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. 2008 Oct;22(10):3736-46.
doi: 10.1096/fj.08-111245. Epub 2008 Jul 7.

Deletion of Go2alpha Abolishes Cocaine-Induced Behavioral Sensitization by Disturbing the Striatal Dopamine System

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Free PMC article

Deletion of Go2alpha Abolishes Cocaine-Induced Behavioral Sensitization by Disturbing the Striatal Dopamine System

Irene Brunk et al. FASEB J. .
Free PMC article

Abstract

The alpha-subunits of the trimeric Go class of GTPases, comprising the splice variants Go1alpha and Go2alpha, are abundantly expressed in brain and reside on both plasma membrane and synaptic vesicles. Go2alpha is involved in the vesicular storage of monoamines but its physiological relevance is still obscure. We now show that genetic depletion of Go2alpha reduces motor activity induced by dopamine-enhancing drugs like cocaine, as repeated injections of cocaine fail to provoke behavioral sensitization in Go2alpha(-/-) mice. In Go2alpha(-/-) mice, D1 receptor signaling in the striatum is attenuated due to a reduced expression of Golf alpha and Gs alpha. Following cocaine treatment, Go2alpha(-/-) mice have lower D1 and higher D2 receptor amounts compared to wild-type mice. The lack of behavioral sensitization correlates with reduced dopamine levels in the striatum and decreased expression of tyrosine hydroxylase. One reason for the neurochemical changes may be a reduced uptake of monoamines by synaptic vesicles from Go2alpha(-/-) mice as a consequence of a lowered set point for filling. We conclude that Go2alpha optimizes vesicular filling which is instrumental for normal dopamine functioning and for the development of drug-induced behavioral sensitization.

