T. Di Paolo

Sex and estrous cycle variations of rat striatal dopamine uptake sites.

The reuptake of dopamine into nerve terminals is the primary mechanism of inactivation of this neurotransmitter in the synaptic cleft. We report sex differences and estrous cycle variations of rat striatal dopamine uptake sites. During the estrous cycle, peak density of striatal dopamine uptake sites labelled with [3H]GBR-12935 occurred in the morning of proestrus in coincidence with peak dopamine, serotonin, dihydroxyphenylacetic acid and 5-hydroxytryptophan levels pointing to a presynaptic effect of gonadal hormones. Striatal homovanillic acid and 5-hydroxyindoleacetic acid levels as well as [3H]GBR-12935 binding affinity remained unchanged throughout the estrous cycle. The density of [3H]GBR-12935 striatal binding sites was lower in ovariectomized rats compared to intact female rats during the estrous cycle, whereas it was similar in gonadectomized male rats, intact male rats and ovariectomized rats. Binding affinity was in general similar for all the groups of rats examined. The affinity of dopamine for striatal [3H]GBR-12935 binding sites was similar in males and ovariectomized females, and did not change during the female estrous cycle. In summary, striatal dopamine uptake site density was lower in male compared to intact female rats and was shown to fluctuate during the female estrous cycle. These results suggest that gonadal hormones could influence the activity of psychoactive drugs acting on neuronal dopamine uptake sites.

Contribution of estrogen receptors alpha and beta to the effects of estradiol in the brain

Clinical and experimental studies show a modulatory role of estrogens in the brain and suggest their beneficial action in mental and neurodegenerative diseases. The estrogen receptors ERalpha and ERbeta are present in the brain and their targeting could bring selectivity and reduced risk of cancer. Implication of ERs in the effect of estradiol on dopamine, opiate and glutamate neurotransmission is reviewed. The ERalpha agonist, PPT, is shown as estradiol to modulate hippocampal NMDA receptors and AMPA receptors in cortex and striatum of ovariectomized rats whereas the ERbeta agonist DPN is inactive. Striatal DPN activity suggests implication of ERbeta in estradiol modulation of D2 receptors and transporters in ovariectomized rats and is supported by the lack of effect of estradiol in ERbeta knockout (ERKObeta) mice. Both ERalpha and ERbeta agonists modulate striatal preproenkephalin (PPE) gene expression in ovariectomized rats. In male mice PPT protects against MPTP toxicity to striatal dopamine; this implicates Akt/GSK3beta signaling and the apoptotic regulators Bcl2 and Bad. This suggests a role for ERalpha in striatal dopamine neuroprotection. ERKOalpha mice are more susceptible to MPTP toxicity and not protected by estradiol; differences in ERKObeta mice are subtler. These results suggest therapeutic potential for the brain of ER specific agonists.

Effect of Chronic Estradiol and Progesterone Treatments of Ovariectomized Rats on Brain Dopamine Uptake Sites

Abstract: Dopamine released from brain nerve terminals is mainly removed from the synaptic cleft by an uptake mechanism. Despite their functional importance, modulation of the dopamine uptake sites is still not well known. Steroid hormones were shown to modulate brain dopamine transmission. The aim of this study was thus to investigate in ovariectomized rats the effects of 17β‐estradiol and progesterone treatments on brain dopamine uptake sites. Treatments consisted of 17β‐estradiol (10 μg/0.2 ml), progesterone (0.72 mg/0.2 ml). 17β‐estradiol + progesterone, or the vehicle (0.3% gelatin in saline solution) twice daily for 2 weeks. The steroid treatments left the affinity of [3H]GBR 12935 binding to striatal homogenates unchanged (ovariectomized rats, 0.823 ± 0.028 nM), whereas the density was increased by these steroids alone or in combination to a similar extent of 16‐23%. Chronic treatment of ovariectomized rats with 17β‐estradiol progesterone, or their combination increased to the same extent and uniformly [3H]‐GBR 12935 binding in the striatum as measured by autoradiography; the increase was similar in the substantia nigra pars compacta, whereas no steroid effect was observed in the nucleus accumbens and in the substantia nigra pars reticulata. In summary, chronic exposure to 17β‐estradiol and/ or progesterone increased dopamine uptake site density in the nigrostriatal dopaminergic system, whereas the nucleus accumbens and the substantia nigra pars reticulata were unaffected.

