Striatal Neuropil Research Articles

Dopamine Receptors in Parkinson's Disease: A Meta‐Analysis of Imaging Studies

AbstractDopamine receptors are abundant along the central nigrostriatal tract and are expressed as 5 subtypes in two receptor families. In PD, compensatory changes in dopamine receptors emerge as a consequence of the loss of dopamine nerve terminals or dopaminergic pharmacotherapy. We performed a systematic review and meta‐analysis of the available PET and single‐photon emission computed tomography studies that have investigated dopamine receptors in PD, PSP and MSA. The inclusion criteria were studies including human PET or single‐photon emission computed tomography imaging; dopamine receptor tracers (D1‐like or D2‐like) and idiopathic PD, PSP, or MSA patients compared with healthy controls. The 67 included D2‐like studies had 1925 patients. Data were insufficient for an analysis of D1‐like studies. PD patients had higher striatal binding early in the disease, but after a disease duration of 4.36 years, PD patients had lower binding values than healthy controls. Striatal D2R binding was highest in unmedicated early PD patients and in the striatum contralateral to the predominant motor symptoms. PSP and MSA‐P patients had lower striatal D2R binding than PD patients (14.2% and 21.8%, respectively). There is initial upregulation of striatal D2Rs in PD, which downregulate on average 4 years after motor symptom onset, possibly because of agonist‐induced effects. The consistent upregulation of D2Rs in the PD striatum contralateral to the predominant motor symptoms indicates that receptor changes are driven by neurodegeneration and loss of striatal neuropil. Both PSP and MSA patients have clearly lower striatal D2R binding values than PD patients, which offers an opportunity for differential diagnostics. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

Developmental Changes in Dendritic Spine Morphology in the Striatum and Their Alteration in an A53T α-Synuclein Transgenic Mouse Model of Parkinson's Disease.

The aging process is accompanied by various neurophysiological changes, and the severity of neurodegenerative disorders such as Parkinson’s disease (PD) increases with aging. However, the precise neuroanatomical changes that accompany the aging process in both normal and pathologic conditions remain unknown. This is in part because there is a lack of high-resolution imaging tool that has the capacity to image a desired volume of neurons in a high-throughput and automated manner. In the present study, focused ion beam/scanning electron microscopy (FIB/SEM) was used to image striatal neuropil in both wild-type (WT) mice and an A53T bacterial artificial chromosome (BAC) human α-synuclein (A53T-BAC-SNCA) transgenic (Tg) mouse model of PD, at 1, 3, 6, and 22 months of age. We demonstrated that spine density gradually decreases, and average spine head volume gradually increases with age in WT mice, suggesting a homeostatic balance between spine head volume and spine density. However, this inverse relationship between spine head volume and spine density was not observed in A53T-BAC-SNCA Tg mice. Taken together, our data suggest that PD is accompanied by an abnormality in the mechanisms that control synapse growth and maturity.

MAP2 Splicing is Altered in Huntington's Disease.

Dendritic alteration of striatal medium spiny neurons is one of the earliest morphological abnormalities in Huntington's disease (HD). The main microtubule-associated protein in dendrites is MAP2. The low-molecular weight isoforms of MAP2 (LMW-MAP2) are the juvenile forms resulting from exclusion of the sequence encoded by exons E7-E9 and are downregulated after the early stages of neuronal development when E7-E9 exon-including high-molecular weight isoforms (HMW-MAP2) are favored. Splicing alteration has recently been proposed to contribute to HD in view of two pathogenic missplicing events resulting in a highly toxic N-terminal version of mutant huntingtin and in a detrimental imbalance in MAP Tau isoforms with three or four tubulin-binding repeats. Both splicing events are postulated targets of the SR splicing factor SRSF6 which has recently been reported to be dramatically altered in HD. SR proteins often regulate functionally related sets of genes and SRSF6 targets are enriched in genes involved in brain organogenesis including several actin-and tubulin-binding proteins. Here we hypothesized that MAP2 might be target of SRSF6 and altered in HD. By SRSF6 knockdown in neuroblastoma cells, we demonstrate that splicing of MAP2 E7-E9 exons is affected by SRSF6. We then show a disbalance in LMW and HMW MAP2 mRNA isoforms in HD striatum in favor of the juvenile LMW forms together with a decrease in total MAP2 mRNA. This is accompanied by a global decrease in total MAP2 protein due to almost total disappearance of HMW-MAP2 isoforms with preservation of LMW-MAP2 isoforms. Accordingly, the predominant dendritic MAP2 staining in striatal neuropil of control subjects is absent in HD cases. In these, MAP2-immunoreactivity is faint and restricted to neuronal cell bodies often showing a sharp boundary at the base of dendrites. Together, our results highlight the importance of splicing alteration in HD and suggest that MAP2 alteration contributes to dendritic atrophy.

