D2 dopamine receptor expression and trafficking is regulated through direct interactions with ZIP.

We have used the yeast two-hybrid system to identify protein kinase C-zeta interacting protein (ZIP) as a novel interacting protein for the D(2) dopamine receptor (DAR). This interaction was identified by screening a rat brain cDNA library using the third intracellular loop of the D(2) DAR as bait. A partial-length cDNA encoding ZIP was isolated and characterized as specifically interacting with the third intracellular loop of the D(2) DAR, but not with the third intracellular loops of other DAR subtypes. Biochemical confirmation of the ZIP-D(2) DAR interaction was obtained by expressing the full-length ZIP and D(2) DAR proteins in mammalian cells and demonstrating that they could be co-immunoprecipitated. We further showed that ZIP and the D(2) DAR could be co-immunoprecipitated from endogenous brain tissues. Immunohistochemical analyses further revealed that ZIP and the D(2) DAR were extensively co-localized within numerous neurons in various brain regions. ZIP exists as three protein isoforms of varying length, which are derived from alternative RNA splicing. All three isoforms were found to interact with the D(2) DAR, which allowed for the delineation of the receptor interacting domain to within 38 residues of ZIP. Functionally, over-expression of ZIP was found to result in decreased expression of the D(2) DAR with a corresponding decrease in receptor modulation of cAMP accumulation. Confocal microscopy revealed that ZIP over-expression also lead to an intracellular accumulation of D(2) DAR protein in lysosome compartments. These results suggest that ZIP can physically interact with the D(2) DAR leading to increased intracellular trafficking to lysosomes with subsequent down-regulation of receptor expression and function.

Ethanol regulation of D(1) dopamine receptor signaling is mediated by protein kinase C in an isozyme-specific manner.

Ethanol consumption potentiates dopaminergic signaling that is partially mediated by the D(1) dopamine receptor; however, the mechanism(s) underlying ethanol-dependent modulation of D(1) signaling is unclear. We now show that ethanol treatment of D(1) receptor-expressing cells decreases D(1) receptor phosphorylation and concurrently potentiates dopamine-stimulated cAMP accumulation. Protein kinase C (PKC) inhibitors mimic the effects of ethanol on D(1) receptor phosphorylation and dopamine-stimulated cAMP levels in a manner that is non-additive with ethanol treatment. Ethanol was also found to modulate specific PKC activities as demonstrated using in vitro kinase assays where ethanol treatment attenuated the activities of lipid-stimulated PKCgamma and PKCdelta in membrane fractions, but did not affect the activities of PKCalpha, PKCbeta(1), or PKCvarepsilon. Importantly, ethanol treatment potentiated D(1) receptor-mediated DARPP-32 phosphorylation in rat striatal slices, supporting the notion that ethanol enhances D(1) receptor signaling in vivo. These findings suggest that ethanol inhibits the activities of specific PKC isozymes, resulting in decreased D(1) receptor phosphorylation and enhanced dopaminergic signaling.

Dopamine reduction of GABA currents in striatal medium-sized spiny neurons is mediated principally by the D(1) receptor subtype.

Dopamine modulates voltage- and ligand-gated currents in striatal medium-sized neurons (MSNs) through the activation of D1- and D2-like family receptors. GABA(A) receptor-mediated currents are reduced by D1 receptor agonists, but the relative contribution of D(1) or D(5 )receptors in this attenuation has been elusive due to the lack of selective pharmacological agents. Here we examined GABA(A) receptor-mediated currents and the effects of D1 agonists on MSNs from wildtype and D(1) or D(5 )receptor knockout (KO) mice. Immunohistochemical and single-cell RT-PCR studies demonstrated a lack of compensatory effects after genetic deletion of D(1) or D(5) receptors. However, the expression of GABA(A )receptor alpha1 subunits was reduced in D(5) KO mice. At the functional level, whole-cell patch clamp recordings in dissociated MSNs showed that GABA peak current amplitudes were smaller in cells from D(5) KO mice indicating that lack of this receptor subtype directly affected GABA(A)-mediated currents. In striatal slices, addition of a D1 agonist reduced GABA currents significantly more in D(5) KO compared to D(1) KO mice. We conclude that D(1) receptors are the main D1-like receptor subtype involved in the modulation of GABA currents and that D(5) receptors contribute to the normal expression of these currents in the striatum.

