Individual differences in ethanol locomotor sensitization are associated with dopamine D1 receptor intra-cellular signaling of DARPP-32 in the nucleus accumbens

Abstract

In mice there are clear individual differences in the development of behavioral sensitization to ethanol, a progressive potentiation of its psychomotor stimulant effect. Variability in the behavioral responses to ethanol has been associated with alcohol preference. Here we investigated if the functional hyperresponsiveness of D1 receptors observed in ethanol sensitized mice leads to an increased activation of DARPP-32, a central regulatory protein in medium spiny neurons, in the nucleus accumbens - a brain region known to play a role in drug reinforcement. Swiss Webster mice received ethanol (2.2 g/kg/day) or saline i.p. administrations for 21 days and were weekly evaluated regarding their locomotor activity. From those treated with ethanol, the 33% with the highest levels of locomotor activity were classified as "sensitized" and the 33% with the lowest levels as "non-sensitized". The latter presented similar locomotor levels to those of saline-treated mice. Different subgroups of mice received intra-accumbens administrations of saline and, 48 h later, SKF-38393, D1 receptor agonist 0.1 or 1 µg/side. Indeed, sensitized mice presented functional hyperresponsiveness of D1 receptors in the accumbens. Two weeks following the ethanol treatment, other subgroups received systemic saline or SKF 10 mg/kg, 20 min before the euthanasia. The nucleus accumbens were dissected for the Western Blot analyses of total DARPP-32 and phospho-Thr34-DARPP-32 expression. D1 receptor activation induced higher phospho-Thr34-DARPP-32 expression in sensitized mice than in non-sensitized or saline. The functionally hyperresponsiveness of D1 receptors in the nucleus accumbens is associated with an increased phospho-Thr34-DARPP-32 expression after D1 receptor activation. These data suggest that an enduring increase in the sensitivity of the dopamine D1 receptor intracellular pathway sensitivity represents a neurobiological correlate associated with the development of locomotor sensitization to ethanol.

Figure 1. Diagram of approximate microinjections hits.

The crosses (+) represent the positions of microinjections in both sides of the Nucleus Accumbens considered correct. The basic diagram is a modified representation of Paxinos and Franklin (2004) atlas.

Figure 2. There is clear individual variability to the development of sensitization to ethanol.

Locomotor activity for 15(means ± S.E.M.) of mice treated with saline or 2.2 g/kg ethanol i.p. (classified as non-sensitized (nsens) or sensitized (sens) based on their locomotor activity in the 21^st day test) in the tests performed on days 1, 7, 14 and 21. (A) Cohort of mice (saline, n = 8; nsens, n = 5; sens, n = 8) that received the lower dose of SKF during the challenge phase. (B) Cohort of mice (saline, n = 11; nsens, n = 6; sens, n = 7) that received the higher dose of SKF during the challenge phase. (C) Cohort of mice (saline, n = 8; nsens, n = 8; sens, n = 9) that was designed for DARPP-32 measures after saline administration. (D) Cohort of mice (saline, n = 6; nsens, n = 7; sens, n = 7) that was designed for DARPP-32 measures after SKF administration. * indicates significantly higher activity levels than those presented by the saline and nsens groups during the same test (p<0.05) and when compared to their own levels in test 1 (p<0.05).

Figure 3. D1 receptor agonist induced hyperresponsive locomotion in sensitized mice.

Locomotor activity (means + S.E.M.) of the saline, sens and nsens in the challenges with intra-NAc administration of saline and SKF-38393 at 0.1 or 1 µg/side. Each challenge was performed 48 h after the previous one. (A) Locomotor activity 20 min before and 60 min after saline intra-NAc administration of saline (n = 8), nsens (n = 5) and sens (n = 8) groups. (B) Locomotor activity 20 min before and 60 min after SKF 0.1 µg/side intra-NAc administration of saline (n = 8), nsens (n = 5) and sens (n = 8) groups, 48 h after the saline challenge (A). (C) Locomotor activity during 15 min (gray backgrounds in figures A and B), after intra-NAc administration of saline and SKF 0.1 µg/side. (D) Locomotor activity 20 min before and 60 min after saline intra-NAc administration of saline (n = 11), nsens (n = 6) and sens (n = 7) groups. (E) Locomotor activity 20 min before and 60 min after SKF 1 µg/side intra-NAc administration of saline (n = 11), nsens (n = 6) and sens (n = 7) groups, 48 h after the saline challenge (D). (F) Locomotor activity during 60 min (gray backgrounds in figures D and E), after intra-NAc administration of saline and SKF 1 µg/side. * indicates significantly higher activity levels than those of the saline and nsens mice and higher locomotor activity levels than their own levels in the saline challenge (p<0.05).

Figure 4. D1 receptor agonist induced accumbal DARPP-32 hyperphosphorylation in sensitized mice.

(A) Protein expression of total DARPP-32, phospho-Thr34-DARPP-32 and phospho-Thr34-DARPP-32/total DARPP-32 in the NAc in saline (n = 8), nsens (n = 8) and sens (n = 9) groups, 20 min after i.p. saline administration. (B) Protein expression of total DARPP-32, phospho-Thr34-DARPP-32 and phospho-Thr34-DARPP-32/total DARPP-32 in the NAc in saline (n = 6), nsens (n = 7) and sens (n = 7) groups, 20 min after 10 mg/kg i.p. SKF-38393 administration. * indicates significantly higher levels than saline and nsens groups (p<0.05). + indicates significantly lower levels than saline group (p<0.05). # indicates significantly higher levels than saline and nsens groups (p<0.05).