Quantitative diffusion tensor imaging detects dopaminergic neuronal degeneration in a murine model of Parkinson's disease.

Early diagnosis of Parkinson's disease (PD) is required to improve therapeutic responses. Indeed, a clinical diagnosis of resting tremor, rigidity, movement and postural deficiencies usually reflect >50% loss of the nigrostriatal system in disease. In a step to address this, quantitative diffusion tensor magnetic resonance imaging (DTI) was used to assess nigrostriatal degeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication model of dopaminergic nigral degeneration. We now demonstrate increased average diffusion (p<0.005) and decreased fractional anisotropy (p<0.03) in the substantia nigra (SN) of 5- to 7-day MPTP-treated animals when compared to saline controls. Transverse diffusivity demonstrated the most significant differences (p < or = 0.002) and correlated with the numbers of SN dopaminergic neurons (r=-0.75, p=0.012). No differences were found in the striatum, corpus callosum, cerebral cortex, or ventricles. These results demonstrate that DTI may be used as a surrogate biomarker of nigral dopaminergic neuronal degeneration.

ROLE OF DOPAMINE OF NUCLEUS ACCUMBENS IN BEHAVIORAL SENSITIZATION TO METHYLPHENIDATE

Behavioral sensitization is defined as the subsequent augmentation of the locomotor response to a drug following repeated administrations of the drug. It is believed to occur due to alterations in the motive circuit in the brain by stressors, central nervous system stimulants, and similar stimuli. The motive circuit (or mesocorticolimbic system) consists of several interconnected nuclei that determine the behavioral response to significant biological stimuli. A final target of the mesocorticolimbic system is the nucleus accumbens (NAc), which is a key structure linking motivation and action. In particular, the dopaminergic innervations of the Nac are considered to be essential in regulating motivated states of behavior such as goal-directed actions, stimulus-reward associations and reinforcement by addictive substances. Therefore, the objective of this study was to investigate the role of dopaminergic afferents of the NAc in the behavioral sensitization elicited by chronic treatment with methylphenidate (MPD), a psychostimulant that is widely used to treat attention deficit hyperactivity disorder. The dopaminergic afferents can be selectively destroyed using catecholamine neurotoxin 6-hydroxydopamine (6-OHDA). In order to determine whether destruction of dopaminergic afferents of the NAc prevents sensitization, I compared locomotor activity in rats that had received infusions of 6-hydroxydopamine (6-OHDA) into the NAc with that of control and sham-operated animals. All groups of rats received six days of single daily MPD injections after measuring their pre and post surgery locomotor baseline. Following the consecutive MPD injections, there was a washout period of 4 days, where no injections were given. Then, a rechallenge injection of MPD was given. Behavioral responses after repeated MPD were compared to those after acute MPD to assess behavioral sensitization. Expression of sensitization to MPD was not prevented by 6-OHDA infusion into the NAc. Moreover, two distinct responses were seen to the acute injection of MPD: one group of rats had essentially no response to acute MPD, while the other had an augmented (‘sensitized’-like) acute response. Among rats with 6-OHDA infusions, the animals with diminished acute response to MPD had intact behavioral sensitization to repeated MPD, while the animals with increased acute response to MPD did not exhibit further sensitization to it. This suggests that the acute and chronic effects of MPD have distinct underlying neural circuitries.

Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin-3.

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein-protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

Chronic exposure to MDMA (Ecstasy) elicits behavioral sensitization in rats but fails to induce cross-sensitization to other psychostimulants.

BACKGROUND: The recreational use of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) among adolescents and young adults has become increasingly prevalent in recent years. While evidence suggests that the long-term consequences of MDMA use include neurodegeneration to serotonergic and, possibly, dopaminergic pathways, little is known about susceptibility, such as behavioral sensitization, to MDMA. METHODS: The objectives of this study were to examine the dose-response characteristics of acute and chronic MDMA administration in rats and to determine whether MDMA elicits behavioral sensitization and whether it cross-sensitizes with amphetamine and methylphenidate. Adult male Sprague-Dawley rats were randomly divided into three MDMA dosage groups (2.5 mg/kg, 5.0 mg/kg, and 10.0 mg/kg) and a saline control group (N = 9/group). All three MDMA groups were treated for six consecutive days, followed by a 5-day washout, and subsequently re-challenged with their respective doses of MDMA (day 13). Rats were then given an additional 25-day washout period, and re-challenged (day 38) with similar MDMA doses as before followed by either 0.6 mg/kg amphetamine or 2.5 mg/kg methylphenidate on the next day (day 39). Open-field locomotor activity was recorded using a computerized automated activity monitoring system. RESULTS: Acute injection of 2.5 mg/kg MDMA showed no significant difference in locomotor activity from rats given saline (control group), while animals receiving acute 5.0 mg/kg or 10.0 mg/kg MDMA showed significant increases in locomotor activity. Rats treated chronically with 5.0 mg/kg and 10.0 mg/kg MDMA doses exhibited an augmented response, i.e., behavioral sensitization, on experimental day 13 in at least one locomotor index. On experimental day 38, all three MDMA groups demonstrated sensitization to MDMA in at least one locomotor index. Amphetamine and methylphenidate administration to MDMA-sensitized animals did not elicit any significant change in locomotor activity compared to control animals. CONCLUSION: MDMA sensitized to its own locomotor activating effects but did not elicit any cross-sensitization with amphetamine or methylphenidate.

