NMDA receptor function in mouse models of Huntington disease.

Huntington disease (HD) is an autosomal dominant disorder in which degeneration of medium-sized spiny striatal neurons occurs. The HD gene and the protein it encodes, huntingtin, have been identified but their functions remain unknown. Transgenic mouse models for HD have been developed and we examined responses of medium-sized striatal neurons recorded in vitro to application of N-methyl-D-aspartate (NMDA) in two of these. The first model (R6/2) expresses exon 1 of the human HD gene with approximately 150 CAG repeats. In the R6/2 an enhancement of currents induced by selective activation of NMDA receptors as well as an enhancement of intracellular Ca(2+) flux occurred in both presymptomatic and symptomatic mice. These alterations appeared specific for the NMDA receptor because alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-mediated currents were reduced in symptomatic R6/2s. In R6/2 animals there were parallel increases in NMDA-R1 and decreases in NMDA-R2A/B subunit proteins as established by immunohistochemistry. The second model (YAC72) contains human genomic DNA spanning the full-length gene and all its regulatory elements with 72 CAG repeats. The phenotypical expression of the disorder develops more gradually than in the R6/2. In YAC72 mice we found similar but less marked increases in responses of medium-sized striatal neurons to NMDA. These findings indicate that alterations in NMDA receptor function may predispose striatal neurons to excitotoxic damage, leading to subsequent neuronal degeneration and underscore the functional importance of NMDA receptors in HD.

Changes in cortical and striatal neurons predict behavioral and electrophysiological abnormalities in a transgenic murine model of Huntington's disease.

Neurons in Huntington's disease exhibit selective morphological and subcellular alterations in the striatum and cortex. The link between these neuronal changes and behavioral abnormalities is unclear. We investigated relationships between essential neuronal changes that predict motor impairment and possible involvement of the corticostriatal pathway in developing behavioral phenotypes. We therefore generated heterozygote mice expressing the N-terminal one-third of huntingtin with normal (CT18) or expanded (HD46, HD100) glutamine repeats. The HD mice exhibited motor deficits between 3 and 10 months. The age of onset depended on an expanded polyglutamine length; phenotype severity correlated with increasing age. Neuronal changes in the striatum (nuclear inclusions) preceded the onset of phenotype, whereas cortical changes, especially the accumulation of huntingtin in the nucleus and cytoplasm and the appearance of dysmorphic dendrites, predicted the onset and severity of behavioral deficits. Striatal neurons in the HD mice displayed altered responses to cortical stimulation and to activation by the excitotoxic agent NMDA. Application of NMDA increased intracellular Ca(2+) levels in HD100 neurons compared with wild-type neurons. Results suggest that motor deficits in Huntington's disease arise from cumulative morphological and physiological changes in neurons that impair corticostriatal circuitry.

Dopamine D4 receptor-deficient mice display cortical hyperexcitability.

The dopamine D(4) receptor (D(4)R) is predominantly expressed in the frontal cortex (FC), a brain region that receives dense input from midbrain dopamine (DA) neurons and is associated with cognitive and emotional processes. However, the physiological significance of this dopamine receptor subtype has been difficult to explore because of the slow development of D(4)R agonists and antagonists the selectivity and efficacy of which have been rigorously demonstrated in vivo. We have attempted to overcome this limitation by taking a multidimensional approach to the characterization of mice completely deficient in this receptor subtype. Electrophysiological current and voltage-clamp recordings were performed in cortical pyramidal neurons from wild-type and D(4)R-deficient mice. The frequency of spontaneous synaptic activity and the frequency and duration of paroxysmal discharges induced by epileptogenic agents were increased in mutant mice. Enhanced synaptic activity was also observed in brain slices of wild-type mice incubated in the presence of the selective D(4)R antagonist PNU-101387G. Consistent with greater electrophysiological activity, nerve terminal glutamate density associated with asymmetrical synaptic contacts within layer VI of the motor cortex was reduced in mutant neurons. Taken together, these results suggest that the D(4)R can function as an inhibitory modulator of glutamate activity in the FC.