Partial dopamine loss enhances activated caspase-3 activity: differential outcomes in striatal projection systems.

Parkinson's disease (PD) is a basal ganglia disorder. Motor symptoms develop insidiously following substantial neurodegeneration of the dopamine (DA) neurons in the nigrostriatal system and produce slowed, infrequent movements, postural instability, and gait changes. A thorough understanding of neurochemical compensations occurring in the striatum during early stages of PD is crucial in identifying components that are altered initially as the DA is depleted. Producing an incomplete lesion of the nigrostriatal DA system in rats would mimic the principal early neurochemical features of human PD. We infused 6-hydroxydopamine unilaterally into the substantia nigra to reach a target of approximately 50% depletion in striatal DA at 4 weeks. This was evaluated by HPLC analysis of tissue DA content and monitored behaviorally by forepaw use reflecting asymmetries in striatal DA levels. DA loss was assessed by using tyrosine hydroxylase immunohistochemical staining, and the data were conjoined with the behavioral assessments. We found that activated caspase-3, its actin cleavage product fractin, and components of the apoptosome were increased significantly in DA-depleted striatum. Thus mobilization of the intrinsic programmed cell death pathway occurred, without cell loss. Elevations in apoptogenic proteins were pronounced in enkephalinergic striatopallidal neurons compared with the substance P-containing striatonigral neurons. Our findings suggest that cellular homeostatic imbalances that accompany even mild striatal DA depletion take time to develop, differentially affect the striatal output pathways, and may be an important feature of early-stage PD. These observations could be capitalized upon to develop therapeutic interventions in the preclinical phases of the disorder.

Neuronal vulnerability in mouse models of Huntington's disease: membrane channel protein changes.

Huntington's disease (HD) is caused by a polyglutamine expansion that results in atrophy of the striatum and frontal cortex during disease progression. HD-susceptible striatal neurons are affected chronologically with initial degeneration of the striatopallidal neurons then the striatonigral projections, whereas large aspiny striatal interneurons (LAN) survive. Two classes of critical membrane proteins were evaluated in transgenic mouse models to determine their association with HD susceptibility, which leads to dysfunction and death in selected striatal neuron populations. We examined potassium (K+) channel protein subunits that form membrane ionophores conducting inwardly and outwardly rectifying K+ currents. K+ channel protein staining was diminished substantially in the HD striatal projection neurons but was not expressed in the HD-resistant LAN. Because loss of K+ channel subunits depolarizes neurons, other voltage-gated ionophores will be affected. N-methyl-D-aspartate (NMDA) receptors and their phosphorylation by cyclic AMP were studied as a mechanism contributing to excitotoxic vulnerability in striatal projection neurons that would lose voltage regulation after diminished K+ channels. NR1 subunits showed significant elevation in the HD transgenic projection systems but were expressed at very low levels in LAN. NR1 subunit phosphorylation by cyclic AMP also was enhanced in striatal projection neurons but not in LAN. Cyclic AMP-driven phosphorylation of NMDA receptors increases the channel open time and elevates neuronal glutamate responsiveness, which may lead to excitotoxicity. Together our data suggest that changes in these proteins and their modification may predispose striatal projection neurons to dysfunction and then degeneratation in HD and provide a mechanism for LAN resistance in the disease.

Striatal potassium channel dysfunction in Huntington's disease transgenic mice.