Mechanisms for the release of dopamine from turtle retina

The retinal circuitry underlying the release of dopamine was examined in the turtle, Pseudemys scripta elegans, using neurochemical release studies, anatomical techniques, and biochemistry. There was a dose- and calcium-dependent release of dopamine from turtle retinas incubated in $\sp3$H-dopamine after perfusion of the GABA antagonist bicuculline. This indicated that dopamine release was tonically inhibited by GABA. Other putative retinal transmitters were examined. Glutamate antagonists selective for hyperpolarizing bipolar cells, such as 2,3-piperidine dicarboxylic acid (PDA), caused dose- and calcium-dependent release of dopamine from the retina. In contrast, release was not observed after perfusion with 4-aminophosphonobutyric acid, a specific antagonist of depolarizing bipolar cells. This indicated that depolarizing bipolar cells were not involved in retinal circuitry underlying the release of dopamine in the turtle retina. The release produced by PDA was blocked by bicuculline, indicating a polysynaptic mechanism of release. None of the other agents tested, which included carbachol, strychnine, dopamine uptake inhibitors, serotonin, tryptamine, muscimol, melatonin, or dopamine itself produced release.^ The cells capable of the release of dopamine were identified using both uptake autoradiography and immunocytochemical localization with dopamine antisera. The simplest circuitry based on these findings is signal transmission from photoreceptors to hyperpolarizing bipolar cells then to GABAergic cells, and finally to dopaminergic amacrine cells. ^

Cytochrome P450 2D18 in the mammalian brain: Recombinant expression, purification, and characterization of xenobiotic and endobiotic metabolism

The cytochromes P450 (P450) comprise a superfamily of hemoproteins that function in concert with NADPH-cytochrome P450 reductase (P450-reductase) to metabolize both endogenous and exogenous compounds. Many pharmacological agents undergo phase I metabolism by this P450 and P450-reductase monooxygenase system. Phase I metabolism ensures that these highly hydrophobic xenobiotics are made more hydrophilic, and hence easier to extrude from the body. While the majority of phase I metabolism occurs in the liver, metabolism in extrahepatic organ-systems like the intestine, kidney, and brain can have important roles in drug metabolism and/or efficacy. ^ While P450-mediated phase I metabolism has been well studied, investigators have only recently begun to elucidate what physiological roles P450 may have. One way to approach this question is to study P450s that are highly or specifically expressed in extrahepatic tissues. In this project I have studied the role of a recently cloned P450 family member, P450 2D18, that was previously shown to be expressed in the rat brain and kidney, but not in the liver. To this end, I have used the baculovirus expression system to over-express recombinant P450 2D18 and purified the functional enzyme using nickel and hydroxylapatite chromatography. SDS-PAGE analysis indicated that the enzyme was purified to electrophoretic homogeneity and Western analysis showed cross-reactivity with rabbit anti-human P450 2D6. Carbon monoxide difference spectra indicated that the purified protein contained no denatured P450 enzyme; this allowed for further characterization of the substrates and metabolites formed by P450 2D18-mediated metabolism. ^ Because P450 2D18 is expressed in brain, we characterized the activity toward several psychoactive drugs including the antidepressants imipramine and desipramine, and the anti-psychotic drugs chlorpromazine and haloperidol. P450 2D18 preferentially catalyzed the N-demethylation of imipramine, desipramine, and chlorpromazine. This is interesting given the fact that other P450 isoforms form multiple metabolites from such compounds. This limited metabolic profile might suggest that P450 2D18 has some unique function, or perhaps a role in endobiotic metabolism. ^ Further analysis of possible endogenous substrates for P450 2D18 led to the identification of dopamine and arachidonic acid as substrates. It was shown that P450 2D18 catalyzes the oxidation of dopamine to aminochrome, and that the enzyme binds dopamine with an apparent KS value of 678 μM, a value well within reported dopamine concentration in brain dopaminergic systems. Further, it was shown that P450 2D18 binds arachidonic acid with an apparent KS value of 148 μM, and catalyzes both the ω-hydroxylation and epoxygenation of arachidonic acid to metabolites that have been shown to have vasoactive properties in brain, kidney, and heart tissues. These data provide clues for endogenous roles of P450 within the brain, and possible involvement in the pathogenesis of Parkinson's disease. ^

