Striatonigral Projection Neurons Research Articles

Regulation of Src family kinases by muscarinic acetylcholine receptors in heterologous cells and neurons.

Five muscarinic acetylcholine (mACh) receptor subtypes are divided into two classes: the M1 class (M1, M3, and M5) and the M2 class (M2 and M4). The former is coupled to Gq proteins, while the latter is coupled to Gi/o proteins. Accumulating evidence indicates that mACh receptors play a significant role in the regulation of the Src family kinase (SFK), a subfamily of non-receptor tyrosine kinases. mACh receptors exert their roles in a subtype-dependent fashion and preferentially target Src and Fyn, two members of SFKs that are expressed in the brain and enriched at synaptic sites. While the M1 receptor positively modulates SFK activity, the M4 receptor inhibits it. By modulating SFKs, mACh receptors are actively involved in the regulation of expression and function of a variety of receptors, structural proteins, and signaling molecules. In particular, the M4 receptor and the dopamine D1 receptor are coexpressed in striatonigral projection neurons of the striatum. Gi/o-coupled M4 and Gq-coupled D1 receptors antagonistically regulate SFK activity, thereby forming a dynamic balance controlling glutamate receptor activity, excitability of neurons, and synaptic plasticity. In summary, mACh receptors play a crucial role in regulating SFK activity in heterologous cells and neurons.

Cell-type- and region-specific modulation of cocaine seeking by micro-RNA-1 in striatal projection neurons

The persistent and experience-dependent nature of drug addiction may result in part from epigenetic alterations, including non-coding micro-RNAs (miRNAs), which are both critical for neuronal function and modulated by cocaine in the striatum. Two major striatal cell populations, the striato-nigral and striato-pallidal projection neurons, express, respectively, the D1 (D1-SPNs) and D2 (D2-SPNs) dopamine receptor, and display distinct but complementary functions in drug-evoked responses. However, a cell-type-specific role for miRNAs action has yet to be clarified. Here, we evaluated the expression of a subset of miRNAs proposed to modulate cocaine effects in the nucleus accumbens (NAc) and dorsal striatum (DS) upon sustained cocaine exposure in mice and showed that these selected miRNAs were preferentially upregulated in the NAc. We focused on miR-1 considering the important role of some of its predicted mRNA targets, Fosb and Npas4, in the effects of cocaine. We validated these targets in vitro and in vivo. We explored the potential of miR-1 to regulate cocaine-induced behavior by overexpressing it in specific striatal cell populations. In DS D1-SPNs miR-1 overexpression downregulated Fosb and Npas4 and reduced cocaine-induced CPP reinstatement, but increased cue-induced cocaine seeking. In DS D2-SPNs miR-1 overexpression reduced the motivation to self-administer cocaine. Our results indicate a role of miR1 and its target genes, Fosb and Npas4, in these behaviors and highlight a precise cell-type- and region-specific modulatory role of miR-1, illustrating the importance of cell-specific investigations.

Regulation of Phosphorylation of AMPA Glutamate Receptors by Muscarinic M4 Receptors in the Striatum In vivo

The acetylcholine muscarinic 4 (M4) receptor is a principal muscarinic receptor subtype present in the striatum. Notably, Gαi/o-coupled M4 receptors and Gαs/Golf-coupled dopamine D1 receptors are coexpressed in striatonigral projection neurons and are thought to interact with each other to regulate neuronal excitability, although underlying molecular mechanisms are poorly understood. In this study, we investigated the role of M4 receptors in the regulation of phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the rat normal and dopamine-stimulated striatum in vivo. We found that a systemic injection of a M4 antagonist tropicamide increased AMPA receptor GluA1 subunit phosphorylation at a protein kinase A-dependent site (S845) in the striatum. The tropicamide-induced S845 phosphorylation was rapid, reversible, and dose-dependent and occurred in the two subdivisions of the striatum, i.e., the caudate putamen and nucleus accumbens. Coadministration of subthreshold doses of tropicamide and a D1 agonist SKF81297 induced a significant increase in S845 phosphorylation. Coadministered tropicamide and a dopamine psychostimulant amphetamine at their subthreshold doses also elevated S845 phosphorylation. Tropicamide alone or coinjected with SKF81297 or amphetamine had no effect on GluA1 phosphorylation at S831. Tropicamide did not affect GluA2 phosphorylation at S880. These results reveal a selective inhibitory linkage from M4 receptors to GluA1 in S845 phosphorylation in striatal neurons. Blockade of the M4-mediated inhibition significantly augments constitutive and dopamine-stimulated GluA1 S845 phosphorylation.