Morphometric analysis of NADPH diaphorase reactive neurons in a rat model of focal excitotoxic striatal injury.

Excitotoxicity is the major component in neuropathological conditions, related to harmful action of imbalanced concentrations of glutamate and its agonists in the nervous tissue, ultimately resulting in cell death. In the present study, we evaluated the effects of an acute striatal lesion induced by a focal N-methyl-D-aspartate (NMDA) microinjection on the morphometry of NADPH diaphorase-reactive neurons (NADPH-d+ ), a subset of cells which release nitric oxide (NO) in the brain and are known by its resistance in pathological conditions. Two hundred and forty NADPH-d neurons from NMDA-lesioned striatum and contralateral counterpart were tridimensionally reconstructed at 1, 3 and 7 post-lesion days (PLDs). Cell body and dendritic field areas, length of dendrites by order and fractal dimension were analyzed. There were no significant morphometric differences when NADPH-d+ neurons from lesioned and control striatal regions were compared among PLDs evaluated. Conversely, a conspicuous pallor in striatal neuropil reactivity was evidenced, especially in latter survival time. In addition, we observed a noticeable inflammatory response induced by NMDA. Our results suggest that NADPH-d+ neurons were spared from deleterious effects of acute NMDA excitotoxic damage in the striatum, reinforcing the notion that this cell group is selectively resistant to injury in the nervous system.

Structural Organization of Striatal Microgliocytes after Transient Focal Ischemia

Microgliocytes are known not to be structurally stable cellular elements; altering the lengths of their processes, they control the synaptic organization of the brain. The aim of the present work was to study the structural organization of the microglia in the striatum and periventricular area of the telencephalon of the rat after transient focal ischemia. Ischemic lesions without cerebral infarction were found to lead to activation of the microglia, promoting reorganization of the striatal neuropil. Activation of microgliocytes was associated with changes in their shape, from the characteristic dendritic shape to the oval and round shapes typical of the ameboid microglia. Microgliocytes were able to proliferate after ischemia.

Regeneration of the MPTP-lesioned dopaminergic system after convection-enhanced delivery of AAV2-GDNF.

Clinical studies to date have failed to establish therapeutic benefit of glial cell-derived neurotrophic factor (GDNF) in Parkinson's disease (PD). In contrast to previous nonclinical neuroprotective reports, this study shows clinically relevant and long-lasting regeneration of the dopaminergic system in rhesus macaques lesioned with 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine 3-6 months before GDNF gene delivery (AAV2-GDNF). The observed progressive amelioration of functional deficits, recovery of dopamine, and regrowth of fibers to the striatal neuropil demonstrate that high GDNF expression in the putamen promotes restoration of the dopaminergic system in a primate model of advanced PD. Extensive distribution of GDNF within the putamen and transport to the severely lesioned substantia nigra, after convection-enhanced delivery of AAV2-GDNF into the putamen, indicates anterograde transport via striatonigral connections and is anticipated to occur in PD patients. Overall, these data demonstrate nonclinical neurorestoration after putaminal infusion of AAV2-GDNF and suggest that clinical investigation in PD patients is warranted.

The intrastriatal injection of thrombin in rat induced a retrograde apoptotic degeneration of nigral dopaminergic neurons through synaptic elimination

We have performed intrastriatal injection of thrombin and searched for distant effects in the cell body region. In striatum, thrombin produced a slight loss of striatal neurons as demonstrated by neural nuclei immunostaining - a non-specific neuronal marker - and the expression of glutamic acid decarboxylase 67 mRNA, a specific marker for striatal GABAergic interneurons, the most abundant phenotype in this brain area. Interestingly, striatal neuropil contained many boutons immunostained for synaptic vesicle protein 2 and synaptophysin which colocalize with tyrosine hydroxylase (TH), suggesting a degenerative process with pre-synaptic accumulation of synaptic vesicles. When we studied the effects on substantia nigra, we found the disappearance of dopaminergic neurons, shown by loss of TH immunoreactivity, loss of expression of TH and dopamine transporter mRNAs, and disappearance of FluoroGold-labelled nigral neurons. The degeneration of substantia nigra dopaminergic neurons was produced through up-regulation of cFos mRNA, apoptosis and accumulation of alpha-synuclein shown by colocalization experiments. Thrombin effects could be mediated by protease-activated receptor 4 activation, as protease-activated receptor 4-activating peptide mimicked thrombin effects. Our results point out the possible relationship between synapse elimination and retrograde degeneration in the nigral dopaminergic system.

