Effect of ShA and LgA amphetamine self-administration on glial
cells in SERT+/+ and SERT-/- rats.
We next investigated the protein expression of the glial glutamate
transporter (GLT-1) that is responsible of the reuptake of glutamate
back into the glial cells from the extracellular space (Roberts-Wolfe &
Kalivas, 2015). In the cNAc two-way ANOVA revealed a significant AMPH
access effect (F(2,57)=10.94, p<0.0001) and an
effect of genotype (F(1,57)=9.349, p=0.0034), whereas in
the sNAc an effect of AMPH access (F(2,56)=5.055,
p=0.0096), but no genotype effect was found. In both cNAc and sNAc,
two-way ANOVA revealed a significant AMPH access x genotype interaction
(cNAc: F(2,57)=3.444, p=0.0387, Fig 5a; sNAc:
F(2,56)=3.347, p=0.0423, Fig 5b). Examining the
individual treatment effects, we found that SERT deletion in both cNAc
and sNAc led to a significant increase of GLT-1 expression in response
to the LgA (cNAc: +29% vs SERT-/--naive, p=0.0115;
+27% vs SERT-/--ShA, p=0.002; sNAc: +29% vs
SERT-/--naive, p=0.0184; +25% vs
SERT-/--ShA, p=0.009), but not to the ShA procedure,
whereas no significant effect was observed in
SERT+/+rats.
Effect of ShA and LgA amphetamine self-administration on NMDA
receptor subunits and the respective scaffolding proteins in the
post-synaptic terminal of SERT+/+ and
SERT-/- rats .
The next step was to investigate the protein expression of the main
subunits of the NMDA receptors. In the cNAc, two-way ANOVA of GluN1
levels, the obligatory subunit of NMDA receptors revealed an effect of
genotype (F(1,57)=4.544, p=0.0374) and a significant
AMPH access x genotype interaction (F(2,57)=8.103,
p=0.0008; Fig 6a). Post-hoc testing revealed that the expression of this
subunit of the NMDA receptor is significantly increased following the
LgA (+40% vs SERT-/--naive, p=0.0056), but not the
ShA procedure in SERT-/- rats. Neither ShA, nor LgA,
affected GluN1 expression in the cNAc of in SERT+/+rats. A different situation was, instead, observed in the sNAc. Two-way
ANOVA revealed a main effect of AMPH access
(F(2,53)=3.314, p=0.044), genotype
(F(1,53)=20.53, p<0.0001) and a significant
AMPH access x genotype interaction (F(2,53)=4.675,
p=0.0135; Fig 6b). Upon sub-testing we found an increased expression of
GluN1 in SERT-/- naïve-rats (+44% vs
SERT+/+-naive, p=0.0006), which was not further
enhanced by the ShA and the LgA procedure. Conversely, both the ShA
(+31% vs SERT-/--naive, p=0.0085) and LgA (+32% vs
SERT-/--naive, p=0.0089) conditions caused an
enhancement in GluN1 protein levels in SERT+/+ rats.
We then investigated the expression of the accessory subunits of the
NMDA receptor, i.e. GluN2A and GluN2B. In the cNAc, two-way ANOVA of
GluN2A levels revealed a main effect of AMPH access
(F(2,57)=5.137, p=0.0089), and an AMPH access x genotype
interaction effect (F(2,57)=11.36, p<0.0001;
Fig 6c); in the same subregion, for the GluN2B subunit, two-way ANOVA
revealed a main effect of genotype (F(1,56)=12.21,
p=0.0009) and an AMPH access x genotype interaction
(F(2,56)=10.41, p=0.0001; Fig 6e). GluN2A subunit
expression was reduced in SERT-/- naïve-rats (-32% vs
SERT+/+-naive, p=0.0433); further, the post-hoc
intergroup comparisons revealed that the removal of SERT caused a
different response to both the ShA and LgA conditions. In fact, GluN2A
protein levels were reduced following both ShA (-36% vs
SERT+/+-naive, p=0.0032) and LgA (-34% vs
SERT+/+-naive, p=0.0063) AMPH self-administration in
SERT+/+ rats, whereas in SERT-/-rats no changes were found for ShA while GluN2A protein levels were
up-regulated following LgA (+28% vs SERT-/--naive,
p=0.0426; +27% vs SERT-/--ShA, p=0.0085). GluN2B
levels were not altered in AMPH-exposed SERT+/+ rats
(ShA: -18% vs SERT+/+-naive, p=0.5623; LgA: -27% vs
SERT+/+-naive, p=0.1727). In SERT-/-rats similarly to GluN2A, we found that the LgA (+42% vs
SERT-/--naive, p=0.0071; +33% vs
SERT-/--ShA, p=0.010), but not the ShA procedure
significantly up-regulated the expression of this subunit.
In the sNAc, two-way ANOVA revealed only a significant genotype effect
(F(1,55)=19.76, p<0.0001; Fig 6d) for GluN2A,
and a significant AMPH access effect (F(2,56)=15.03,
p<0.0001; Fig 6f) for GluN2B.
Following the analysis of the NMDA receptor subunits, we investigated
the expression of the main scaffolding protein of NMDA receptors, SAP102
(Won et al., 2017). In the cNAc, two-way ANOVA revealed a main effect of
AMPH access (F(2,56)=9.318, p=0.0003) and an AMPH access
x genotype interaction (F(2,56)=4.476, p=0.0157; Fig
7a). Post-hoc testing of the main treatment effects showed that both ShA
(-20% vs SERT+/+-naive, p=0.0033) and LgA (-27% vs
SERT+/+-naive, p=0.0001) AMPH self-administration
resulted in reduced SAP102 expression in SERT+/+ rats,
whereas no changes were observed under both experimental conditions in
SERT-/- rats. In the sNAc, no significant changes in
SAP102 levels were observed (Fig 7b).
Effect of ShA and LgA amphetamine self-administration on AMPA
receptor subunits and their respective scaffolding proteins in the
post-synaptic terminal of SERT+/+ and
SERT-/- rats .
We then moved to the protein expression analysis of the main subunits of
the AMPA receptor, i.e. GluA1 and GluA2. In the cNAc, for both GluA1 and
GluA2 subunits, two-way ANOVA revealed a significant AMPH access x
genotype interaction (GluA1: F(2,57)=16.03,
p<0.0001, Fig 8a; GluA2: F(2,57)=13.79,
p<0.0001, Fig 8c). For GluA2 two-way ANOVA revealed also a
main effect of genotype (F(2,57)=13.79, p=0.0006). SERT
knock out reduced GluA1 (-31% vs SERT+/+-naive,
p=0.0477), but not GluA2, levels. The LgA condition reduced GluA1 levels
in SERT+/+ (-37% vs SERT+/+-naive,
p=0.0017) while increasing it in SERT-/- (+34% vs
SERT-/--naive, p=0.0048). GluA2 levels were reduced
following both ShA (-26% vs SERT+/+-naive, p=0.0447)
and LgA (-32% vs SERT+/+-naive, p=0.0083) AMPH
self-administration in SERT+/+, while deletion of SERT
increased GluA2 levels following both ShA (+27% vs
SERT-/--naive, p=0.0329) and LgA (+32% vs
SERT-/--naive, p=0.0071) AMPH self-administration. In
the sNAc, two-way ANOVA of both GluA1 and GluA2 AMPA subunits revealed a
main effect of AMPH access (GluA1: F(2,57)=17.17,
p<0.0001; GluA2: F(2,58)=4.995, p=0.010),
genotype (GluA1: F(1,57)=17.81, p<0.0001;
GluA2: F(1,58)=15.09, p=0.0003) and an AMPH access x
genotype interaction effect (GluA1: F(2,57)=3.571,
p=0.0346, Fig 8b; GluA2: F(2,58)=3.301, p=0.0439, Fig
8d). Both ShA and LgA conditions resulted in an elevation in GluA1 and
GluA2 protein levels in SERT+/+ rats (GluA1: ShA +34%
vs SERT+/+-naive, p=0.0329; LgA +37% vs
SERT+/+-naive, p=0.0185; GluA2: ShA +46% vs
SERT+/+-naive, p=0.0259; LgA +44% vs
SERT+/+-naive, p=0.0411) whereas, in
SERT-/- rats, no changes were observed with the
exception of the increased expression of GluA1 following LgA (+54% vs
SERT-/--naive, p=0.0001; -37% vs
SERT-/--ShA, p=0.0017).
We then examined the expression of the scaffolding proteins specific for
AMPA receptor subunits GluA1 and GluA2, i.e. SAP97 and GRIP,
respectively (Won et al., 2017). In the cNAc, for both SAP97 and GRIP,
two-way ANOVA revealed a main effect of AMPH access (SAP97:
F(2,57)=4.464, p=0.0158; GRIP:
F(1,58)=5.822, p=0.0050), and an AMPH access x genotype
interaction (SAP97: F(2,57)=5.152, p=0.0088, Fig 9a;
GRIP: F(2,58)=4.385, p=0.0169, Fig 9c). In
SERT+/+ rats, we found a significant reduction of
SAP97 under LgA AMPH self-administration conditions only (-30% vs
SERT+/+-naive, p=0.0011), whereas GRIP levels were
reduced under both ShA (-27% vs SERT+/+-naive,
p=0.0108) and LgA conditions (-32% vs SERT+/+-naive,
p=0.0019). Conversely, under both experimental conditions, no
significant changes were shown in SERT-/- rats. In the
sNAc, two-way ANOVA of both SAP97 and GRIP levels revealed a main effect
of AMPH access (SAP97: F(2,57)=7.647, p=0.0011; GRIP:
F(1,57)=3.795, p=0.0284) and an AMPH access x genotype
interaction (SAP97: F(2,57)=15.86, p<0.0001,
Fig 9b; GRIP: F(2,57)=7.230, p=0.0016, Fig 9d). Post-hoc
testing of the main treatment effects in SERT+/+ rats
revealed reduced expression of SAP97 following LgA (-25% vs
SERT+/+-naive, p=0.0429), but not ShA, procedures
whereas GRIP was similarly reduced by both experimental conditions (ShA:
-23% vs SERT+/+-naive, p=0.0150; LgA: -31% vs
SERT+/+-naive, p=0.0005). Conversely, in
SERT-/- rats, we found an increase in the expression
of SAP97 following the LgA (-30% vs SERT-/--naive,
p=0.0093; -41% vs SERT-/--ShA, p<0.0001),
but not the ShA condition, whereas GRIP expression was decreased in
SERT-/- naïve-rats (-29% vs
SERT+/+-naive, p=0.0059) without further changes
following both ShA and LgA AMPH self-administration.