Retinoic Acid Receptor β Controls Development of Striatonigral Projection Neurons through FGF-Dependent and Meis1-Dependent Mechanisms.

The mammalian striatum controls sensorimotor and psychoaffective functions through coordinated activities of its two striatonigral and striatopallidal output pathways. Here we show that retinoic acid receptor β (RARβ) controls development of a subpopulation of GABAergic, Gad65-positive striatonigral projection neurons. In Rarb(-/-) knock-out mice, concomitant reduction of Gad65, dopamine receptor D1 (Drd1), and substance P expression at different phases of prenatal development was associated with reduced number of Drd1-positive cells at birth, in contrast to normal numbers of striatopallidal projection neurons expressing dopamine receptor D2. Fate mapping using BrdU pulse-chase experiments revealed that such deficits may originate from compromised proliferation of late-born striosomal neurons and lead to decreased number of Drd1-positive cells retaining BrdU in postnatal day (P) 0 Rarb(-/-) striatum. Reduced expression of Fgf3 in the subventricular zone of the lateral ganglionic eminence (LGE) at embryonic day 13.5 may underlie such deficits by inducing premature differentiation of neuronal progenitors, as illustrated by reduced expression of the proneural gene Ascl1 (Mash1) and increased expression of Meis1, a marker of postmitotic LGE neurons. In agreement with a critical role of FGF3 in this control, reduced number of Ascl1-expressing neural progenitors, and a concomitant increase of Meis1-expressing cells, were observed in primary cell cultures of Rarb(-/-) LGE. This defect was normalized by addition of fibroblast growth factor (FGF). Such data point to role of Meis1 in striatal development, also supported by reduced neuronal differentiation in the LGE of Meis1(-/-) embryos. Our data unveil a novel mechanism of development of striatonigral projection neurons involving retinoic acid and FGF, two signals required for positioning the boundaries of Meis1-expressing cells.

Loss of the preferential control over the striato-nigral direct pathway by striatal NMDA receptors in a rat model of Parkinson's disease.

By using multi-probe microdialysis we previously demonstrated that endogenous glutamate differentially regulates the activity of the striatal output pathways in vivo, through N-methyl-d-aspartate (NMDA) receptors containing the GluN2A or GluN2B subunits. Using the same approach, we presently investigate whether reverse dialysis of NMDA in the striatum differentially affects GABA release in the striatum and in striatal target areas, i.e. globus pallidus (GP) and substantia nigra reticulata (SNr). Moreover, we ask whether this control is altered under parkinsonian conditions. Intrastriatal NMDA perfusion (10 min) evoked GABA release more potently in SNr (1-100 μM) than in other regions (10-100 μM), suggesting preferential control over striato-nigral projection neurons. Intrastriatal NMDA more potently stimulated glutamate levels in the striatum (1-100 μM) and SNr (1-10 μM) than in GP (10 μM). Striatal dopamine denervation with 6-hydroxydopamine caused a leftward shift in the NMDA concentration-response curve. Intrastriatal NMDA elevated GABA levels at 0.1 μM (all regions) and 1 μM (striatum and GP only), but not at higher concentrations, indicating that, compared to naïve animals, the GABA response in SNr was attenuated. Attenuation of the glutamate response was also observed in SNr (NMDA effective only at 0.1 μM). Conversely, the glutamate response in GP was widened (NMDA effective in the 0.1-1 μM range). We conclude that NMDA preferentially stimulates the activity of the striato-nigral direct pathway under physiological conditions. In Parkinson's disease, dopamine loss compromises the NMDA ability to stimulate striato-nigral neurons, thus shifting the NMDA control towards the striato-pallidal ones.