Cellular Localization of AMPA Type Glutamate Receptor Subunits in the Basal Ganglia of Pigeons (Columba livia)

Corticostriatal and thalamostriatal projections utilize glutamate as a neurotransmitter in mammals and birds. The influence on striatum is mediated, in part, by ionotropic AMPA-type glutamate receptors, which are heteromers composed of GluR1–4 subunits. Although the cellular localization of AMPA-type subunits has been well characterized in mammalian basal ganglia, their localization in avian basal ganglia has not. We thus carried out light microscopic single- and double-label and electron microscopic single-label immunohistochemical studies of GluR1–4 distribution and cellular localization in pigeon basal ganglia. Single-label studies showed that the striatal neuropil is rich in GluR1, GluR2, and GluR2/3 immunolabeling, suggesting the localization of GluR1, GluR2 and/or GluR3 to the dendrites and spines of striatal projection neurons. Double-label studies and perikaryal size distribution determined from single-label material indicated that about 25% of enkephalinergic and 25% of substance P-containing striatal projection neuron perikarya contained GluR1, whereas GluR2 was present in about 75% of enkephalinergic neurons and all substance-P -containing neurons. The perikaryal size distribution for GluR2 compared to GluR2/3 suggested that enkephalinergic neurons might more commonly contain GluR3 than do substance P neurons. Parvalbuminergic and calretininergic striatal interneurons were rich in GluR1 and GluR4, a few cholinergic striatal interneurons possessed GluR2, but somatostatinergic striatal interneurons were devoid of all subunits. The projection neurons of globus pallidus all possessed GluR1, GluR2, GluR2/3 and GluR4 immunolabeling. Ultrastructural analysis of striatum revealed that GluR1 was preferentially localized to dendritic spines, whereas GluR2/3 was found in spines, dendrites, and perikarya. GluR2/3-rich spines were generally larger than GluR1 spines and more frequently possessed perforated post-synaptic densities. These results show that the diverse basal ganglia neuron types each display different combinations of AMPA subunit localization that shape their responses to excitatory input. For striatal projection neurons and parvalbuminergic interneurons, the combinations resemble those for the corresponding cell types in mammals, and thus their AMPA responses to glutamate are likely to be similar.

Anatomical basis of glial cell line-derived neurotrophic factor expression in the striatum and related basal ganglia during postnatal development of the rat.

There is increasing evidence that glial cell line-derived neurotrophic factor (GDNF) plays a role as a limiting, striatal target-derived neurotrophic factor for dopamine neurons of the substantia nigra pars compacta (SNpc) by regulating the magnitude of the first phase of postnatal natural cell death which occurs in these neurons. While it has been shown that GDNF mRNA is relatively abundant in postnatal striatum, the cellular basis of its expression has been unknown. We therefore used nonradioactive in situ hybridization and immunohistochemistry to examine the cellular basis of GDNF mRNA and protein expression, respectively, in postnatal striatum and related structures. We found that GDNF mRNA is expressed within medium-sized striatal neurons. Expression in glia was not observed. At the protein level, regionally, GDNF expression in striatum was observed in striosomal patches, as previously described. At a cellular level a few neurons were observed, but they do not account for the striosomal pattern. This pattern is predominantly due to GDNF-positive neuropil. Some of this neuropil arises from tyrosine hydroxylase-positive nigro-striatal dopaminergic afferents. Astrocytic processes do not appear to contribute to the striosomal pattern. GDNF-positive fibers are identified not only within intrinsic striatal neuropil, but also in fibers within the major striatal efferent targets: the globus pallidus, the entopeduncular nucleus, and the SN pars reticulata. We conclude that during normal postnatal development, medium-sized neurons are the principal source of GDNF within the striatum.