Huntington's disease (HD) is a neurodegenerative disorder that mainly affects the projection neurons of the striatum and cerebral cortex. Genetic mouse models of HD have shown that neurons susceptible to the mutation exhibit morphological and electrophysiological dysfunctions before and during development of the behavioral phenotype. We used HD transgenic mouse models to examine inwardly and outwardly rectifying K+ conductances, as well as expression of some related K+ channel subunits. Experiments were conducted in slices and dissociated cells from two mouse models, the R6/2 and TgCAG100, at the beginning and after full development of overt behavioral phenotypes. Striatal medium-sized spiny neurons (MSNs) from symptomatic transgenic mice had increased input resistances, depolarized resting membrane potentials, and reductions in both inwardly and outwardly rectifying K+ currents. These changes were more dramatic in the R6/2 model than in the TgCAG100. Parallel immunofluorescence studies detected decreases in the expression of K+ channel subunit proteins, Kir2.1, Kir2.3, and Kv2.1 in MSNs, which contribute to the formation of the channel ionophores for these currents. Attenuation in K+ conductances and channel subunit expression contribute to altered electrophysiological properties of MSNs and may partially account for selective cellular vulnerability in the striatum.

Increased GABAergic function in mouse models of Huntington's disease: reversal by BDNF.

Huntington's disease (HD) is characterized by loss of striatal gamma-aminobutyric acid (GABA)ergic medium-sized spiny projection neurons (MSSNs), whereas some classes of striatal interneurons are relatively spared. Striatal interneurons provide most of the inhibitory synaptic input to MSSNs and use GABA as their neurotransmitter. We reported previously alterations in glutamatergic synaptic activity in the R6/2 and R6/1 mouse models of HD. In the present study, we used whole-cell voltage clamp recordings to examine GABAergic synaptic currents in MSSNs from striatal slices in these two mouse models compared to those in age-matched control littermates. The frequency of spontaneous GABAergic synaptic currents was increased significantly in MSSNs from R6/2 transgenics starting around 5-7 weeks (when the overt behavioral phenotype begins) and continuing in 9-14-week-old mice. A similar increase was observed in 12-15-month-old R6/1 transgenics. Bath application of brain-derived neurotrophic factor, which is downregulated in HD, significantly reduced the frequency of spontaneous GABAergic synaptic currents in MSSNs from R6/2 but not control mice at 9-14 weeks. Increased GABA current densities also occurred in acutely isolated MSSNs from R6/2 animals. Immunofluorescence demonstrated increased expression of the ubiquitous alpha1 subunit of GABA(A) receptors in MSSNs from R6/2 animals. These results indicate that increases in spontaneous GABAergic synaptic currents and postsynaptic receptor function occur in parallel to progressive decreases in glutamatergic inputs to MSSNs. In conjunction, both changes will severely alter striatal outputs to target areas involved in the control of movement.

Morphological and electrophysiological characterization of abnormal cell types in pediatric cortical dysplasia.

The mechanisms responsible for seizure generation in cortical dysplasia (CD) are unknown, but morphologically abnormal cells could contribute. We examined the passive and active membrane properties of cells from pediatric CD in vitro. Normal- and abnormal-appearing cells were identified morphologically by using infrared videomicroscopy and biocytin in slices from children with mild to severe CD. Electrophysiological properties were assessed with patch clamp recordings. Four groups of abnormal-appearing cells were observed. The first consisted of large, pyramidal cells probably corresponding to cytomegalic neurons. Under conditions that reduced the contribution of K(+) conductances, these cells generated large Ca(2+) currents and influx when depolarized. When these cells were acutely dissociated, peak Ca(2+) currents and densities were greater in cytomegalic compared with normal-appearing pyramidal neurons. The second group included large, nonpyramidal cells with atypical somatodendritic morphology that could correspond to "balloon" cells. These cells did not display active voltage- or ligand-gated currents and did not appear to receive synaptic inputs. The third group included misoriented and dysmorphic pyramidal neurons, and the fourth group consisted of immature-looking pyramidal neurons. Electrophysiologically, neurons in these latter two groups did not display significant abnormalities when compared with normal-appearing pyramidal neurons. We conclude that there are cells with abnormal intrinsic membrane properties in pediatric CD. Among the four groups of cells, the most abnormal electrophysiological properties were displayed by cytomegalic neurons and large cells with atypical morphology. Cytomegalic neurons could play an important role in the generation of epileptic activity.