Circuit-Selective Striatal Synaptic Dysfunction in the Sapap3 Knockout Mouse Model of Obsessive-Compulsive Disorder

Synapse-associated protein 90/postsynaptic density protein 95-associated protein 3 (SAPAP3) is an excitatory postsynaptic protein implicated in the pathogenesis of obsessive-compulsive behaviors. In mice, genetic deletion of Sapap3 causes obsessive-compulsive disorder (OCD)-like behaviors that are rescued by striatal expression of Sapap3, demonstrating the importance of striatal neurotransmission for the OCD-like behaviors. In the striatum, there are two main excitatory synaptic circuits, corticostriatal and thalamostriatal. Neurotransmission defects in either or both of these circuits could potentially contribute to the OCD-like behaviors of Sapap3 knockout (KO) mice. Previously, we reported that Sapap3 deletion reduces corticostriatal alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid-type glutamate receptor-mediated synaptic transmission. Whole-cell electrophysiological recording techniques in acute brain slices were used to measure synaptic transmission in the corticostriatal and thalamostriatal circuits of Sapap3 KO mice and littermate control animals. Transgenic fluorescent reporters identified striatopallidal and striatonigral projection neurons. SAPAP isoforms at corticostriatal and thalamostriatal synapses were detected using immunostaining techniques. In contrast to corticostriatal synapses, thalamostriatal synaptic activity is unaffected by Sapap3 deletion. At the molecular level, we find that another SAPAP family member, SAPAP4, is present at thalamostriatal, but not corticostriatal, synapses. This finding provides a molecular rationale for the functional divergence we observe between thalamic and cortical striatal circuits in Sapap3 KO mice. These findings define the circuit-level neurotransmission defects in a genetic mouse model for OCD-related behaviors, focusing attention on the corticostriatal circuit for mediating the behavioral abnormalities. Our results also provide the first evidence that SAPAP isoforms may be localized to synapses according to circuit-selective principles.

Bcl-2 enhances the formation of newborn striatal long-projection neurons in adult rat brain after a transient ischemic stroke

It has been reported that B-cell lymphoma 2 (Bcl-2) enhances neurogenesis as well as supporting axonal growth after injury. In the present study, we investigated whether Bcl-2 overexpression plays a role in the formation of newborn striatonigral projection neurons in the adult rat brain after transient middle cerebral artery occlusion (MCAO). We infused human Bcl-2-expressing plasmid (pBcl-2) into the lateral ventricle immediately after 30 min of MCAO, injected 5'-bromodeoxyuridine (BrdU) intraperitoneally to label proliferative cells, and microinjected fluorogold (FG) into the substantia nigra at 11 weeks of reperfusion followed by multiple immunostaining of striatonigral projection neurons at 12 weeks. We found that pBcl-2 treatment significantly increased the number of newborn neurons (BrdU(+)-NeuN(+)) in the striatum ipsilateral to the MCAO. We further detected newborn striatonigral projection neurons (BrdU(+)-FG(+)-NeuN(+)) in the ipsilateral striatum at 12 weeks. More interestingly, the number of newborn striatonigral projection neurons (BrdU(+)-FG(+)) was significantly increased by pBcl-2 treatment compared to that by pEGFP, a control plasmid. Taken together, we found that Bcl-2 overexpression in the brain enhanced the generation of newborn striatonigral projection neurons. This provides a potential strategy for promoting the reestablishment of neural networks and brain repair after ischemic injury.