Connectivity and cytoarchitecture of the ventral telencephalon in the salamander Plethodon shermani

The cytoarchitecture and axonal connection pattern of centers in the ventral telencephalon of the salamander Plethodon shermani were studied using biocytin for anterograde and retrograde labeling of cell groups, as well as by intracellular injections. Application of biocytin to the main and accessory olfactory bulbs identified the olfactory pallial regions and the vomeronasal portion of the amygdala, respectively. According to our results, the amygdala of Plethodon is divided into (1) a rostral part projecting to visceral and limbic centers and receiving afferents from the dorsal thalamus, and (2) a caudal part receiving accessory olfactory input. The striatopallial transition area (SPTA) lies rostrodorsally to the caudal (vomeronasal) amygdala and is similar in connections and possibly in function. The rostral striatum has few descending projections to the medulla, whereas the intermediate striatum sends strong projections to the tegmentum and medulla. The caudal striatum has strong ascending projections to the striatum and descending projections to the ventral hypothalamus. The dendritic trees of neurons labeled below the striatum and in the SPTA spread laterally from the soma, whereas dendrites of striatal neurons converge into the laterally situated striatal neuropil. In the caudal amygdala, three distinct types of neurons are found differing in dendritic arborization. It is concluded that, hodologically, the rostral part of the urodele amygdala corresponds to the central and basolateral amygdala and the caudal part to the cortical/medial amygdala of mammals. The urodele striatum is divided into a rostral striatum proper, an intermediate dorsal pallidum, and a caudal part, with distinct connections described here for the first time in a vertebrate.

Subependymal Zone: Immunohistochemically Distinct Compartment in the Adult Mammalian Forebrain

The subependymal zone (SEZ) lining lateral walls of the lateral cerebral ventricles represents the site of active neurogenesis in the brain of adult mammals. Peroxidase immunohistochemistry performed in paraffin-embedded sections reveals that structural organization of the SEZ differs from other regions in the brain. The SEZ is devoid of synapses that are abundant in the adjacent striatal neuropil. Therefore immunostaining of synaptophysin detects sharp borders of the SEZ. Using immunophenotypization, we identified cell types constituting the SEZ in the intact rat forebrain. The presence of neural progenitor/stem cells was confirmed by finding of nestin-immunopositive cells. Detection of the astroglial marker GFAP confirmed that astrocytes represented major supporting elements responsible for creating a unique microenvironment of the SEZ. One type of the astroglia participated in covering surfaces of the blood vessels and boundaries of the SEZ. The second astroglial cell type formed branched elongated tubes that enwrapped other SEZ cell types with their cytoplasmic extensions. The interior of astrocytic channels was occupied with small densely aggregated NCAM-immunoreactive neuroblasts. Bipolar morphology indicated that these cells probably underwent migration. Immunodetection of other neuronal markers like beta-III tubulin, MAP-2 and Pan neurofilaments identified positive cells in the neighbouring brain parenchyma but not in the SEZ. The rostral migratory stream (RMS) linked with the anterior SEZ had a similar structural arrangement. It contained a large amount of nestin+ and vimentin+ cells. The RMS consisted of GFAP+ astrocytic tubes ensheathing NCAM+ neuroblasts. On the contrary to the SEZ, the RMS neuroblasts expressed beta-III tubulin. However, markers of postmitotic neurons MAP-2, Pan neurofilaments and synaptophysin were not expressed in the RMS. Our study describes a complex histological structure of the rat SEZ, identifies its individual cell types and demonstrates a usefulness of immunohistochemical detection of cell-specific markers in a study of microenvironment forming neurogenic zones in the mammalian brain.

Immunohistochemical localization of PDE10A in the rat brain

PDE10A is a newly identified cAMP/cGMP phosphodiesterase for which mRNA is highly expressed in the mammalian striatum. In the present study, PDE10A protein and mRNA expression throughout the rat brain were determined, using a monoclonal antibody (24F3.F11) for Western blot and immunohistochemical analyses and an antisense riboprobe for in situ hybridization. High levels of mRNA are observed in most of the neuronal cell bodies of striatal complex (caudate n, n. accumbens and olfactory tubercle), indicating that PDE10A is expressed by the striatal medium spiny neurons. PDE10A-like immunoreactivity is dense throughout the striatal neuropil, as well as in the internal capsule, globus pallidus, and substantia nigra. These latter regions lack significant expression of PDE10A mRNA. Thus, PDE10A is transported throughout the dendritic tree and down the axons to the terminals of the medium spiny neurons. These data suggest a role for PDE10A in regulating activity within both the striatonigral and striatopallidal pathways. In addition, PDE10A immunoreactivity and mRNA are found at lower levels in the hippocampal pyramidal cell layer, dentate granule cell layer and throughout the cortex and cerebellar granule cell layer. Immunoreactivity is detected only in cell bodies in these latter regions. This more restricted subcellular localization of PDE10A outside the striatum suggests a second, distinct function for the enzyme in these regions.

