Voltage-dependency of the dopamine transporter in rat brain.
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Animals↗
Biomedical subjects
N R Zahniser
Publications and source records attributed to N R Zahniser.
Differences in NMDA receptor antagonist-induced locomotor activity and [3H]MK-801 binding sites in short-sleep and long-sleep mice.
Short-Sleep (SS) and Long-Sleep (LS) mice differ in initial sensitivity to ethanol. Ethanol acts as an antagonist at N-methyl D-aspartate receptors (NMDARs). Therefore, we tested whether SS and LS mice also differ in initial sensitivity to NMDAR antagonists. Systemic injection (intraperitoneal) of either the noncompetitive NMDAR antagonist MK-801 (dizocilpine) or the competitive NMDAR antagonist 2-carboxypiperazin-4-yl-propyl-1-phosphonic acid (CPP) produced similar results. At lower drug doses, SS mice showed greater locomotor activation than LS mice; and at higher doses, SS mice continued to be activated whereas LS mice became sedated. Brain levels of [3H]MK-801 were 40% higher in SS, compared with LS, mice. However, blood levels of [3H]MK-801 and [3H]CPP and brain levels of [3H]CPP were similar in the two lines. NMDARs were measured using quantitative autoradiographic analysis of in vitro [3H]MK-801 binding to SS and LS mouse brains. Significantly higher (20 to 30%) receptor densities were observed in the hippocampus and cerebral cortex of SS mice. Our results support the hypothesis that SS and LS mice differ in initial sensitivity to NMDAR antagonists and suggest that the line differences in the dose-response relationships for MK-801- and CPP-induced locomotor activity are qualitatively similar to those reported for ethanol. Differences in pharmacokinetics and number of NMDARs may contribute to, but are unlikely to entirely account for, the differential behavioral responsiveness of SS and LS mice to MK-801 and CPP.
Animals↗
Multiple ionic conductances of the human dopamine transporter: the actions of dopamine and psychostimulants.
Electrophysiological and pharmacological studies of a cloned human dopamine transporter (hDAT) were undertaken to investigate the mechanisms of transporter function and the actions of drugs at this target. Using two-electrode voltage-clamp techniques with hDAT-expressing Xenopus laevis oocytes, we show that hDAT can be considered electrogenic by two criteria. (1) Uptake of hDAT substrates gives rise to a pharmacologically appropriate "transport-associated" current. (2) The velocity of DA uptake measured in oocytes clamped at various membrane potentials was voltage-dependent, increasing with hyperpolarization. Concurrent measurement of transport-associated current and substrate flux in individual oocytes revealed that charge movement during substrate translocation was greater than would be expected for a transport mechanism with fixed stoichiometry of 2 Na+ and 1 Cl- per DA+ molecule. In addition to the transport-associated current, hDAT also mediates a constitutive leak current, the voltage and ionic dependencies of which differ markedly from those of the transport-associated current. Ion substitution experiments suggest that alkali cations and protons are carried by the hDAT leak conductance. In contrast to the transport-associated functions, the leak does not require Na+ or Cl-, and DAT ligands readily interact with the transporter even in the absence of these ions. The currents that hDAT mediates provide a functional assay that readily distinguishes the modes of action of amphetamine-like "DA-releasing" drugs from cocaine-like translocation blockers. In addition, the voltage dependence of DA uptake suggests a mechanism through which presynaptic DA autoreceptor activation may accelerate the termination of dopaminergic neurotransmission in vivo.
Animals↗
Activation of protein kinase C inhibits uptake, currents and binding associated with the human dopamine transporter expressed in Xenopus oocytes.
Activation of protein kinase C (PKC) regulates the activity of a number of neurotransmitter transporters. When Xenopus oocytes expressing the cloned human dopamine transporter (hDAT) were pretreated with bath-applied phorbol 12-myristate 13-acetate (PMA), a PKC activator, [3H]DA uptake decreased irreversibly in a time- and dose-dependent manner (IC50 = 22 nM; maximal inhibition = 63-85%). The inhibition appeared to be PKC-specific because incubation with the inactive form of phorbol ester 4alpha-phorbol-12,13-didecanoate (400 nM) did not change the uptake activity and PMA (100 nM) inhibition could be partially blocked by the selective PKC inhibitor bisindolylmaleimide I (1 microM). Saturation studies of [3H]DA uptake showed that PMA-induced inhibition was due to a decrease in V(max) with no change in K(T). Similar to uptake, PMA pretreatment inhibited both the hDAT transport-associated and substrate-independent leak currents. PMA also decreased membrane capacitance (C(m)) by 40%, selectively in hDAT-expressing oocytes. In addition, PMA pretreatment resulted in a 77% decrease in B(max) of [3H]mazindol binding to intact oocytes. In contrast, binding to whole homogenates of PMA-pretreated oocytes was not significantly altered. These results suggest that PMA regulates hDAT expressed in Xenopus oocytes by altering cell surface trafficking of hDAT.