Metabotropic glutamate receptors mGluR1alpha and mGluR2/3 display dynamic expression patterns in developing rat striatum.

The developmental expression of two metabotropic glutamate receptors (mGluR), mGluR1alpha and mGluR2/3 was evaluated in the rat striatum from birth to adulthood. The mGluR1alpha 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.

Facilitated glutamatergic transmission in the striatum of D2 dopamine receptor-deficient mice.

Dopamine (DA) receptors play an important role in the modulation of excitability and the responsiveness of neurons to activation of excitatory amino acid receptors in the striatum. In the present study, we utilized mice with genetic deletion of D2 or D4 DA receptors and their wild-type (WT) controls to examine if the absence of either receptor subtype affects striatal excitatory synaptic activity. Immunocytochemical analysis verified the absence of D2 or D4 protein expression in the striatum of receptor-deficient mutant animals. Sharp electrode current- and whole cell patch voltage-clamp recordings were obtained from slices of receptor-deficient and WT mice. Basic membrane properties were similar in D2 and D4 receptor-deficient mutants and their respective WT controls. In current-clamp recordings in WT animals, very little low-amplitude spontaneous synaptic activity was observed. The frequency of these spontaneous events was increased slightly in D2 receptor-deficient mice. In addition, large-amplitude depolarizations were observed in a subset of neurons from only the D2 receptor-deficient mutants. Bath application of the K+ channel blocker 4-aminopyridine (100 microM) and bicuculline methiodide (10 microM, to block synaptic activity due to activation of GABA(A) receptors) markedly increased spontaneous synaptic activity in receptor-deficient mutants and WTs. Under these conditions, D2 receptor-deficient mice displayed significantly more excitatory synaptic activity than their WT controls, while there was no difference between D4 receptor-deficient mice and their controls. In voltage-clamp recordings, there was an increase in frequency of spontaneous glutamate receptor-mediated inward currents without a change in mean amplitude in D2 receptor-deficient mutants. In WT mice, activation of D2 family receptors with quinpirole decreased spontaneous excitatory events and conversely sulpiride, a D2 receptor antagonist, increased activity. In D2 receptor-deficient mice, sulpiride had very little net effect. Morphologically, a subpopulation of medium-sized spiny neurons from D2 receptor-deficient mice displayed decreased dendritic spines compared with cells from WT mice. These results provide evidence that D2 receptors play an important role in the regulation of glutamate receptor-mediated activity in the corticostriatal or thalamostriatal pathway. These receptors may function as gatekeepers of glutamate release or of its subsequent effects and thus may protect striatal neurons from excessive excitation.

Fluorescent in situ transcription in cells and tissues.

This method assesses cellular mRNA transcripts in tissue sections and cell cultures using unique short anti-sense primers directed against sequences in particular protein(s). The unlabeled synthetic cDNA oligonucleotide primers are extended complementary to a sense mRNA transcript using reverse transcriptase and labeled through incorporation of a fluorescent-labeled dUTP nucleotide base. The new cDNA will be synthesized upstream from the point of primer hybridization, and has a specific activity of fluorescent labeling dependent upon the length of the template mRNA from the primer location to the 5'-terminus. This procedure provides rapid detection of low abundance mRNA messages that can be related to other cellular protein components, labeled experimentally with alternative fluorochromes.

Striatal ionotropic glutamate receptor ontogeny in the rat.

