Altered Sexual Behavior in Dopamine Transporter (DAT) Knockout Male Rats: A Behavioral, Neurochemical and Intracerebral Microdialysis Study

Abstract

Central dopamine plays a key role in sexual behavior. Recently, a Dopamine Transporter knockout (DAT KO) rat has been developed, which displays several behavioral dysfunctions that have been related to increased extracellular dopamine levels and altered dopamine turnover secondary to DAT gene silencing. This prompted us to characterize the sexual behavior of these DAT KO rats and their heterozygote (HET) and wild type (WT) counterparts in classical copulatory tests with a sexually receptive female rat and to verify if and how the acquisition of sexual experience changes along five copulatory tests in these rat lines. Extracellular dopamine and glutamic acid concentrations were also measured in the dialysate obtained by intracerebral microdialysis from the nucleus accumbens (Acb) shell of DAT KO, HET and WT rats, which underwent five copulatory tests, when put in the presence of an inaccessible sexually receptive female rat and when copulation was allowed. Markers of neurotropism (BDNF, trkB), neural activation (Δ-FosB), functional (Arc and PSA-NCAM) and structural synaptic plasticity (synaptophysin, syntaxin-3, PSD-95) were also measured in the ventral tegmental area (VTA), Acb (shell and core) and medial prefrontal cortex (mPFC) by Western Blot assays. The results indicate that the sexual behavior of DAT KO vs. HET and WT rats shows peculiar differences, mainly due to a more rapid acquisition of stable sexual activity levels and to higher levels of sexual motivation and activity. These differences occurred with differential changes in dopamine and glutamic acid concentrations in Acb dialysates during sexual behavior, with lower increases of dopamine and glutamic acid in DAT KO vs. WT and HET rats, and a lower expression of the markers investigated, mainly in the mPFC, in DAT KO vs. WT rats. Together these findings confirm a key role of dopamine in sexual behavior and provide evidence that the permanently high levels of dopamine triggered by DAT gene silencing cause alterations in both the frontocortical glutamatergic neurons projecting to the Acb and VTA and in the mesolimbic dopaminergic neurons, leading to specific brain regional changes in trophic support and neuroplastic processes, which may have a role in the sexual behavior differences found among the three rat genotypes.

Keywords: Arc; BDNF/trkB; D-FosB; DAT knockout rats; dopamine; glutamic acid; sexual behavior; synaptic proteins.

Figure 1

Schematic representation of a coronal section of the rat brain showing the track of the microdialysis probe in the Acb shell (Paxinos and Watson, 2004). Insert: the square bracket in the micro-photograph indicates the portion of the Neutral Red-stained section showing the active part of the dialyzing membrane of the microdialysis probe into the Acb shell. Abbreviations: CPu, caudate-putamen; ac, anterior commissure; AcbS, nucleus accumbens shell; AcbC, nucleus accumbens core.

Figure 2

Percentage of male WT, HET and DAT KO rats (8–10 per group) engaged in sexual behavior in copulatory tests 1–5. During each test, male rats were put together with a sexually receptive female rat for 60 min as described in the “Materials and Methods” section. The percent of rats showing mounts, intromissions and that reached ejaculation is reported (Chi-square test, not significant).

Figure 3

Sexual behavior of male WT, HET and DAT KO rats in the first series of the copulatory test 1, 2, 3, 4 and 5. Male rats were put together with a sexually receptive female rat and observed in order to measure copulatory parameters as described in the “Materials and Methods” section. Copulatory parameters were measured directly or calculated as described in the “Material andMethods” section. Values are means ± SEM of 8/10 rats per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 with respect to WT; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001, DAT KO with respect to HET; ^§P < 0.05, ^§§P < 0.01, ^§§§§P < 0.0001, with respect to T1 (two-way ANOVA for repeated measures followed by Tukey’s or Bonferroni’s pairwise comparisons).

