Phosphorylation cascades control the actions of ethanol on cell cAMP signalling.

Our studies indicate that, in the presence of particular isoforms of adenylyl cyclase (i.e., type 7 AC), moderately intoxicating concentrations of ethanol will significantly potentiate transmitter-mediated activation of the cAMP signaling cascade. Activation of this signaling cascade may have important implications for the mechanisms by which ethanol produces intoxication, and/or for the mechanisms of neuroadaptation leading to tolerance to, and physical dependence on, ethanol. We initiated a series of studies to investigate the phosphorylation of AC7 by PKC, the role of this phosphorylation in modulating the sensitivity of AC7 to activation by Gsalpha, and the PKC isotype(s) involved in the phosphorylation of AC7. The T7 epitope-tagged AC7 expressed in Sf9 and HEK293 cells was found to be phosphorylated in vitro by the catalytic subunit of PKC. Treatment of AC7-transfected HEK293 cells with phorbol dibutyrate (PDBu) or ethanol increased the phosphorylation of AC7 and its responsiveness to Gsalpha. In human erythroleukemia (HEL) cells, which endogeneously express AC7, ethanol and PDBu increased AC activity stimulated by PGE(1). The potentiation by both PDBu and ethanol was found to be sensitive to the PKC delta-selective inhibitor, rottlerin. The potentiation of AC activity by ethanol in HEL cells was also selectively attenuated by the RACK inhibitory peptide specific for PKC delta, and by expression of the dominant negative, catalytically inactive, form of PKC delta. These data demonstrate that AC7 can be phosphorylated by PKC, leading to an increase in functional activity, and ethanol can potentiate AC7 activity through a PKC delta-mediated phosphorylation of AC7.

Human adenylyl cyclase type 7 contains polymorphic repeats in the 3' untranslated region: investigations of association with alcoholism.

Platelet adenylyl cyclase activity has been proposed as a trait marker for alcoholism [Tabakoff et al. (1988): N Engl J Med 318:134-13;9; Parsian et al. (1996): Alcohol Clin Exp Res 20:745-751]. Human adenylyl cyclase type 7 (ADCY7) is a member of the adenylyl cyclase gene family, and it may be the major form of adenylyl cyclase expressed in human platelets. The published cDNA sequence of ADCY7 indicated the presence of potentially polymorphic regions in the 3' untranslated region of ADCY7. PCR techniques combined with fluorescently labeled primers were used to amplify two separate tetranucleotide repeat regions [(AACA)n] in the 3' untranslated region of ADCY7 from the genomic DNA of 62 unrelated individuals. The upstream (AACA)4-repeat was not polymorphic. Five different genotypes were found in the downstream (AACA)5-7 tetranucleotide repeat region. We also tested the association of the tetranucleotide polymorphism to alcohol dependence. When 30 alcoholic and 17 control individuals were compared, no difference was found in the ADCY7 tetranucleotide polymorphism between alcohol-dependent and control groups. Nevertheless, to our knowledge these are the first polymorphisms reported in an adenylyl cyclase gene. Adenylyl cyclases are important receptor-G protein-coupled effectors and are involved in numerous neuronal functions in the central nervous system. Whether variations in ADCY7 and possible variations in other members of this gene family are underlying other psychiatric disorders remains to be studied.

Adenylyl cyclases: mRNA and characteristics of enzyme activity in three areas of brain.

