Androgen decreases dopamine neurone survival in rat midbrain.

Clinical studies show that men are more likely to develop disorders affecting midbrain dopaminergic pathways, such as drug addiction and Parkinson's disease (PD). Although a great deal of focus has been given to the role of oestrogen in the maintenance of midbrain dopaminergic pathways, little is known about how testosterone influences these pathways. In the present study, we used stereological analysis of tyrosine hydroxylase-immunoreactive (TH-IR) cell bodies to determine how testosterone influences the dopaminergic cell bodies of the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA). Rats and mice were castrated at postnatal day (PN) 60, and these midbrain cell populations were counted on PN 90. One month after castration, TH-IR cell number had increased in the SNpc and VTA of rats and mice. Replacement with testosterone or the non-aromatisable analogue dihydrotestosterone (DHT) in castrated animals reduced TH-IR cell number in the SNpc and VTA in rats. In mice, the decrease of TH-IR cell number with testosterone or DHT replacement was observed only in the SNpc. The apparent increase in TH-IR neurone number after castration is not explained by an increase in TH expression because the number of nondopaminergic cells (TH-immunonegative, TH-IN) did not decrease proportionally after castration. TH-IN cell number did not change after castration or hormone replacement in rat or mouse SNpc or VTA. These findings suggest that testosterone may play a suppressive role in midbrain dopaminergic pathways.

Oestrogen receptors enhance dopamine neurone survival in rat midbrain.

Previous findings in our laboratory and elsewhere have shown that ovariectomy of rats in adulthood attenuates cocaine-stimulated locomotor behaviour. Ovarian hormones enhance both cocaine-stimulated behaviour and increase dopamine overflow after psychomotor stimulants. The present study aimed to determine whether ovarian hormones have these effects in part by maintaining dopamine neurone number in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) and to investigate the roles of specific oestrogen receptors (ERs) in the maintenance of mesencephalic dopamine neurones. To accomplish this goal, we used unbiased stereological techniques to estimate the number of tyrosine hydroxylase-immunoreactive (TH-IR) cell bodies in midbrain regions of intact, ovariectomised and hormone-replaced female rats and mice. Animals received active or sham gonadectomy on postnatal day 60 and received vehicle, 17beta-oestradiol (E(2)) or selective ER agonists propyl-pyrazole-triol (PPT, ERalpha) or diarylpropionitrile (DPN, ERbeta) for 1 month post-surgery. In both rats and mice, ovariectomy reduced the number of TH-IR cells in the SNpc and VTA. Replacement with E(2), PPT or DPN prevented or attenuated the loss observed with ovariectomy in both rats and mice. An additional study using ER knockout mice revealed that adult female mice lacking ERalpha had fewer TH-IR cells in midbrain regions than wild-type mice, whereas mice lacking ERbeta had TH-IR cell counts comparable to wild-type. These findings suggest that, although both ER subtypes play a role in the maintenance of TH-IR cell number in the SNpc and VTA, ERalpha may play a more significant role.

Sex, steroids, and stimulant sensitivity.

The current study investigated ovarian modulation of the locomotor response to cocaine in rats. Ovariectomy in females lowered the response to cocaine (10 mg/kg i.p.), whereas castration did not change that of males. The locomotor responses of prepubertal males and females to cocaine were similar. However, the postpubertal sex difference resulted from a fall in cocaine-stimulated locomotion in males rather than a rise in females. Neonatal testosterone treatment of female rat pups decreased the response to cocaine in adulthood. These findings suggest that both the activational and organizational effects of gonadal steroids contribute to the greater response of females to cocaine.

Sex differences in cocaine-stimulated motor behavior: disparate effects of gonadectomy.

Sex differences in biological substrates of drug use and addiction are poorly understood. The present study investigated sexual dimorphisms in motor behavior following acute cocaine administration (10, 20, or 40 mg/kg, i.p.). Cocaine increased stereotypy rating, horizontal and vertical activity in both sexes, and effects were always greater in females than males. A population analysis using data from multiple experiments indicated that horizontal activity scores were normally distributed in males but not in females. Gonadectomy induced disparate effects on cocaine-stimulated motor behavior. Population analysis indicated that castrated males exhibited more horizontal activity and stereotypy than shams. Ovariectomy did not affect cocaine-stimulated stereotypy but did attenuate horizontal activity in a subset of rats that had not been vaginally lavaged. In summary, gonadectomy effects were sex and behavioral topography specific and indicate that activational effects of gonadal hormones partially mediate the robust sex differences in cocaine-stimulated open-field behavior.