Transient and progressive electrophysiological alterations in the corticostriatal pathway in a mouse model of Huntington's disease.

Alterations in the corticostriatal pathway may precede symptomatology and striatal cell death in Huntington's disease (HD) patients. Here we examined spontaneous EPSCs in striatal medium-sized spiny neurons in slices from a mouse model of HD (R6/2). Spontaneous EPSC frequency was similar in young (3-4 weeks) transgenics and controls but decreased significantly in transgenics when overt behavioral symptoms began (5-7 weeks) and was most pronounced in severely impaired transgenics (11-15 weeks). These differences were maintained after bicuculline or tetrodotoxin, indicating they were specific to glutamatergic input and likely presynaptic in origin. Decreases in presynaptic and postsynaptic protein markers, synaptophysin and postsynaptic density-95, occurred in 11-15 week R6/2 mice, supporting the electrophysiological results. Furthermore, isolated, large-amplitude synaptic events (>100 pA) occurred more frequently in transgenic animals, particularly at 5-7 weeks, suggesting additional dysregulation of cortical inputs. Large events were blocked by tetrodotoxin, indicating a possible cortical origin. Addition of bicuculline and 4-aminopyridine facilitated the occurrence of large events. Riluzole, a compound that decreases glutamate release, reduced these events. Together, these observations indicate that both progressive and transient alterations occur along the corticostriatal pathway in experimental HD. These alterations are likely to contribute to the selective vulnerability of striatal medium-sized spiny neurons.

Mice lacking D5 dopamine receptors have increased sympathetic tone and are hypertensive.

Dopamine is an important transmitter in the CNS and PNS, critically regulating numerous neuropsychiatric and physiological functions. These actions of dopamine are mediated by five distinct receptor subtypes. Of these receptors, probably the least understood in terms of physiological functions is the D5 receptor subtype. To better understand the role of the D5 dopamine receptor (DAR) in normal physiology and behavior, we have now used gene-targeting technology to create mice that lack this receptor subtype. We find that the D5 receptor-deficient mice are viable and fertile and appear to develop normally. No compensatory alterations in other dopamine receptor subtypes were observed. We find, however, that the mutant mice develop hypertension and exhibit significantly elevated blood pressure (BP) by 3 months of age. This hypertension appears to be caused by increased sympathetic tone, primarily attributable to a CNS defect. Our data further suggest that this defect involves an oxytocin-dependent sensitization of V1 vasopressin and non-NMDA glutamatergic receptor-mediated pathways, potentially within the medulla, leading to increased sympathetic outflow. These results indicate that D5 dopamine receptors modulate neuronal pathways regulating blood pressure responses and may provide new insights into mechanisms for some forms of essential hypertension in humans, a disease that afflicts up to 25% of the aged adult population in industrialized societies.

D1 dopamine receptor stimulation increases GluR1 surface expression in nucleus accumbens neurons.

The goal of this study was to understand how dopamine receptors, which are activated during psychostimulant administration, might influence glutamate-dependent forms of synaptic plasticity that are increasingly recognized as important to drug addiction. Regulation of the surface expression of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit GluR1 plays a critical role in long-term potentiation, a well-characterized form of synaptic plasticity. Primary cultures of rat nucleus accumbens neurons were used to examine whether dopamine receptor stimulation influences cell surface expression of GluR1, detected using antibody to the extracellular portion of GluR1 and fluorescence microscopy. Surface GluR1 labeling on processes of medium spiny neurons and interneurons was increased by brief (5-15 min) incubation with a D1 agonist (1 microm SKF 81297). This effect was attenuated by the D1 receptor antagonist SCH 23390 (10 microm) and reproduced by the adenylyl cyclase activator forskolin (10 microm). Labeling was decreased by glutamate (10-50 microm, 15 min). These results are the first to demonstrate modulation of AMPA receptor surface expression by a non-glutamatergic G protein-coupled receptor. Normally, this may enable ongoing regulation of AMPA receptor transmission in response to changes in the activity of dopamine projections to the nucleus accumbens. When dopamine receptors are over-stimulated during chronic drug administration, this regulation may be disrupted, leading to inappropriate plasticity in neuronal circuits governing motivation and reward.