Rat striatal N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate (KA) receptor staining were evaluated postnatally in the rat. Immunohistochemistry was used to detect subunit proteins of the three glutamate receptor subtypes. The glutamate receptors displayed distinct developmental expression patterns in the striatum. Morphological distributions for the NMDA R1 subunit (representative of NMDA receptors), Glu R1 and Glu R2/3 subunits (indicative of AMPA receptors), and Glu R5/6/7 subunits (demonstrating KA receptors) attained adult expression patterns and levels at different postnatal time points. The ontogenic maturation sequence of striatal glutamate receptor expression was KA, then AMPA and lastly NMDA. Staining patterns for NMDA and AMPA subunit proteins were detected initially as dense patches in the neuropil, which changed to a homogeneous stain of the striatum by the second week of life. Cellular staining for the three subtypes was intense within the highly reactive neuropil patches, but less intensely stained in neurons located outside these zones. The KA receptor subunit did not exhibit neuropil heterogeneity, but was distributed evenly at birth. All three glutamate receptor subtypes were visible within the striatal neuron populations. Populations of striatal neurons that expressed the three differential glutamate receptor subtypes overlap, exhibit different growth patterns and dendritic staining. These results support a functional emergence of different glutamate receptor activation within the striatum and provide a potential therapeutic means to isolate developmental disorders specifically associated with excitatory circuits of the basal ganglia.

Electrophysiological and morphological analyses of cortical neurons obtained from children with catastrophic epilepsy: dopamine receptor modulation of glutamatergic responses.

The present study examined the electrophysiological effects produced by activation of specific dopamine (DA) receptors and the distribution of DA receptor subtypes and glutamate receptor subunits [N-methyl-D-aspartate (NMDAR1) and GluR1] in cortical tissue samples obtained from children (ages 3 months to 16 years) undergoing epilepsy surgery. DA receptor activation produced differential effects depending on the receptor subtype that was activated. D1 receptor family agonists generally enhanced cortical excitability and favored the emergence of epileptogenic activity. In contrast, D2 receptor family agonists had more variable effects on cortical excitability and the expression of epileptiform discharges. Activation of D1 or D2 receptors decreased the amplitude of non-NMDA-mediated excitatory postsynaptic potentials. In contrast, DA and D1 agonists increased the amplitude of NMDA-mediated potentials. Immunohistochemical analysis showed that the DA receptor subtypes and glutamate receptor subunits examined were present in all cortical layers and areas throughout development. Whole-cell voltage clamp recordings of pyramidal neurons visualized with differential interference contrast optics and infrared videomicroscopy indicated that these neurons displayed a persistent Na(+) current, followed by an outward current. DA reduced the outward current but had little effect on the persistent Na(+) current. These results suggest a dual role for DA's actions in the human cerebral cortex. Activation of D2 receptors or antagonism of D1 receptors may help control seizures in children.

Striatal excitatory amino acid receptor subunit expression in the D1A-dopamine receptor-deficient mouse.

The influence of dopamine receptor deletion on the expression and distribution of striatal excitatory amino acid (EAA) receptor subunits comprising the N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtypes were examined in the D1A dopamine (DA) receptor-deficient mouse. EAA receptor subunit immunofluorescent staining was altered by the DA receptor genetic mutation. The NMDA-R1 subunit was used as a marker for NMDA-type receptors. The number of striatal neurons expressing this subunit decreased and there was a modest attenuation in the neuropil staining in the mutants in contrast to littermate controls. The R1 subunit for the glutamate receptor (GluR1) was used as an indicator of the AMPA receptor subtype. Immunostaining for this subunit also showed changes induced by deletion of the DA receptor subtype. In contrast to the NMDA-R1 subunit, neuropil staining for the GluR1 subunit was elevated in the mutant in comparison to littermate controls, such that the immunofluorescent reaction obscured detection of the subunit protein in striatal interneurons. The results are discussed in relation to the potential impact on functional interactions between the EAA and DA systems in the striatum.

Coexpression of striatal dopamine receptor subtypes and excitatory amino acid subunits.