Partial, graded losses of dopamine terminals in the rat caudate-putamen: an animal model for the study of compensatory adaptation in preclinical parkinsonism.

Procedures to lesion dopamine (DA) neurons innervating the rat caudate-putamen (CP) in a partial, graded fashion are described in this study. The goal is to provide a lesion model that supports intra-animal comparisons of voltammetric recordings used to investigate compensatory adaptation of DA neurotransmission. Lesions exploited the topography of mesostriatal DA neurons, microinjections of the neurotoxin 6-hydroxydopamine (6-OHDA) into the medial and lateral edges of the ventral mesencephalon containing DA cell bodies and microdissection of the CP into six regions. Analysis of tissue DA content in these regions by HPLC-EC demonstrated that 6-OHDA injected into the lateral substantia nigra results in a significantly greater loss of DA in lateral versus medial regions of the CP. The direction of the graded loss of DA was reversed (i.e. a medial to lateral lesion gradient) by the injection of 6-OHDA into the ventral tegmental area near the medial SN. Extracellular concentrations of electrically evoked DA could be measured across the mediolateral axis of the CP in a single animal using the technique of in vivo voltammetry. More importantly, graded decreases in the amplitude of evoked DA levels generally followed the direction of the tissue DA gradient in lesioned animals. These results suggest that the graded loss of DA terminals in the CP, coupled to a spatially and temporally resolved technique for monitoring extracellular DA, is a viable tool for investigating compensatory adaptation in the mesostriatal DA system.

Effect of ovarian hormones and estrous cycle on stimulation of the hypothalamo-pituitary-adrenal axis by cocaine.

Cocaine is known to exert sexually dimorphic HPA axis effects in rats and to disrupt estrous cyclicity and/or fertility in rats, nonhuman primates, and humans. The present studies investigated the reciprocal interactions between ovarian hormones and HPA axis responses to cocaine. Thirty minutes after injection, cocaine (15 mg/kg i.p.) increased serum ACTH and corticosterone more in cycling than ovariectomized females or male rats. ACTH and corticosterone were highest in proestrus when estradiol was elevated. Cocaine did not alter serum estradiol in females or testosterone in males but did stimulate progesterone secretion in both sexes. Cocaine-stimulated progesterone secretion was significantly greater in females than in males or ovariectomized females, and greater in proestrous than diestrous 1 rats. Cocaine stimulated corticosterone and progesterone secretion in sham-adrenalectomized, but not adrenalectomized rats, indicating that the adrenal gland and not the ovary is the source of cocaine-stimulated progesterone. Estrogen influenced cocaine-stimulated progesterone secretion more than corticosterone, suggesting different release mechanisms for the two steroids in the adrenal. These results suggest that adrenally derived progesterone could contribute to cocaine-induced physiological changes, including inhibited gonadotropin release.

Dopamine release and uptake are greater in female than male rat striatum as measured by fast cyclic voltammetry.

The present studies investigated sexual dimorphisms in dopamine release and uptake using fast-scan cyclic voltammetry in anesthetized rats and in brain slices. Electrical stimulation of the medial forebrain bundle of anesthetized rats at high frequency (60 Hz) elicited significantly more extracellular dopamine in the caudate nucleus of females than males. This sex difference was apparent over a range of current intensities applied to the stimulating electrode. Local electrical stimulation of brain slices in vitro verified in vivo results as more extracellular dopamine was elicited by single and 10 pulse stimulations in the caudate nucleus of females. Kinetic analysis of in vivo and in vitro dopamine overflow data indicated that dopamine release (the concentration of dopamine released per stimulus pulse) and the maximal velocity of dopamine uptake are greater in female rats, but the affinity of the transporter for dopamine was the same in males and females. None of these three parameters varied across the female estrous cycle. Linear regression analysis of dopamine release versus maximal uptake velocity data indicated a significant association of release and uptake sites in each sex and regression lines for males and females virtually overlapped. One explanation for these results is greater dopamine neuron terminal density in female caudate nucleus. These sexual dimorphisms in dopaminergic neurotransmission provide a novel, plausible mechanism to explain robust sex differences in behavioral responses of rats to psychostimulant drugs and may have implications for human neurological disorders and drug abuse.

