luis granero, valencia

in the presence of ethanol. Which suggests that ethanol and spermidine produce their
effects by acting at different sites within the NMDA receptor complex. In contrast, the
inhibitory effect of ethanol on the maximally neurotoxic action of NMDA was
significantly and in a concentration-dependent manner reduced by co-application of
spermidine, suggesting that spermidine-induced potentiation of NMDA receptor
function in this situation is sufficiently powerful to mask the inhibitory action of
ethanol on other sites within the NMDA receptor. For example, polyamines may act
by shielding the NMDA receptor proton sensor thereby reversing the tonic inhibition
of protons and increasing the frequency of channel opening. Similarly to protons,
ethanol decreases the frequency of NMDA receptor channel openings indicating that
regions that control channel gating are also the most likely sites of ethanol action.
Therefore, our data seems to suggest that ethanol may act on or upstream of the
proton sensor thereby enhancing its inhibitory coupling to the gating mechanism of
the channel. In the presence of spermidine, when the proton sensor is covered, such
action would be abolished.

INTRA-ACCUMBENS ACAMPROSATE INCREASES
EXTRACELLULAR DOPAMINE IN THE RAT
NUCLEUS ACCUMBENS IN VIVO:
A MICRODIALYSIS STUDY

Cano-Cebrián MJ, Zornoza T, Polache A and Granero L.

Department of Pharmaceutics, University of Valencia, 46100 Burjassot, Spain

The nucleus accumbens (NAc) is an important site of interaction between the
neurotransmitters dopamine (DA) and glutamate (Glu). Interactions among these
transmitters may play an important role in relapse on alcoholism. Acamprosate is an
anticraving drug used in relapse prevention of alcoholism. Recently, some authors
have described a weak antagonist effect of this drug on NMDA receptors. We have
studied the influence of acamprosate on DA levels in NAc of the rat by using in vivo
microdialysis in order to test if the effects of this drug are compatible with those
observed with well recognized antagonist of NMDA receptors when these drugs are
administered directly by retrodialysis in NAc.
Dialysis experiments were performed 24-48 hours after stereotaxic implantation (A/
P: +2.5, L: -1.4, V: 8.1; coordinates relative to bregma and skull surface) of a dialysis
probe with 2 mm of permeable membrane (Hospal, AN69). At this time, dialysis
probes were perfused at 3.5 µL/ min with a solution containing in mM: NaCl: 147;
KCl: 3.0; CaCl2: 1.3; MgCl2: 1.0 in 0.1 mM sodium phosphate buffer (pH= 7.4).
Experimental treatment was initiated after the establishment of a baseline that was
defined as five consecutive samples with less than 10% of variation in DA content.
Treatment involved the local application of Acamprosate for 40 min through the
dialysis probe in the NAc. The drug was dissolved in the perfusion solution at three
concentrations: 0.05 mM, 0.5 mM and 5 mM. DA concentrations in the NAc were on-
line analyzed once every 20 min (65 µL samples) by HPLC with electrochemical
detection (potential set at +0.7 V). At the end of experiments rats were overdosed

alexander kosowski, stockholm

with chloral hydrate, brains removed and cresyl violet staining was used to verify
probe placement. The average concentration of five stable samples was considered the
control and was defined as 100%. All values were expressed as percent of controls.
Basal values of DA in NAc did not differ significantly between experimental groups.
The mean basal dialysate concentration of DA was 1.92 ± 0.64 fmol/ min. Local
application of the higher doses of Acamprosate (500 µM and 5 mM) statistically
increased extracellular DA concentrations in NAc in a dose dependent manner,
whereas the lower concentration (50 µM) had no effect (see figure).
These effects are similar to those observed with kynurenic acid, a well established
antagonist of the NMDA receptors. This broad spectrum antagonist applied by reverse
dialysis in NAc also induces a dose dependent increase on DA release in this region.

This research was supported by funds from Ministerio del Interior: Delegación del
Gobierno para el Plan Nacional sobre Drogas. MJCC is supported by a grant from
Ministerio del Interior of Spain; TZ is supported by a grant from Ministerio de
Educación, Cultura y Deporte of Spain.

THE NOVEL COMPETITIVE AMPA ANTAGONIST
ZK200775 DECREASES NICOTINE-INDUCED
DOPAMINE RELEASE IN THE NUCLEUS ACCUMBENS

Alexander Kosowski, Gvido Cebers and Sture Liljequist

Department of Clinical Neuroscience, Division of Drug Dependence Research, Karolinska
Institutet, Stockholm, Sweden