Neurofilament-M interacts with the D1 dopamine receptor to regulate cell surface expression and desensitization.

We used the yeast two-hybrid assay to identify novel proteins that interact with the D(1) dopamine receptor. The third cytoplasmic loop (residues 217-273) of the rat D(1) receptor was used as bait to identify clones encoding interacting proteins from a rat brain cDNA library. This identified two clones encoding the C terminus of rat neurofilament-M (NF-M) (residues 782-846). The NF-M clone did not interact with the third cytoplasmic loops of the rat D(2), D(3), or D(4) receptors, but showed weak interaction with that of the D(5) receptor. Coexpression of full-length NF-M with the D(1) receptor in HEK-293 cells resulted in >50% reduction of receptor binding accompanied by a reduction in D(1) receptor-mediated cAMP accumulation. NF-M had no effect on the expression of other dopamine receptor subtypes. Using a D(1) receptor-green fluorescent protein chimera and confocal fluorescence microscopy, we found that NF-M reduced D(1) receptor expression at the cell surface and promoted accumulation of the receptor in the cytosol. Interestingly, the D(1) receptors that were expressed at the cell surface in the presence of NF-M were resistant to agonist-induced desensitization. Cellular colocalization of NF-M and the D(1) receptor in the rat brain was examined by epifluorescence microscopy. These experiments showed that approximately 50% of medium-sized striatal neurons expressed both proteins. Colocalization was also observed in pyramidal cells and interneurons within the frontal cortex. Similar immunohistochemical analyses using NF-M-deficient mice showed decrements in D(1) receptor expression compared with control mice. These results suggest that NF-M interacts with the D(1) receptor in vivo and may modify its expression and regulation.

Striatal neurochemical changes in transgenic models of Huntington's disease.

Transgenic mouse models of Huntington's disease (HD) were examined following the onset of overt behavioral symptoms. The HD transgenic mice demonstrated profound striatal losses in D1, D2, and D3 dopamine (DA) receptor proteins in comparison with their nonsymptomatic, age-matched littermate controls. In parallel, a robust increase in the striatal D5 DA receptor subtype occurred in the transgenic compared with the wild-type control mice. This receptor elevation was accompanied by heightened cyclic AMP levels, which may be induced by the adenylyl cyclase-linked D5 receptor. This is a unique result; normal striatal D5 protein levels are modest and not thought to contribute substantially to cyclic AMP-mediated DA signaling mechanisms. Simple compensatory up-regulation of D5 DA receptors in response to D1 receptor subtype loss does not explain our findings, because genetic inactivation of the D1 DA receptor does not alter levels of D5 DA receptor expression. Immunofluorescent detection of tyrosine hydroxylase showed that nigrostriatal DA containing terminals were reduced, further supporting that disturbances in DA signaling occurred in HD transgenic models. The substance P-containing striatal efferent pathway was more resistant to the HD mutation than met-enkephalin-producing striatal projection neurons in the transgenics, based on neuropeptide immunofluorescent staining. Analogous findings in multiple transgenic models suggest that these changes are due to the presence of the transgene and are not dependent on its composition, promotor elements, or mouse strain background. These findings suggest modifications in the striatal DA system and that its downstream signaling through cyclic AMP mechanisms is disrupted severely in HD following onset of motor symptoms.