Dopamine D 4 Receptor-Deficient Mice Display Cortical Hyperexcitability

The dopamine D(4) receptor (D(4)R) is predominantly expressed in the frontal cortex (FC), a brain region that receives dense input from midbrain dopamine (DA) neurons and is associated with cognitive and emotional processes. However, the physiological significance of this dopamine receptor subtype has been difficult to explore because of the slow development of D(4)R agonists and antagonists the selectivity and efficacy of which have been rigorously demonstrated in vivo. We have attempted to overcome this limitation by taking a multidimensional approach to the characterization of mice completely deficient in this receptor subtype. Electrophysiological current and voltage-clamp recordings were performed in cortical pyramidal neurons from wild-type and D(4)R-deficient mice. The frequency of spontaneous synaptic activity and the frequency and duration of paroxysmal discharges induced by epileptogenic agents were increased in mutant mice. Enhanced synaptic activity was also observed in brain slices of wild-type mice incubated in the presence of the selective D(4)R antagonist PNU-101387G. Consistent with greater electrophysiological activity, nerve terminal glutamate density associated with asymmetrical synaptic contacts within layer VI of the motor cortex was reduced in mutant neurons. Taken together, these results suggest that the D(4)R can function as an inhibitory modulator of glutamate activity in the FC.Fil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Cepeda, Carlos. University of California at Los Angeles; Estados UnidosFil: Hurst, Raymond S.. University of California at Los Angeles; Estados UnidosFil: Flores Hernandez, Jorge. University of California at Los Angeles; Estados UnidosFil: Ariano, Marjorie A.. The Chicago Medical School; Estados UnidosFil: Falzone, Tomas Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Kozell, Laura B.. Oregon Health Sciences University; Estados UnidosFil: Meshul, Charles K.. Oregon Health Sciences University; Estados UnidosFil: Bunzow, James R.. Oregon Health Sciences University; Estados UnidosFil: Low, Malcolm J.. Oregon Health Sciences University; Estados UnidosFil: Levine, Michael S.. University of California at Los Angeles; Estados UnidosFil: Grandy, David K.. Oregon Health Sciences University; Estados Unido

Differential coupling of dopaminergic D2 receptors expressed in different cell types. Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in LtK- fibroblasts, hyperpolarization, and cytosolic-free Ca2+ concentration decrease in GH4C1 cells.

Dopaminergic D2 receptors are widely regarded as typical inhibitory receptors, as they both inhibit adenylyl cyclase and decrease the cytosolic free Ca2+ concentration ([Ca2+]i) by activating K+ channels. A D2 receptor has recently been cloned (Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M. D., Machida, C. A., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787) and expressed in two different cell lines, pituitary GH4C1 cells and Ltk- fibroblasts, where it has been shown to induce inhibition of adenylyl cyclase. We have investigated the additional effector systems coupled to this receptor. The responses observed in the two cells lines, which express similar levels of receptors (0.5-1 x 10(5)/cell), were surprisingly different. In GH4C1 cells D2 receptors failed to affect phosphoinositide hydrolysis and induced a decrease of [Ca2+]i. This latter effect appears to be mediated by hyperpolarization, most likely due to the activation of K+ channels. In striking contrast, in Ltk- fibroblasts the D2 receptor induced a rapid stimulation of inositol(1,4,5)-trisphosphate (+73% at 15 s) followed by the other inositol phosphates, and an immediate increase of [Ca2+]i due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. In both GH4C1 and Ltk- cells, the D2 receptor response was mediated by G protein(s) sensitive to pertussis toxin. The increases of inositol trisphosphate and [Ca2+]i observed in Ltk- cells required dopamine concentrations only slightly higher than those inhibiting adenylyl cyclase (EG50 = 25, 29, and 11 nM, respectively) and were comparable in magnitude to the responses induced by the endogenous stimulatory receptor agonists, thrombin and ATP. The results demonstrate that in certain cells D2 receptors are efficiently coupled to the stimulation of phosphoinositide hydrolysis. The nature of receptor responses appears therefore to depend on the specific properties not only of the receptor molecule but also of the cell type in which it is expressed