anna larsson, göteborg

The reinforcing properties of nicotine are thought to be mediated through the
mesocorticolimbic dopamine (DA) system and systemic nicotine stimulates the
release of dopamine (DA) in the nucleus accumbens (NAcc). Accumulated evidence
show that nicotine facilitates glutamatergic transmission in the in the ventral
tegmental area (VTA) where N-methyl-D-aspartate (NMDA) glutamate receptors
appear to play an important role. However, less is known about the involvement of the
other ionotropic glutamate receptor subtype, a-amino-3-hydroxy-5-methylisoxazole-
4-propionate (AMPA) receptors. Although recent data suggest that AMPA receptor
antagonism in the VTA does not affect the nicotine induced DA release in the NAcc,
the role of the AMPA receptors in nicotine addiction needs further clarification. In
this study we examined the effects of the novel water-soluble AMPA receptor
antagonist [1,2,3,4-tetrahydro-7-morpholinyl-2,3-dioxo-6-(fluoromethyl)quinoxalin-
1-yl] methylphosphonate (ZK200775) on nicotine-induced DA release in the NAcc
using in vivo microdialysis in freely moving alert rats. Nicotine dose dependently (0.3
and 0.6 mg/kg; s.c.) stimulated DA release in NAcc (p<0.05). Administration of
ZK200775 (30 minutes prior to nicotine; 0.6 mg/kg; s.c.) significantly decreased the
NAcc DA release at 3 mg/kg (p<0.001) but not at 6 mg/kg. ZK200775 (6 mg/kg) had
no effect on DA release when administered alone. Selectivity of ZK200775 for
AMPA receptors vs NMDA receptors was assessed using cell viability test in primary
cerebellar granule cell cultures. These experiments showed that although ZK200775
was 30 times more potent at the AMPA receptors, higher doses of this compound
would no longer be AMPA-selective and also block the NMDA receptors. To clarify
this issue the effects of a competitive NMDA receptor antagonist CGP39551 and a
competitive AMPA receptor antagonist NBQX on the nicotine-induced DA release in
NAcc were investigated. When administered 30 minutes prior to nicotine 0.6 mg/kg,
CGP39551 (10mg/kg; i.p.) decreased the DA release (p<0.05) whereas NBQX
(10mg/kg; i.p) had no effect. These findings suggest that a combined blockade of both
AMPA and NMDA receptors produces more pronounced inhibition of the nicotine
induced DA release in Nacc compared to a selective blockade of either NMDA or
AMPA receptors.

THE ROLE OF DIFFERENT NICOTINIC ACETYL-
CHOLINE RECEPTOR SUBUNITS IN MEDIATING
BEHAVIORAL AND NEUROCHEMICAL
EFFECTS OF ETHANOL IN MICE

A.Larsson, L. Svensson, B. Söderpalm, J.A. Engel

Institute of Physiology and Pharmacology, Department of Pharmacology, Göteborg University, Box
431, SE-405 30 Göteborg, Sweden.

Ethanol and nicotine are the most abused drugs and it is well-known that co-abuse of
ethanol and nicotine is frequent in man. Our research group has previously obtained
results indicating that the ethanol-induced stimulation of the mesolimbic dopamine

elin löf, göteborg

system and of locomotor activity may involve activation of central nicotinic
acetylcholine receptors (nAChR) especially those located in the ventral tegmental
area. Since there are different subpopulations of nAChR expressed in the central
nervous system we have performed a series of studies investigating the respective role
of some receptor subtypes for the ethanol-nicotine interactions. In these experiments,
the effects of various, more or less specific, nAChR antagonists on the stimulation of
dopamine overflow in the nucleus accumbens (N. Acc.) and on the locomotor
stimulation induced by ethanol mice have been studied.
Locomotor activity was measured in an open field arena using photocell detection.
The mice (male, NMRI) were allowed to habituate for 1 hour before drug treatment,
then they were treated with a nicotinic antagonist and 10 minutes later ethanol (2 g/kg
i.p.) was administered. In vivomicrodialysis was used to determine dopamine
overflow in the N. Acc. induced by ethanol. The ethanol-induced DA overflow was
challenged with different nicotinic antagonists. Dopamine was detected by HPLC
with electrochemical detection. The nicotinic antagonists used were mecamylamine
(negative allosteric modulator of nAChR), methyllycaconitine (MLA, a7antagonist, 2
mg/kg i.p.), dihydro-b-erythroidine (DHbE, a4b2antagonist, 0.5 mg/kg s.c.) and a-
conotoxin Mll (a3b2, b3and a6 –antagonist, 5 nmol locally administered into the
ventral tegmental area).
Mecamylamine blocked the ethanol-induced stimulation of both locomotor activity
and accumbal dopamine overflow. Neither MLA nor DHbE did significantly reduce
the ethanol-induced locomotor stimulation or the dopamine overflow in the N.Acc.
whereas a-conotoxin Mll antagonized the ethanol-induced locomotor stimulation as
well as dopamine overflow in the N.Acc..
The present results suggest that the stimulatory effects of ethanol on locomotion and
dopamine overflow in the N.Acc. do not involve the a4b2 or a7subunit compositions
of the nAChR and that the antagonizing effects of mecamylamine are mediated via a
site not directly associated with the a4b2 or a7subunits. The results from the a-
conotoxin Mll experiments suggest that the ethanol enhancement of locomotor
activity and accumbal dopamine overflow indicate that nicotinic subunit composition
containing a3b2, b3and a6 are involved in mediating those effects of ethanol.

PHARMACOLOGICAL MANIPULATIONS
MODELLING INTERMITTENT STRESS EXPOSURE
INCREASE ALCOHOL CONSUMPTION AND
SENSITIVITY IN THE RAT

Löf E.1), Engel J.A.1)and Soderpalm B.1)2)

1)

Inst. Physiology and Pharmacology, Dept. Pharmacology, Sahlgrenska Academy, Göteborg
University, Sweden. 2) Inst. Clinical Neuroscience, Section of Psychiatry, Sahlgrenska
Academy, Göteborg University, Sweden.
2)