Alanine↗
Opposing actions of adenosine A2a and dopamine D2 receptor activation on GABA release in the basal ganglia: evidence for an A2a/D2 receptor interaction in globus pallidus.
There is increasing evidence that adenosine (ADO) and dopamine (DA) interact directly in the basal ganglia via actions at ADO A2a and DA D2 receptors, respectively. The purpose of this study was to determine 1) the extent to which these receptors modulate endogenous GABA release in discrete regions of the rat basal ganglia and 2) whether GABA release is modulated by a direct and opposing interaction between ADO A2a and DA D2 receptors. Tissue slices of striatum (STR) containing globus pallidus (GP; STR/GP) and micropunches of STR, GP, and substantia nigra pars reticulata (SNr) were studied. Radioligand binding demonstrated that ADO A1, ADO A2a, and DA D2 receptors were present in each of the tissue preparations with the exception of SNr, in which ADO A2a receptors were not detected. Stimulation of ADO A2a receptors with CGS 21680 (1-10 nM) increased electrically stimulated GABA release in STR/GP slices and GP micropunches. Consistent with the lack of A2a receptors in SNr, CGS 21680 had no effect on GABA release from this region. In contrast, stimulation of DA D2 receptors with N-0437 (1-100 nM) inhibited evoked GABA release from STR/GP slices and both GP and SNr micropunches. The D2-mediated inhibition of GABA release in GP was abolished in the presence of CGS 21680 (10 nM). These experiments demonstrate that stimulation of ADO A2a and DA D2 receptors has opposing effects on endogenous GABA release in STR and GP. These opposing actions may explain the antagonistic interactions between ADO and DA that have been observed in behavioral studies and support the hypothesis that the striatopallidal efferent system is an important anatomical substrate for the A2a/D2 receptor interaction.
Adenosine↗
Effects of dietary restriction on motor learning and cerebellar noradrenergic dysfunction in aged F344 rats.
Fisher 344 rats were fed either ad libitum or with a diet containing a 40% reduction of calories beginning at 4 months of age. At 14 months and 22 months male rats were tested for their ability to learn a complex motor skill. At both ages the diet restricted rats reached criterion of performing 10 successful crosses in 10 min at an earlier time than ad libitum fed controls. At 22 months of age the diet restricted rats showed improved acquisition of running times for the task. Male rats at 14 and 22 months and female rats at 24 months were examined electrophysiologically for the ability of isoproterenol to augment the action of GABA in the cerebellum when both substances were applied iontophoretically from an extracellular multibarreled glass electrode. In all 3 age and sex groups there was an improvement in the beta-adrenergic receptor modulation of GABA responses in the dietary restricted vs. ad libitum rats. However, no difference was observed between dietary restricted and ad libitum rats when the number and affinity of cerebellar beta-adrenergic receptors was assessed with 125I-iodopindolol binding. Overall, there was a significant improvement in cerebellar noradrenergic function in the dietary restricted rats and this was accompanied by an improvement in motor learning.
Age Factors↗
Medial dorsal striatum is more sensitive than lateral dorsal striatum to cocaine inhibition of exogenous dopamine clearance: relation to [3H]mazindol binding, but not striosome/matrix.
Previous studies have shown that acute systemic cocaine inhibits dopamine (DA) transport and thereby produces dose-dependent changes in exogenous DA clearance in the brain. This measure reflects the dynamic activity of the DA transporter. There are also differential effects of cocaine on DA clearance in dorsal and ventral striatum, and evidence from many studies suggests that even within the dorsal striatum, the interaction of cocaine with the DA transporter may not be homogeneous. A greater understanding of how cocaine interacts with the striatal DA transporter will help clarify the role of the striatum in mediating effects of cocaine. In these studies we used in vivo electrochemical recording to examine the effect of ip cocaine on exogenous DA clearance in the medial and lateral dorsal striatum with respect to both DA transporter binding sites and electrode localization to striosomes or matrix. Baseline exogenous DA clearance was different in medial and lateral dorsal striatum in the absence of cocaine. Systemic cocaine produced a more pronounced inhibition of DA clearance in medial dorsal striatum than in lateral dorsal striatum. [3H]Mazindol binding to DA transporters was lower in medial dorsal striatum, but was not in register with striosome or matrix compartments. There was no notable difference between cocaine's effects on DA clearance in striosome or matrix that was due to this compartmentalization. Others have demonstrated that medial dorsal striatum receives proportionally more innervation from the mesolimbic, as opposed to the nigrostriatal, DA system. Taken together, these and previous in vivo results suggest that this differential sensitivity to cocaine reflects the unique properties of the ascending mesolimbic DA projection and the lower density of DA transporters associated with it.
