Gaynor A. Larson

Dopamine D2 Receptor-Deficient Mice Exhibit Decreased Dopamine Transporter Function but No Changes in Dopamine Release in Dorsal Striatum

Abstract : Presynaptic D2 dopamine (DA) autoreceptors, which are well known to modulate DA release, have recently been shown to regulate DA transporter (DAT) activity. To examine the effects of D2 DA receptor deficiency on DA release and DAT activity in dorsal striatum, we used mice genetically engineered to have two (D2+/+), one (D2+/‐), or no (D2‐/‐) functional copies of the gene coding for the D2 DA receptor. In vivo microdialysis studies demonstrated that basal and K+‐evoked extracellular DA concentrations were similar in all three genotypes. However, using in vivo electrochemistry, the D2‐/‐ mice were found to have decreased DAT function, i.e., clearance of locally applied DA was decreased by 50% relative to that in D2+/+ mice. In D2+/+ mice, but not D2‐/‐ mice, local application of the D2‐like receptor antagonist raclopride increased DA signal amplitude, indicating decreased DA clearance. Binding assays with the cocaine analogue [3H]WIN 35,428 showed no genotypic differences in either density or affinity of DAT binding sites in striatum or substantia nigra, indicating that the differences seen in DAT activity were not a result of decreased DAT expression. These results further strengthen the idea that the D2 DA receptor subtype modulates activity of the striatal DAT.

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.

Amphetamine and Methamphetamine Differentially Affect Dopamine Transporters in Vitro and in Vivo

The psychostimulants d-amphetamine (AMPH) and methamphetamine (METH) release excess dopamine (DA) into the synaptic clefts of dopaminergic neurons. Abnormal DA release is thought to occur by reverse transport through the DA transporter (DAT), and it is believed to underlie the severe behavioral effects of these drugs. Here we compare structurally similar AMPH and METH on DAT function in a heterologous expression system and in an animal model. In the in vitro expression system, DAT-mediated whole-cell currents were greater for METH stimulation than for AMPH. At the same voltage and concentration, METH released five times more DA than AMPH and did so at physiological membrane potentials. At maximally effective concentrations, METH released twice as much [Ca2+]i from internal stores compared with AMPH. [Ca2+]i responses to both drugs were independent of membrane voltage but inhibited by DAT antagonists. Intact phosphorylation sites in the N-terminal domain of DAT were required for the AMPH- and METH-induced increase in [Ca2+]i and for the enhanced effects of METH on [Ca2+]i elevation. Calmodulin-dependent protein kinase II and protein kinase C inhibitors alone or in combination also blocked AMPH- or METH-induced Ca2+ responses. Finally, in the rat nucleus accumbens, in vivo voltammetry showed that systemic application of METH inhibited DAT-mediated DA clearance more efficiently than AMPH, resulting in excess external DA. Together these data demonstrate that METH has a stronger effect on DAT-mediated cell physiology than AMPH, which may contribute to the euphoric and addictive properties of METH compared with AMPH.

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.

Dopamine transport by the serotonin transporter: a mechanistically distinct mode of substrate translocation

The serotonin transporter (SERT) is the principal mechanism for terminating serotonin (5-HT) signals in the nervous system and is a site of action for a variety of psychoactive drugs including antidepressants, amphetamines, and cocaine. Here we show that human SERTs (hSERTs) and rat SERTs are capable of robust dopamine (DA) uptake through a process that differs mechanistically from 5-HT transport in several unanticipated ways. DA transport by hSERT has a higher maximum velocity than 5-HT transport, requires significantly higher Na+ and Cl− concentrations to sustain transport, is inhibited noncompetitively by 5-HT, and is more sensitive to SERT inhibitors, including selective serotonin reuptake inhibitors. We use a thiol-reactive methane thiosulfonate (MTS) reagent to modify a conformationally sensitive cysteine residue to demonstrate that hSERT spends more time in an outward facing conformation when transporting DA than when transporting 5-HT. Cotransfection of an inactive or an MTS-sensitive SERT with wild-type SERT subunits reveals an absence of cooperative interactions between subunits during DA but not 5-HT transport. To establish the physiological relevance of this mechanism for DA clearance, we show using in vivo high-speed chronoamperometry that SERT has the capacity to clear extracellularly applied DA in the hippocampal CA3 region of anesthetized rats. Together, these observations suggest the possibility that SERT serves as a DA transporter in vivo and highlight the idea that there can be distinct modes of transport of alternative physiological substrates by SERT.

Modulation of endogenous GABA release by an antagonistic adenosine A1 / dopamine D1 receptor interaction in rat brain limbic regions but not basal ganglia

Behavioral and biochemical studies suggest that a negative interaction exists between adenosine A1 and dopamine D1 receptors in the brain and that this may contribute to the psychomotor effects of adenosine receptor agonists and antagonists. We examined the functional significance of A1 and D1 receptor subtypes in modulating electrically evoked endogenous GABA release from slices/punches of rat basal ganglia (striatum, globus pallidus, striatum containing globus pallidus, and substantia nigra reticulata) and limbic regions (ventral pallidum and nucleus accumbens). In basal ganglia, stimulation of A1 receptors with the selective agonist R‐PIA (1–100 nM) resulted in a concentration‐dependent decrease in GABA release. The selective A1 antagonist DPCPX (10–100 nM) increased GABA release, suggesting that endogenous adenosine tonically inhibits GABA release. However, in basal ganglia, consistent dopamine D1 receptor modulation of GABA, release was not observed in response to either D1 agonists or antagonists. Furthermore, the A1 receptor‐mediated inhibition of GABA release was not changed by concurrent activation of D1 receptors, thus confirming the lack of D1 receptor modulation under these conditions. In contrast, in ventral pallidum and nucleus accumbens, stimulation of D1 receptors with SKF‐82958 (1 μM) increased GABA release significantly. The D1 receptor‐mediated increase in GABA release was attenuated by concurrent activation of adenosine A1 receptors. These results are consistent with the hypothesis that an antagonistic A1/D1 receptor interaction may be important in modulating GABA release in limbic regions. Synapse 33:274–281, 1999.

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 cocaines 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.

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.

Rats classified as low or high cocaine locomotor responders: A unique model involving striatal dopamine transporters that predicts cocaine addiction-like behaviors

Individual differences are a hallmark of drug addiction. Here, we describe a rat model based on differential initial responsiveness to low dose cocaine. Despite similar brain cocaine levels, individual outbred Sprague-Dawley rats exhibit markedly different magnitudes of acute cocaine-induced locomotor activity and, thereby, can be classified as low or high cocaine responders (LCRs or HCRs). LCRs and HCRs differ in drug-induced, but not novelty-associated, hyperactivity. LCRs have higher basal numbers of striatal dopamine transporters (DATs) than HCRs and exhibit marginal cocaine inhibition of in vivo DAT activity and cocaine-induced increases in extracellular DA. Importantly, lower initial cocaine response predicts greater locomotor sensitization, conditioned place preference and greater motivation to self-administer cocaine following low dose acquisition. Further, outbred Long-Evans rats classified as LCRs, versus HCRs, are more sensitive to cocaines discriminative stimulus effects. Overall, results to date with the LCR/HCR model underscore the contribution of striatal DATs to individual differences in initial cocaine responsiveness and the value of assessing the influence of initial drug response on subsequent expression of addiction-like behaviors.