AB1558 Sigma-AldrichAnti-Dopamine D2 Receptor Antibody

Female sexual behavior is an established model of a naturally motivated behavior which is regulated by activity within the mesolimbic dopamine system. Repeated activation of the mesolimbic circuit by female sexual behavior elevates dopamine release and produces persistent postsynaptic alterations to dopamine D1 receptor signaling within the nucleus accumbens. Here we demonstrate that sexual experience in female Syrian hamsters significantly increases spine density and alters morphology selectively in D1 receptor-expressing medium spiny neurons within the nucleus accumbens core, with no corresponding change in dopamine receptor binding or protein expression. Our findings demonstrate that previous life experience with a naturally motivated behavior has the capacity to induce persistent structural alterations to the mesolimbic circuit that can increase reproductive success and are analogous to the persistent structural changes following repeated exposure to many drugs of abuse.

Injury to the central nervous system (CNS) can result in lifelong loss of function due in part to the regenerative failure of CNS neurons. Inhibitory proteins derived from myelin and the astroglial scar are major barriers for the successful regeneration of injured CNS neurons. Previously, we described the identification of a novel compound, F05, which promotes neurite growth from neurons challenged with inhibitory substrates in vitro, and promotes axonal regeneration in vivo (Usher et al., 2010). To identify additional regeneration-promoting compounds, we used F05-induced gene expression profiles to query the Broad Institute Connectivity Map, a gene expression database of cells treated with greater than 1300 compounds. Despite no shared chemical similarity, F05-induced changes in gene expression were remarkably similar to those seen with a group of piperazine phenothiazine antipsychotics (PhAPs). In contrast to antipsychotics of other structural classes, PhAPs promoted neurite growth of CNS neurons challenged with two different glial derived inhibitory substrates. Our pharmacological studies suggest a mechanism whereby PhAPs promote growth through antagonism of calmodulin signaling, independent of dopamine receptor antagonism. These findings shed light on mechanisms underlying neurite-inhibitory signaling, and suggest that clinically approved antipsychotic compounds may be repurposed for use in CNS injured patients.

It is well known that dopamine imbalances are associated with many psychiatric disorders and that the dopaminergic receptor D₂ is the main target of antipsychotics. Recently it was shown that levels of two proteins implicated in dopaminergic signaling, Neuronal calcium sensor-1 (NCS-1) and DARPP-32, are altered in the prefrontal cortex (PFC) of both schizophrenic and bipolar disorder patients. NCS-1, which inhibits D₂ internalization, is upregulated in the PFC of both patients. DARPP-32, which is a downstream effector of dopamine signaling, integrates the pathways of several neurotransmitters and is downregulated in the PFC of both patients. Here, we used PC12 cells stably overexpressing NCS-1 (PC12-NCS-1 cells) to address the function of this protein in DARPP-32 signaling pathway in vitro. PC12-NCS-1 cells displayed downregulation of the cAMP/PKA pathway, with decreased levels of cAMP and phosphorylation of CREB at Ser133. We also observed decreased levels of total and phosphorylated DARPP-32 at Thr34. However, these cells did not show alterations in the levels of D₂ and phosphorylation of DARPP-32 at Thr75. These results indicate that NCS-1 modulates PKA/cAMP signaling pathway. Identification of the cellular mechanisms linking NCS-1 and DARPP-32 may help in the understanding the signaling machinery with potential to be turned into targets for the treatment of schizophrenia and other debilitating psychiatric disorders.

Dopamine receptors (DRs) are members of seven transmembrane domain trimeric guanosine 5'-triphosphate (GTP)-binding protein-coupled receptor family. Through dopamine receptor activation, dopamine plays a significant role in regulating gene expression, such as induced tumor cell migration.

here is considerable evidence that dopamine D2 receptors can modulate AMPA receptor-mediated neurotoxicity. However, the molecular mechanism underlying this process remains essentially unclear. Here we report that D2 receptors inhibit AMPA-mediated neurotoxicity through two pathways: the activation of phosphoinositide-3 kinase (PI-3K) and downregulation of AMPA receptor plasma membrane expression, both involving a series of protein-protein coupling/uncoupling events. Agonist stimulation of D2 receptors promotes the formation of the direct protein-protein interaction between the third intracellular loop of the D2 receptor and the ATPase N-ethylmaleimide-sensitive factor (NSF) while uncoupling the NSF interaction with the carboxyl tail (CT) of the glutamate receptor GluR2 subunit of AMPA receptors. Previous studies have shown that full-length NSF directly couples to the GluR2CT and facilitates AMPA receptor plasma membrane expression. Furthermore, the CT region of GluR2 subunit is also responsible for several other intracellular protein couplings, including p85 subunit of PI-3K. Therefore, the direct coupling of D2-NSF and concomitant decrease in the NSF-GluR2 interaction results in a decrease of AMPA receptor membrane expression and an increase in the interaction between GluR2 and the p85 and subsequent activation of PI-3K. Disruption of the D2-NSF interaction abolished the ability of D2 receptor to attenuate AMPA-mediated neurotoxicity by blocking the D2 activation-induced changes in PI-3K activity and AMPA receptor plasma membrane expression. Furthermore, the D2-NSF-GluR2-p85 interactions are also responsible for the D2 inhibition of ischemia-induced cell death. These data may provide a new avenue to identify specific targets for therapeutics to modulate glutamate receptor-governed diseases, such as stroke.