Animals↗
Pharmacological characterization of [3H]idazoxan, [3H]RX821002 and p-[125I]iodoclonidine binding to alpha 2-adrenoceptors in rat cerebral cortical membranes.
Binding characteristics of alpha 2-adrenoceptors in rat cerebral cortical membranes were compared using the antagonist radioligands [3H]idazoxan, [3H]2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline ([3H]RX821002), and the partial agonist radioligand [125I]2-[2,6-(dichloro-4-iodophenyl)imino]imidazoline ([125I]iodoclonidine). With [3H]RX821002 and alpha 2-adrenoceptor subtype-selective competitors, both alpha 2A/D- and alpha 2C-adrenoceptor subtypes were detected, suggesting rat cortical membranes contain approximately 90% alpha 2A/D-adrenoceptor subtype and 10% alpha 2C-adrenoceptor subtype. Only alpha 2A/D-adrenoceptors were detected with [3H]idazoxan and [125I]iodoclonidine. All three radioligands bound to a single high affinity site (Kd = 0.3-1.6 nM). However, the densities of sites labeled by [3H]idazoxan and [125I]iodoclonidine were 50% greater than the density labeled by [3H]RX821002, likely representing non-adrenoceptor binding sites. The density of [125I]iodoclonidine binding sites in glycylglycine buffer was similar to that labeled by [3H]RX821002. These results suggest that: (1) alpha 2A/D-adrenoceptors are the predominant subtype in rat cerebral cortex, (2) demonstrate that the small number of alpha 2C-adrenoceptors in this tissue can be detected using prazosin to displace [3H]RX821002 binding, and (3) non-adrenoceptor binding with [125I]iodoclonidine can be minimized with the use of glycylglycine buffer.
Adrenergic alpha-Antagonists↗
Repeated intravenous cocaine administration to rats produces behavioral sensitization without changing brain cocaine levels.
Repeated intraperitoneal (i.p.) administration of cocaine to rats results in behavioral sensitization. However, augmented brain cocaine levels are also produced by this treatment. In the present study, a 43% increase in cocaine levels was observed in striatum in response to eight once-daily i.p. injections of cocaine (10 mg/kg). It has been suggested that this dispositional change does not occur with intravenous (i.v.) cocaine administration. In agreement with this suggestion, the striatal cocaine levels observed following either a single i.v. injection or eight once-daily i.v. injections of cocaine (1 mg/kg) were similar. Nonetheless, the rats became behaviorally sensitized in response to the repeated i.v. cocaine administration. These results suggest that the increased brain levels of cocaine observed following repeated i.p. cocaine administration cannot completely account for behavioral sensitization.
Animals↗
Cocaine levels in striatum and nucleus accumbens: augmentation following challenge injection in rats withdrawn from repeated cocaine administration.
There is general agreement that the brain level of cocaine achieved in response to an intraperitoneal (i.p.) cocaine challenge is higher in animals that have been treated repeatedly with cocaine. However, whether this change in drug disposition persists following withdrawal from repeated treatment is controversial. Rats received either single or repeated injections of cocaine (10 mg/kg, i.p.), and levels of cocaine were measured in dorsal striatum and nucleus accumbens 20 min after the last injection. Cocaine levels were 60-70% higher in both brain regions of rats that had been treated with cocaine once daily for 7 days compared to rats that had received only a single injection. On the 7th day of withdrawal, similar elevations were observed after a challenge injection of cocaine in animals that had been treated repeatedly with cocaine but not in animals that had been treated repeatedly with saline. This persistent pharmacokinetic change should be taken into account in studies of cocaine-induced behavioral sensitization.
Animals↗
Endogenous GABA release from rat striatal slices: effects of the GABAB receptor antagonist 2-hydroxy-saclofen.