Shingosine-1-phosphate regulates PKA/DARPP-32 signaling in striatal medium spiny neurons via GPR6-independent mechanisms

The vast majority of striatal neurons are GABAergic medium-sized spiny neurons. These cells receive glutamatergic input from the cortex, thalamus and limbic areas and dopaminergic input from the mesencephalon. Most relevant evidence indicates that dopamine D1 receptors are located on striatonigral projection neurons,5, 7 and that adenosine A2A receptors4, 12, 14 and most dopamine D2 receptors5, 7, 14 are located on striatopallidal projection neurons (see, however, 1, 13). Here we have utilized regulation of the phosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein of mol. wt 32,000 (DARPP-32) to study the possible interactions among nigrostriatal dopaminergic neurons and the two classes of dopaminoceptive target neurons. We show that, in striatal slices, the D2 receptor agonist, quinpirole, strongly inhibits the phosphorylation of DARPP-32 induced by either the D1 receptor agonist, SKF 81297, or the A2A receptor agonist, CGS 21680. Tetrodotoxin abolished the effect of quinpirole on the D1 agonist-induced but not the A2A agonist-induced phosphorylation of DARPP-32. These data indicate that: (i) adenosine A2A and dopamine D2 receptors interact within the same striatopallidal neurons, and (ii) D2 receptors present on the striatopallidal neurons modulate the effects of D1 receptors on the striatonigral neurons. Thus, a single neurotransmitter is capable of activating distinct classes of receptors on distinct populations of target neurons, which, in turn, interact with each other through intercellular communication.

An Improved BAC Transgenic Fluorescent Reporter Line for Sensitive and Specific Identification of Striatonigral Medium Spiny Neurons

The development of BAC transgenic mice expressing promoter-specific fluorescent reporter proteins has been a great asset for neuroscience by enabling detection of neuronal subsets in live tissue. For the study of basal ganglia physiology, reporters driven by type 1 and 2 dopamine receptors have been particularly useful for distinguishing the two classes of striatal projection neurons – striatonigral and striatopallidal. However, emerging evidence suggests that some of the transgenic reporter lines may have suboptimal features. The ideal transgenic reporter line should (1) express a reporter with high sensitivity and specificity for detecting the cellular subset of interest and that does not otherwise alter the biology of the cells in which it is expressed, and (2) involve a genetic manipulation that does not cause any additional genetic effects other than expression of the reporter. Here we introduce a new BAC transgenic reporter line, Drd1a-tdTomato line 6, with features that approximate these ideals, offering substantial benefits over existing lines. In this study, we investigate the integrity of dopamine-sensitive behaviors and test the sensitivity and specificity of tdTomato fluorescence for identifying striatonigral projection neurons in mice. Behaviorally, hemizygous Drd1a-tdTomato line 6 mice are similar to littermate controls; while hemizygous Drd2-EGFP mice are not. In characterizing the sensitivity and specificity of line 6 mice, we find that both are high. The results of this characterization indicate that line 6 Drd1a-tdTomato+/− mice offer a useful alternative approach to identify both striatonigral and striatopallidal neurons in a single transgenic line with a high degree of accuracy.

The GTP-binding protein Rhes modulates dopamine signalling in striatal medium spiny neurons

Rhes is a small GTP-binding protein prominently localized in the striatum. Previous findings obtained in cell culture systems demonstrated an involvement of Rhes in cAMP/PKA signalling pathway, at a level proximal to the activation of heterotrimeric G-protein complex. However, its role in the striatum has been, so far, only supposed. Here we studied the involvement of Rhes in dopaminergic signalling, by employing mice with a null mutation in the Rhes gene. We demonstrated that the absence of Rhes modulates cAMP/PKA signalling in both striatopallidal and striatonigral projection neurons by increasing Golf protein levels and, in turn, influencing motor responses challenged by dopaminergic agonist/antagonist. Interestingly, we also show that Rhes is required for a correct dopamine-mediated GTP binding, a function mainly associated to stimulation of dopamine D2 receptors. Altogether, our results indicate that Rhes is an important modulator of dopaminergic transmission in the striatum.