We characterized undifferentiated (UN) and three differentiation conditions of the SH-SY5Y neuroblastoma cell line for phenotypic markers of dopaminergic cells, sensitivity to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium ion (MPP+), the requirement to utilize the dopamine (DA) transporter (DAT) for MPP+ toxicity, and the neuroprotective effects of pramipexole. Cells were differentiated with retinoic acid (RA), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and RA followed by TPA (RA/TPA). RA/TPA treated cells exhibited the highest levels of tyrosine hydroxylase and DAT but lower levels of vesicular monoamine transporter. The kinetics of [3H]DA uptake and [3H]MPP+ uptake to DAT in RA/TPA differentiated cells were similar to that of rat and mouse caudate-putamen synaptosomes. RA/TPA differentiated cells evidenced high sensitivity to the neurotoxic effects of MPP+ (0.03 to 3.0 mM), and the neurotoxic effects of MPP+ were blocked with the DAT inhibitor 1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine (GBR 12909). DA-induced cell death was not more sensitive in RA vs RA/TPA differentiated cells and was not inhibited by transporter inhibitors. RA/TPA differentiated cells exhibited 3-fold and 6-fold higher levels, respectively, of DA D2 and D3 receptors than UN or RA differentiated cells. Pretreatment with pramipexole was protective against MPP+ in the RA/TPA differentiated cells but not in undifferentiated or RA differentiated cells. The neuroprotective effect of pramipexole was concentration-dependent and dopamine D2/D3 receptor dependent. In contrast, protection by pramipexole against DA was not DA receptor dependent. Further characterization of the neuroprotective effects of DA agonists in this model system can provide unique information about DA receptor dependent and independent mechanisms of neuroprotection.

Human immunodeficiency virus (HIV) infection continues to increase at alarming rates in drug abusers, especially in women. Drugs of abuse can cause long-lasting damage to the brain and HIV infection frequently leads to a dementing illness. To determine how these drugs interact with HIV to cause CNS damage, we used an in vitro human neuronal culture characterized for the presence of dopaminergic receptors, transporters and estrogen receptors. We determined the combined effects of dopaminergic drugs, methamphetamine, or cocaine with neurotoxic HIV proteins, gp120 and Tat.Acute exposure to these substances resulted in synergistic neurotoxic responses as measured by changes in mitochondrial membrane potential and neuronal cell death. Neurotoxicity occurred in a sub-population of neurons. Importantly, the presence of 17beta-estradiol prevented these synergistic neurotoxicities and the neuroprotective effects were partly mediated by estrogen receptors.Our observations suggest that methamphetamine and cocaine may affect the course of HIV dementia, and additionally suggest that estrogens modify the HIV-drug interactions.

Dopaminergic cells in the retina express the receptor for brain-derived neurotrophic factor (BDNF) (). To investigate whether BDNF can influence the development of the retinal dopaminergic pathway, we performed intraocular injections of BDNF during the second or third postnatal week and visualized the dopaminergic system with tyrosine hydroxylase (TH) immunohistochemistry. Both regimens of BDNF treatment caused an increase in TH immunoreactivity in stratum 1 and stratum 3 of the inner plexiform layer (IPL). D2 dopamine receptor immunoreactivity, a presynaptic marker of dopaminergic cells (), was also increased in stratum 1 and stratum 3 of the inner plexiform layer. These data suggest that BDNF causes sprouting of dopaminergic fibers in the inner plexiform layer. Other neurochemical systems, for example, the cholinergic amacrine cells, remained unaffected. Similar effects were observed after injections of neurotrophin-3 and neurotrophin-4, but not nerve growth factor. Analysis of whole-mounted TH-immunolabeled retinae revealed hypertrophy of dopaminergic cells (+41% in soma areas; p < 0.01) and an increase of labeled dopaminergic varicosities in stratum 1 of the IPL (+51%; p < 0.01) after BDNF treatment. The opposite was observed in mice homozygous for a null mutation of the bdnf gene: dopaminergic cells were atrophic (-22.5% in soma areas; p < 0.05), and the density of TH-positive varicosities in stratum 1 was reduced (57%; p < 0.01). We conclude that BDNF controls the development of the retinal dopaminergic network and may be particularly important in determining the density of dopaminergic innervation in the retina.