The reproducibility of endogenous GABA release evoked by multiple periods of electrical field stimulation was examined in rat striatal slices. In these experiments, NO-328 was used to block GABA uptake, and evoked GABA release (overflow) was completely Ca2+ dependent. A seemingly invariant observation in these experiments was that spontaneous GABA release (outflow) progressively decreased as a function of superfusion time and that GABA overflow decreased 25-30% in response to the second of two periods of stimulation (S2/S1 ratios = 0.70 to 0.75). The attenuation of GABA release was not explained by the amount of GABA lost to the superfusion buffer (fractional release), direct depletion of releasable pools of GABA, or slice viability. Furthermore, the decreases in GABA release were not dependent on stimulation frequency (5-15 Hz) or the absolute amount of GABA evoked by electrical stimulation. However, the GABAB receptor antagonist 2-hydroxy-saclofen (2-OH-saclofen; 316 microM) not only enhanced GABA overflow, when superfused throughout both periods of stimulation, but also resulted in S2/S1 ratios of unity. When 2-OH-saclofen was superfused throughout the second stimulation period only, GABA overflow was almost two-fold greater than that evoked by the initial period of stimulation (2-OH-saclofen-free). In addition, these S2 responses were approximately 30% greater than S1 responses that were observed when 2-OH-saclofen was present throughout the entire superfusion period. These results indicate that activation of GABAB receptors was involved in the progressive attenuation of GABA release and further emphasize that GABAB receptors play an important role in modulating endogenous GABA release from striatal slices.
Animals↗
Adenosine A2a receptor modulation of electrically evoked endogenous GABA release from slices of rat globus pallidus.
Adenosine A2a receptors have been localized to GABAergic striatopallidal neurons, but their functional role is unknown. To address this question, the modulation of endogenous GABA release by adenosine A2a receptors was examined in slices of rat globus pallidus. The selective adenosine A2a receptor agonist CGS-21680 (3.0-10 nM) significantly increased electrically stimulated release (overflow) of GABA, with 10 nM CGS-21680 resulting in a 44% increase compared with the control. Both the nonselective adenosine receptor antagonist 8-phenyltheophylline (10 microM) and the selective A2a receptor antagonist KF-17837 (100 nM) abolished the CGS-21680-induced increase in GABA overflow. Higher concentrations of CGS-21680 (0.10-1.0 microM) decreased GABA overflow by approximately 25%. 8-Phenyltheophylline (10 microM) antagonized these effects, whereas KF-17837 (100 nM) did not, suggesting actions of CGS-21680 on other adenosine receptors at these concentrations. These results demonstrate that activation of adenosine A2a receptors augments electrically stimulated release of GABA from globus pallidus slices and suggest a mechanism by which adenosine may modulate GABAergic output from the striatopallidal efferent system.
Adenosine↗
Reduced clearance of exogenous dopamine in rat nucleus accumbens, but not in dorsal striatum, following cocaine challenge in rats withdrawn from repeated cocaine administration.
We investigated whether changes in the dopamine transporter in the nucleus accumbens or striatum are involved in cocaine-induced behavioral sensitization by using in vivo electrochemistry to monitor the clearance of locally applied dopamine in anesthetized rats. Rats were injected with cocaine-HCl (10 mg/kg i.p.) or saline daily for 7 consecutive days and then withdrawn for 7 days. Pressure ejection of a finite amount of dopamine at 5-min intervals from a micropipette adjacent to the electrochemical recording electrode produced transient and reproducible dopamine signals. After a challenge injection of cocaine (10 mg/kg i.p.), the signals in the nucleus accumbens of cocaine-treated animals became prolonged and the clearance rate of the dopamine decreased, indicating significant inhibition of the dopamine transporter. In contrast, simultaneous measurements in the dorsal striatum indicated a transient increase in both the amplitude of the signals and the clearance rate of the dopamine. The signals in both brain regions in the saline-treated animals given the cocaine challenge were similar to those in untreated animals given an acute injection of cocaine (10 mg/kg i.p.) or saline. Behaviorally, not all of the cocaine-treated animals were sensitized; however, both sensitized and nonsensitized animals displayed similar changes in dopamine clearance rate. Quantitative autoradiography with [3H]mazindol revealed that the affinity of the dopamine transporter for cocaine and the density of binding sites were similar in cocaine- and saline-treated rats. The decrease in dopamine clearance rate observed in the nucleus accumbens of the cocaine-treated rats after a challenge injection of cocaine is consistent with increased dopaminergic transmission, but does not appear to be sufficient in itself for producing behavioral sensitization.
Animals↗
Clearance of exogenous dopamine in rat dorsal striatum and nucleus accumbens: role of metabolism and effects of locally applied uptake inhibitors.