RNase protection assays were used in a comparative analysis of the quantities of mRNA for five "calcium-sensitive" (types I, III, V, VI, and VIII) adenylyl cyclases and one "calcium-insensitive" (type II) adenylyl cyclase in mouse cerebral cortex, cerebellum, and nucleus accumbens. The mRNA levels for type V adenylyl cyclase were dominant in the nucleus accumbens. Type V adenylyl cyclase mRNA was also found in the cerebral cortex and at low levels in the cerebellum. Type I adenylyl cyclase mRNA was the major form in the cerebellum with 15-50-fold higher levels compared with other adenylyl cyclase mRNAs. Type I adenylyl cyclase mRNA was also the most prominent adenylyl cyclase mRNA in the cerebral cortex, although the mRNA levels of other adenylyl cyclase forms were more comparable to those of the type I enzyme in this brain area. The mRNA levels for adenylyl cyclase types II, III, VI, and VIII were intermediate to low depending on the brain area. Cell membranes from the nucleus accumbens demonstrated adenylyl cyclase activity that was synergistically activated by concomitant addition of GTP and forskolin to assay mixtures, reflecting a characteristic of type V adenylyl cyclase protein. Calcium/calmodulin stimulated adenylyl cyclase activity in membranes from all three brain areas. However, synergistic activation of adenylyl cyclase activity by GTP and calcium/calmodulin was noted only with cortical membranes, and this characteristic may reflect the presence of type VIII adenylyl cyclase mRNA in the cortex. Although mRNA for type VIII adenylyl cyclase was almost equivalent in the cortex and cerebellum, the lack of a synergistic effect of GTP plus calcium/calmodulin on the cerebellar enzyme activity may be a result of the significant dominance of type I adenylyl cyclase mRNA (and protein) in the cerebellum. In general, the mRNA levels for the various adenylyl cyclases were predictive of the regulatory characteristics of adenylyl cyclase activity in membranes of the brain areas studied.

The characterization of a novel human adenylyl cyclase which is present in brain and other tissues.

We characterized a human cDNA clone which encodes a novel adenylyl cyclase. Data from Southern and Northern blot analysis, and analysis of sequence similarity with a recently cloned mouse adenylyl cyclase (10), indicated that the human adenylyl cyclase was a species variant of type VII adenylyl cyclase. The sequence of the novel human adenylyl cyclase indicated it was a member of the type II adenylyl cyclase family, and we compared the regulatory characteristics of the novel human enzyme with those of type II adenylyl cyclase. The human type VII and rat type II adenylyl cyclases, expressed in human embryonic kidney 293 cells, were activated by prostaglandin E1 (PGE1), but only type VII was activated by isoproterenol. The stimulation of type VII adenylyl cyclase by PGE1 and isoproterenol was attenuated by pretreatment of the cells with staurosporine. Phorbol 12,13-dibutyrate synergistically enhanced the stimulation of both type VII and type II enzyme activity by PGE1 and by the constitutively active Gs mutant Gs (Q227L). The human type VII adenylyl cyclase activity was unresponsive to capacitatively induced changes in intracellular Ca2+. The functional characteristics of human type VII adenylyl cyclase resemble those of the rat type II enzyme, but the enzymes may respond differently to in vivo phosphorylation conditions. While the mRNA for adenylyl cyclase type II was found in several brain areas, the message for type VII adenylyl cyclase was localized primarily to the cerebellar granule cell layer.

Localization of the gene for a novel human adenylyl cyclase (ADCY7) to chromosome 16.

A novel form of human adenylyl cyclase (ADCY7) has been discovered in the human erythroleukemia cell line (HEL). This cell line has been widely used as a model for studies of the characteristics of human platelets. Data from HEL cells suggests that ADCY7 may be the major AC form in human platelets. In the current study polymerase chain reaction (PCR) techniques coupled with use of human/rodent somatic hybrid panels and a yeast artificial chromosome (YAC) library were used to determine the chromosomal localization of the gene (adcy7) for ADCY7 enzyme. A 251-bp product from the 3' untranslated region of human adcy7 was amplified for PCR mapping and the results localize the adcy7 gene to region 16q12-16q13 of the human genome. The AC enzyme family is characterized by the presence of 12 membrane-spanning domains in its sequences, and this chromosomal region is known to contain other genes coding for proteins characterized by 12 membrane-spanning domains.

The 5-HT3 antagonist MDL-72222 exacerbates ethanol withdrawal seizures in mice.

Ethanol-dependent mice were treated with the 5-HT3 antagonist MDL 72222 after withdrawal from ethanol. Treatment with unit doses (0, 5.6, 10, and 17.0 mg/kg) of MDL 72222 at 0, 4, and 7 hr after withdrawal dose-dependently exacerbated the severity of ethanol withdrawal seizures. Treatment with a single dose (17 mg/kg) of MDL 72222 at 5 hr after withdrawal also exacerbated the severity of ethanol withdrawal seizures. Ethanol naive mice treated with MDL 72222 (56 mg/kg) did not display any seizures. Treatment with another 5-HT3 antagonist, ICS 205-930 (23 and 46 mg/kg), or the 5-HT2 receptor antagonist ketanserin, did not affect ethanol withdrawal seizures. The findings suggest MDL 72222 selectively enhances sensitivity to withdrawal seizures following chronic ethanol exposure.