The striatal cellular coexpression patterns for the D(1A) and D2 dopamine (DA) receptor subtypes and the ionotropic excitatory amino acid (EAA) subunits of the N-methyl-D-aspartate (NMDA-R1) and the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) (GluR1 and GluR2/3) receptor subunits were examined morphologically. Their coincidence was assessed by visualization of mRNA transcripts, localization of encoded receptor proteins, and binding analysis using concurrently paired methods of fluorescence detection. The findings indicated that 1) mRNA transcripts for both receptor systems were detected in the medium-sized neuron population, and the distribution of receptor message closely reflected protein and binding patterns, with the exception of the GluR1 subunit; 2) both DA receptor mRNA transcripts were coexpressed with each ionotropic EAA receptor subunit examined and with each other, and NMDA and AMPA receptor subunits also showed coincident expression; 3) D(1A) DA receptor protein was detected in neurons which coexpressed EAA subunit proteins; and 4) GluR2/3 and NMDA-R1 subunit proteins were coexpressed in medium-sized neurons which also demonstrated D2 DA receptor binding sites. These findings suggest morphological receptor "promiscuity" since the coexpression patterns between DA and EAA receptors were found in all permutations. The results provide a spatial framework for physiological findings describing functional interactions between the two DA receptor types and between specific DA and EAA receptors in the striatum.

Cellular distribution of the rat D4 dopamine receptor protein in the CNS using anti-receptor antisera.

A polyclonal antiserum was generated against a unique peptide fragment in the rat D4 dopamine (DA) receptor. The titer was monitored using solid-phase ELISA and once it was established, specificity was assessed using Chinese Hamster Ovary (CHO) cells, stably transfected with the full-length cDNA for the rat D4 DA receptor. Immunofluorescent staining produced by incubation with the anti-D4 DA receptor antiserum was selective for D4 DA receptor-transfected CHO cells, and was expressed at their cell membranes and cytoplasm. Attenuated staining for D4 DA receptor protein was visible in untransfected, K1 CHO cells, and in D2 or D3 DA receptor-transfected CHO cells. The regional and cellular CNS distribution patterns for the D4 DA receptor subtype were examined, and illustrated significant protein levels within the frontal (FCx) and parietal cortices. Lesser amounts of receptor protein staining occurred in the thalamus, globus pallidus, hippocampus, cerebellar vermis, and very low expression was detected in the striatum (CPu). D4 DA receptor protein staining was correlated with the cellular expression of its mRNA transcripts in these same brain regions using concurrent fluorescent analyses. The homologous coincidence in staining patterns for the D4 DA receptor transcripts and encoded proteins in identified neurons of the FCx and CPu showed variations in receptor expression in these identified basal ganglia pathways.

Cellular distribution of the rat D1B receptor in central nervous system using anti-receptor antisera.

Polyclonal antisera have been generated against two unique polypeptide fragments in the rat D1B dopamine (DA) receptor, as deduced from the cDNA sequence. Antisera titers were monitored using solid-phase ELISA. Once the titers were established, antisera specificity was determined using Chinese Hamster ovary (CHO) cells, stably transfected with the full-length cDNA for the rat D1B DA receptor. Immunoreactivity following staining with either anti-D1B DA receptor antisera was equivalent, selective for the D1B DA receptor-transfected CHO cells, and expressed at their membrane and within the cell cytoplasm. Minimal immunofluorescent staining for D1B DA receptor proteins was detected in untransfected CHO cells, or in D1A DA receptor-transfected CHO cells. The regional and cellular distribution patterns for the D1B DA receptor subtype were examined in various brain areas and illustrated significant protein levels within the frontal and parietal cortices and in the hippocampus and dentate gyrus. Lesser amounts of receptor protein staining were seen in the dorsal striatum, olfactory tubercle, and cerebellar vermis. D1B DA receptor protein staining was correlated with the cellular expression of D1B DA receptor mRNA transcripts in these same brain regions using concurrent fluorescent analyses. The homologous coincidence in staining patterns for the D1B DA receptor transcripts and encoded proteins in identified neurons of the frontal cortex and striatum showed variations in receptor expression in these identified basal ganglia pathways.

Agonist-induced morphologic decrease in cellular D1A dopamine receptor staining.