NR2A and NR2B subunit containing NMDA receptors differentially regulate striatal output pathways

Triple probe microdialysis was employed to investigate whether striatal NR2A and NR2B subunit containing NMDA receptors regulate the activity of striato-pallidal and striato-nigral projection neurons. Probes were implanted in the striatum, ipsilateral globus pallidus and substantia nigra reticulata. Intrastriatal perfusion with the NR2A subunit selective antagonist (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) reduced pallidal GABA and increased nigral glutamate (GLU) release whereas perfusion with the NR2B subunit selective antagonist (R-(R*,S*)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidinepropanol (Ro 25-6981) reduced nigral GABA and elevated striatal and pallidal GLU release. To confirm that changes in GABA levels were because of blockade of (GLUergic-driven) tonic activity of striatofugal neurons, tetrodotoxin was perfused in the striatum. Tetrodotoxin reduced both pallidal and nigral GABA release without changing GLU levels. To investigate whether striatal NR2A and NR2B subunits were also involved in phasic activation of striatofugal neurons, NVP-AAM077 and Ro 25-6981 were challenged against a NMDA concentration able to evoke GABA release in the three areas. Both antagonists prevented the NMDA-induced striatal GABA release. NVP-AAM077 also prevented the NMDA-induced surge in GABA release in the globus pallidus, whereas Ro 25-6981 attenuated it in the substantia nigra. We conclude that striatal NMDA receptors containing NR2A and NR2B subunits preferentially regulate the striato-pallidal and striato-nigral projection neurons, respectively.

Central administration of the neurotensin receptor antagonist sr48692 attenuates vacuous chewing movements in a rodent model of tardive dyskinesia

Tardive dyskinesia is a movement disorder that develops in 20–30% of patients treated with chronic neuroleptics. Whilst the pathogenesis of tardive dyskinesia remains unclear, altered expression of neuropeptides in the basal ganglia has been implicated in its emergence. The peptide neurotensin is expressed in both dopamine D1 receptor-bearing neurons of the direct striatonigral pathway and dopamine D2 receptor-bearing neurons of the indirect striatopallidal pathway. Increased levels of striatal neurotensin messenger RNA (mRNA) are reported following chronic neuroleptic therapy. Chronic treatment with the typical antipsychotic haloperidol elicits neurotensin immunoreactivity in a large number of striatopallidal and a modest number of striatonigral projection neurons, whilst treatment with the potent dopamine releaser, methamphetamine, induces intense neurotensin immunoreactivity in striatonigral projection neurons. In order to determine whether increased levels of striatal neurotensin mRNA in the direct striatonigral or the indirect striatopallidal pathway play a more influential role in the development of tardive dyskinesia, we explored the effects of a specific neurotensin antagonist in a rodent model (vacuous chewing movements [VCMs] induced by chronic neuroleptics). Three groups of animals received injections of fluphenazine decanoate (25 mg/kg) or its vehicle sesame oil every 3 weeks for at least 18 weeks. They were then surgically implanted with bilateral guide cannulae aimed at the striatum, the substantia nigra pars reticulata, or the globus pallidus respectively. After recovery, animals were infused with 2-[(1-(7-chloro-4-quinolinyl)-5-(2,6-imethoxyphenyl)pyrazol-3-yl)carbonylamino]tricyclo(3.3.1.1. 3.7)decan-2-carboxylic acid (SR48692; 0.25, 0.50, and 1.0 nmol/μl), or its vehicle (10% dimethyl sulfoxide [DMSO] in saline) and observed for 60 min. Intra-striatal, intra-nigral or intra-pallidal infusion of SR48692 attenuated neuroleptic-induced VCMs. These findings lend further support to a role for neurotensin in the development of VCMs but do not clarify which pathway plays a more important role. Thus, treatments that reduce or prevent the effects of increased neurotensin expression and release may be useful in the management of tardive dyskinesia.

Analysis of mRNA expression using double in situ hybridization labeling with isotopic and nonisotopic probes.