Effects of sex and gonadectomy on cocaine metabolism in the rat.

The purpose of the current study is to determine whether sex differences in metabolism of cocaine (COC) exist that could contribute to the greater behavioral sensitivity of females to COC administration. To investigate this question, concentrations of COC and its two principle metabolites benzoylecgonine (BE) and ecgonine methyl ester (EME) were measured by gas chromatography/mass spectroscopy in brain and plasma collected from male and female rats that were sacrificed between 5 and 90 min after injection COC (15 mg/kg i.p.). COC concentrations did not differ in plasma or brain tissue of males and females, but sex-specific patterns of metabolite distribution were detected. BE was 2-fold higher in plasma and brain of males than females, whereas EME was much higher in brain and plasma of females. The influence of gonadal hormones on COC metabolite patterns were determined using gonadectomized and prepubertal rats. Castration of male or female rats did not alter brain or plasma COC, but did decrease BE concentrations. Seven-day-old pups injected with 15 mg/kg of COC had higher blood and brain COC than adults and relatively low levels of metabolites. No sex differences were found for COC, BE, or EME in brain or plasma of pups. These findings indicate that although gonadal steroids influence COC metabolism, these effects do not explain sex differences in COC-induced behaviors.

Dissociation of dopamine release in the nucleus accumbens from intracranial self-stimulation.

Mesolimbic dopamine-releasing neurons appear to be important in the brain reward system. One behavioural paradigm that supports this hypothesis is intracranial self-stimulation (ICS), during which animals repeatedly press a lever to stimulate their own dopamine-releasing neurons electrically. Here we study dopamine release from dopamine terminals in the nucleus accumbens core and shell in the brain by using rapid-responding voltammetric microsensors during electrical stimulation of dopamine cell bodies in the ventral tegmental area/substantia nigra brain regions. In rats in which stimulating electrode placement failed to elicit dopamine release in the nucleus accumbens, ICS behaviour was not learned. In contrast, ICS was acquired when stimulus trains evoked extracellular dopamine in either the core or the shell of the nucleus accumbens. In animals that could learn ICS, experimenter-delivered stimulation always elicited dopamine release. In contrast, extracellular dopamine was rarely observed during ICS itself. Thus, although activation of mesolimbic dopamine-releasing neurons seems to be a necessary condition for ICS, evoked dopamine release is actually diminished during ICS. Dopamine may therefore be a neural substrate for novelty or reward expectation rather than reward itself.

Dopamine release and uptake rates both decrease in the partially denervated striatum in proportion to the loss of dopamine terminals.

The present study tested the hypothesis that normal concentrations of extracellular dopamine are preserved in the partially denervated striatum without active compensatory changes in dopamine uptake or release. One to four weeks after adult rats were unilaterally lesioned with 6-hydroxydopamine, fast-scan cyclic voltammetry at Nafion-coated, carbon-fiber microelectrodes was used to monitor extracellular dopamine levels in vivo, under urethane anesthesia. Simultaneous voltammetric recordings were collected in the lesioned and contralateral control striata. Extracellular dopamine was elicited by bilateral electrical stimulation of the medial forebrain bundle. A 20 Hz stimulation evoked similar concentrations of extracellular dopamine in both lesioned and control striata, although tissue dopamine was decreased 30-70% in lesioned striata, as determined subsequently by HPLC-EC. However, kinetic analysis of the voltammetric recordings revealed that the concentration of dopamine released per stimulus pulse and Vmax for dopamine uptake decreased in proportion to the magnitude of the lesion. These data support the hypothesis that normal extracellular dopamine levels can be generated in the partially lesioned striatum in the absence of active neuronal compensation. These results also suggest that passive mechanisms involved in the regulation of extracellular dopamine play an important role in maintaining function during the preclinical or presymptomatic phase of Parkinson's disease.

