In recent years, the distribution of dopamine receptor subtypes among the principal neurons of the neostriatum has been the subject of debate. Conventional anatomical and physiological approaches have yielded starkly different estimates of the extent to which D1 and D2 class dopamine receptors are colocalized. One plausible explanation for the discrepancy is that some dopamine receptors are present in physiologically significant numbers, but the mRNA for these receptors is not detectable with conventional techniques. To test this hypothesis, we examined the expression of DA receptors in individual neostriatal neurons by patch-clamp and RT-PCR techniques. Because of the strong correlation between peptide expression and projection site, medium spiny neurons were divided into three groups on the basis of expression of mRNA for enkephalin (ENK) and substance P (SP). Neurons expressing detectable levels of SP but not ENK had abundant mRNA for the D1a receptor. A subset of these cells (approximately 50%) coexpressed D3 or D4 receptor mRNA. Neurons expressing detectable levels of ENK but not SP had abundant mRNA for D2 receptor isoforms (short and long). A subset (10-25%) of these neurons coexpressed D1a or D1b mRNAs. Neurons coexpressing ENK and SP mRNAs consistently coexpressed D1a and D2 mRNAs in relatively high abundance. Functional analysis of neurons expressing lower abundance mRNAs revealed clear physiological consequences that could be attributed to these receptors. These results suggest that, although colocalization of D1a and D2 receptors is limited, functional D1 and D2 class receptors are colocalized in nearly one-half of all medium spiny projection neurons.

Dopaminergic neurons exert a major modulatory effect on the forebrain. Dopamine and adenosine 3′,5′-monophosphate–regulated phosphoprotein (32 kilodaltons) (DARPP-32), which is enriched in all neurons that receive a dopaminergic input, is converted in response to dopamine into a potent protein phosphatase inhibitor. Mice generated to contain a targeted disruption of the DARPP-32 gene showed profound deficits in their molecular, electrophysiological, and behavioral responses to dopamine, drugs of abuse, and antipsychotic medication. The results show that DARPP-32 plays a central role in regulating the efficacy of dopaminergic neurotransmission.

Unlike other neostriatal neurons, cholinergic interneurons exhibit spontaneous, low-frequency, repetitive firing. To gain an understanding of the K+ channels regulating this behavior, acutely isolated adult rat cholinergic interneurons were studied using whole-cell voltage-clamp and single-cell reverse transcription-PCR techniques. Cholinergic interneurons were identified by the presence of choline acetyltransferase (ChAT) mRNA. Depolarization-activated potassium currents in cholinergic interneurons were dominated by a rapidly inactivating, K+-selective A current that became active at subthreshold potentials. Depolarizing prepulses inactivated this component of the current, leaving a delayed, rectifier-like current. Micromolar concentrations of Cd2+ dramatically shifted the voltage dependence of the A current without significantly affecting the delayed rectifier. The A-channel antagonist 4-aminopyridine (4-AP) produced a voltage-dependent block (IC50, approximately 1 mM) with a prominent crossover at millimolar concentrations. On the other hand, TEA preferentially blocked the sustained current component at concentrations <10 mM. Single-cell mRNA profiling of subunits known to give rise to rapidly inactivating K+ currents revealed the coexpression of Kv4.1, Kv4.2, and Kv1.4 mRNAs but low or undetectable levels of Kv4.3 and Kv3.4 mRNAs. Kv1.1, beta1, and beta2 subunit mRNAs, but not beta3, were also commonly detected. The inactivation recovery kinetics of the A-type current were found to match those of Kv4.2 and 4.1 channels and not those of Kv1.4 or Kv1. 1 and beta1 channels. Immunocytochemical analysis confirmed the presence of Kv4.2 but not Kv1.4 subunits in the somatodendritic membrane of ChAT-immunoreactive neurons. These results argue that the depolarization-activated somatodendritic K+ currents in cholinergic interneurons are dominated by Kv4.2- and Kv4. 1-containing channels. The properties of these channels are consistent with their playing a prominent role in governing the slow, repetitive discharge of interneurons seen in vivo.

