.

Physiological consequences of selective suppression of synaptic transmission in developing cerebral cortical networks in vitro: Differential effects on intrinsically generated bioelectric discharges in a living ‘model’ system for slow-wave sleep activity

“…SAP-dependent maturation has recently been documented for a variety of central nervous regions, including the corpus striatum (Gustafson et al, 2006), hypothalamus (Belousov et al, 2002(Belousov et al, , 2004, retina (Butts et al, 2007;Grzywacz and Sernagor, 2000;Harris et al, 2002), superior colliculus (Chandrasekaran et al, 2007;Meier et al, 2002Meier et al, , 2003Razak and Pallas, 2007), lower brainstem (Kotak and Sanes, 2003), cerebellum (Andjus et al, 2003;Liu and Cull-Candy, 2002;Nakanishi and Okazawa, 2006;Ouardouz and Sastry, 2005) and spinal cord (Galante et al, 2001;Rosato-Siri et al, 2002) but the present updated survey -which is complete in principle through December 2007 -will be essentially restricted to the cerebral (paleo-and archi-as well as neo-) cortex.…”

“…Acetylcholine receptors (muscarinic but not nicotinic) take over as the main source of excitatory synaptic interactions within the network (see Section 2). Such a replacement of glutamate by acetylcholine as the major excitatory neurotransmitter, after the former had been chronically inactivated, was already on record for primary cultures of hypothalamus and spinal cord but not for neocortical neurons (Belousov et al, 2001(Belousov et al, , 2002. Indeed, it was only when we allowed two organotypic explants to cross-innervate one another (Baker and van Pelt, 1997) that cholinergic plasticity manifested itself in occipital cortex tissue.…”

Crosstalk among electrical activity, trophic factors and morphogenetic proteins in the regulation of neurotransmitter phenotype specification

“…The switch from noradrenergic to cholinergic upon innervation of the footpad sweat glands reviewed above represents the first evidence of the plasticity of the cholinergic phenotype specification. This is not restricted to the peripheral nervous system, mouse and rat glutamatergic hypothalamic neurons induce expression of the cholinergic phenotype when glutamate signaling is inhibited through NMDAR-mediated and Ca 2+ /CREB/NF-κB-dependent mechanism (Belousov et al, 2001; Belousov et al, 2002; Liu et al, 2008). In contrast, excessive stimulation of brain excitability by stress or by inhibiting the acetylcholine esterase in vivo and in hippocampal slices respectively, downregulates expression of the cholinergic gene locus through a Ca 2+ and c-Fos-mediated pathway (Kaufer et al, 1998).…”

Use of calcium imaging for analysis of neuronal gap junction coupling

“…We previously demonstrated [4, 5] that neuronal disinhibition with bicuculline, a GABA A receptor antagonist, induces cytoplasmic Ca 2+ increases in neurons in primary hypothalamic cultures. In control (non-treated) cultures, these increases are glutamate-dependent as are completely suppressed by ionotropic glutamate receptor antagonists, AP5 plus 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (100/10 µM).…”

“…In contrast, in cultures that are chronically (2–3 weeks) treated with AP5 (100 µM) or AP5+CNQX (100/10 µM), neurons display the bicuculline-induced Ca 2+ increases despite the continued presence of glutamate receptor antagonists. These glutamate-independent Ca 2+ increases are suppressed by acetylcholine receptor antagonists and represent up-regulation of cholinergic functions in neurons by chronic NMDA receptor blockade [4, 5]. Moreover, in AP5+CNQX-chronically treated cultures, gap junctions contribute to the generation of bicuculline-induced Ca 2+ increases as a significant amount of these Ca 2+ transients also is suppressed by carbenoxolone, a general gap junction blocker [3].…”