Levodopa or D2 agonist induced dyskinesia in MPTP monkeys: correlation with changes in dopamine and GABAA, receptors in the striatopallidal complex

Dopamine D1 and D2 receptors as well as the GABA/benzodiazepine receptor complex in the striatum and the globus pallidus (internal: GPi and external: GPe) were studied by autoradiography using [3H]SCH 23390, [3H]spiperone, and [3H]flunitrazepam ([3H]FNZ) respectively, in five groups of cynomolgus monkeys. These included (i) untreated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-monkeys; (ii) MPTP monkeys treated chronically with levodopa injections; (iii) MPTP monkeys treated chronically with injections of the novel D2 agonist U91356A; (iv) MPTP monkeys treated chronically with U91356A delivered through an osmotic mini-pump; and (5) naive controls. Animals treated in a pulsatile mode with U91356A or levodopa injections showed progressive sensitization to their respective drug and developed choreic dyskinesia. In contrast, animals treated in a continuous mode with U91356A showed behavioral tolerance but did not develop dyskinesia. A trend for a down-regulation of putaminal D2 receptors was observed following D2 agonist stimulation with U913356A. Striatal [3H]FNZ binding was significantly decreased only in animals treated in a continuous mode with U91356A. The dopamine receptor decrease in the striatum could be implicated with the development of tolerance but cannot explain the appearance of dyskinesia. Denervation by MPTP was associated with a decrease of the GPe/GPi [3H]FNZ binding ratio which reflects an imbalance of striatal output pathways; this ratio was not reversed by any of the treatments although changes were observed in the GPe and GPi. Indeed, pulsatile U91356A treatment restored the decreased [3H]FNZ binding in the GPe near control values and levodopa showed a similar tendency. A significant increase of [3H]FNZ binding in the GPi only of dyskinetic monkeys, namely those treated with pulsatile U91356A or levodopa was seen compared to untreated MPTP or naive controls. This GABAA receptor up-regulation might lead to a supersensitive state of the GPi to gabaergic input which may be involved in the mechanism underlying the development of dopaminomimetic-induced dyskinesia.

Effects of chronic estrogen treatment on dopamine concentrations and turnover in discrete brain nuclei of ovariectomized rats

We investigated the effects of chronic estrogen treatment in ovariectomized rats on the concentration of dopamine in 33 discrete brain nuclei. In order to assess estrogens influence on dopamine turnover, some of the rats were administered alpha-methyl-p-tyrosine. Estrogen treatment reduced the concentrations of dopamine in the nucleus accumbens septi, striatum, median eminence, nucleus anterior hypothalami, nucleus suprachiasmaticus, nucleus arcuate IV-V, area ventralis tegmenti, interpeduncular nucleus and nucleus interstitialis striae terminalis. Treatment was without effect on dopamine turnover in all areas studied.

Gonadal hormones modulate 5-hydroxytryptamine2A receptors : Emphasis on the rat frontal cortex

The effect of short-term (Sprague-Dawley rats, two weeks) and long-term ovariectomy (Sprague-Dawley and Fischer rats, three months) on serotonin 5-hydroxytryptamine2A receptors in different regions of the brain and its possible correction with an 17 beta-estradiol treatment (10 micrograms, b.i.d., two weeks) were studied in comparison to intact rats. Saturation binding assays were performed using [3H]ketanserin to estimate 5-hydroxytryptamine2A receptor density and affinity in tissue homogenates of frontal cortex of Fischer rats and quantitative autoradiography was performed to evaluate receptor specific binding in frontoparietal cortex, nucleus accumbens, striatum and dorsal raphe nucleus of Fischer rats, and in frontal cortex of the two strains of rats. Messenger RNA levels of 5-hydroxytryptamine2A receptors were measured by in situ hybridization in frontal cortex of the two strains of rats. An overall decrease of 5-hydroxytryptamine2A receptor densities was found in all the brain regions of ovariectomized Fischer rats assayed, and this could be restored towards control levels by estradiol treatment. No change in the 5-hydroxytryptamine2A receptor affinity was measured in the frontal cortex. A similar pattern of changes was observed for the messenger RNA levels encoding the 5-hydroxytryptamine2A receptors and receptor density, suggesting the implication of a genomic mechanism. Experiments in Sprague-Dawley rats confirmed and extended the results obtained with Fischer rats. By analogy, in humans, this 5-hydroxytryptamine2A receptor modulation may underlie the mood and movement disorders associated with menopause.