Group I Metabotropic Glutamate Receptors in the Monkey Striatum: Subsynaptic Association with Glutamatergic and Dopaminergic Afferents

Group I metabotropic glutamate receptors (mGluRs) are involved in long-term synaptic plasticity and neuroprotection in the striatum, but the specific role(s) of mGluR1 and mGluR5 remain poorly understood. In this study, we used electron-microscopic immunocytochemistry to compare the pattern of subsynaptic and subcellular distribution of mGluR1a and mGluR5 in relation to putative glutamatergic and dopaminergic inputs to the monkey striatum. At the light-microscopic level, both group I mGluRs are expressed in the striatal neuropil. In addition, numerous perikarya of striatal output neurons are immunostained for mGluR5, but much less frequently for mGluR1a. At the electron-microscopic level, immunoreactivity for both receptor subtypes is primarily expressed postsynaptically in dendrites and spines, although presynaptic mGluR1a labeling of glutamatergic thalamostriatal boutons and, less frequently, dopaminergic and corticostriatal terminals is also seen. In contrast to mGluR1a, mGluR5 immunoreactivity is rarely encountered presynaptically. In postsynaptic elements, 40-70% of immunoreactivity for both receptor subtypes is expressed intracellularly, whereas 30-60% is apposed to the plasma membrane. More than 80% of the labeling apposed to the plasma membrane is extrasynaptic. The remaining 20% is located at the edges of putative glutamatergic synapses or in the active zone of symmetric synapses. In mGluR5-, but not mGluR1a-immunostained sections, approximately 70% of dopaminergic symmetric synapses are labeled perisynaptically. These data emphasize the differential pattern of subsynaptic localization of the two group I mGluRs and provide various presynaptic and postsynaptic sites whereby mGluR1 and mGluR5 could mediate different, but complementary, effects on glutamatergic and dopaminergic transmission in the primate striatum.

Striatal monoaminergic terminals in Lewy body and Alzheimer's dementias

Vesicular monoamine transporter type 2 and benzodiazepine binding site expressions were examined with quantitative autoradiography in postmortem striata from 19 patients with dementia with Lewy bodies, seven patients with dementia with Lewy bodies and Alzheimer's disease, 12 patients with Alzheimer's disease, and eight neurologically normal subjects. Striatal vesicular monoamine transporter type 2 expression in dementia with Lewy bodies and in dementia with Lewy bodies plus Alzheimer's disease was reduced significantly, indicating degeneration of nigrostriatal projections. Striatal benzodiazepine binding site expression was unchanged, indicating preserved intrinsic striatal neuropil. Vesicular monoamine transporter type 2 and benzodiazepine binding site expressions were each preserved in Alzheimer's disease striatum. We conclude that dementia with Lewy bodies may be distinguished from Alzheimer's disease by postmortem examination or by future in vivo measurements of the striatal vesicular monoamine transporter type 2.

The Potential for Circuit Reconstruction by Expanded Neural Precursor Cells Explored through Porcine Xenografts in a Rat Model of Parkinson's Disease

Neural precursors with the properties of neural stem cells can be isolated from the developing brain, can be expanded in culture, and have been suggested as a potential source of cells for neuronal replacement therapies in degenerative disorders such as Parkinson's disease (PD). Under such conditions an improved spectrum of functional benefit may be obtained through homotypic reconstruction of degenerated neural circuitry, and to this end we have investigated the potential of expanded neural precursor cells (ENPs) to form long axonal projections following transplantation in the 6-hydroxydopamine-lesioned rat model of PD. ENPs have been isolated from the embryonic pig, since implantation in a xenograft environment is thought to favor axonal growth. These porcine ENPs possessed similar properties in vitro to those described in other species: they proliferated in response to epidermal and fibroblast growth factor-2, expressed the neuroepithelial marker nestin, and differentiated into neurons, astrocytes, and occasional oligodendrocytes on mitogen withdrawal. The use of pig-specific markers following xenotransplantion into cyclosporin A-immunosuppressed rats revealed that many cells differentiated into neurons and displayed extensive axogenesis, such that when placed in the region of the substantia nigra fibers projected throughout the striatal neuropil. These neurons were not restricted in the targets to which they could project since following intrastriatal grafting fibers were seen in the normal striatal targets of the pallidum and substantia nigra. Staining for a pig-specific synaptic marker suggested that synapses were formed in these distant sites. A small number of these cells differentiated spontaneously to express a catecholaminergic phenotype, but were insufficient to mediate behavioral recovery. Our results suggest that when the efficiency of neurochemical phenotype induction is increased, ENP-derived neurons have the potential to be a uniquely flexible source of cells for therapeutic cell replacement where anatomical reconstruction is advantageous.

