Dopamine input to the striatum is required for voluntary motor movement, behavioral reinforcement, and responses to drugs of abuse. It is speculated that these functions are dependent on either excitatory or inhibitory modulation of corticostriatal synapses onto medium spiny neurons (MSNs). While dopamine modulates MSN excitability, a direct presynaptic effect on the corticostriatal input has not been clearly demonstrated. We combined optical monitoring of synaptic vesicle exocytosis from motor area corticostriatal afferents and electrochemical recordings of striatal dopamine release to directly measure effects of dopamine at the level of individual presynaptic terminals. Dopamine released by either electrical stimulation or amphetamine acted via D2 receptors to inhibit the activity of subsets of corticostriatal terminals. Optical and electrophysiological data suggest that heterosynaptic inhibition was enhanced by higher frequency stimulation and was selective for the least active terminals. Thus, dopamine, by filtering less active inputs, appears to reinforce specific sets of corticostriatal synaptic connections.

Five adenosines within the coding sequence of the serotonin 2C receptor (5-HT2C) pre-mRNA are converted to inosines by RNA editing (named A, B, C' (E), C, and D sites). In human prefrontal cortex (PFC), the most abundant 5-HT2C mRNA sequences result from editing at the A site, or from the editing combinations AC'C, ABCD, and ABD. In suicide victims with a history of major depression, C' site editing is significantly increased, D site editing is significantly decreased, and the C site shows a trend toward increased editing. Treatment of mice with the antidepressant drug fluoxetine (Prozac) causes changes in C', C, and D site editing that are exactly opposite to those seen in suicide victims. Thus, one outcome of fluoxetine treatment may be to reverse the abnormalities in 5-HT2C pre-mRNA editing seen in depressed suicide victims.

The existence of G protein-coupled receptor (GPCR) dimers and/or oligomers has been demonstrated in heterologous systems using a variety of biochemical and biophysical assays. While these interactions are the subject of intense research because of their potential role in modulating signaling and altering pharmacology, evidence for the existence of receptor interactions in vivo is still elusive because of a lack of appropriate methods to detect them. Here, we adapted and optimized a proximity ligation assay (PLA) for the detection in brain slices of molecular proximity of two antigens located on either the same or two different GPCRs. Using this approach we were able to confirm the existence of dopamine D2 and adenosine A2A receptor complexes in the striatum of mice ex vivo.

Amphetamine (AMPH) is known to raise extracellular dopamine (DA) levels by inducing stimulation-independent DA efflux via reverse transport through the DA transporter and by inhibiting DA re-uptake. In contrast, recent studies indicate that AMPH decreases stimulation-dependent vesicular DA release. One candidate mechanism for this effect is the AMPH-mediated redistribution of DA from vesicles to the cytosol. In addition, the inhibition of stimulation-dependent release may occur because of D2 autoreceptor activation by DA that is released via reverse transport. We used the D2 receptor antagonist sulpiride and mice lacking the D2 receptor to address this issue. To evaluate carefully AMPH effects on release and uptake, we recorded stimulated DA overflow in striatal slices by using continuous amperometry and cyclic voltammetry. Recordings were fit by a random walk simulation of DA diffusion, including uptake with Michaelis-Menten kinetics, that provided estimates of DA concentration and uptake parameters. AMPH (10 M) promoted the overflow of synaptically released DA by decreasing the apparent affinity for DA uptake (K m increase from 0.8 to 32 M). The amount of DA released per pulse, however, was decreased by 82%. This release inhibition was prevented partly by superfusion with sulpiride (47% inhibition) and was reduced in D2 mutant mice (23% inhibition). When D2 autoreceptor activation was minimal, the combined effects of AMPH on DA release and uptake resulted in an enhanced overflow of exocytically released DA. Such enhancement of stimulation-dependent DA overflow may occur under conditions of low D2 receptor activity or expression, for example as a result of AMPH sensitization.

No abstract

Dysregulation of dopamine transmission is thought to contribute to schizophrenic psychosis and drug dependence. Dopamine release is regulated by D2 dopamine autoreceptors, and D2 receptor ligands are used to treat psychosis and addiction. To elucidate the long-term effects of D2 autoreceptor activity on dopamine signaling, dopamine overflow evoked by single or paired-pulse stimulation was compared in striatal slices from D2-null mutant and wild-type mice. Quinpirole, a D2/D3 receptor agonist, had no effect on evoked dopamine release in D2 mutant mice, indicating that D2 receptors are the only release-regulating receptors at the axon terminal. Dopamine release inhibition by GABA(B) receptor activation was unchanged in D2 mutant mice, suggesting that other G-protein-coupled pathways remained normal in the absence of D2 autoreceptors. Paired-pulse stimulation revealed that autoinhibition of dopamine release was maximal 500 msec after stimulation and lasted <5 sec. In D2-null mutants, dopamine overflow in response to single stimuli was severely decreased. Experiments with the uptake inhibitor nomifensine indicated that this was caused by enhanced dopamine uptake rather than reduced release. Analysis of dopamine overflow kinetics using a simulation model suggested that the enhanced uptake was caused by an increase in the maximal velocity of uptake, V(max). These results from D2-null mutant mice support the suggestion that D2 autoreceptors and dopamine transporters interact to regulate the amplitude and timing of dopamine signals.