In vivo electrochemistry was used to investigate the mechanisms contributing to the clearance of locally applied dopamine in the dorsal striatum and nucleus accumbens of urethane-anesthetized rats. Chronoamperometric recordings were continuously made at 5 Hz using Nafion-coated carbon fiber electrodes. When a finite amount of dopamine was pressure-ejected at 5-min intervals from a micropipette adjacent to the electrode, transient and reproducible dopamine signals were detected. Substitution of L-alpha-methyldopamine, a substrate for the dopamine transporter but not for monoamine oxidase, for dopamine in the micropipette did not substantially alter the time course of the resulting signals. This indicates that metabolism of locally applied dopamine to 3,4-dihydroxy-phenylacetic acid is not responsible for the decline in the dopamine signal. Similarly, changing the applied oxidation potential from +0.45 to +0.80 V, which allows for detection of 3-methoxytyramine formed from dopamine via catechol-O-methyltransferase, had little effect on signal amplitude or time course. In contrast, lesioning the dopamine terminals with 6-hydroxydopamine, or locally applying the dopamine uptake inhibitors cocaine or nomifensine before pressure ejection of dopamine, significantly increased the amplitude and time course of the dopamine signals in both regions. The effects of cocaine and nomifensine were greater in the nucleus accumbens than in the dorsal striatum. Local application of lidocaine and procaine had no effect on the dopamine signals. Initial attempts at modeling resulted in curves that were in qualitative agreement with our experimental findings. Taken together, these data indicate that (1) uptake of dopamine by the neuronal dopamine transporter, rather than metabolism or diffusion, is the major mechanism for clearing locally applied dopamine from the extracellular milieu of the dorsal striatum and nucleus accumbens, and (2) the nucleus accumbens is more sensitive to the effects of inhibitors of dopamine uptake than is the dorsal striatum.
3,4-Dihydroxyphenylacetic Acid↗
Cocaine administration: D1 dopamine receptor function and dopamine clearance/diffusion in rat striatum and nucleus accumbens.
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Adenylyl Cyclases↗
Cocaine-induced behavioral sensitization and D1 dopamine receptor function in rat nucleus accumbens and striatum.
Based on electrophysiological data showing that repeated cocaine administration produces persistent enhancement of D1 dopamine (DA) receptor-mediated responses in nucleus accumbens (NAc), we investigated whether changes in neurochemical properties of these receptors resulted when rats were injected with cocaine (15 mg/kg) for 6 days followed by a 7-day abstinence period. D1 DA receptor density and affinities for either [3H]SCH 23390 or DA were similar between NAc and striatum and between saline and cocaine treatment groups. DA-stimulated adenylyl cyclase activity was 1.5-fold higher in striatum than in NAc; however, repeated cocaine treatment produced no persistent changes in enzyme activity in either brain area.
Analysis of Variance↗
GABAA receptor function and regional analysis of subunit mRNAs in long-sleep and short-sleep mouse brain.
The greater sensitivity of long-sleep (LS), as compared with short-sleep (SS), mice to ethanol is due in part to differences in GABAA receptor function in specific brain regions. To determine if differences in subunit composition of GABAA receptors contribute to this differential sensitivity, we measured alpha 1 and gamma 2 subunit mRNAs with Northern analysis and in situ hybridization and gamma 2S, gamma 2L and alpha 6 subunit mRNAs with polymerase chain reaction (PCR) amplification. No differences in mRNAs in whole brain were apparent by Northern analysis. In situ hybridization revealed that alpha 1 and gamma 2 subunit mRNAs were co-localized in many brain regions but that they still had distinct patterns of hybridization. However, the few differences observed between LS and SS mice in the levels of hybridization for these subunits did not show a regional distribution consistent with ethanol sensitivity differences. Similar ratios of gamma 2L, and gamma 2S subunit mRNAs were found in LS and SS mouse cerebral cortex and hippocampus, and both mouse lines expressed essentially only gamma 2L subunit mRNA in cerebellum. mRNA for the alpha 6 subunit was detected only in cerebellum and also was qualitatively similar between LS and SS mice. Studies of muscimol-stimulated 36Cl- uptake by cortical membrane vesicles confirmed earlier findings that ethanol does not enhance function of GABAA receptors in SS mice when assayed at 30 degrees C. However, at 34 degrees C ethanol did increase this function in SS mice although the enhancement remained greater in LS mice. These functional results, together with the results showing similar levels of alpha 1, gamma 2S, gamma 2L and alpha 6 subunits in LS and SS mice, suggest that the ethanol-insensitivity of SS mouse GABAA receptors cannot be due solely to lack of subunits required for ethanol action and further suggest that differences in catalytic mechanisms affecting post-translational processing may account for some genetic differences in ethanol sensitivity of GABAA receptors.
Animals↗
Signal transduction pathways involved in presynaptic receptor-mediated inhibition of dopamine release in rat striatum.
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Animals↗