In recent years, the distribution of dopamine receptor subtypes among the principal neurons of the neostriatum has been the subject of debate. Conventional anatomical and physiological approaches have yielded starkly different estimates of the extent to which D 1 and D 2 class dopamine receptors are colocalized. One plausible explanation for the discrepancy is that some dopamine receptors are present in physiologically significant numbers, but the mRNA for these receptors is not detectable with conventional techniques. To test this hypothesis, we examined the expression of DA receptors in individual neostriatal neurons by patch-clamp and RT-PCR techniques. Because of the strong correlation between peptide expression and projection site, medium spiny neurons were divided into three groups on the basis of expression of mRNA for enkephalin (ENK) and substance P (SP The signaling pathways activated by dopamine in the neostriatum have been the subject of intense study since it was discovered that the loss of dopamine leads to the psychomotor symptoms of Parkinson's disease (Hornykiewcz, 1973). Subsequently, several other common psychomotor disorders, including schizophrenia and Tourette's syndrome, have been linked to alterations in neostriatal dopaminergic signaling (Nemeroff and Bissette, 1988;Erenberg, 1992). In recent years, significant progress has been made in characterizing the membrane receptors transducing the signals of dopamine in the neostriatum and the brain in general. How these receptors are distributed among the principal neuronal cell types in the neostriatum has been the subject of debate (Surmeier et al., 1993). This controversy stems primarily from discrepancies in the results obtained from functional studies on the one hand and anatomical studies on the other. The most compelling anatomical data are in situ hybridization studies suggesting that D 1a and D 2 mRNA are segregated primarily in the two major efferent neostriatal populations (Gerfen, 1992; LeMoine and Bloch, 1995). In particular, D 1a receptor mRNA is found in substance P-expressing (SP) neurons projecting to the substantia nigra, whereas D 2 receptor mRNA is found in enkephalin-expressing (ENK) neurons projecting exclusively to the globus pallidus. More recent immunocytochemical work supports this conclusion (Hersch et al., 1995), although others have reported significant degrees of receptor protein colocalization .Functional studies, on the other hand, repeatedly have observed responses to D 1 and D 2 class agonists that are difficult to explain if these receptor classes are not colocalized (Uchimura et al., 1986;Akaike et al., 1987;Cepeda et al., 1993; for review, see Surmeier et al., 1993). The most compelling evidence comes from patch-clamp studies of acutely isolated neostriatal neuronswhere synaptic interactions have been removed-showing neuromodulatory effects of both D 1 and D 2 class agonists in the same cell (Surmeier et al., 1992). In this study, it was also shown that neurons projecting axons to the substantia nigra coexp...

Dopamine has long been known to regulate the activity of striatal cholinergic interneurons and the release of acetylcholine. Yet, the cellular mechanisms by which this regulation occurs have not been elucidated. One way in which dopamine might act is by modulating voltage-dependent Ca2+ channels. To test this hypothesis, the impact of dopaminergic agonists on Ca2+ channels in neostriatal cholinergic interneurons was studied by combined whole cell voltage-clamp recording and single-cell reverse transcription-polymerase chain reactions. Cholinergic interneurons were identified by the presence of choline acetyltransferase mRNA. Nearly, all interneurons tested (90%, n = 17) coexpressed D2 (short and long isoforms) and D1b (D5) dopamine receptor mRNAs. D1a receptor mRNA was found in only a small subset (20%) of the sample and D3 and D4 receptor mRNAs were undetectable. D2 receptor agonists rapidly and reversibly reduced N-type Ca2+ currents. D1b/D1a receptor activation had little or no effect on Ca2+ currents. The D2 receptor antagonist sulpiride blocked the effect of D2 agonists. Dialysis with guanosine-5'-O-(2-thiodiphosphate) or brief exposure to the G protein (Gi/o) alkylating agent N-ethylmaleimide also blocked the D2 modulation. The reduction in N-type currents was neither accompanied by kinetic slowing nor significantly reversed by depolarizing prepulses. The D2 receptor effects were mediated by a membrane-delimited pathway, because the modulation was not seen in cell-attached patches when agonist was applied to the bath and was not disrupted by perturbations in cytosolic signaling pathways known to be linked to D2 receptors. Activation of M2 muscarinic receptors occluded the D2 modulation, suggesting a shared signaling element. However, activation of protein kinase C attenuated the M2 modulation without significantly affecting the D2 modulation. Taken together, our results suggest that activation of D2 dopamine receptors in cholinergic interneurons reduces N-type Ca2+ currents via a membrane-delimited, Gi/o class G protein pathway that is not regulated by protein kinase C. This signaling pathway may underlie the ability of D2 receptors to reduce striatal acetylcholine release.