Oral Communication 11, Tuesday Sept. 25th 5.00 pm-6.00 pm; Room: Lecture Hall 2: Hormones and neurotransmitter in alcoholism

Presentation OC11-1 Enhanced conditioned place preference and voluntary ethanol consumption in mice lacking the prodynorphin gene is associated to reduce opioid functional activity in the brain Femenia T, García-Gutierrez S, Sanguino E, Manzanares J (Spain) Aims. The aim of this study was to examine the effects of targeted disruption of the kappa-opioid endogenous ligand dynorphin on alcohol consumption and proenkephalin (PENK) gene expression and mu-opioid receptor function in wild type (WT) and prodynorphin knock out (PDYN-KO) mice. Methods. Conditioned place preference (CPP) was examined in prodynorphin KO and WT mice. Ethanol was administered (2 g/kg, p.o.) and the subject was placed in a test chamber that contains the drug-related environmental cues in one compartment and neutral cues in another. The time that mice spend in each chamber was examined. Voluntary consumption of ethanol in mice was examined as follows: PDYN-KO and WT mice were given continual free choice access to an ethanol solution or water and food at libitum for 4 weeks. The ethanol concentration was gradually increased (2, 4, 6 and 8% v/v) every 2 days. In the following maintenance ethanol concentration (8%) was kept constant for 3 weeks. Ethanol intake was expressed in g/ethanol/kg/day. Coronal brain sections from naíve PDYN-KO and WT mice were cut at different levels containing the regions of interest: caudate-putamen (Cpu), cingulated cortex (Cg) and nucleus accumbens (Acb). Proenkephalin (PENK) gene expression was measured by in situ hybridisation. The functional activity of mu-receptors was determined using DAMGO-stimulated [ 35 S]-GTPgS binding autoradiography. Results. The results showed that the deletion of PDYN gene increased (29%) the time spent in the place conditioned of ethanol compared to WT mice. Furthermore, voluntary ethanol consumption is higher in PDYN KO (3.60 g/kg/day) compared to WT mice (2.40 g/kg/day). The neurochemical studies revealed that the PENK gene expression was lower in CPu (20.69%) ( P = 0.013), AcbC (25.69%) ( P = 0.002) and AcbS (34.05%) ( P = 0.002) in PDYN-KO compared to WT mice. The analyses of DAMGO-stimulated [ 35 S]-GTPgS binding autoradiography showed decreased functional activity in Cpu (27%) ( P = 0.008), AcbC (20.7%) ( P = 0.067) AcbS (13.45%) ( P = 0.291) and Cg (43.7%) ( P = 0.006) in PDYN-KO compared to WT mice. Conclusions. Taken together, these results suggest that the deletion of the prodynorphin gene increased the vulnerability for ethanol consumption by decreasing, at least in part, PENK gene expression and μ-opioid receptor function in brains areas closely related to ethanol reinforcement. (Supported by grant from Spanish Ministry of Health FIS 05/0429). Presentation OC11-2 Does the benzodiazepine receptor ligand Ro 15-4513 bind to specific ethanol-sites on δ-subunit containing GABA-A receptors? Korpi E-R, Debus F, Leppae E, Linden A-M, Rabe H, Boehme I, Lueddens H (Finland) Aims. In rodent models, GABAA receptors with the α4/6 and δ subunits, expressed in the cerebellum and forebrain, have been linked to ethanol sensitivity and voluntary ethanol drinking. Here, we attempted to identify the putative ethanol-binding sites in native and recombinant GABAA receptors. Methods. We examined the claimed δ subunit-dependent/γ2 subunit-independent ethanol/[ 3 H]Ro 15-4513 binding sites on GABAA receptors by studying [ 3 H]Ro 15-4513 binding in HEK 293 cell membrane homogenates containing rat recombinant α 6/4β3δ receptors and by [ 3 H]Ro 15-4513 autoradiography in mouse brain sections. Results. Specific high-affinity [ 3 H]Ro 15-4513 binding could not be detected in recombinant receptors or in cerebellar or thalamic sections of γ2(F77I) knockin mice (which have impaired γ2 subunit-dependent benzodiazepine site binding), nor was this binding in brain sections of wild-type C57BL/6 mice inhibited by 1–100 mM ethanol. Conclusions. No evidence was obtained for ethanol-displaceable binding of the putative alcohol-antagonist ligand [ 3 H]Ro 15-4513. Therefore, high-affinity ethanol binding site(s) on the GABAA receptors, if exist(s) at all, may be dependent on specific intracellular modifications or cell membrane conditions, which are not operative in brain cryostat sections or recombinant receptors. Presentation OC11-3 Ethanol stimulates growth hormone release in GH3 pituitary tumor cells via calcium and swelling dependent mechanisms Langelueddecke C, Weiger TM, Jakab M, Handlechner A, Schmidt S, Kainz V, Oberascher K, Ritter M, Hermann A (Austria) Aims. Calcium activated big potassium channels (BK) are involved in the regulation of a number of processes like action potential repolarisation, blood pressure control or hormone secretion to name a few. BK channels are a main target of ethanol. Growth hormone (GH) secreted by pituitary cells is known to be reduced under the influence of ethanol in adults and is subnormal in short fetal alcohol syndrome children. The mechanism by which ethanol acts on pituitary cells is unknown. In addition there are conflicting reports if the GH release is affected by ethanol at the hypothalamic or endocrine cellular level. We investigated the GH release and its mechanisms in GH3 pituitary tumor cells. Methods. GH release was measured with an enzyme immuno assay. GH3 cells were incubated either for 24 hours in MEM medium supplemented with 30 mM ethanol or for spontaneous GH release studies 30 mM ethanol was applied in a Ringer solution under isoosmolar conditions. Swelling of cells was determined by measuring the cell circumference before and during ethanol application. Relative changes in free intracellular calcium were measured in 96 well plates in a photometer after loading cells with Fluo3. BK single channel activity was recorded from outside out patches. Results. Incubating GH3 cells for 24 h with 30 mM ethanol increased GH secretion compared to 24 h controls. Addition of 30 mM sucrose did not have any significant effect on GH secretion. Instantaneous application of 30 mM isoosmolar ethanol also increased GH release. Isoosmolar applied ethanol induced cell swelling followed by a regulatory volume decrease (RVD). Iberiotoxin a BK channel blocker delayed RVD. Free intracellular calcium was increased by ethanol. Exchanging calcium by cobalt in the extracellular solution to block calcium influx diminished calcium levels in the presence of ethanol. Thyrotropin-releasing-hormone showed a similar action like ethanol. BK channels were activated in outside out patches by application of ethanol. Conclusions. Our data indicate that ethanol induces a GH release from pituitary cells by a mechanism transmitted via an increase in free intracellular calcium as well as by an induction of cell swelling. Intracellular calcium is not mobilized by ethanol. Cell swelling might also induce BK activity which in turn may be responsible for RVD. Our results suggest that the reduction of GH secretion as observed in humans after alcohol ingestion is modulated at hypothalamic levels while at the pituitary level ethanol appears to have a stimulating effect on GH release.