Metabotropic Glutamate Receptors mGluR1α and mGluR2/3 Display Dynamic Expression Patterns in Developing Rat Striatum

The developmental expression of two metabotropic glutamate receptors (mGluR), mGluR1α and mGluR2/3 was evaluated in the rat striatum from birth to adulthood. The mGluR1α receptor subtype displayed a patchy organization perinatally that became more homogeneous after the first postnatal week. The adult pattern of receptor expression consisted of homogeneous punctate profiles spread throughout the striatum. The mGluR2/3 receptor subtype exhibited a unique pattern of ontogenic expression, being associated exclusively with fibers of the internal capsule that penetrate the striatum, during the perinatal period. The protein localization for this subtype spread into the striatal neuropil after the first postnatal week, in parallel to the development of afferent terminations and arborizations to the nucleus. Unlike the ionotropic GluR subunits that are associated with somata and dendrites, neither subtype of metabotropic receptor was associated with neuronal cell bodies within the striatum.

α-Actinin-2 in rat striatum: localization and interaction with NMDA glutamate receptor subunits

Alpha-actinin (α-actinin-2) is a protein which links the NR1 and NR2B subunits of N-methyl- d-aspartate (NMDA) glutamate receptors to the actin cytoskeleton. Because of the importance of NMDA receptors in modulating the function of the striatum, we have examined the localization of α-actinin-2 protein and mRNA in striatal neurons, and its biochemical interaction with NMDA receptor subunits present in the rat striatum. Using an α-actinin-2-specific antibody, we found intense immunoreactivity in the striatal neuropil and within striatal neurons that also expressed parvalbumin, calretinin and calbindin. Conversely, α-actinin-2 immunoreactivity was not detected in neurons expressing choline acetyltransferase and neuronal nitric oxide synthase. Dual-label in situ hybridization revealed that the highest expression of α-actinin-2 mRNA is in substance P-containing striatal projection neurons. The α-actinin-2 mRNA is also present in enkephalinergic projection neurons and interneurons expressing parvalbumin, choline acetyl transferase and the 67-kDa isoform of glutamic acid decarboxylase, but was not detected in somatostatin-expressing interneurons. Immunoprecipitation of membrane protein extracts showed that α-actinin-2 is present in heteromeric complexes of NMDA subunits, but is not associated with AMPA receptors in the striatum. A subunit-specific anti-NR1 antibody co-precipitated major fractions of NR2A and NR2B subunits, but only a minor fraction of striatal α-actinin-2. Conversely, α-actinin-2 antibody immunoprecipitated only modest fractions of striatal NR1, NR2A and NR2B subunits. These data demonstrate that α-actinin-2 is a very abundant striatal protein, but exhibits cellular specificity in its expression, with very high levels in substance-P-containing projection neurons, and very low levels in somatostatin and neuronal nitric oxide synthase interneurons. Despite the high expression of this protein in the striatum, only a minority of NMDA receptors are linked to α-actinin-2. This interaction may identify a subset of receptors with distinct anatomical and functional properties.