Distinct regulation of dopamine D2S and D2L autoreceptor signaling by calcium

D2 autoreceptors regulate dopamine release throughout the brain. Two isoforms of the D2 receptor, D2S and D2L, are expressed in midbrain dopamine neurons. Differential roles of these isoforms as autoreceptors are poorly understood. By virally expressing the isoforms in dopamine neurons of D2 receptor knockout mice, this study assessed the calcium-dependence and drug-induced plasticity of D2S and D2L receptor-dependent G protein-coupled inwardly rectifying potassium (GIRK) currents. The results reveal that D2S, but not D2L receptors, exhibited calcium-dependent desensitization similar to that exhibited by endogenous autoreceptors. Two pathways of calcium signaling that regulated D2 autoreceptor-dependent GIRK signaling were identified, which distinctly affected desensitization and the magnitude of D2S and D2L receptor-dependent GIRK currents. Previous in vivo cocaine exposure removed calcium-dependent D2 autoreceptor desensitization in wild type, but not D2S-only mice. Thus, expression of D2S as the exclusive autoreceptor was insufficient for cocaine-induced plasticity, implying a functional role for the co-expression of D2S and D2L autoreceptors

MT-7716, a potent NOP receptor agonist, preferentially reduces ethanol seeking and reinforcement in post-dependent rats

Dysregulation of the nociceptin (N/OFQ) system has been implicated in alcohol abuse and alcoholism, and growing evidence suggests that targeting this system may be beneficial for treating alcoholism. To further explore the treatment target potential of the N/OFQ system, the novel non-peptide, small-molecule N/OFQ (NOP) agonist MT-7716, (R)-2-3-[1-(Acenaphthen-1-yl)piperidin-4-yl]-2-oxo-2,3-dihydro-1H-benzimidazol-1-yl-N-methylacetamide hydrochloride hydrate, was examined for its effects on ethanol self-administration and stress-induced reinstatement of alcohol seeking in non-dependent and post-dependent rats. Male Wistar rats were trained to self-administer ethanol and then made ethanol dependent via repeated intragastric ethanol intubation. The effects of MT-7716 (0.3 and 1 mg/kg; PO) on alcohol self-administration were determined 2 weeks following dependence induction, when baseline self-administration was restored. Effects of MT-7716 on stress-induced reinstatement were tested in separate cohorts of rats, 1 and 3 weeks post-withdrawal. MT-7716 reduced alcohol self-administration and stress-induced reinstatement of alcohol seeking in post-dependent rats, but was ineffective in non-dependent animals. Moreover, the prevention of stress-induced reinstatement by MT-7716 was more pronounced at 3 weeks post-dependence. The results further confirm treatment target potential for the NOP receptor and identify non-peptide NOP agonists as promising potential treatment drugs for alcohol abuse and relapse prevention. The findings also support dysregulation of the N/OFQ system as a factor in alcohol seeking and reinforcement

Endogenous Nociceptin / Orphanin FQ System Involvement in Hypothalamic-Pituitary-Adrenal Axis Responses: Relevance to Models of Inflammation

