Amphetamine effects in Nucleus Accumbens shell (sNAc)
Twenty-four hours into withdrawal from AMPH self-administration, we found that vGluT1 expression was reduced at baseline in the sNAc of SERT-/- rats, compared to SERT+/+rats, suggesting reduced vesicular release of glutamate in SERT-/- rats with consequently lower extracellular levels in the synapse. Of note, SERT+/+ rats showed increased vGluT1 expression after both ShA and LgA, whereas SERT-/- rats exhibited an increase of the vesicular transporter following ShA and no change following LgA. Since the extracellular levels of glutamate are determined by a balance between its release and reuptake, the evidence that the accumbal expression of the plasmalemmal glutamate transporter GLT-1 is not altered by AMPH in SERT+/+ rats might be indicative of an increased AMPH-induced overflow of glutamate in the sNAc of these animals. Instead, GLT-1 expression is increased in SERT-/- LgA rats, possibly reducing the extracellular levels of glutamate, whereas it is not changed following ShA. This suggests that SERT deletion may be associated with an increased glutamate overflow as a consequence of a ShA-induced increase of vGluT1. On the other hand, SERT deletion may reduce glutamate overflow as a consequence of a LgA-induced increase of GLT-1 expression. Since the sNAc is critical for the reinforcing properties of psychostimulants (Guillem et al., 2014), the possibility exists that altered extracellular glutamate levels in this brain subregion of SERT-/- rats may affect drug seeking.
The analysis of the expression of glutamate receptors in the sNAc revealed differences between NMDA and AMPA receptors. At baseline, a significant change was represented by the increased expression of GluN1 in SERT-/- rats, perhaps resulting from the reduced glutamate release caused by the decreased expression of vGluT1. Following both ShA and LgA AMPH self-administration, we found increased expression of GluN1 in SERT+/+ rats, suggesting that the proposed higher release of glutamate in SERT+/+rats is not compensated by a significant reduction in the main NMDA subunit. Conversely, in SERT-/- rats, neither ShA nor LgA are able to further enhance the expression of GluN1, suggesting that SERT deletion-induced changes in GluN1 expression may be maximal. Notably, the observed increase in GluN1 expression were not accompanied by an increase in the levels of the accessory subunits GluN2A and GluN2B and of its anchoring protein SAP102, suggesting receptor instability following both ShA and LgA.
Regarding AMPA receptors, we found no significant changes at baseline between the two genotypes. GluA1 and GluA2 expression was up-regulated following either ShA or LgA in SERT+/+ rats. However, when analyzing the main scaffolding proteins for these two AMPA subunits, we found reduced expression of both SAP97 and GRIP in SERT+/+ rats. These findings suggest that GluA1 and GluA2 receptors lose their stability following both drug regimens in SERT+/+ rats. In SERT-/- rats, both GluA1 and SAP97 expression was significantly increased following LgA drug exposure, suggesting that LgA to AMPH increased the stability of at least the GluA1 receptor in these animals.
In SERT+/+ rats, the proposed AMPH-induced increased release of glutamate, not compensated by changes in the expression of GLT-1, leads to an increased expression of the main glutamate receptors. Instead, the localization and, presumably, the functionality of these receptors is attenuated by the uncoupling of their respective auxiliary proteins independently from the duration of the AMPH exposure. Conversely, in SERT-/- rats, the proposed reduced glutamate release is accompanied by enhanced reuptake only following LgA drug exposure, a combination that leads to a functional compensation only for GluA1 but not for GluA2 AMPA subunit nor for the different NMDA receptor subunits. Of note, the increased expression of GluA1, but not GluA2, AMPA subunit following LgA may lead to the formation of GluA2-lacking and Ca2+-permeable AMPARs, a mechanism known to drive addiction (Wolf, 2016).