Immunoblot and immunohistochemical comparison of murine monoclonal antibodies specific for the rat D1a and D1b dopamine receptor subtypes

The two D1-like dopamine receptor subtypes, D1a and D1b, are structurally similar and pharmacologically indistinguishable using currently available ligands. To differentiate between the D1-like dopamine receptor subtypes, murine monoclonal antibodies to the rat D1a and the rat D1b dopamine receptor have been prepared. Rat D1-like and D2-like dopamine receptors expressed in Sf9 cells were used to verify the immunospecificity of the monoclonal anti-(D1a dopamine receptor) and anti-(D1b dopamine receptor) antibodies using immunoblot and immunohistochemical techniques. These two antibodies were used to compare the temporal dynamics of D1-like dopamine receptors expressed in Sf9 cells following infection with recombinant baculovirus and to monitor the partial purification of detergent solubilized receptors following ion exchange chromatography. Immunoreactivity of the anti-(D1a receptor) antibody was observed in the striatum and cortical regions of the rat brain using immunoblot techniques. No reactivity on immunoblots was observed for the anti-(D1b receptor) antibody using rat brain tissue, probably due to the low levels of receptor expression. For immunohistochemical studies using rat brain slices, the anti-(D1a receptor) antibody heterogeneously labeled cells and punctate processes within the striatal neuropil while labeling in the adjacent cerebral cortex was weak. Anti-(D1b receptor) antibody immunoreactivity was weak in the striatum and generally limited to sparse perikarya in the dorsal region. However, immunoreactivity was observed in numerous cells within the vertical and horizontal limbs of the diagonal band and in the ventral pallidum. Immunoreactivity of the anti-(D1b receptor) antibody was also observed in layer V pyramidal neurons of the frontal sensorimotor cortex.

Immunohistochemical Localization of NMDA- and AMPA-Type Glutamate Receptor Subunits in the Basal Ganglia of Red-Eared Turtles

Corticostriatal and thalamostriatal projection systems have been shown to utilize glutamate as a neurotransmitter in mammals and birds. Although corticostriatal and thalamostriatal projection systems have been demonstrated in turtles, it is uncertain whether they too use glutamate as their neurotransmitter. Immunohistochemical localization of glutamate and of NMDA- and AMPA-type ionotropic glutamate receptor subunits (NMDAR2A/B, GluR1, GluR2/3, and GluR4) were used to address this issue. Numerous medium-sized neurons that were rich in NMDAR2A/B and GluR2/3 were observed in the striatal part of the basal ganglia of red-eared turtles. Smaller numbers of medium-sized neurons and some large neurons rich in the GluR1 and GluR4 subunits were also observed in the striatum. The striatal neuropil was notably rich in GluR1, GluR2/3 and NMDAR2A/B subunits. The pallidal region was specifically rich in large neurons possessing GluR4 subunits. Consistent with the glutamate receptors on striatal and pallidal neurons, sources of input to the striatum and pallidum in turtle such as the dorsomedial and dorsolateral thalamic nuclei (which appear to correspond to intralaminar thalamic nuclei), telencephalic pallial cell groups, and the apparent subthalamic nucleus homologue were rich in glutamatergic neurons. The results show that the thalamostriatal, corticostriatal and subthalamo-pallidal projection systems of turtles are glutamatergic and that similar basal ganglia cell types in turtles and mammals have largely similar glutamate receptor characteristics.

Cloning and Characterization of RGS9-2: A Striatal-Enriched Alternatively Spliced Product of the RGS9 Gene

Regulators of G-protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) for alpha subunits of heterotrimeric G-proteins. Previous in situ hybridization analysis of mRNAs encoding RGS3-RGS11 revealed region-specific expression patterns in rat brain. RGS9 showed a particularly striking pattern of almost exclusive enrichment in striatum. In a parallel study, RGS9 cDNA, here referred to as RGS9-1, was cloned from retinal cDNA libraries, and the encoded protein was identified as a GAP for transducin (Galphat) in rod outer segments. In the present study we identify a novel splice variant of RGS9, RGS9-2, cloned from a mouse forebrain cDNA library, which encodes a striatal-specific isoform of the protein. RGS9-2 is 191 amino acids longer than the retinal isoform, has a unique 3' untranslated region, and is highly enriched in striatum, with much lower levels seen in other brain regions and no expression detectable in retina. Immunohistochemistry showed that RGS9-2 protein is restricted to striatal neuropil and absent in striatal terminal fields. The functional activity of RGS9-2 is supported by the finding that it, but not RGS9-1, dampens the Gi/o-coupled mu-opioid receptor response in vitro. Characterization of a bacterial artificial chromosome genomic clone of approximately 200 kb indicates that these isoforms represent alternatively spliced mRNAs from a single gene and that the RGS domain, conserved among all known RGS members, is encoded over three distinct exons. The distinct C-terminal domains of RGS9-2 and RGS9-1 presumably contribute to unique regulatory properties in the neural and retinal cells in which these proteins are selectively expressed.