Figure 4

(A) Extracellular dopamine and (B) glutamic acid concentrations in the Acb shell dialysates obtained from WT, HET and DAT KO rats during sexual activity. Rats from each line, which underwent five copulatory tests with a sexually receptive female rat in the 3 weeks preceding the experiment, stereotaxically implanted with a microdialysis probe aimed at the Acb shell, were placed individually into the mating cage and perfused with the dialysis buffer as described in the “Materials and Methods” section. An inaccessible receptive female rat was then placed inside the small cage of the mating cage (time = 0). After 30 min, copulation was allowed by removing the small cage for 60 min, after which the female rat was removed from the mating cage. During the experiment, NCPEs were counted and copulatory parameters measured, and dialysate aliquots collected every 15 min and analyzed for dopamine and glutamic acid as described in the “Materials and Methods” section. Values are means ± SEM of the values obtained by 8/10 rats per group. ^§P < 0.05, ^§§P < 0.01, with respect to basal values (no female rat); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 with respect to WT; ^#P < 0.05, ^##P < 0.01, DAT KO with respect to HET rats (two-way ANOVA for repeated measures followed by Tukey’s or Bonferroni’s pairwise comparisons).

Figure 5

Differences in the number of NCPEs, mounts, intromissions, and ejaculations recorded from WT, HET and DAT KO rats during the microdialysis experiments reported in Figure 4. The experimental conditions were identical to those described in the legend of Figure 4. Values are means ± SEM of the values obtained by 8/10 rats per group. *P < 0.05, **P < 0.01, ****P < 0.0001 with respect to WT; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001, DAT KO with respect to HET rats (two-way ANOVA for repeated measures followed by Tukey’s or Bonferroni’s pairwise comparisons).

Figure 6

(A) Percent of mounts, intromissions, and ejaculations followed by genital self-grooming; (B) frequency (i.e., number of episodes of genital self-grooming after mounts, intromissions and ejaculations) and (C) duration (s) of genital self-grooming episodes in WT, HET and DAT KO rats after mounts, intromissions and ejaculations in the first series of sexual activity during the microdialysis experiment reported in Figure 4. The experimental conditions were identical to those described in the legend of Figure 4. Values are means ± SEM of the values obtained by 8/10 rats per group. *P < 0.05, **P < 0.01, ****P < 0.0001 vs. WT rats; ^#P < 0.05, ^###P < 0.001, ^####P < 0.0001, DAT KO with respect to HET rats (A: Chi-square test; B,C: one-way ANOVA followed by Tukey’s pairwise comparisons). M, mounts; I, intromissions; E, ejaculations.

Figure 7

Western Blot analysis of trkB, BDNF, Δ-FosB, Arc, synaptophysin, syntaxin-3, PSD-95 and PSA-NCAM in the mPFC and VTA of WT, HET and DAT KO rats after the microdialysis experiment. Histograms on the left are the densitometric analyses of the marker/GAPDH band gray optical density (O.D.) ratios. Blot lines on the right are representative samples of two animals from each experimental group. Values are means ± SEM of the values obtained by the reported number of rats per group. *P < 0.05, **P < 0.01 with respect to WT rats; ^#P < 0.05 DAT KO with respect to HET rats (one-way ANOVA followed by Tukey’s pairwise comparisons).

Figure 8

Western Blot analysis of trkB, BDNF, Δ-FosB, Arc, synaptophysin, syntaxin-3, PSD-95 and PSA-NCAM in the Acb shell and Acb core of WT, HET and DAT KO rats after the microdialysis experiment. Histograms on the left are the densitometric analyses of the marker/GAPDH band gray optical density (O.D.) ratios. Blot lines on the right are representative samples of two animals from each experimental group. Values are means ± SEM of the values obtained by the reported number of rats per group. *P < 0.05 with respect to WT rats (one-way ANOVA followed by Tukey’s pairwise comparisons).