AbstractA large number of in vivo studies in the last decade have confirmed that abused drugs are able to up-regulate gene expression in striatal neurons. For example, acute or chronic exposures to psychostimulants, cocaine and amphetamine, increased basal levels of mRNA and protein products of immediate early genes (c-fos, zif/268, ΔFosB, etc.) in the rodent striatum (1–3). Similarly, acute administration of cocaine and amphetamines induced robust mRNA expression of preprodynorphin (PPD) and substance P (SP) in striatonigral projection neurons and preproenkaphalin (PPE) in striatopallidal projection neurons (4–6). The increased immediate early gene and neuropeptide expression is mediated via selective activation of dopamine receptors (primary D1 subtype) (7,8). Glutamatergic transmission also significantly contributes to the genomic effect of stimulants (9,10). The altered gene expression has been considered to be an essential molecular step for the development of psychoplasticity in the striatum related to addictive properties of drugs of abuse (11,12).KeywordsHybridization SignalStriatal NeuronIsotopic ProbeNitroblue Tetrazolium ChlorideAddictive PropertyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Increased Response to Intrastriatal L(+)-2-Amino-4-Phosphonobutyrate (L-AP4) in Unilateral 6-Hydroxydopamine-Lesioned Rats

Unilateral intrastriatal injection of the prototype Group III metabotropic glutamate receptor agonist L(+)-2-amino-4-phosphonobutyrate (L-AP4) induces rotation in rats with ipsilateral unilateral 6-hydroxydopamine lesions of the nigrostriatal projection but not in unlesioned control animals [Kearney et al., (1998) Neuroscience 87:881–891]. We studied differences in striatal neuron Fos-like immunoreactivity expression, striatal neuron Fos-like immunoreactivity localization, and regional [14C]-2-deoxyglucose uptake after unilateral intrastriatal injection of L-AP4 between control and unilateral 6-hydroxydopamine lesioned rats. There was a left shift of the dose response curve with more striatal neurons expressing Fos-like immunoreactivity at lower doses of L-AP4 in 6-hydroxydopamine lesioned animals. In unlesioned striata, L-AP4 injections tended to induce Fos-like immunoreactivity in striato-pallidal projection neurons. In lesioned animals, the majority of striatal neurons expressing Fos-like immunoreactivity were striatonigral projection neurons. In both control and lesioned animals, intrastriatal injection of L-AP4 produced widespread decreases in [14C]-2-deoxyglucose uptake in basal ganglia nuclei and related regions, but the magnitude of this effect increased markedly in lesioned animals. Striatal dopamine denervation enhances the response of striatal neurons to intrastriatally injected L-AP4 with an apparent shift in the type of striatal projection neuron responding to L-AP4.

The distribution of dynorphinergic terminals in striatal target regions in comparison to the distribution of substance P-containing and enkephalinergic terminals in monkeys and humans

Single- and double-label immunohistochemical techniques using several different highly specific antisera against dynorphin peptides were used to examine the distribution of dynorphinergic terminals in globus pallidus and substantia nigra in rhesus monkeys and humans in comparison to substance P-containing and enkephalinergic terminals in these same regions. Similar results were observed in monkey and human tissue. Dynorphinergic fibers were very abundant in the medial half of the internal pallidal segment, but scarce in the external pallidal segment and the lateral half of internal pallidal segment. In substantia nigra, dynorphinergic fibers were present in both the pars compacta and reticulata. Labeling of adjacent sections for enkephalin or substance P showed that the dynorphinergic terminals overlapped those for substance P in the medial half of the internal pallidal segment, but showed only slight overlap with enkephalinergic terminals in the external pallidal segment. The substance P-containing fibers were moderately abundant along the borders of the external pallidal segment, and enkephalinergic fibers were moderately abundant in parts of the internal pallidal segment. Dynorphinergic and substance P-containing terminals overlapped extensively in the nigra, and both extensively overlapped enkephalinergic fibers in medial nigra. Immunofluorescence double-labeling studies revealed that dynorphin co-localized extensively with substance P in individual fibers and terminals in the medial half of the internal pallidal segment and in substantia nigra. Thus, as has been found in non-primates, dynorphin within the striatum and its projection systems appears to be extensively localized to substance P-containing striatopallidal and striatonigral projection neurons. Nonetheless, our results also raise the possibility that a population of substance P-containing neurons that projects to the internal pallidal segment and does not contain dynorphin is present in primate striatum. Our results also suggest the possible existence of populations of striatopallidal and striatonigral projection neurons in which substance P and enkephalin or dynorphin and enkephalin, or all three, are co-localized. Thus, striatal projection neurons in primates may not consist of merely two types, one containing substance P and dynorphin and the other enkephalin.

