Molecular Brain Research 83 (2000) 63–71 www.elsevier.com / locate / bres Research report Multiple calmodulin genes exhibit systematically differential responses to chronic ethanol treatment and withdrawal in several regions of the rat brain * Sandor Vizi, Arpad Palfi, Karoly Gulya Department of Zoology and Cell Biology, University of Szeged,2 Egyetem St., POB 659, Szeged, H-6722 Hungary Accepted 25 July 2000 Abstract 21 Ethanol induces profound alterations in the neuronal signaling systems, including the calcium (Ca ) signaling. Prolonged exposure to ethanol evokes adaptive changes in the affected systems as they strive to restore the normal neuronal function. We investigated the 21 involvement of calmodulin (CaM) genes, coding for the major mediator protein of intracellular Ca signals, in these adaptive processes at the mRNA level. The changes induced in the regional abundances of the CaM I, II, and III mRNA classes by chronic ethanol treatment 35 and withdrawal were examined by means of quantitative in situ hybridization, employing gene-specific [ S]cRNA probes on rat brain cryostat sections. Regional analysis of the resulting changes in mRNA levels highlighted brain areas that belong in neuronal systems known to be especially sensitive to the action of ethanol. The results revealed systematically differential regulation for the three mRNA classes: the CaM I and CaM III mRNA levels displayed increases, and CaM II levels decreases in the affected brain regions, in both chronic ethanol- and withdrawal-treated animals. As regards the numbers of brain regions undergoing significant alterations in mRNA content, the CaM I mRNA levels exhibited changes in most brain areas, the CaM II levels did so in a lower number of brain regions, and the CaM III levels changed in only a few brain areas. These results suggest a differential regulation for the CaM genes in the rat brain and may help towards elucidation of the functional significance of the multiple CaM genes in the mammalian genome.  2000 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Gene structure and function: general Keywords: Chronic ethanol treatment; Withdrawal; Rat brain; Gene expression; Calmodulin mRNAs; Quantitative in situ hybridization 1. Introduction and glycine receptors), plasma membrane ion channels and 1 21 pumps (voltage-dependent Na and Ca channels, 1 1 21 Ethanol, a sedative-hypnotic agent, remains the most Na ,K -ATPase and Ca -ATPase), and enzymes in- widely abused drug worldwide. Research aiming to reveal volved in neuronal signal transduction, such as phospholip- its mechanism of action led to the theory of the ‘fluidiza- ase A and adenylyl cyclase, see Refs. [5,8,11]. Thus, 2 tion’ of biological membranes, with concomitant perturba- ethanol can produce profound changes in neuronal ex- tion of the biological function of membrane-bound proteins citability and signal transduction, leading to dysfunctions 21 via disruption of their lipid surroundings [8]. A number of in the second messenger systems, including Ca sig- recent studies have pinpointed the proteins themselves as naling. 21 the primary target molecules for ethanol. The target sites Calmodulin (CaM), the major Ca -binding protein in include ionotropic and metabotropic receptors of the the mammalian brain, plays a central role in mediating 21 neuronal plasma membrane (NMDA, kainate, nicotinic intracellular Ca signals. Besides its involvement in the acetylcholine, a- and b-adrenergic, 5-HT , opiate, GABA local regulation of various ion channels [16], CaM is also 3 A crucial for long-distance intracellular signaling and, via its nuclear transport, for transcriptional regulation [7]. Since *Corresponding author. Tel.: 136-62-544-048; fax: 136-62-544-049. E-mail address: gulyak@bio.u-szeged.hu (K. Gulya). these functions require the mobilization and translocation 0169-328X / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0169-328X(00)00185-6