Nociceptin/orphanin FQ (N/OFQ) peptide and its receptor (NOP) function in the neuromodulation of anxiety, stress and hypothalamic-pituitary-adrenal (HPA) axis activity. We investigated the endogenous NOP system using the selective NOP antagonist, UFP-101, during the HPA axis response to bacterial endotoxin, lipopolysaccharide (LPS). Although i.c.v. N/OFQ (1 μg/rat) had no significant effect on LPS-induced (250 μg/rat i.p) plasma corticosterone release at 30 or 60 min post-i.c.v. injection, i.c.v. UFP-101 (1 μg/rat)/LPS significantly attenuated plasma adrenocorticotrophic hormone and corticosterone at the 30-min time-point compared to i.c.v saline (0.9%)/LPS. Parvocellular paraventricular nucleus (PVN) corticotrophin-releasing factor (CRF) and corticotrophic pro-opiomelanocortin (POMC), but not parvocellular PVN arginine vasopressin (AVP), mRNA expression was significantly increased by LPS compared to non-LPS control. Intracerebroventricular UFP-101/LPS treatment was associated with increased POMC mRNA expression 4 h after injection and a clear trend towards increased parvocellular CRF mRNA. Furthermore, i.c.v. UFP-101 was selectively associated with an LPS-induced increase in parvocellular AVP mRNA, an effect that was absent in the i.c.v saline/LPS group. To determine whether LPS challenge was associated with compensatory changes in N/OFQ precursor or NOP receptor mRNAs, in a separate study, we undertook reverse transcriptase-polymerase chain reaction analysis of preproN/OFQ and NOP transcripts. In support of an endogenous role for central N/OFQ in inflammatory stress, we found that LPS significantly increased preproN/OFQ transcript expression in the hypothalamus 4 h after injection compared to the saline control. No changes in preproN/OFQ mRNA level in the hippocampus or basal forebrain (including bed nucleus of stria terminalis) were seen, albeit at 4 h. LPS was associated with a significant attenuation of NOP mRNA in the basal forebrain at 4 h, possibly as a compensatory response to increased N/OFQ release. Although the exact mechanisms require elucidation, the findings obtained in the present study provide evidence indicating that the endogenous NOP system is involved in the acute HPA axis response to immune challenge

Decreased plasma nociceptin/orphanin FQ levels after acute coronary syndromes

Foregoing researches made on the N/OFQ system brought up a possible role for this system in cardiovascular regulation. In this study we examined how N/OFQ levels of the blood plasma changed in acute cardiovascular diseases. Three cardiac patient groups were created: enzyme positive acute coronary syndrome (EPACS, n = 10), enzyme negative ACS (ENACS, n = 7) and ischemic heart disease (IHD, n = 11). We compared the patients to healthy control subjects (n = 31). We found significantly lower N/OFQ levels in the EPACS [6.86 (6.21–7.38) pg/ml], ENACS [6.97 (6.87–7.01) pg/ml and IHD groups [7.58 (7.23–8.20) pg/ml] compared to the control group [8.86 (7.27–9.83) pg/ml]. A significant correlation was detected between N/OFQ and white blood cell count (WBC), platelet count (PLT), creatine kinase (CK), glutamate oxaloacetate transaminase (GOT) and cholesterol levels in the EPACS group.Decreased plasma N/OFQ is closely associated with the presence of acute cardiovascular disease, and the severity of symptoms has a significant negative correlation with the N/OFQ levels. We believe that the rate of N/OFQ depression is in association with the level of ischemic stress and the following inflammatory response. Further investigations are needed to clarify the relevance and elucidate the exact effects of the ischemic stress on the N/OFQ system

Repeated amphetamine treatment induces neurite outgrowth and enhanced amphetamine-stimulated dopamine release in rat pheochromocytoma cells (PC12 cells) via a protein kinase C- and mitogen activated protein kinase-dependent mechanism

Repeated intermittent treatment with amphetamine (AMPH) induces both neurite outgrowth and enhanced AMPH-stimulated dopamine (DA) release in PC12 cells. We investigated the role of protein kinases in the induction of these AMPH-mediated events by using inhibitors of protein kinase C (PKC), mitogen activated protein kinase (MAP kinase) or protein kinase A (PKA). PKC inhibitors chelerythrine (100 nm and 300 nm), Ro31-8220 (300 nm) and the MAP kinase kinase inhibitor, PD98059 (30 µm) inhibited the ability of AMPH to elicit both neurite outgrowth and the enhanced AMPH-stimulated DA release. The direct-acting PKC activator, 12- O -tetradecanoyl phorbol 13-acetate (TPA, 250 nm) mimicked the ability of AMPH to elicit neurite outgrowth and enhanced DA release. On the contrary, a selective PKA inhibitor, 100 µm Rp-8-Br-cAMPS, blocked only the development of AMPH-stimulated DA release but not the neurite outgrowth. Treatment of the cells with acute AMPH elicited an increase in the activity of PKC and MAP kinase but not PKA. These results demonstrated that AMPH-induced increases in MAP kinase and PKC are important for induction of both the enhancement in transporter-mediated DA release and neurite outgrowth but PKA was only required for the enhancement in AMPH-stimulated DA release. Therefore the mechanisms by which AMPH induces neurite outgrowth and the enhancement in AMPH-stimulated DA release can be differentiated.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66040/1/j.1471-4159.2003.02127.x.pd

