1-4
5-8
9-12
13-16
17-20
21-24
25-28
29-32
33-36
37-40
41-44
underlying stress-induced alcohol abuse have however not yet been elucidated,
although some evidence indicate that glucocorticoids are involved. The stress
response involves several neuronal and endocrine alterations besides those related to
the hypothalamic-pituitary-adrenal gland axis. The aim of the present experiments
was to investigate in the rat whether intermittent activation of the sympathetic nervous
system and consequences thereof alter voluntary ethanol (EtOH) consumption as well
as EtOH sensitivity in the mesolimbic dopamine (DA) system, an important part of
the brain reward system. Intermittent administrations of tyramine (TYR) and
adrenaline (A) were used to mimic stress-induced sympathetic neuronal activation and
A release from the adrenal medulla, respectively. Since intermittent sympathetic
activation increases blood insulin (INS) levels, we included a third model in which
rats were treated with INS. Thus, EtOH low- and medium-preferring rats, in a free
choice between water and EtOH, were deprived of EtOH and subsequently treated
with saline (0.2 ml/kg i.p., 0.2 ml/kg s.c.), saline (0.2 ml/kg i.p.) and INS (1-4 U/day
s.c.), TYR (40 mg/kg i.p.) and saline (0.2 ml/kg s.c.) or A (0.2 mg/kg i.p.) and saline
(0.2 ml/kg s.c.) daily for 15 consecutive days. Thereafter, EtOH consumption was
reexamined, followed by in vivo microdialysis where extracellular accumbal DA
levels were measured after acute EtOH (1 g/kg i.p.) challenge. All three stress models
increased EtOH intake and preference and the extracellular accumbal DA levels after
EtOH were significantly higher in the TYR and A pretreated rats, compared to the
saline and INS pretreated ones. Hence, three manipulations aimed at modelling
peripheral responses to stress all increased alcohol preference and intake. The results
from the TYR and A pretreated rats are very similar to those previously observed after
pretreatment with hexamethonium (HEX), a peripheral antagonist for nicotinic
acetylcholine receptors (nAChRs), both regarding EtOH preference and EtOH-
induced accumbal DA release. It is tempting to suggest a similar mechanism for the
nicotine (NIC) induced increase in EtOH consumption observed both in laboratory
animals and in human smokers, since NIC activates the sympathetic nervous system
followed by a functional blockade of peripheral nAChRs, and since smokers display
elevated blood levels of INS as well as INS resistance. The similarity in EtOH-
induced DA response between INS treated animals and controls, does not exclude an
increased sensitivity of the mesolimbic DA system to EtOH in INS treated rats, since
only the presynaptic response to EtOH was investigated. Thus a tentative
augmentation of postsynaptic DA D2 receptor sensitivity, as previously observed after
intermittent NIC, could not be excluded.
RECEPTORS AND THE REINFORCING AND
DOPAMINE-ACTIVATING EFFECTS OF ETHANOL
Jörgen A. Engel, 1, 2 Bo Söderpalm
Institute of Physiology and Pharmacology, Department of pharmacology and 2Institute of Clinical
Neuroscience, Section of Psychiatry, Sahlgrenska Academy, Göteborg University
ethanol (EtOH) intake in the rat, a response that may be involved in mediating the
positive reinforcing effects of EtOH. The mechanisms underlying this DA activation
remain to be elucidated. Recent data indicate that ventral tegmental nicotinic
acetylcholine receptors are indirectly involved, secondary to some EtOH-induced
effect in the nAc. This latter effect may involve interference with accumbal
strychnine-sensitive glycine receptors (SSGRs), since accumbal application of
strychnine prevents the DA elevation observed after accumbal or intraperitoneal
EtOH administration. Here this hypothesis was further challenged by examining the
influence of bilateral accumbal application of strychnine, a competitive antagonist for
the SSGRs, or glycine on EtOH intake and preference, as well as on the concomitant
DA overflow in the nAc, in EtOH high-preferring Wistar rats. During the course of
these and other experiments we have observed that outbreed Wistar rats may be
bimodally distributed with respect to their DA response to accumbal perfusion with
glycine or strychnine. Consequently the results below are presented separately for
strychnine-sensitive and non-sensitive rats, as well as for glycine-sensitive and non-
sensitive rats.
>60 % during continuous access to a bottle of 6% EtOH solution and a water bottle)
male Wistar rats were limited to drink only one hour/day during one week. Thereafter
the animals were equipped bilaterally with microdialysis probes aimed at the nAc. On
the experimental day the rats were both water and EtOH deprived when connected to
the microdialysis system. Baseline samples were collected during 60 minutes prior to
bilateral perfusion with Ringer, strychnine or glycine and 60 minutes after drug
perfusion the animals were presented with the EtOH and water bottles and allowed to
drink for 60 minutes. The amounts of fluids consumed were registered. Each rat was
treated with strychnine or glycine on one of the two experimental days and with
Ringer on the other. The order of treatment was balanced and only rats with an EtOH
preference >50 % when receiving Ringer were included in the study.