THE EFFECT OF GABAMIMETICS ON NEUROTRANSMITTER RECEPTOR BINDING AND FUNCTION IN RAT BRAIN

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and alterations in central GABAergic transmission may contribute to the symptoms of a number of neurological and psychiatric disorders. Because of this relationship, numerous laboratories are attempting to develop agents which will selectively enhance GABA neurotransmission in brain. Due to these efforts, several promising compounds have recently been discovered. Should these drugs prove to be clinically effective, they will be used to treat chronic neuropsychiatric disabilities and, therefore, will be administered for long periods of time. Accordingly, the present investigation was undertaken to determine the neurochemical consequences of chronic activation of brain GABA systems in order to better define the therapeutic potential and possible side-effect liability of GABAmimetic compounds.^ Chronic (15 day) administration to rats of low doses of amino-oxyacetic acid (AOAA, 10 mg/kg, once daily), isonicotinic acid hydrazide (20 mg/kg, b.i.d.), two non-specific inhibitors of GABA-T, the enzyme which catabolizes GABA in brain, or (gamma)-acetylenic GABA (10 mg/kg, b.i.d.) a catalytic inhibitor of this enzyme, resulted in a significant elevation of brain and CSF GABA content throughout the course of treatment. In addition, chronic administration of these drugs, as well as the direct acting GABA receptor agonists THIP (8 mg/kg, b.i.d.) or kojic amine (18 mg/kg, b.i.d.) resulted in a significant increase in dopamine receptor number and a significant decrease in GABA receptor number in the corpus striatum of treated animals as determined by standard in vitro receptor binding techniques. Changes in the GABA receptor were limited to the corpus striatum and occurred more rapidly than did alterations in the dopamine receptor. The finding that dopamine-mediated stereotypic behavior was enhanced in animals treated chronically with AOAA suggested that the receptor binding changes noted in vitro have some functional consequence in vitro.^ Coadministration of atropine (a muscarinic cholinergic receptor antagonist) blocked the GABA-T inhibitor-induced increase in striatal dopamine receptors but was without effect on receptor alterations seen following chronic administration of direct acting GABA receptor agonists. Atropine administration failed to influence the drug-induced decreases in striatal GABA receptors.^ Other findings included the discovery that synaptosomal high affinity ('3)H-choline uptake, an index of cholinergic neuronal activity, was significantly increased in the corpus striatum of animals treated acutely, but not chronically, with GABAmimetics.^ It is suggested that the dopamine receptor supersensitivity observed in the corpus striatum of animals following long-term treatment with GABAmimetics is a result of the chronic inhibition of the nigrostriatal dopamine system by these drugs. Changes in the GABA receptor, on the other hand, are more likely due to a homospecific regulation of these receptors. An hypothesis based on the different sites of action of GABA-T inhibitors vis-a-vis the direct acting GABA receptor agonists is proposed to account for the differential effect of atropine on the response to these drugs.^ The results of this investigation provide new insights into the functional interrelationships that exist in the basal ganglia and suggest that chronic treatment with GABAmimetics may produce extrapyramidal side-effects in man. In addition, the constellation of neurochemical changes observed following administration of these drugs may be a useful guide for determining the GABAmimetic properties of neuropharmacological agents. ^

