Male and female Sprague-Dawley rats were obtained from Charles River Laboratories at 15 days of age and their brains were removed for autoradiographic analysis at 25, 35, 40, 60, 80, 100, and 120 days of age. Females were sacrificed on the second day of diestrous (determined by a vaginal smear) ±2 days of the same ages. An average of six subjects were studied at each age. Preliminary studies utilized subjects gonadectomized at 28 days of age (n=4). Subjects were treated in accordance with all relevant institutional, state and federal guidelines.

Sections were obtained and treated as previously described (Andersen et al., 1997). Briefly, frozen brains were sectioned coronally in 10 µm slices at -15^oC, thaw-mounted on gelatin-coated slides, and stored at -70^oC. Slides were thawed to room temperature the day of processing 5 min before incubation in D₁ or D₂ buffers. Saturation analyses were obtained using seven concentrations of each ligand. D₁ receptors were determined using ³H-SCH-23390 (0.05-5 nM; 72.8 Ci/mmol) with 2 µM cis-flupenthixol as the blank. D₂ receptor binding was determined with ³H-nemonapride (0.05-3.0 nM; 81.4 Ci/mmol) and 2 µM haloperidol. Specific binding at various concentrations of free ligand were analyzed with LIGAND (Munson and Robard, 1980) to derive estimates of B_max and K_d, using non-linear regression to a single-site model for each subject. Protein concentrations were determined with the Commassie Blue method in each region across age. Final values for B_maxwere expressed as fmol/mg protein.

As illustrated in Figure 1, males and females differed markedly in their pattern of dopamine receptor development in striatum (Age x Gender Interaction D₁: F6,48 = 11.0, p< 0.0001; D₂: F6,42 = 5.60, p< 0.0001). Male D₁ and D₂ receptor density had a much more prominent rise than in females between 25 days of age and the onset of puberty at 40 days. For example, ₂ B_max increased 144±26% in males vs 31±7% in females. The decline in male receptor density was also more marked between 40 days and adult levels (120 days). D₁ B_max decreased 34±4% in males, but by only 7±8% in females. In short, male rats had more extensive over production and elimination in striatum, though adult values were nearly identical by 120 days. K_d values did not differ between males and females or across age in the striatum.

Fig. 1: Mean (±S.E.) B_max values (expressed as fmol/mg protein) for density of D₁ and D₂ receptors in male and female rats between 25 and 120 days of age in the striatum and nucleus accumbens based on receptor autoradiography.

We also observed differences in the laterality of D₁ and D₂ receptors in male striatum, but not in female striatum. D₁ receptor density was approximately 20-30% higher in the left striatum than the right until 100 days of age, when laterality reversed with right becoming 20% higher. A similar phenomenon was observed for D₂ receptors in male striatum, however, receptor asymmetries were no longer observed by 60 days. In contrast, females D₁ and D₂ receptor density did not differ between right and left hemisphere at all ages.

There were enduring gender differences in D₁ receptor density in nucleus accumbens (F1,44 = 109.1, p< 0.0001). Despite comparable densities at 25 days of age, D₁ B_max rose faster and higher in males than females (Age X Gender interaction: F6,44 = 3.31, p< 0.01). The male and female density curves were parallel after 40 days of age, with each demonstrating a slight dip at 80 days. At 120 days D₁ B_max was 57.8± 21.2% greater in males than females.

Overall, there was no gender difference in D₂ density in the nucleus accumbens (F1,42 = 0.13, p > 0.7). Males had a greater increase from 25 to 40 days (147±24%) than females (18±9%), but both groups showed no significant variations from 40 to 120 days. Again, no sex or age differences in K_d were observed in nucleus accumbens.

Gonadectomy at 28 days of age, as pubertal hormones are beginning to rise and exert their activational effects, did not significantly effect D₁ and D₂ receptor density in the striatum of male or female rats (see Table 1). Data are expressed as a percentage of intact control for both castrated (Cx) males and ovariectomized (Ovx) females.

TABLE 1.
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        Cx Males*     Ovx Females*
D1     83.3 ± 15.0     92.9 ± 23.2
D2     76.3 ± 10.0#    86.2 ± 8.5 
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* data are expressed as a % of control values at P40
# p=0.08

The observation of more elaborate over production and elimination of D₁ and D₂ family receptors in striatum of males than females across periadolescence has important implications for our understanding of ADHD. We hypothesize that the extensive over production of dopamine receptors in striatum and accumbens during prepubertal development may help explain why males are more often afflicted by ADHD as dopaminergic increases in these regions can produce hyperactivity and stereotypies (Niemegeers and Janssen, 1974; Giros et al., 1996). The extensive pruning of dopamine receptors in striatum after puberty is consistent with the observation that ADHD often recedes or diminishes in severity in males. The possibility that either failure of striatal dopamine receptor density to recede in females, or an aberrant overproduction in the females, may explain why ADHD symptoms are more likely to persist after puberty in afflicted females (Ernst et al., 1994). Data from Ernst et al (1994) suggests that ADHD females may actually effected more severely than ADHD males. It is also possible that the enduring differences in D1 density in nucleus accumbens may have some relation to the greater incidence of substance abuse in males given the putative role of the accumbens D₁ system in addictive behaviors (Maldonado et al., 1993).

These preclinical data are also consistent with clinical (human autopsy specimens) that demonstrated marked over production and elimination of D₁ and D₂ receptors in striatal dopamine receptor density during childhood and adolescence (Seeman et al. 1987). Unfortunately, they did not examine gender differences in that study. However, gender differences in periadolescent D₁ and D₂ receptor changes may be consonant with recent observations with magnetic resonance imaging that shows that the human striatum shrinks during adolescence in boys but not in girls (Giedd et al. 1997). Overall, the present results provide further support for sex differences in brain structure and suggest that the developmental processes of over production and competitive elimination may be involved in the emergence of some of these differences.

The mechanism underlying these sex difference in the overproduction and elimination of dopamine receptor density during periadolescence is not straight forward. The most likely explanation underlying gender differences in dopamine receptors is due to pubertal changes in gonadal hormones, where they take on an activational role. However, as Table 1 illustrates, gonadectomy at postnatal day 28 (as hormone levels are beginning to surge) did not have a dramatic effect on this process. Two hypotheses were discounted: 1) either testosterone stimulates the overproduction in males, which is not the case, as castration did not markedly suppress the receptor density at day 40; or 2) estrogen inhibits the overproduction in females — also not true as no disinhibition of periadolescent receptor overproduction was observed. Thus, the question as to what is the mechanism underlying these gender differences remains to be answered. The answer may lie in the original organizing effects of hormones during early brain development. The current direction of this line of research in our laboratory is to document the effects of chronic methylphenidate on periadolescent changes in dopamine receptor density in males and females.

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