Microelectrodes for the measurement of catecholamines in biological systems.

Many of the molecules involved in biological signaling processes are easily oxidized and have been monitored by electrochemical methods. Temporal response, spatial considerations, and sensitivity of the electrodes must be optimized for the specific biological application. To monitor exocytosis from single cells in culture, constant potential amperometry offers the best temporal resolution, and a low-noise picoammeter improves the detection limits. Smaller electrodes, with 1-micron diameters, provided spatial resolution sufficient to identify the locations of release sites on the surface of single cells. For the study of neurotransmitter release in vivo, larger cylindrical microelectrodes are advantageous because the secreted molecules come from multiple terminals near the electrode, and the greater amounts lead to a larger signal that emerges from the Johnson noise of the current amplifier. With this approach, dopamine release elicited by two electrical stimulus pulses at 10 Hz was detected with fastscan cyclic voltammetry in vivo. Nafion-coated elliptical electrodes have previously been shown to be incapable of detecting such concentration changes without extensive signal averaging. In addition, we demonstrate that high-pass filtering (200 Hz) of cyclic voltammograms recorded at 300 V/s decreases the background current and digitization noise at these microelectrodes, leading to an improved signal. Also, high-pass filtering discriminated against ascorbic acid, DOPAC, and acidic pH changes, three common interferences in vivo.

Triadimefon and triadimenol: effects on monoamine uptake and release.

Acute administration of the agricultural fungicide triadimefon produced a neurotoxic syndrome in rats characterized by increased motor activity, stereotyped behaviors, and altered monoamine metabolism. Triadimenol, a metabolite of triadimefon in mammals, plants, and soil, also increased motor activity in rodents. To test the hypothesis that triadimefon and triadimenol are indirect-acting dopamine agonists, the present studies examined their abilities to inhibit monoamine uptake, bind to the dopamine transporter, and stimulate dopamine efflux in rat brain tissue, in vitro. Both triazoles inhibited the uptake of dopamine in striatal synaptosomal preparations. Triadimefon was 100-fold less potent than GBR12909, a prototypical inhibitor of dopamine uptake (IC50 = 4.7 microM vs. 37.2 nM, respectively), and triadimenol was about three-fold less potent than triadimefon. Triadimefon also weakly inhibited the uptake of norepinephrine in cortical synaptosomes (IC50 = 22.4 microM), but neither compound blocked the uptake of serotonin in cortical synaptosomes (IC50s > 100 microM). Triadimefon and triadimenol had similar affinity for [3H]mazindol binding sites on the dopamine transporter (IC50s approximately 1-1.5 microM, only two- to threefold greater than GBR12909). Neither triadimefon nor triadimenol (0.01-100 microM) increased basal efflux of [3H]DA that had been preloaded into striatal minces in vitro. An unexpected result was that GBR12909 (10 microM) increased basal efflux of [3H]DA by 71%, suggesting that this compound has DA releasing properties. These data suggest that increased synaptic concentrations of dopamine due to inhibition of dopamine uptake may play an important role in the neurobehavioral effects of triadimefon and triadimenol.

Overoxidized polypyrrole-coated carbon fiber microelectrodes for dopamine measurements with fast-scan cyclic voltammetry.

Thin films of overoxidized polypyrrole have been electro-chemically coated onto carbon fiber microelectrodes and used for dopamine measurements with background-substracted, fast-scan cyclic voltammetry at a scan rate of 300 V/s. The films were stable on the electrode surface only when the electrodes were scanned to high potentials (1400 mV vs SSCE) in pH 7.4 aqueous buffer. Dopamine sensitivity and ascorbate and dihydroxyphenylacetic acid (DOPAC) rejection at the overoxidized polypyrrole-coated electrode were compared to those at carbon fiber electrodes coated with Nafion, a perfluorinated ion-exchange material. At 300 V/s, the overoxidized polypyrrole-coated electrode was almost 3 times more sensitive to dopamine than an uncoated disk electrode. Furthermore, the films were as effective as Nafion in the attenuation of the response to ascorbate and DOPAC, common interferences of dopamine in vivo. Overoxidized polypyrrole-coated electrodes maintained a stable response to dopamine for several hours when implanted in the rat brain. The electrochemical deposition procedure was effective at both elliptical and cylindrical electrodes. This is in contrast to the dip-coating procedures employed with Nafion films that lead to nonuniform coatings at cylindrical electrodes.