Influence of oestrogenic compounds on monoamine transporters in rat striatum.

Oestrogens have been reported to modulate rat membrane (DAT) and vesicular (VMAT2) dopamine transporters. A recent pilot study of postmenopausal women showed that chronic oestrogen replacement therapy increases striatal DAT. In the present study, we first investigated whether the oestrogen receptors α and β mediate the effects of oestradiol on DAT and VMAT2. Two days after ovariectomy, Sprague‐Dawley rats were treated for 2 weeks with oestradiol or specific ligands for oestrogen receptor α, 4,4′,4′′‐(4‐propyl‐[1H]‐pyrazole‐1,3,5‐triyl)trisphenol (PPT) or oestrogen receptor β, 2,3‐bis(4‐hydroxyphenyl)‐propionitrile (DPN). Ovariectomy caused a decrease in [125I]‐3β‐(4‐iodophenyl)‐tropane‐2β‐carboxylic acid isopropyl ester ([125I] RTI‐121) specific binding to DAT transporters in the middle striatum compared to values for intact rats, and this was reversed by oestradiol replacement therapy. DPN, but not PPT, mimicked the effect of oestradiol. [125I] RTI‐121 specific binding in the anterior and posterior striatum was not affected by ovariectomy or any of the drug treatments. Second, we investigated whether oestradiol increased DAT specific binding after a longer period of hormonal withdrawal (a model of hormonal withdrawal at menopause) and whether the selective oestrogen receptor modulators (SERMs), tamoxifen and raloxifene, could reproduce the oestradiol‐induced increase of [125I] RTI‐121 specific binding in long‐term ovariectomised rats. Four months after ovariectomy, Sprague‐Dawley rats were treated for 2 weeks with oestradiol, tamoxifen or raloxifene, and then killed. Ovariectomy decreased [3H] RTI‐121 specific binding to DAT transporters in the middle striatum compared to values for intact rats. Treatment with oestradiol, tamoxifen and raloxifene reversed this effect. [125I] RTI‐121 specific binding in anterior and posterior striatum was not affected by ovariectomy or treatment with oestrogen receptor ligands. In both experiments, neither ovariectomy nor the oestrogenic treatments modulated striatal [3H] tetrahydrobenazine specific binding to VMAT2. Overall, these results suggest that oestrogen receptor β mediates the oestradiol‐induced increase of striatal DAT and that oestradiol can increase DAT density even after long‐term steroid withdrawal. The results also support the premise that the SERMs tamoxifen and raloxifene exert oestrogenic agonist effects in the brain.

Oestrogen Receptors and Signalling Pathways: Implications for Neuroprotective Effects of Sex Steroids in Parkinson's Disease