Activation of dopamine D 2 receptors decreases DARPP-32 phosphorylation in striatonigral and striatopallidal projection neurons via different mechanisms

The vast majority of striatal neurons are GABAergic medium-sized spiny neurons. These cells receive glutamatergic input from the cortex, thalamus and limbic areas and dopaminergic input from the mesencephalon. Most relevant evidence indicates that dopamine D 1 receptors are located on striatonigral projection neurons, [5, 7] and that adenosine A 2A receptors [4, 12, 14] and most dopamine D 2 receptors [5, 7, 14] are located on striatopallidal projection neurons (see, however, [1, 13]). Here we have utilized regulation of the phosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein of mol. wt 32,000 (DARPP-32) to study the possible interactions among nigrostriatal dopaminergic neurons and the two classes of dopaminoceptive target neurons. We show that, in striatal slices, the D 2 receptor agonist, quinpirole, strongly inhibits the phosphorylation of DARPP-32 induced by either the D 1 receptor agonist, SKF 81297, or the A 2A receptor agonist, CGS 21680. Tetrodotoxin abolished the effect of quinpirole on the D 1 agonist-induced but not the A 2A agonist-induced phosphorylation of DARPP-32. These data indicate that: (i) adenosine A 2A and dopamine D 2 receptors interact within the same striatopallidal neurons, and (ii) D 2 receptors present on the striatopallidal neurons modulate the effects of D 1 receptors on the striatonigral neurons. Thus, a single neurotransmitter is capable of activating distinct classes of receptors on distinct populations of target neurons, which, in turn, interact with each other through intercellular communication.

Targeted Expression of a Toxin Gene to D1 Dopamine Receptor Neurons byCre-Mediated Site-Specific Recombination

Idiopathic Parkinson's disease involves the loss of midbrain dopaminergic neurons, resulting in the presynaptic breakdown of dopaminergic transmission in the striatum. Huntington's disease and some neurodegenerative diseases with Parkinsonian features have postsynaptic defects caused by striatal cell death. Mice were generated in which an attenuated form of the diphtheria toxin gene (tox-176) was expressed exclusively in D1 dopamine receptor (D1R)-positive cells with the aim of determining the effect of this mutation on development of the basal ganglia and on the locomotor phenotype. Transgenic mice expressing Cre, a site-specific DNA recombinase, were crossed with a second line in which a transcriptionally silenced tox-176 gene was inserted into the D1R gene locus by homologous recombination. Young doubly transgenic mutant mice expressing the tox-176 gene displayed bradykinesia, dystonia, and had falls caused by myoclonic jerks. The mutant brain had evidence of apoptosis and reactive gliosis and, consistent with the D1R expression pattern, the striatum was reduced in volume, and the Islands of Calleja were absent. In contrast, the cortex was of normal thickness. D1Rs were not detectable in mutants by in situ hybridization or ligand autoradiography, whereas D2 dopamine receptor (D2R) mRNA and protein was present in the striatum. In addition, substance P and dynorphin, neuropeptides known to be expressed in D1R-positive striatonigral projection neurons were not detectable. Enkephalin, a marker found in D2-positive striatopallidal projection neurons was expressed in the mutant brain. The mutant represents a novel neurodegenerative disease model with a dramatic extrapyramidal phenotype.