% in EtOH high-preferring strychnine-sensitive rats, and these animals consumed
more EtOH during strychnine than during Ringer perfusion. In line with previous
results the EtOH intake failed to elevate accumbal DA levels in these animals. In
animals not responding to strychnine perfusion per se (strychnine non-sensitive rats)
there was no significant alteration of EtOH intake and EtOH produced a similar
elevation of accumbal DA levels as during Ringer treatment. The difference between
strychnine sensitive and non-sensitive rats may be due to limited excovery of the drug
or to some innate difference in the number or sensitivity of SSGRs. Alternatively,
EtOH intake and EtOH-induced DA release are governed by different mechanisms in
strychnine-sensitive and non-sensitive rats. We hypothesize that strychnine-sensitive
rats consume more EtOH in order to compensate for the lower accumbal DA levels
and/or in an attempt to overcome the strychnine-induced blockade of EtOH‚s DA
activating effect. We are currently testing the hypothesis that repeated accumbal
strychnine treatment may produce an extinction phenomenon with respect to EtOH
intake in strychnine-sensitive rats.
nAc by approx. 50% in EtOH high-preferring glycine-sensitive rats. During glycine,
as compared to Ringer perfusion, these animals displayed a lower EtOH preference
and tended to decrease their ethanol intake, which was compensated for by an
increase in water intake. EtOH intake produced a slight, non-significant further
elevation of accumbal DA output in these glycine-sensitive rats. In animals not
responding to glycine with a DA elevation (glycine non-sensitive rats) the DA levels
were either unaffected or slightly decreased. In these animals neither EtOH preference
nor EtOH intake was affected by the accumbal glycine perfusion. EtOH intake
produced a similar elevation of accumbal DA levels after glycine and Ringer
perfusion in glycine non-sensitive rats, when compared to the pre-EtOH DA levels.
Since glycine by itself raised the accumbal DA levels and since the glycine treated
rats apparently consumed just the right amount of EtOH to obtain DA levels almost
identical to those observed under control conditions, we suggest that the decreased
EtOH preference in glycine treated glycine-sensitive rats reflects a substitution
phenomenon.
produced an increase in accumbal DA output in both experiments. Interestingly,
EtOH intake produced a significantly larger increase in accumbal DA output in
glycine sensitive rats (approx. 50%) than in glycine non-sensitive rats (approx. 25%),
whereas there appeared to be no difference in the amount of EtOH consumed.
positive reinforcing and DA activating effects of EtOH. The possibility of developing
selective agonists or antagonists for this receptor population should be explored. Such
compounds could be interesting candidates for a future pharmacological treatment of
alcoholism.
PHOSPHOLIPASE D1 (PLD1) AND PLD2
mRNA EXPRESSION IN THE RAT BRAIN
Lund University Hospital, S-221 85, Lund, Sweden.
vesicle recycling and neurotransmission in the central nervous system. The PLD-
subtypes yet cloned, PLD1 and PLD2 are phosphodiesterases that hydrolyse
phosphatidylcholine (PC) into the potent lipid second messenger phosphatidic acid
(PA) and the head-group choline. Most downstream events of PLD-activation depend
upon PA. Chronic ethanol is known to impose several effects on mediators of
intracellular signal transduction. Levine et al. (Alcohol. Clin. Exp. Res. 24:1, 93-
rat PC-12 cell line. This prompted us to explore if long-term ethanol exposure would
affect the gene expression of PLD1 and PLD2 in the rat brain.
Materials and Methods: Adult male Sprague-Dawley rats received a liquid ethanol
diet or an isocaloric control diet (without ethanol) for 40 days. The level of ethanol
given was gradually increased so that the rats finally received 9% ethanol during the
final 30 days. The cerebral cortex, cerebellum and mid-brain of each rat was collected
and extracted for total-RNA. This material was reverse transcribed and used as the
template for real-time quantitative PCR-analysis of PLD1 and PLD2 mRNA
expression. For these analyses the ABI PRISM 7700 (TaqMan) Detector System
(Applied Biosystems) was employed. Results: In the cerebellum an upregulation in
both PLD1- and PLD2 mRNA was observed in the ethanol exposed animals.
However, in the cerebral cortex, the expression of both transcriptsshowed a decline.
In the midbrain no change in the gene-expression of either PLD-subtype was seen.
Discussion: This study shows that PLD1- and PLD2 mRNA expression is affected by
long-term ethanol exposure. An explanation is pending why PLD1 and PLD2 mRNA
are upregulated in the cerebellum whereas these transcripts are downregulated in the
cortex. It is conceivable that the differences in PLD mRNA expression are caused by
neuroadaptive processes in response to ethanol, presumably operating at the
transcriptional level
NOVEL MODEL OF ALCOHOL CRAVING
Stockholm, Sweden
which encompasses environmental cues. Our group has now developed a model to
study this behaviour in animals. Naïve male Wistar rats were trained to press a lever
and orally consume ethanol in an operant chamber. Each day, the same pattern of
transporting, handling and weighing was followed and a priming dose of solution was
available in the operant box prior to the start of the session. Both active and inactive
lever responses led to cue light illumination and thus provided no additional cue. Rats
were given 0.2% saccharin for 6 days followed by 20 days of 5% alcohol/0.2%
saccharin solution. Rats were then given a free choice between the alcohol solution
and water until stable. The next day, rats were brought to the room as usual but were
kept in their cage before entering the self-administration boxes. Therefore, rats were
exposed to environmental cues associated with their drinking. Rats receiving 5
minutes of cue responded significantly more on the active lever when compared to
baseline (p<0.0001) while no change in response was observed after 10 minutes
(p=0.15) or 15 minutes (p=0.35) of cue. Blood alcohol level was 41.1±6.0 mg/dL for
the 5 minute cue group compared to 28.4±3.7 mg/dL in no cue controls (p=0.02).
Subcutaneous injection of naltrexone dose-dependently decreased active lever