A novel adenylyl cyclase sequence cloned from the human erythroleukemia cell line.

The polymerase chain reaction (PCR) was used to detect several forms of adenylyl cyclase (AC's) expressed in human erythroleukemia (HEL) cells. Degenerate oligonucleotide primers were synthesized based on the conserved sequences in the C2a area of the AC's. HEL cells were found to contain mRNA for type III and type VI AC. In addition, a novel AC message was identified. The cloned sequence, excluding primer areas, represented 69 amino acids with most similarity to rat AC's II and IV. Northern analysis of RNA from HEL cells demonstrated a 6.7 kilobase message. RNase protection assays revealed that in HEL cells the novel AC mRNA was dominant compared to types III and VI. Human embryonic kidney cells (HEK293) were also used a source of mRNA to synthesize cDNA for PCR reactions. The HEK293 cells were found to contain message corresponding to type II, III, VI AC as well as the novel AC message. The novel AC message was also detected in human brain tissue and was most abundant in the caudate, cerebellum and hippocampus. The smallest amount of novel AC mRNA in the tested brain tissue was found in the cortex. The mRNA for the novel AC was relatively abundant in human liver.

Ethanol fails to modify [3H]GR65630 binding to 5-HT3 receptors in NCB-20 cells and in rat cerebral membranes.

Low concentrations of ethanol have been found to enhance the electrophysiologic effect of serotonin (5-HT) acting at 5-HT3 receptors on NCB-20 cells. To determine whether this action of ethanol reflects a change in the agonist-receptor interaction, the effect of ethanol (100 mM) on agonist and antagonist binding to 5-HT3 receptor was studied in vitro in membrane from NCB-20 cells and from cortex plus hippocampus of rat. The antagonist [3H]GR65630 was used to label 5-HT3 recognition sites. Ethanol did not change the characteristics of saturable [3H]GR65630 binding in either membrane preparation. In competition studies, the agonists 5-HT and 2-methyl-5-HT completely inhibited the binding of [3H]GR65630 to NCB-20 cell membranes, while in brain membranes the maximum displacement of specific [3H]GR65630 binding by 5-HT was approximately 30%. Ethanol decreased the affinity of the receptor for 2-methyl-5-HT, but not to 5-HT in NCB-20 cells, and had no effect on agonist binding in brain membranes. The results indicate that enhancement of 5-HT responses at 5-HT3 receptors by ethanol is not a result of changes in the equilibrium binding characteristics of the agonist.

Plus-maze behavior and susceptibility to 3-mercaptopropionate-induced seizures in rat lines selected for high and low alcohol sensitivity.

Selective outbreeding for high and low acute alcohol sensitivity has produced two rat lines (alcohol-sensitive ANT and alcohol-insensitive AT lines) that also differ in their sensitivity to GABAergic drugs, benzodiazepines and barbiturates. These rats were now compared in two behavioral tests believed to involve central GABAergic mechanisms, in elevated plus-maze test and in 3-mercaptopropionate-induced seizure test. The AT animals spent more time in the open arms of the plus-maze than the ANT rats, suggesting that the AT's behave less anxiously. The ANT's were more susceptible to seizures induced by 3-mercaptopropionate (50 mg/kg, IP) than the AT's, suggesting the ANT's having greater sensitivity to a decrease in brain GABA concentration. At the time of the first seizure signs, there was a tendency, though a nonsignificant one, to greater decreases in brain GABA in the ANT's than AT's. These results suggest that there are differences in GABA-related behaviors between ethanol-naive rats of the lines produced by selective outbreeding for differences in alcohol sensitivity. In theory, these behavioral line differences might physiologically counteract alcohol effects in the ANT's and enhance them in the AT's. In elevated plus-maze test, however, an acute dose of ethanol (1 g/kg, IP) significantly changed the behavior of the ANT animals, but only up to level of the AT rats. The apparent sensitivity to ethanol may thus be dependent on the naive behavior of the alcohol-insensitive AT and alcohol-sensitive ANT rats.

Effect of ethanol on brain catecholamines in rat lines developed for differential ethanol-induced motor impairment.