The distribution of D1A dopamine (DA) receptor proteins was assessed by using subtype specific antireceptor antisera after acute DA exposure. The immunofluorescent staining of D1A DA receptor protein expression was examined in (1) stably transfected Chinese hamster ovary (CHO) cells, (2) primary striatal cell cultures, and (3) rat striatal brain slices. After agonist exposure as brief as 2 min and as long as 60 min, profound loss of immunofluorescent D1A receptor protein staining occurred in each paradigm. Additionally in the tissue slice, immunofluorescent neuropil staining for the receptor protein also was attenuated. The DA-induced alteration in receptor protein staining was blocked by the antagonist (+)-butaclamol and by the selective D1-family antagonist SCH 23390. Receptor staining patterns reverted back to the control immunofluorescent distribution within 15 min after removing the agonist from the bath. Immunofluorescence for the second-messenger cyclic AMP increased at all DA exposure times in the three experimental paradigms, was blocked by D1-family antagonists, and decreased to basal staining after brief recovery periods. This demonstrated the functional integrity of the D1A receptor in target cells. Pretreatment with the mitogenic plant lectin concanavalin A blocked the immunofluorescent decrease in receptor staining but not the elevation of the second messenger, indicating a morphologic distinction in these two events, parallel to other biochemical reports. The data suggested that a morphologic basis of acute homologous D1A DA receptor desensitization may be transposition of membrane-surface receptors to a transiently unavailable, intracellular compartment. This finding is supported by specific fluorescence incorporation of FM1-43, used as a marker of endocytosis, in CHO cells treated with DA.

Modulatory actions of dopamine on NMDA receptor-mediated responses are reduced in D1A-deficient mutant mice.

The role of D1 dopamine (DA) receptors in mediating the ability of DA to modulate responses attributable to activation of NMDA receptors was examined in mice lacking D1A dopamine receptors. Specifically, experiments were designed to test the hypothesis that the ability of DA to potentiate responses mediated by activation of NMDA receptors was attributable to activation of D1 receptors. Based on this hypothesis, we would predict that in the D1A mutant mouse, either DA would not induce enhancement of NMDA-mediated responses, or the enhancement would be severely attenuated. The results provided evidence to support the hypothesis. In mutant mice, DA and D1 receptor agonists did not potentiate responses mediated by activation of NMDA receptors. In contrast, in control mice, both DA and D1 receptor agonists markedly potentiated responses mediated by activation of NMDA receptors. The effects of DA in attenuating responses mediated by activation of non-NMDA receptors also were altered in the mutant, suggesting that this action of DA may require coupling or interactions between D1 and D2 receptors. The present studies also provided an opportunity to assess some of the basic electrophysiological and morphological properties of neostriatal neurons in mice lacking D1A DA receptors. Resting membrane potential, action potential parameters, input resistance, excitability, somatic size, dendritic extent, and estimates of spine density in mutants and controls were similar, suggesting that these basic neurophysiological and structural properties have not been changed by the loss of the D1A DA receptor.

Co-expression of receptor mRNA and protein: striatal dopamine and excitatory amino acid subtypes.

Dopamine (DA) is known to modulate the post-synaptic response of the excitatory amino acid (EAA) neurotransmitters in the striatum. Thus the intrinsic neurons in this nucleus are potential sites of cross-interaction between these two systems. The recent isolation of 5 different DA receptor subtypes and more than 20 EAA subunits argues for a complicated functional role for the protein products encoded by these transcripts. The simultaneous detection of cellular mRNA distributions and translated protein products was an initial step to determine differences in post-translational expression at the cellular level of resolution for two of these receptors. The cloned D2 DA receptor subtype and the ionotropic GluR1 EAA receptor subunit were examined by fluorescence in situ transcription (FIST) following hybridization of specific cDNA primers, complementary to the mRNA transcripts encoding these receptors. Nascent extension of the annealed primer using reverse transcriptase was detected after incorporation of fluorescently labeled dUTP. Protein products were visualized by standard immunofluorescence after incubation with anti-peptide antisera that were selective for each receptor protein. The experimental data corroborate previous work describing the regional expression of ligand binding and in situ hybridization detected with radiolabeled probes for the DA and EAA receptor systems in the striatum. The dual fluorescence method can be completed within 2 days and may be adapted to cellular localization of many novel mRNA/protein combinations to examine post-translational processing within thin tissue slices.