TIME DEPENDENT ALTERATIONS IN CENTRAL NERVOUS SYSTEM DOPAMINE RECEPTORS INDUCED BY DOPAMINE AGONIST TREATMENT

Levodopa, the precursor of dopamine, is currently the drug of choice in the treatment of Parkinson's disease. Recently, two direct dopamine agonists, bromocriptine and pergolide, have been tested for the treatment of Parkinson's disease because of reduced side effects compared to levodopa. Few studies have evaluated the effects of long-term treatment of dopamine agonists on dopamine receptor regulation in the central nervous system. Thus, the purpose of this study was to determine whether chronic dopamine agonist treatment produces a down-regulation of striatal dopamine receptor function and to compare the results of the two classes of dopaminergic drugs.^ Levodopa with carbidopa, a peripheral decarboxylase inhibitor, was administered orally to rats whereas bromocriptine and pergolide were injected intraperitoneally once daily. Several neurochemical parameters were examined from 1 to 28 days.^ Levodopa minimally decreased striatal D-1 receptor activity but increased the number of striatal D-2 binding sites. Levodopa increased the V(,max) of tyrosine hydroxylase (TH) in all brain regions tested. Protein blot analysis of striatal TH indicated a significant increase in the amount of TH present. Dopamine-beta-hydroxylase (DBH) activity was markedly decreased in all brain regions studied and mixing experiments of control and drug-treated cortices did not show the presence of an increased level of endogenous inhibitors.^ Bromocriptine treatment decreased the number of D-2 binding sites. Striatal TH activity was decreased and protein blot analysis indicated no change in TH quantity. The specificity of bromocriptine for striatal TH suggested that bromocriptine preferentially interacts with dopamine autoreceptors.^ Combination levodopa-bromocriptine was administered for 12 days. There was a decrease in both D-1 receptor activity and D-2 binding sites, and a decrease in brain HVA levels suggesting a postsynaptic receptor action. Pergolide produced identical results to the combination levodopa-bromocriptine studies.^ In conclusion, combination levodopa-bromocriptine and pergolide treatments exhibited the expected down-regulation of dopamine receptor activity. In contrast, levodopa appeared to up-regulate dopamine receptor activity. Thus, these data may help to explain, on a biochemical basis, the decrease in the levodopa-induced side effects noted with combination levodopa-bromocriptine or pergolide therapies in the treatment of Parkinson's disease. ^

Function of histaminergic retinopetal axons in rat and primate retinas

Mammalian retinas receive input from histaminergic neurons in the posterior hypothalamus. These neurons are most active during the waking state of the animal, but their role in retinal information processing is not known. To determine the function of these retinopetal axons, their targets in the rat and monkey retina were identified. Using antibodies to three histamine receptors, HR1, HR2, and HR3, the immunolabeling was analyzed by confocal and electron microscopy. These experiments showed that mammalian retinas possess histamine receptors. In macaques and baboons, diurnal species, HR3 receptors were found at the apex of ON-bipolar cell dendrites in cone pedicles and rod spherules, sclerad to the other neurotransmitter receptors that have been localized there. In addition, HR1 histamine receptors were localized to large puncta in the inner plexiform layer, a subset of ganglion cells and retinal blood vessels. In rats, a nocturnal species, the localization of histamine receptors in the retina was markedly different. Most HR1 receptors were localized to dopaminergic amacrine cells and on elements in the rod spherule. To determine how histaminergic retinopetal axons contribute to retinal information processing, responses of retinal ganglion cells to histamine were analyzed. The effects of histamine on the maintained and light-evoked activity of retinal ganglion cells were analyzed. In monkeys, histamine and the HR3 agonist, methylhistamine, increased or decreased the maintained activity of most ganglion cells, but a few did not respond. The responses of a subset of ganglion cells to light stimuli were decreased by histamine, a finding suggesting that histaminergic retinopetal axons contribute to light adaptation during the day. In rats, histamine nearly always increased the maintained activity and produced both increases and decreases in the light responses. The effects of histamine on maintained activity of ganglion cells in the rat can be partially attributed to HR1-mediated changes in the activity of dopaminergic amacrine cells, at night. Together, these experiments provide the first indication of the function of retinopetal axons in mammalian retinas. ^

Functional differences of beta-tubulin isotypes: A study of microtubule assembly and antimitotic drug resistance

Mammalian cells express 7 β-tubulin isotypes in a tissue specific manner. This has long fueled the speculation that different isotypes carry out different functions. To provide direct evidence for their functional significance, class III, IVa, and VI β-tubulin cDNAs were cloned into a tetracycline regulated expression vector and stably transfected Chinese hamster ovary cell lines expressing different levels of ectopic β-tubulin were compared for effects on microtubule organization, microtubule assembly and sensitivity to antimitotic drugs. It was found that all three isotypes coassembled with endogenous β-tubulin. βVI expression caused distinct microtubule rearrangements including microtubule dissociation from the centrosome and accumulation at the cell periphery; whereas expression of βIII and βVIa caused no observable changes in the interphase microtubule network. Overexpression of all 3 isotypes caused spindle malformation and mitotic defects. Both βIII and βIVa disrupted microtubule assembly in proportion to their abundance and thereby conferred supersensitivity to microtubule depolymerizing drugs. In contrast, βVI stabilized microtubules at low stoichiometry and thus conferred resistance to many microtubule destabilizing drugs but not vinblastine. The 3 isotypes caused differing responses to microtubule stabilizing drugs. Expression of βIII conferred paclitaxel resistance while βVI did not. Low expression of βIVa caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that βIVa may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of βVIa expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by increased paclitaxel binding, and drug supersensitivity is lost. From this study, I concluded that β-tubulin isotypes behave differently from each other in terms of microtubule organization, microtubule assembly and dynamics, and antimitotic drug sensitivity. The isotype composition of cell can impart subtle to dramatic effects on the properties of microtubules leading to potential functional consequences and opening the opportunity to exploit differences in microtubule isotype composition for therapeutic gain. ^