The importance of the central catecholamines, with the emphasis on the noradrenergic neurons in the differential sensitivity to ethanol between the AT (alcohol-tolerant) rats selected for low and the ANT (alcohol-nontolerant) rats selected for high sensitivity to ethanol-induced (2 g/kg) motor impairment, was clarified by studying the effects of ethanol (2 and 4 g/kg, IP) on the utilization of norepinephrine (NA) and dopamine (DA), and on the metabolism of NA. The utilization of the catecholamines was estimated from the disappearance of the amines after inhibition of the brain tyrosine hydroxylase by alpha-methyl-p-tyrosine (200 mg/kg, IP), given 15 min after the administration of ethanol. The formation of 3-methoxy-4-hydroxy-phenylglycol (MHPG) was used as an estimate of NA metabolism, and was measured 30 min after the administration of ethanol. The basal utilization rate of NA and DA was similar between the two rat lines, but the increased formation of MHPG suggested that the naive AT rats had a higher noradrenergic activity in the limbic forebrain, hypothalamus, and cerebellum than did ANT rats. In the brain of both lines, ethanol accelerated the utilization and metabolism of NA in the same manner. Ethanol also increased the utilization of DA in the limbic forebrain of the AT and ANT rats. The higher sensitivity of the ANT rats' DA neurons to ethanol in the limbic forebrain and striatum was revealed by the significant rat line X ethanol interaction. The present findings suggest that the AT and ANT rats differ in the dopaminergic, but not in the noradrenergic responses to ethanol.

The alcohol tolerant and alcohol nontolerant rat lines selected for differential sensitivity to ethanol: a tool to study mechanisms of the actions of ethanol.

Genetic selection work conducted in the Research Laboratories of State Alcohol Company (Alko Ltd), Helsinki, Finland, has resulted in the establishment of the ethanol sensitive Alcohol Nontolerant (ANT) and ethanol insensitive Alcohol Tolerant (AT) rat lines which differ in their sensitivity to ethanol induced motor impairment. These lines have been used in attempts to identify the mechanisms controlling ethanol induced motor impairment. The Alcohol Tolerant rats show a lower sensitivity to ethanol induced motor impairment on a tilting plane over a wide range of doses, but the lines do not differ in all behavioral measures of ethanol sensitivity. Furthermore, the Alcohol Tolerant line shows a higher capacity to develop acute tolerance and less calm behaviour, which may contribute to the line difference. Neurochemical work has shown differences in the functioning and sensitivity to ethanol of the catecholaminergic and GABAergic systems in the two lines, suggesting a role for both of these systems in the control of ethanol induced motor impairment.

GABA turnover in the brain of rat lines developed for differential ethanol-induced motor impairment.

The role of GABAergic neurons in the differential sensitivity to ethanol between the AT (Alcohol Tolerant) and ANT (Alcohol Nontolerant) rat lines developed for low and high degree of motor impairment from ethanol, was studied by comparing the effect of ethanol (2 or 4 g/kg, IP) on GABA turnover in different regions of the brain in these rat lines. GABA turnover was estimated from the accumulation of GABA after inhibition of GABA aminotransferase with aminooxyacetic acid (AOAA, 50 mg/kg, IP) given 10 min after administration of ethanol. The rats were killed two hours after the AOAA treatment with focused microwaves. The concentrations of GABA, aspartate, glutamate, glutamine and taurine were analyzed with HPLC. The saline-treated ANT rats were found to have a higher concentration of GABA in the striatum and a higher rate of GABA accumulation in the cerebellum than the AT rats. Ethanol suppressed the accumulation of GABA in both lines, but the suppression was significantly greater in the AT rats than in the ANT rats. In specific regions, this line difference was significant in the cerebral cortex and cerebellum with the higher ethanol dose. No line differences were found in the brain or tail blood ethanol concentration. AOAA increased the concentration of glutamine, decreased that of aspartate and glutamate, and did not modify that of taurine. The AOAA-induced changes in the concentrations of these amino acids were, however, minor relative to those found in the concentrations of GABA. The results that GABAergic mechanisms are involved in the differential sensitivity to the motor-impairing effects of ethanol between the AT and ANT rats.

Effect of GABAergic drugs on motor impairment from ethanol, barbital and lorazepam in rat lines selected for differential sensitivity to ethanol.