Activation of adenosine A 2A and dopamine D 1 receptors stimulates cyclic AMP-dependent phosphorylation of DARPP-32 in distinct populations of striatal projection neurons

In the striatum, adenosine A 2A and dopamine D 1 receptors are segregated in striatopallidal and striatonigral projection neurons, respectively. In this study, we have examined the effects of activating adenosine A 2A and dopamine D 1 receptors on the state of phosphorylation of DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein of mol. wt 32,000), a potent endogenous regulator of protein phosphatase-1 that is highly expressed in striatal medium-sized spiny neurons. In rat striatal slices, the D 1 receptor agonist SKF 81297 and the A 2A receptor agonist CGS 21680 transiently increased the levels of phosphorylated DARPP-32 in a concentration-dependent manner. In the same preparation, the two agonists were also able to induce a significant increase in cyclic AMP formation. When striatal slices were incubated with a combination of CGS 21680 and SKF 81297, the effects of the two agonists on both DARPP-32 phosphorylation and cyclic AMP formation were additive. The maximal effects of SKF 81297 and CGS 21680 on DARPP-32 phosphorylation were of similar magnitude, and were completely abolished by the cyclic AMP-dependent protein kinase inhibitor, Rp-cAMPS. The present results show that DARPP-32 phosphorylation in the striatum is stimulated by adenosine, acting on A 2A receptors, and dopamine, acting on D 1 receptors, and that cyclic AMP is the mediator in both cases. Our data also suggest that dopamine and adenosine regulate the state of phosphorylation of DARPP-32 in distinct sub-populations of medium-sized spiny neurons expressing dopamine D 1 and adenosine A 2A receptors, respectively.

Localization of mGluR1a-like immunoreactivity and mGluR5-like immunoreactivity in identified populations of striatal neurons

Metabotropic glutamate receptors are important mediators of excitatory amino acid neurotransmission in the striatum. Two-color immunofluorescence histochemistry and immunohistochemistry in combination with retrograde tract-tracing techniques were used to examine the distribution of metabotropic glutamate receptor subtypes 1a and 5 (mGluR1a and mGluR5) among identified subpopulations of striatal projection neurons and interneurons. The majority of striatopallidal and striatonigral neurons were double-labeled for both mGluR1a or mGluR5. Approximately 60% to 70% of either striatonigral or striatopallidal neurons expressed mGluR1a- or mGluR5-like immunoreactivity. The percentage of double-labeled striatopallidal or striatonigral projection neurons did not differ among striatal quadrants. Striatal interneurons expressing parvalbumin or somatostatin or choline acetyltransferase exhibited varying degrees of expression of mGluR1a or mGluR5. Virtually all (94%) parvalbumin-immunoreactive striatal neurons expressed mGluR1a-like immunoreactivity with a majority (79%) of these neurons expressing mGluR5-like immunoreactivity. A high percentage (89%) of striatal choline acetyltransferase-immunoreactive neurons were double-labeled for mGluR1a-like immunoreactivity. Approximately 65% of striatal choline acetyltransferase-immunoreactive neurons expressed mGluR5-like immunoreactivity. A majority (65%) of somatostatin-immunoreactive striatal interneurons expressed mGluR1a-like immunoreactivity with a slightly lower percentage (55%) expressing mGluR5-like immunoreactivity. These findings indicate considerable heterogeneity among striatal projection and interneurons with respect to mGluR1a and mGluR5 expression. There may be subpopulations of striatonigral and striatopallidal projection neurons. These results are consistent as well with prior data indicating subpopulations of the different classes of striatal interneurons.

The in vivo release of taurine in the striatonigral pathway.

Cortical information is processed in the neostriatum (STR) and then transmitted to the output nuclei of the basal ganglia, the entopeduncular nucleus (EP) and the substantia nigra pars reticulata (SNr). In the mammalian neostriatum the majority of neurones are the medium-size spiny neurons, sending their axons to the globus pallidus (GP) or the EP/SNr, by either the “direct” or the “indirect” pathways26. The “direct” pathway consists of a direct projection from the neostriatum to the EP/SNr, while the “indirect” pathways consist of a striatal projection to the GP and thence to the output nuclei directly or via the subthalamic nucleus (STN). Striatonigral projection neurons, neurons of the GP, SNr and EP are GABAergic, whereas those of the STN use glutamate. Activation of the “direct” pathway thus leads to inhibition in the EP/SNr whereas activation of the “indirect” pathways produces the opposite effect i.e. disinhibition; the imbalance between the two is thought to underlie disorders of movement1,6,12,15.