Interhemispheric modulation of dopamine receptor interactions in unilateral 6-OHDA rodent model.

A critical assumption in the unilateral 6-hydroxydopamine (6-OHDA) model is that interactions between the intact and denervated hemispheres do not influence the response to insult. The present study examined this issue by assessing the effects of unilateral substantia nigra 6-OHDA lesions in rats that previously had received corpus callosum transections, a treatment designed to minimize interhemispheric influences. Quantitative autoradiography in the caudate-putamen ipsilateral to the lesion revealed that corpus callosum transection did not alter the increase in D2-like receptors ([125I]-epidepride-labeled sites) that is induced by unilateral 6-OHDA lesion. There were no effects of either 6-OHDA lesion or transection on D1 receptor density ([125I]-SCH23982 autoradiography). As a functional endpoint, dopamine-stimulated cAMP efflux was measured in superfused striatal slices. In this paradigm, the net effect of dopamine (DA) represents a combination of D1 receptor-mediated stimulation and D2 receptor-mediated inhibition. 6-OHDA lesion increased cAMP efflux induced by exposure to 100 microM DA alone; corpus callosum transection did not alter this effect. An interaction between 6-OHDA lesion and transection status was revealed, however, by comparison of results obtained with DA alone vs. DA plus the D2 antagonist sulpiride (to block the D2 inhibitory effects of 100 microM DA). This comparison revealed two important effects of 6-OHDA lesion in rats with an intact corpus callosum: 1) a moderate decrease in dopamine D1 receptor-mediated stimulation; and 2) a dramatic decrease in the ability of D2 receptors to inhibit this stimulation. Corpus callosum transection prevented these effects of 6-OHDA. These results provide a biochemical demonstration of D1:D2 receptor uncoupling in unilateral 6-OHDA lesioned rats, and suggest that interhemispheric influences (e.g., contralateral cortico-striatal glutamatergic projections) may contribute to lesion-induced alterations in D1:D2 receptor interactions.

Triadimefon, a triazole fungicide, induces stereotyped behavior and alters monoamine metabolism in rats.

Triadimefon, a triazole fungicide, has been observed to increase locomotion and induce stereotyped behavior in rodents. The present experiments designed to characterize the stereotyped behavior induced by triadimefon used a computer-supported observational method, and tested the hypothesis that these observed effects involved central dopaminergic systems. Adult male and female Sprague-Dawley rats were injected with triadimefon (0, 50, 100, and 200 mg/kg) in corn oil (2 ml/kg ip) 4 hr prior to behavioral assessment. The two lowest doses of triadimefon increased the frequency of locomotion and rearing, while the highest dose induced highly stereotyped behaviors, including backward locomotion, circling, and head weaving. Immediately after behavioral testing, the rats were sacrificed, and the striata and olfactory tubercles, terminal fields of the nigrostriatal and mesolimbic dopamine systems, respectively, were removed. Steady-state concentrations of the monoamines dopamine and serotonin and their metabolites were determined by HPLC-EC. In independent experiments, the direct effects of triadimefon on dopamine (D1 and D2) receptor binding and dopamine-sensitive adenylate cyclase activity were assessed in vitro using rat striata. Dopamine concentrations were increased in olfactory tubercles, but decreased in striatum. Concentrations of 5-hydroxyindoleacetic acid (the major metabolite of serotonin) were increased only in striatum, and only in animals treated with 200 mg/kg triadimefon. In vitro, triadimefon neither competed with D1 or D2 dopaminergic radioligands nor affected dopamine-stimulated adenylate cyclase activity. Together these behavioral and biochemical data lend support to the hypothesis that triadimefon may have actions similar to those produced by indirect-acting dopamine agonists.