In two inbred strains of mice, C57BL/6 and 129Sv, the majority of forebrain neocortical pre-mRNA encoding the serotonin 2C (5-HT 2C ) receptor is altered by adenosine-to-inosine editing. As a result, Ͼ60% of all mRNAs encode receptors with reduced constitutive and agonist-stimulated activity. However, in the BALB/c strain, a genetically distinct inbred strain with lower forebrain serotonin levels, spontaneously elevated anxiety, and increased stress reactivity, the majority of 5-HT 2C mRNA is nonedited and encodes receptors with the highest constitutive activity and the highest agonist affinity and potency. Neither acute stress (the forced swim test) nor chronic treatment with the serotonin-selective reuptake inhibitor fluoxetine elicit significant changes in 5-HT 2C pre-mRNA editing in C57BL/6 mice. In contrast, exposure of BALB/c mice to acute stress and chronic treatment of nonstressed BALB/c mice with fluoxetine elicit significant, site-specific increases in 5-HT 2C pre-mRNA editing that increase the pool of mRNA encoding receptors with reduced function. These changes in 5-HT 2C pre-mRNA editing resemble those detected previously in the prefrontal cortex of subjects with major depression. However, when chronic fluoxetine treatment is combined with stress exposure of BALB/c mice, these changes in 5-HT 2C pre-mRNA editing are no longer detected. These findings illustrate that 5-HT 2C pre-mRNA editing responses to stress and chronic fluoxetine are modulated by the genetic background, as well as the behavioral state of the animal. They suggest further that the changes in 5-HT 2C pre-mRNA editing found in major depression reflect a previously unrecognized molecular response to stress that can be prevented by chronic antidepressant treatment.

Serotonin 2C (5-HT 2C ) receptor pre-mRNA is a substrate for RNA editing enzymes that convert five adenosines (named A, B, CЈ, C, and D editing sites) to inosines. Editing of two of these sites (CЈ and C) is crucial for decreasing the efficiency of the receptor to activate G-protein. Nucleotide sequence analysis of mouse forebrain neocortical 5-HT 2C mRNA isoforms revealed that editing at these two sites is regulated in a serotonindependent manner. In serotonin-depleted mice, CЈ-and C-site editing is significantly decreased. This results in an increased expression of 5-HT 2C mRNA isoforms encoding receptors with higher sensitivity to serotonin. In contrast, a 4 d treatment with the 5-HT 2A/2C agonist (Ϯ)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane significantly increases the editing frequency at the CЈ site and leads to increased expression of 5-HT 2C mRNA isoforms encoding receptors that activate G-protein least efficiently. None of the drug treatments led to alterations in cytoplasmic 5-HT 2C mRNA levels. These data indicate that editing of 5-HT 2C pre-mRNA is a mechanism that retains basic response properties of 5-HT 2C receptors in the face of changing synaptic input to keep receptor activation within an optimal range for information processing. Key words: serotonin; 5-HT 2C receptor; RNA editing; forebrain neocortex; 5-HT depletion; 5-HT 2A/2C agonistThe conversion of adenosine (A) to inosine (I) by ADARs (adenosine deaminases that act on RNA) is the most widespread editing in higher eukaryotes (Bass, 2002). Although the total inosine content of rat brain poly(A ϩ ) RNA predicts that one inosine occurs approximately once every 17,000 nucleotides (Paul and Bass, 1998), only a few neuronal substrates for A to I editing have been identified. They include the serotonin 2C (5-HT 2C ) receptor, the first G-protein-coupled receptor known to be edited. In 5-HT 2C pre-mRNA, a total of five closely spaced adenosines (named A, B, CЈ, C, and D editing sites) located within a sequence that encodes the second intracellular loop of the receptors protein can be converted to inosines (Burns et al., 1997;Niswender et al., 1999). This editing can change up to three triplet codons and has the potential to generate 24 different protein isoforms. Compared with nonedited 5-HT 2C receptors, the receptor isoform that results from editing at the ABCD sites and other partially edited isoforms that are edited at the CЈsite but not the C site exhibit a fourfold reduction in the efficiency to activate G-protein in response to agonist stimulation. This reduction is even higher (15-to 25-fold) for completely edited isoforms and other partially edited isoforms that are edited at both CЈ and C sites. Other partially edited isoforms that are not edited at the CЈ and/or C site appear to be fully functional (Niswender et al., 1999;Wang et al., 2000).At present, the significance of 5-HT 2C pre-mRNA editing in vivo is still unknown. However, a recent study indicates that this editing is regulated and suggests that this regulation is sensitive ...