The effect of GABAergic drugs on the motor-impairing effects of ethanol, barbital, and lorazepam were studied in the ethanol-sensitive ANT (Alcohol Nontolerant) and ethanol-insensitive AT (Alcohol Tolerant) rat lines, selected for differential ethanol-induced motor impairment on the tilting plane. The basic population from which these rat lines were derived, the mixed (M) line, was also included in the study. The ANT rats were more sensitive to the intoxicating effects of ethanol, barbital, and lorazepam than the AT and M rats at the dose ranges tested. Picrotoxin antagonized motor impairment from all three drugs. Flumazenil (Ro 15-1788) antagonized only the effects of lorazepam, and isoniazid did not modify motor impairment induced by any of the three drugs. These results confirm that the selection of AT and ANT lines has not been specific to ethanol, and that it has increased sensitivity to ethanol, barbital, and lorazepam in the ANT rats rather than decreasing it in the AT rats relative to the M rats. The finding that picrotoxin counteracted motor impairment from ethanol, barbital, and lorazepam support the view that the GABAA receptor complex is important in mediating the intoxicating effects of these drugs. These results also suggest that the genetically-determined difference in sensitivity to ethanol between the rat lines involves GABAergic mechanisms, but it remains to be determined whether any part of the GABAA receptor itself has been affected by the selection program.

Brain regional and adrenal monoamine concentrations and behavioral responses to stress in alcohol-preferring AA and alcohol-avoiding ANA rats.

The concentrations of monoamines, precursors and metabolites in various brain regions and the levels of catecholamines in the adrenal glands were determined from naive rats of the AA and ANA lines, and from ones immediately after an escapable shock test. The brain determinations were made with a new step-gradient ion-pair elution method on a reversed phase column and coulometric detection. Several significant differences were observed in the amine concentrations, largely confirming and extending the findings made before the genetic revitalization of the lines: in particular, the AAs, unlike other alcohol-preferring rodents, had higher 5-hydroxytryptamine concentrations. The AA rats tended to have smaller changes than the ANAs in brain aminergic systems and had significantly less change in adrenal epinephrine and dopamine levels after the shock test. The AAs were consistently found to be less active than ANAs in this shock test and in a warm-water swim test, but whether this was a cause or an effect of their brain and adrenal changes could not be determined. Our behavioral results might suggest a reduced reaction of the alcohol-preferring rats to aversive stimulation.

Subchronic treatment with vanadate does not potentiate the toxicity of cardiac glycosides.

Since it has been claimed that vanadate is an endogenous regulator of Na/K-ATPase activity and that it potentiates the toxicity of cardiac glycosides, we were alarmed to discover that certain Finnish physicians were prescribing vanadate in combination with other trace minerals to elderly patients for many different chronic diseases (e.g., cancer, rheumatism). To study the interaction of vanadate and cardiac glycosides, we fed vanadate in the drinking water (25 micrograms/mL) to guinea pigs for 20 d, and studied either their sensitivity to the acute toxicity of the cardiac glycoside ouabain or whether the vanadate would influence the subacute toxicity of ouabain. Vanadate had no influence on the toxicity of ouabain either acute or subchronically administered, nor was there any sign of inhibition of Na/K-ATPase activity as measured by 86Rb-uptake into intact erythrocytes (RBCs), RBC content of sodium or potassium or Na/K-ATPase activity in RBC membranes prepared from the vanadate-treated guinea pigs. Vanadate had been absorbed in substantial quantities from the gastrointestinal tract, since serum, heart, liver, and especially kidney contained measurable amounts of vanadium in contrast to controls, but it is concluded that this vanadate is not in a biologically active form.

Failure of Ro 15-4513 to antagonize ethanol in rat lines selected for differential sensitivity to ethanol and in Wistar rats.

An imidazobenzodiazepine, Ro 15-4513, acting as a partial inverse agonist at the central benzodiazepine receptors has been recently reported to reverse efficiently the intoxicating effects of ethanol. In studies designed to delineate the role of benzodiazepine receptors in the ethanol-induced motor impairment difference between two rat lines selectively bred for high and low sensitivity to ethanol, however, we could not antagonize the effects of ethanol by Ro 15-4513 in the tilting plane and horizontal wire tests. Neither could we observe any consistent antagonism of ethanol actions in Han:Wistar rats, although we used a wide range of Ro 15-4513 doses, injected the drug intraperitoneally or intragastrically and before or after ethanol administration, and carried out the tests for motor impairment (rotarod, horizontal wire test and intoxication rating) at various times after the drug administration. The ex vivo assay of flunitrazepam binding in brain homogenates revealed the presence of compound(s) inhibiting the binding after administration of Ro 15-4513. Ro 15-4513 antagonized the motor impairing effects of lorazepam. In conclusion, Ro 15-4513 failed to function as a specific antagonist of moderate doses of ethanol in several tests for motor impairment in different rat lines.

