ABSTRACTderived from the 129/sv strain (Clontech) was screened using a mouse D3 cDNA probe (17). A positive clone encompassing exon 2 of the murine D3 gene was isolated and further characterized. A 7-kb Xho I-Asp718 fragment was engineered for targeted mutagenesis by introducing the GKNeo cassette (16) in antisense orientation at the Sal I site in exon 2 (17). Integration of sequences derived from the pGKNeo cassette generates a novel open reading frame, resulting in the following peptide sequence appended after Arg-148: PASDGIRT-WQNNTENEVYVEQRLLISFFRL Opal (Stop). The sequence of the mutant allele was confirmed by direct sequencing of reverse transcription-PCR (rPCR) products derived from brain mRNAs of -/-and +/-mice (data not shown).Transfection ofES Cells and Embryo Manipulations. J-1 ES cells (a kind gift of R. Jaenisch, Massachussetts Institute of Technology) at passage 13 were grown on mitomycin C-treated embryonic fibroblasts derived from a homozygous neomycin (Neo)-resistant transgenic mouse (16). Cells (2 x 107) were electroporated in a 1-ml cuvette (path length-0.2 cm) at 0.4 kV and 25 ,uF. Cells were plated onto 40 gelatin-coated Petri dishes (6 cm) on embryonic feeder cells. Selection with G418 (0.3 mg/ml; active concentration of 0.66 ,vg/mg of dry powder; GIBCO) was applied 24 hr after plating and was continued for 7-9 days. Individual Neo-resistant colonies were picked using a dissection microscope and expanded as described (16). Genomic DNA was prepared from an aliquot of cells for each clone using previously described techniques and analyzed by Southern blotting (18). Recovery, microinjection, and transfer of 3.5 day postcoitus embryos was performed as described (16).

Since dopamine receptors are important in the regulation of renal and cardiovascular function, we studied the cardiovascular consequences of the disruption of the D 3 receptor, a member of the family of D 2 -like receptors, expressed in renal proximal tubules and juxtaglomerular cells. Systolic and diastolic blood pressures were higher ( ‫ف‬ 20 mmHg) in heterozygous and homozygous than in wild-type mice. An acute saline load increased urine flow rate and sodium excretion to a similar extent in wild-type and heterozygous mice but the increase was attenuated in homozygous mice. Renal renin activity was much greater in homozygous than in wild-type mice; values for heterozygous mice were intermediate. Blockade of angiotensin II subtype-1 receptors decreased systolic blood pressure for a longer duration in mutant than in wild-type mice. Thus, disruption of the D 3 receptor increases renal renin production and produces renal sodium retention and renin-dependent hypertension. ( J .

We have determined the crystal structure at 1.8 A resolution of a complex of alpha-bungarotoxin with a high affinity 13-residue peptide that is homologous to the binding region of the alpha subunit of acetylcholine receptor. The peptide fits snugly to the toxin and adopts a beta hairpin conformation. The structures of the bound peptide and the homologous loop of acetylcholine binding protein, a soluble analog of the extracellular domain of acetylcholine receptor, are remarkably similar. Their superposition indicates that the toxin wraps around the receptor binding site loop, and in addition, binds tightly at the interface of two of the receptor subunits where it inserts a finger into the ligand binding site, thus blocking access to the acetylcholine binding site and explaining its strong antagonistic activity.

Dopamine release is activated by ethanol and addicting drugs, but molecular mechanisms linking dopaminergic signaling to neuronal responses and drinking behavior are poorly understood. We report that dopamine-D2 receptors induce PKA Calpha translocation and increase CRE-regulated gene expression. Ethanol also activates PKA signaling. Subthreshold concentrations of the D2 agonist NPA and ethanol, without effect alone, together cause synergistic PKA translocation and CRE-mediated gene transcription. D2 or adenosine A2 receptor blockade, pertussis toxin, Rp-cAMPS, or overexpression of dominant-negative peptides that sequester betagamma dimers prevent synergy. Importantly, overexpression of a betagamma inhibitor peptide in the nucleus accumbens strikingly reduces sustained alcohol consumption. We propose that synergy of D2 and A2 confers ethanol hypersensitivity and that betagamma dimers are required for voluntary drinking.

Neuro-immune interactions enable mutual regulation of the nervous and immune systems. To date, evidence exists for manipulations of immune cells by neurotransmitters in the periphery. In this study, we suggest the existence of a pathway by which the brain affects immune cells. The pathway we describe here is mediated by dopamine receptors expressed on activated T cells, termed blasts. Blasts can cross the blood brain barrier regardless of antigen specificity and can therefore encounter neurotransmitters in the brain. We show that blasts have a unique response to dopaminergic activation, which has no counterpart in resting T cells. Dopaminergic activation of blasts induces a Th1 bias in their cytokine profile and causes changes in surface marker expression. We further suggest that these changes can subsequently be transferred to peripheral T cells. We have tested this pathway in two in vivo systems: in rats exogenously administered with L-dopa, and in schizophrenia, which is characterized by a central nervous system-restricted increase in dopamine. In both models, peripheral T cells exhibit similar features to those of dopaminergically activated blasts. The existence of such a pathway by which the brain can regulate immune cells opens a conceptually new direction in neuro-immune interactions.