Neurophysin expression is stimulated by dopamine D1 agonist in dispersed hypothalamic cultures.

We have exposed primary dispersed hypothalamic cultures from 14-day-old fetal Sprague-Dawley rats to substances known to either elevate adenosine 3',5'-cyclic monophosphate (cAMP) levels or increase vasopressin (VP) secretion. The levels of VP in the medium collected from the cultures were determined by radioimmunoassay, and the number of neurophysin (NP)-positive cells after immunohistochemistry was counted. cAMP-elevating agents, 3-isobutyl-1-methylxanthine (200 microM) and forskolin (25 microM), in combination (I-F) maintained NP synthesis and VP secretion in 19-day cultures. I-F replacement by K+ (28 mM), isoproterenol (10 microM), glutamate (10 microM), or bicuculline (10 microM) during the last week of culture resulted in maintenance of NP expression and transient stimulation of VP secretion, but these agents did not induce NP expression independently of I-F treatment. In contrast, exposure to the dopamine D1 agonist SKF-38393 (10 microM) significantly increased NP expression independently and after replacement of I-F. Dopamine D1A receptors were detected by immunofluorescence on NP-expressing cells, providing a morphological basis for this response. These results suggest a role for D1A receptors in the regulation of VP gene expression.

D3 and D2 dopamine receptors: visualization of cellular expression patterns in motor and limbic structures.

The distribution of the D3 and D2 dopamine receptor subtypes in forebrain regions of the basal ganglia and mesocorticolimbic system was determined. This was assessed through combined fluorescent visualization of subtype selective anti-peptide antibodies for these cloned receptors and detection of their ligand recognition sites using the D2 subfamily antagonist,N-(p-aminophenethyl) spiperone (NAPS fluoroprobe). The double-labeling technique enabled direct comparison of the cloned receptor proteins and NAPS fluoroprobe binding in vitro. The application of these two methods together produced results comparable to single-labeling paradigms. Functional D3 receptors, defined as the coincident fluorescence of the D3 receptor antisera and fluoroprobe binding, were detected in the core region of the nucleus accumbens and exhibited a laminated expression pattern in the frontal cortex. D3 receptor protein was expressed robustly in neurons of the dorsolateral striatum, but showed an intense neuropil reaction in the globus pallidus. Functional D2 receptors, defined as the coincident fluorescence of the D2 receptor antisera and fluoroprobe binding, were detected in the frontal cortex and the medial shell of the nucleus accumbens. Thus, heterogeneities occurred in the cellular expression of functional D3 and D2 receptors in forebrain dopaminoceptive areas. D3 appears more related to basal ganglia and structures involved with motoric behavior, while D2 was associated with regions associated with cognitive/affective functions.

Localization of dopamine D1A receptor protein in rat kidneys.

The dopamine D1A receptor subtype was identified in rat kidney with both light microscopic immunohistochemistry and electron microscopic immunocytochemistry. Antipeptide polyclonal antisera were directed to both extracellular and intracellular regions of the native receptor. The use of such receptor-subtype-selective antibodies allows for the identification of specific dopamine receptor subtype clones that are not distinguished by current pharmacological or receptor-ligand binding technology. Selectivity of the antipeptide antisera was validated by their ability to recognize native receptor protein expressed in permanently transfected mouse LTK- cells. In the rat kidney, D1A receptor protein was localized to the juxtaglomerular apparatus (JGA), proximal tubule, distal tubule, cortical collecting duct, and renal vasculature. In the JGA, the receptor was predominantly located in the arteriolar smooth muscle layer within cytoplasmic granules previously shown to contain renin. In the proximal tubules, staining was localized both on the brush-border and basolateral membranes. The D1A receptor, which is present in the central nervous system, is now identified in the rat kidney at those sites previously labeled as DA1 receptor sites on the basis of pharmacological binding studies. These results suggest that at least some of the renal dopamine DA1 receptors correspond structurally to the central dopamine D1A receptor.