Early postnatal treatment with propranolol affects development of brain amines and behavior.

The present study examined the effects of early postnatal treatment with a beta-adrenoceptor antagonist propranolol (5 mg/kg IP daily) on concomitant and subsequent behavior and central aminergic transmission in rats. During propranolol exposure from the 7th to the 20th postnatal days sleep-wake recordings, carried out with the static charge sensitive bed (SCSB) method, showed a decrease in the percentage of active sleep and an increase in waking. When the animals were 1-3 months of age, the open field behavior was changed, immobility time in the Porsolt's swim test was lengthened, and voluntary alcohol consumption was increased in the propranolol-treated rats. Neither motor reactivity to auditory stimuli nor spontaneous alternation behavior was affected. At the age of 4 months concentrations of brain amines and their metabolites were measured from several brain regions. In the propranolol-treated rats the noradrenaline levels were increased in the limbic forebrain and cerebellum. The results suggest that in rats the exposure to propranolol during the rapid growth period of cerebral catecholamine systems, and the concomitant alterations in sleep are related to later changes in behavior and to increased noradrenaline content in the limbic forebrain and cerebellum.

Effects of ethanol, barbital, and lorazepam on brain monoamines in rat lines selectively outbred for differential sensitivity to ethanol.

The acute effects of ethanol, barbital, and lorazepam on the synthesis and metabolism of brain monoamines were studied in the AT (Alcohol Tolerant) and ANT (Alcohol Nontolerant) lines of rats, which have been selected for differential motor impairment after ethanol administration. The ethanol-sensitive ANT rats are also more sensitive than the ethanol-insensitive AT rats to the motor impairment caused by barbital and lorazepam. Ethanol increased, whereas barbital and lorazepam decreased, the synthesis of catecholamines in several regions of the brain. Ethanol did not affect the formation of DOPAC, whereas barbital and lorazepam reduced it. Similarly, the accumulation of 5-HTP was increased after administration of ethanol, but was decreased after administration of barbital or lorazepam. Ethanol, barbital and lorazepam decreased the formation of 5-HIAA. The rat lines did not differ in any of these responses. Some differences could, however, be demonstrated between the AT and ANT rats in the effects of the three drugs on the levels of the brain monoamines. Although the importance of these differences in the differential sensitivity to these drugs between the two lines is difficult to determine, the role of central monoaminergic mechanisms cannot be excluded. These findings also suggest that the motor impairment induced by ethanol, barbiturates, and benzodiazepines is probably not primarily based on the monoaminergic systems.

Effect of prenatal alcohol exposure on neonatal sleep-wake behaviour and adult alcohol consumption in the AA and ANA rat lines.

To study the role of genetic factors in the effects of prenatal alcohol exposure on behaviour, dams of two rat lines developed to differ in voluntary alcohol intake, alcohol-preferring (AA) and alcohol-avoiding (ANA) rats were given a 5-10% alcohol solution mixed with a 1% sucrose solution as a sole drinking liquid throughout gestation. Sleep-wake behaviour of the offspring was studied at the ages of 7, 14 and 20 days, using a movement-sensitive mattress. In ANA rats, sleep recordings showed that prenatal alcohol exposure increased the percentage of waking but decreased the percentage of active sleep. Sleep-wake behaviour of the AA rats was not affected by alcohol exposure in utero. Prenatal alcohol exposure did not change open field behaviour in 1 month old rats, except that the alcohol-exposed AA rats' defaecation was decreased. When rats were 3 months old, voluntary intake of 10% (v/v) alcohol increased for alcohol-exposed ANA rats and decreased for alcohol-exposed AA rats as compared to the controls. The results indicate that AA rats may compensate for the effects of prenatal alcohol exposure on behaviour, whereas offspring of the alcohol-exposed ANA dams suffer from severe behavioural disturbances. These findings further suggest that genetic factors are responsible for the differences in the susceptibility of rat foetuses to alcohol-induced long-term effects on behaviour.