Parkinson’s disease (PD) is an age‐related neurodegenerative disorder with a higher incidence in the male population. In the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model of PD, 17β‐oestradiol but not androgens were shown to protect dopamine (DA) neurones. We report that oestrogen receptors (ER)α and β distinctly contribute to neuroprotection against MPTP toxicity, as revealed by examining the membrane DA transporter (DAT), the vesicular monoamine transporter 2 (VMAT2) and tyrosine hyroxylase in ER wild‐type (WT) and knockout (ERKO) C57Bl/6 male mice. Intact ERKOβ mice had lower levels of striatal DAT and VMAT2, whereas ERKOα mice were the most sensitive to MPTP toxicity compared to WT and ERKOβ mice and had the highest levels of plasma androgens. In both ERKO mice groups, treatment with 17β‐oestradiol did not provide neuroprotection against MPTP, despite elevated plasma 17β‐oestradiol levels. Next, the recently described membrane G protein‐coupled oestrogen receptor (GPER1) was examined in female Macaca fascicularis monkeys and mice. GPER1 levels were increased in the caudate nucleus and the putamen of MPTP‐monkeys and in the male mouse striatum lesioned with methamphetamine or MPTP. Moreover, neuroprotective mechanisms in response to oestrogens transmit via Akt/glycogen synthase kinase‐3 (GSK3) signalling. The intact and lesioned striata of 17β‐oestradiol treated monkeys, similar to that of mice, had increased levels of pAkt (Ser 473)/βIII‐tubulin, pGSK3 (Ser 9)/βIII‐tubulin and Akt/βIII‐tubulin. Hence, ERα, ERβ and GPER1 activation by oestrogens is imperative in the modulation of ER signalling and serves as a basis for evaluating nigrostriatal neuroprotection.

Oestrogens Prevent Loss of Dopamine Transporter (DAT) and Vesicular Monoamine Transporter (VMAT2) in Substantia Nigra of 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine Mice

Previous results from our laboratory have shown that 17β‐oestradiol prevents 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) striatal dopamine depletion. 17β‐oestradiol, oestriol and oestrone are the naturally occurring oestogens in humans. Using various dopamine markers, the present study investigated whether oestrone and oestriol such as 17β‐oestradiol have neuroprotective activity in MPTP‐treated mice. Male mice were treated with 17β‐oestradiol, oestriol or oestrone for 5 days before and after MPTP administration, and were compared with nonlesioned mice receiving the same treatment. Striatal concentrations of dopamine and its metabolites, 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were assayed by high‐performance liquid chromatography. Dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) specific binding were measured by autoradiography. DAT, VMAT2 and tyrosine hydroxylase mRNA levels were measured by in situ hybridisation. MPTP induced a loss of DAT and VMAT2 specific binding in the striatum and substantia nigra, as well as a decrease of VMAT2 mRNA in the substantia nigra. 17β‐oestradiol treatment prevented the loss of these dopaminergic markers, as well as striatal concentrations of dopamine, DOPAC and HVA. Mice receiving oestriol and oestrone showed catecholamine concentrations comparable to MPTP mice. Oestriol treatment had no effect on dopaminergic markers in MPTP mice whereas oestrone prevented striatal DAT loss and the decrease of VMAT2 mRNA in the substantia nigra. In nonlesioned mice, 17β‐oestradiol, oestriol or oestrone had no effect on all the dopaminergic markers investigated. In conclusion, a weak or a lack of effect of oestriol and oestrone was observed compared to 17β‐oestradiol in MPTP mice and none of these steroids had an effect in nonlesioned mice. A DAT and VMAT2 specific binding decrease after MPTP in the striatum and substantia nigra, as well as a decrease of substantia nigra VMAT2 mRNA, was observed and could be prevented by oestradiol.

Relation between brain dopamine loss and D2 dopamine receptor density in MPTP monkeys.

The relationship between dopamine (DA), dihydroxyphenylacetic acid (DOPAC) or homovanillic acid (HVA) concentrations and [3H]spiperone binding to D2 DA receptors in caudate nucleus, putamen and nucleus accumbens following DAergic lesion with MPTP in monkeys was investigated. The level of DA depletion varied from 37% to 100% while a mean elevation in [3H]spiperone binding density of 114.8 +/- 4.7% of control (P less than 0.01) was observed when DA depletion was at least 90% of control values. A logarithmic correlation (y = -7.19 In x +111.15; r = -0.54, P less than 0.01) between DA concentrations (x) and the density of [3H]spiperone binding sites (y) was observed in all brain regions. The correlation between DOPAC or HVA concentrations and [3H]spiperone binding was best represented by linear rather than logarithmic equations. These results indicate that supersensitivity of DA receptors develops after MPTP lesion in monkeys when the number of remaining nerve fibers becomes too few to compensate for the DA loss.