D2 dopamine receptor protein location: Golgi impregnation-gold toned and ultrastructural analysis of the rat neostriatum.

The neostriatal distribution of D2 dopamine receptor protein has been assessed using subtype-selective polyclonal antibodies generated against three unique polypeptide sequences of the receptor. The experimental tissues were processed by peroxidase based immunohistochemical procedures for routine light microscopy, Golgi impregnation-gold toned morphological characterization, and correlative light/electron microscopy. The results demonstrated a regional gradient of D2-like dopamine receptor expression in the neostriatum, where lateral portions in the nucleus exhibited more reactive cell bodies than medial portions. D2-like expression was detected in the three populations of neostriatal neurons, i.e., the medium-sized spiny projection neurons, and the medium- and large-sized aspiny interneuron types. Morphometric measurements of labeled neurons verified that medium and large diameter neurons expressed the D2-like receptor subtype. D2-like immunoreactivity was distributed throughout the cytoplasm in dendritic processes, and in presynaptic terminal boutons. Immunoreactivity for the receptor protein was also detected in small, thinly myelinated axons, suggesting the possibilities of anterograde transport of the receptor from cell bodies in the substantia nigra to their neostriatal terminal fields, as well as from local axon collaterals of neostriatal projections neurons. These findings provide evidence of widespread distribution of the D2-like receptor protein in neostriatal neurons, and showed that the presynaptic D2 receptors contain analogous epitopes to the postsynaptic receptor subtype.

Dopamine receptor distribution in the rat CNS: elucidation using anti-peptide antisera directed against D1A and D3 subtypes.

Anti-peptide antibodies were generated against amino acid sequences of intracellular and extracellular portions of the native proteins for the cloned rat D1A and D3 dopamine receptor subtypes in order to determine the cellular distribution of these specific forms in the brain. These polyclonal antisera exhibited high specific titers, assessed by ELISA and immunofluorescent detection of functional recombinant receptor proteins expressed in stably transfected Chinese hamster ovary (CHO) cells. Central nervous system (CNS) areas of the male rat were examined using standard immunofluorescent methods in fresh frozen tissues. This paradigm detected D1A-like and D3-like dopamine receptor staining primarily in larger-sized neurons throughout layers 3 and 5 of the cortex, in medium-diameter somata of the striatum, and in the densely packed cells of the olfactory tubercle and hippocampal formation. More attenuated immunoreactivity for both dopamine receptor subtypes was noted in the substantia nigra, not associated with perikarya. Differences in cellular staining patterns and intensity were evident between the D1A-like and D3-like dopamine receptor subtypes. Equivalent morphological elements exhibited dopamine receptor expression following incubation using antisera generated against either extracellular or intracellular epitopes of either the D1A or D3 native proteins. Dopamine receptor immunoreactivity could not be detected in the cerebellum at equivalent antisera dilutions used to discriminate cellular staining patterns within the forebrain. Fluorescent-labeled latex microspheres were infused into the substantia nigra terminal fields to retrogradely identify the cell bodies of the striatonigral projection system. This paradigm showed that 80% of striatonigral neurons expressed D1A-like receptors, while 65% demonstrated D3-like dopamine receptor staining. This distribution for the D1A-like and D3-like receptor subtypes suggests that overlap may occur in the expression of the receptors in the striatonigral neuron population. Our previous results localizing cellular D2-like receptor expression patterns in this projection system of the rat neostriatum implies that all three of these dopamine receptor subtypes may be co-expressed in this efferent system.