Figures

Figure 1.
Figure 1.
Cocaine-induced behavior in wild-type (wt) and Go2α−/− mice. A) Effects of acute and repeated intermittent injections of cocaine on locomotor activity of wt (n=16) and Go2α−/− (n=15) littermates. Single administration of cocaine increased locomotor activity in both genotypes (cocaine F1, 27=44.77, P<0.0001); however, there were only slight, but not significant, differences between the genotypes (genotype F1, 27=1.43, P=0.24). Subsequently, daily injections of cocaine led to an increase in locomotor activity (behavioral sensitization) in wild-type mice (Trial F3, 81=0.04, P<0.0001). In contrast, Go2α−/− mice showed no development of sensitization (genotype F1, 27=7.79, P<0.01). *P < 0.05. B) Both genotypes did not differ in the conditioned place preference induced by cocaine (T26=0.059, P=0.95). The dashed line indicates the time spent in the drug-paired side before drug administration.
Figure 2.
Figure 2.
In vitro effects of cocaine on synaptosomes from wild-type (wt) and Go2α−/− mice. A) Synaptosomes from wt or Go2α−/− mouse brains were loaded with [3H]dopamine for 10 min at 37°C, either in the absence or presence of 5 μM cocaine. Nonspecific uptake using 20 μM GBR 12909, a specific inhibitor of DAT, was subtracted. As expected, cocaine completely blocked uptake into synaptosomes with no difference between the genotypes. *P < 0.05. B) Synaptosomes from both wild-type and Go2α−/− mice were permeabilized by SLO, and vesicular uptake was determined in the absence or presence of 5 μM cocaine. Nonspecific uptake in the presence of reserpine was subtracted. Vesicular uptake was not affected by cocaine, and there was no difference between the genotypes. Data represent means ± sd of 3 determinations.
Figure 3.
Figure 3.
Expression of trimeric G-protein subunits and D1- and D2-receptor proteins in wild-type (wt) and Go2α−/− mice. A) Ratio of Go1α and Go2α expression. Blots: synaptosomal fractions (P2) from whole brains of Go1α−/− (P20), Goα−/− (P30), or Go2α−/− (adult) and of the corresponding wt littermates were analyzed using the monoclonal antibody clone 101.1 (recognizing both splice variants), the Go2α-specific clone 101.4 (only applicable for Western blot analysis), and the sc13532 Goα antibody preferentially recognizing Go1α. Note that in Goα−/− preparations, none of the antibodies gives a signal, whereas signals for clone 101.4 or sc13532 antibodies are almost or completely absent in Go2α−/− or Go1α−/− mice, respectively. Go2α represents one-third of Goα in P20 (left bars) or adult (right bars) brains, as revealed by quantification using synaptobrevin (Syb) as an internal standard. Go1α is not up-regulated in Go2α−/− mice compared to wt littermates. Immunofluorescence: cerebellar sections were labeled by an antibody (clone 101.1), recognizing both Goα subunits (15). The mean immunofluorescence/100 μm2 of Goα subunits checked was significantly reduced in the molecular layer from 105.2 ± 6.8 in wt to 72.8 ± 9.4 in Go2α−/− mice and in the granular cell layer from 41.7 ± 9.0 (wt) to 21.9 ± 1.8 (knockout) (means ± sd). B) Quantification was performed with synaptosomal preparations from striata of four pairs of wt and Go2α−/− mice. The amounts of Golfα and Gsα, but not of Gqα or Gβ subunits, in general (Gβ1–4), were reduced in Go2α−/− mice. C) There was no significant difference in the amounts of D1- and D2-receptor protein between wt and Go2α−/− mice. For each quantification in B and C, 5, 10, and 15 μg of protein of the respective sample was loaded. Quantification is given as relative optical density (OD) and was performed using synaptophysin (Syp) or synaptobrevin (Syb) as an internal control. Graphs show the quantification from the 10 μg protein load. Values are expressed as means ± sd. *P < 0.05.
Figure 4.
Figure 4.
Expression of dopamine (D1, D2) receptor proteins and G-protein α-subunits in wild-type (wt) and Go2α−/− mice following cocaine treatment. Western blot analyses were performed from postnuclear membrane preparation from frozen striata of both genotypes using cocaine-treated animals presented in Fig. 1. A) After treatment with cocaine, amounts of D1 receptor were lower and of D2 receptor were higher in striata of Go2α−/− mice compared to wt littermates. B) The expression of Golfα and Gsα leveled out between the genotypes following cocaine treatment. For each quantification, 5, 10, and 15 μg of protein of the respective sample was loaded; the graphs show the quantification from the 10-μg protein load. Quantification is given as relative OD and was performed using either Syp or Syb as an internal control. Values are expressed as means ± sd. *P < 0.05.
Figure 5.
Figure 5.
Monoamine levels in brain areas of wild-type (wt) and Go2α−/− mice. Serotonin, noradrenaline, and dopamine were analyzed in extracts from frozen brains or brain areas using HPLC. A) Until day P8, there are no significant differences in whole brain monoamine concentrations between wt and Go2α−/− mice. B) In the striatum of P12 and adult Go2α−/− animals, dopamine amounts are decreased compared to wt littermates. Values represent means ± se; n = 6. *P < 0.05.
Figure 6.
Figure 6.
Dopamine synthesizing and metabolizing enzymes, as well as transporters in wild-type (wt) and Go2α−/− mice. A) Amounts of TH and DAT were analyzed in synaptosomal preparations (whole brain) from wt and Go2α−/− mice. Amount of TH is reduced in Go2α−/− mice. Values represent means ± se; n = 4. B) Determination of MAO A + B activity in synaptosomes from wt and Go2α−/− mice revealed no difference between the genotypes. Values represent the means ± se; n = 3. C) VMAT2 expression in wt and Go2α−/− mice. Subcellular fractionation [H, homogenate; P2, synaptosomes; LP1, lysed synaptosomal pellet 1 at 29,000 g; LP2, lysed synaptosomal pellet 2 (SVs) at 360,000 g] of wt and Go2α−/− mice revealed that VMAT2 is enriched in the SV fraction (LP2) in both genotypes. Surprisingly, more VMAT2 is expressed in the SV fraction of Go2α−/− mice compared to wt littermates. For each quantification, 5, 10, and 15 μg of protein of the respective sample was loaded. Quantification is given as relative optical density (OD) and was performed using Syp as a reference. Graphs show the quantification from 10-μg protein load. Values represent means ± se; n = 4 animals/genotype. *P < 0.05.
Figure 7.
Figure 7.
Go2α modulates [3H] monoamine uptake into VMAT2-containing SVs. A, B) SLO-permeabilized synaptosomes (A) or isolated SVs (B) of either wt or Go2α−/− mice were subjected to [3H] serotonin uptake in the absence or presence of 5 or 100 μM of GMP-P(NH)P, respectively. GMP-P(NH)P failed to modulate vesicular uptake in knockout animals. C) SVs from either wt, heterozygous, or knockout animals were subjected to serotonin uptake in the absence or presence of 100 μM GMP-P(NH)P. Values are expressed as the GMP-P(NH)P-mediated inhibition (%). Note that there is no inhibition in the knockout-derived SVs. D) [3H]serotonin uptake into permeabilized synaptosomes or isolated SVs is moderately decreased in Go2α−/− animals compared to wt littermates. Values were taken from 10 different experiments, each run in triplicate. The reserpine-sensitive uptake of wt animals was set at 100%. Bars indicate means ± sd. E) The amount of the vacuolar ATPase in various subcellular fractions analyzed using an antibody against the 116-kDa subunit revealed an enrichment in the SV fraction (LP2), with no difference between wt- and Go2α−/−-derived membranes. Graphs show the quantification from 10-μg protein load. Data were normalized to Syp; means ± sd; n = 4 animals/genotype. *P < 0.05.
Figure 8.
Figure 8.
Effects of D1- and D2-receptor activation on motor activity. Dopamine enhances motor activity both by promoting the direct pathway via the stimulatory D1 receptor signaling and by slowing down the indirect pathway via the inhibitory D2-receptor signaling.

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