Dopamine receptor mRNA expression patterns by opioid peptide cells in the nucleus accumbens of the rat: A double in situ hybridization study

Colocalization of proenkephalin and prodynorphin mRNAs with each other as well as with D1, D2, and D3 dopamine receptor mRNAs was analyzed in the nucleus accumbens of the rat. Distinct combinations were detected in the rostral pole, core, and shell subdivisions of the nucleus accumbens. Proenkephalin and prodynorphin mRNAs were principally localized in separate cells in the core. All detectable prodynor cells in the core expressed D1 mRNA but not D2 mRNA. Conversely, approximately 95% of the proenkephalin-positive cells in this region expressed D2 mRNA but not D1 mRNA. This pattern was identical to that observed in the caudate putamen. In the rostral pole and the shell, embedded in a background of this “typical” colocalization pattern, clusters of cells expressing a distinct configuration were found. In these clusters, proenkephalin-positive cells expressed both prodynorphin and D1 mRNAs, but they did not express D2 mRNA. D3 and prodynorphin mRNAs were colocalized in “limbic” striatal areas, including the ventromedial caudate putamen, the rostral pole, and the medial shell. In contrast, D3 mRNA was not detected in any proenkephalin-positive cells. Together with the prodynorphin/D1 data, this suggests that a subset of prodynorphin cells expresses both D1 and D3 mRNAs. It is concluded that (1) clusters of cells that coexpress proenkephalin, prodynorphin, and D1 mRNAs overlap extensively with previously defined cytoarchitectural cell clusters in the nucleus accumbens and (2) a subset of the prodynorphin cells in the ventromedial caudate putamen and the nucleus accumbens contains both D1 and D3 mRNAs. © 1995 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50067/1/903610106_ftp.pd

Structural and Functional Evolution of the Trace Amine-Associated Receptors TAAR3, TAAR4 and TAAR5 in Primates

The family of trace amine-associated receptors (TAAR) comprises 9 mammalian TAAR subtypes, with intact gene and pseudogene numbers differing considerably even between closely related species. To date the best characterized subtype is TAAR1, which activates the Gs protein/adenylyl cyclase pathway upon stimulation by trace amines and psychoactive substances like MDMA or LSD. Recently, chemosensory function involving recognition of volatile amines was proposed for murine TAAR3, TAAR4 and TAAR5. Humans can smell volatile amines despite carrying open reading frame (ORF) disruptions in TAAR3 and TAAR4. Therefore, we set out to study the functional and structural evolution of these genes with a special focus on primates. Functional analyses showed that ligands activating the murine TAAR3, TAAR4 and TAAR5 do not activate intact primate and mammalian orthologs, although they evolve under purifying selection and hence must be functional. We also find little evidence for positive selection that could explain the functional differences between mouse and other mammals. Our findings rather suggest that the previously identified volatile amine TAAR3–5 agonists reflect the high agonist promiscuity of TAAR, and that the ligands driving purifying selection of these TAAR in mouse and other mammals still await discovery. More generally, our study points out how analyses in an evolutionary context can help to interpret functional data generated in single species

Differential Modulation of Beta-Adrenergic Receptor Signaling by Trace Amine-Associated Receptor 1 Agonists

Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes