Effects of fluoxetine on methylphenidate-induced open-field behavior
Administration of methylphenidate increased ambulation (MP2 vs. V, P<0.01; MP5 vs. V, P<0.001) and stereotypy counts (MP2 vs. V, P<0.001; MP5 vs. V, P<0.01) in the first (0–20 min) and second half (20–40 min) of the test (). Fluoxetine alone had no effect on these two parameters (FLX vs. V, P>0.05). The methylphenidate + fluoxetine combinations increased ambulation counts in a manner similar to methylphenidate alone (MP2+FLX vs. MP2 and MP5+FLX vs. MP5, P>0.05) and more than fluoxetine alone (MP2+FLX vs. FLX and MP5+FLX vs. FLX, P<0.001). As with methylphenidate alone, the methylphenidate + fluoxetine combinations induced stereotypies (MP2+FLX vs. V or FLX, and MP5+FLX vs. V or FLX, P<0.001). This increase in stereotypy counts was similar in the MP2 and MP2+FLX groups (P>0.05). However, the MP5+FLX group showed higher stereotypy counts than the MP5 alone group (P<0.001; ) early and late in the test, demonstrating that fluoxetine potentiated methylphenidate-induced stereotypies for this higher methylphenidate dose.
Figure 2 Drug effects on open-field behavior. Ambulation (A) and stereotypy counts (B) are shown for animals that received a systemic injection of vehicle (V), fluoxetine (5 mg/kg; FLX), methylphenidate (2 or 5 mg/kg; MP2, MP5), or methylphenidate + fluoxetine (more ...)
Effects of fluoxetine on methylphenidate-induced gene expression in the striatum
Administration of methylphenidate alone induced a dose-dependent increase in zif 268
expression in the striatum on all three rostrocaudal levels (– and and ), consistent with our previous findings (Brandon & Steiner, 2003
; Yano & Steiner, 2005a
). For zif 268
, a significant increase in expression was observed in five (MP2) and 17 (MP5) of the 23 striatal sectors, and for c-fos
, in three (MP2) and 11 (MP5) sectors (P<0.05 vs. V) (). Gene regulation varied considerably between different striatal regions. For both zif 268
, the most robust increase was observed on middle and caudal striatal levels, in dorsal/central and medial sectors (–) that receive sensorimotor and cingulate cortical inputs (). In contrast, the nucleus accumbens displayed more modest drug effects. No statistically significant changes in gene expression were seen with 2 mg/kg of methylphenidate alone (P>0.05 vs. V). The 5 mg/kg dose significantly increased zif 268
expression in the lateral shell only (MP5 vs. V, P<0.001, and ). In order to compare the regional patterns of methylphenidate-induced zif 268
expression across the 23 striatal sectors, we performed a correlation analysis. This analysis confirmed that the regional distribution of increases (vs. vehicle-treated controls) was highly correlated between zif 268
expression (zif 268
: MP2, r=0.844, P<0.001; MP5, r=0.931, P<0.001; not shown).
Figure 6 Topography of fluoxetine-potentiated gene regulation by methylphenidate. Maps depict the distribution of zif 268 (A) and c-fos expression (B) in the rostral, middle and caudal striatum after an injection of fluoxetine (5 mg/kg; FLX), methylphenidate (2 (more ...)
Effects of fluoxetine, methylphenidate and methylphenidate + fluoxetine on zif 268 expression in the striatum.
Effects of fluoxetine, methylphenidate and methylphenidate + fluoxetine on c-fos expression in the striatum.
Figure 4 Fluoxetine potentiation of methylphenidate-induced zif 268 expression in specific striatal sectors. Mean density values (mean±SEM) for zif 268 expression in rats that received an injection of vehicle (V), fluoxetine (5 mg/kg; FLX), methylphenidate (more ...)
In contrast to methylphenidate, fluoxetine (5 mg/kg) alone did not modify gene expression, neither in the caudate-putamen nor in the nucleus accumbens (–). None of the 23 sectors showed significant changes in zif 268 or c-fos expression (P>0.05 vs. V, and , and ).
However, when given in conjunction with methylphenidate, fluoxetine potentiated methylphenidate-induced IEG expression in the striatum. Correlation analyses show that the regional distribution of this potentiation (POT; i.e., the difference between MP+FLX and MP) was similar for zif 268 and c-fos expression (zif 268 x c-fos: methylphenidate 2 mg/kg, POT2, r=0.775, P<0.001; 5 mg/kg, POT5, r=0.840, P<0.001; ). Moreover, despite relatively modest potentiation for 2 mg/kg of methylphenidate, principally the same sectors were affected as for 5 mg/kg (POT2 x POT5: zif 268, r=0.659, P<0.05; c-fos, r=0.759, P<0.01; not shown).
Figure 7 The potentiation of gene induction displays a similar regional distribution in the striatum for zif 268 and c-fos. Scatterplots show the correlations between zif 268 and c-fos potentiation for 2 mg/kg (r=0.775, POT2, top) and 5 mg/kg (r=0.840, POT5, bottom) (more ...)
The fluoxetine potentiation was reflected in a higher proportion of the 23 striatal sectors displaying significantly increased zif 268 and c-fos expression after the methylphenidate + fluoxetine treatment, compared with methylphenidate alone (zif 268: MP2+FLX vs. MP2, 10 sectors vs. 5 sectors; MP5+FLX vs. MP5, 19 vs. 17; c-fos: 6 vs. 3 and 16 vs. 11; ). Direct statistical comparisons showed that, for the 2 mg/kg dose of methylphenidate, c-fos induction was significantly more robust in the MP2+FLX group than in the MP2 group in one sector (middle level, dorsal sector; , POT2). For the 5 mg/kg dose, the potentiation (MP5+FLX vs. MP5, POT5) was statistically significant in 15 and 13 of the 23 striatal sectors, for zif 268 and c-fos, respectively. Further analysis showed that the magnitude of the fluoxetine potentiation was principally related to the magnitude of gene induction produced by methylphenidate alone (5 mg/kg, MP5 x POT5: zif 268, r=0.638, P<001; c-fos, r=0.814, P<001; ; 2 mg/kg: P>0.05; not shown). However, a more pronounced potentiation than predicted by the methylphenidate response was seen in the dorsolateral and ventrolateral (sensorimotor) sectors of the middle striatum for both zif 268 and c-fos expression (). This finding confirms that the lateral (sensorimotor) striatum displayed a more pronounced fluoxetine potentiation of gene regulation than the medial (associative) striatum; this is also apparent in the potentiation maps ().
Figure 8 Relationship between the gene induction by methylphenidate alone and the fluoxetine potentiation in the different striatal sectors. Scatterplots depict the correlations between gene induction by 5 mg/kg methylphenidate (MP5) and the fluoxetine potentiation (more ...)
Fluoxetine also potentiated methylphenidate-induced gene expression in selective regions of the nucleus accumbens, predominantly in the lateral part of the shell ( and ). After MP2+FLX, but not MP2 alone, zif 268 expression was significantly increased in the lateral shell, as was c-fos expression after MP5+FLX, but not MP5 alone. For zif 268, a significant potentiation in MP5+FLX vs. MP5 animals (POT5) was seen in the lateral shell as well as in the medial core ().
Effects of fluoxetine on methylphenidate-induced zif 268 expression in the cortex
Administration of methylphenidate alone induced a dose-dependent up-regulation of zif 268 expression in the cortex on all four rostrocaudal levels (, ). A statistically significant increase in zif 268 mRNA levels was observed in 7 (MP2) and 11 (MP5) of the 22 cortical areas (P<0.05 vs. V). However, this effect was restricted to dorsomedial cortical regions, including the cingulate, medial agranular and motor cortex (mainly on rostral to caudal levels), as well as the prelimbic and insular/lateral orbital cortex (frontal level). These are mostly limbic and associative areas. In contrast, the somatosensory cortex and insular cortex (except frontal level) were not affected by methylphenidate on any rostrocaudal levels (MP2 or MP5 vs. V, P>0.05).
Figure 9 Effects of methylphenidate + fluoxetine combination treatment on zif 268 expression in the cortex. Maps show the distribution of zif 268 expression in the cortex on frontal, rostral, middle and caudal levels after an injection of fluoxetine (5 mg/kg; (more ...)
Effects of fluoxetine, methylphenidate and methylphenidate + fluoxetine on zif 268 expression in the cortex.
Fluoxetine given alone tended to increase zif 268 mRNA levels in many cortical areas, but this effect was statistically significant only in the cingulate (frontal level) and motor cortex (caudal level) (FLX vs. V, P<0.05 and P<0.01, respectively, and ). After the methylphenidate + fluoxetine treatment, significantly enhanced zif 268 expression was found in eight (MP2+FLX) and 14 (MP5+FLX) of the 22 cortical areas (as compared to 7 and 11, respectively, for methylphenidate only, see above). However, the methylphenidate + fluoxetine treatment did not produce significantly higher zif 268 mRNA levels than methylphenidate alone in any cortical area (MP+FLX vs. MP; P>0.05), except in the infralimbic cortex on the frontal level for 2 mg/kg of methylphenidate (; MP2+FLX vs. MP2; P<0.01; MP5+FLX vs. MP5; P>0.05). Therefore, in contrast to the striatum, this dose of fluoxetine did not robustly potentiate cortical gene regulation by methylphenidate.
Our earlier findings (Yano & Steiner, 2005a
; Cotterly et al., 2007
) showed that there is a positive correlation between psychostimulant-induced gene expression in cortical areas and gene induction in the striatal sectors targeted by these cortical areas, indicating coordinated molecular changes in cortical and striatal nodes of corticostriatal circuits. We assessed whether such a relationship existed in the present study and whether it was affected by the present SSRI treatments. Thus, drug-induced increases in zif 268
expression in cortical areas were compared with those in their respective striatal target sectors (see ). When a striatal sector received input from more than one cortical area, the values of these cortical areas were averaged. Our results show that, overall, methylphenidate-induced zif 268
expression in the cortical areas was positively correlated with that in their connected 23 striatal sectors (MP2, r=0.493, P<0.05; MP5, r=0.436, P<0.05), confirming our earlier findings (Yano & Steiner, 2005a
; Cotterly et al., 2007
). This effect was more robust when only the 18 sectors of the caudate-putamen were included (MP2, r=0.556, P<0.05; MP5, r=0.676, P<0.01). The addition of fluoxetine to methylphenidate weakened this correlation (23 sectors: MP2+FLX, r=0.326, P>0.05; MP5+FLX, r=0.307, P>0.05; 18 sectors: MP2+FLX, r=0.503, P<0.05; MP5+FLX, r=0.567, P<0.05).
Effects of citalopram on methylphenidate-induced IEG expression in the striatum
We also assessed whether the potentiation of methylphenidate-induced gene regulation generalized to other SSRIs. Our results demonstrate that this is the case. Administration of the SSRI citalopram (5 mg/kg) together with methylphenidate (5 mg/kg) potentiated methylphenidate-induced expression of zif 268 () and c-fos (not shown) in the striatum. The regional distribution of this potentiation was similar to that produced by fluoxetine.