Ultrastructural localization of cyclic GMP and cyclic AMP in rat striatum.

The subcellular localization of cyclic GMP and cyclic AMP in the rat caudate-putamen has been studied using horseradish peroxidase immunocytochemistry. Both of the putative neurotransmitter second messengers were visualized in neurons and glial cells at light microscopic resolutions, but not all cells of either category gave detectable staining. This was confirmed at the ultrastructural level where both stained and unstained elements of the same cell type were found within the same field. A striking variation was seen in cyclic nucleotide staining intensity within individual neural and glial cells. Both of the cyclic nucleotides were detected within postsynaptic terminal boutons and within astroglial processes. Cyclic GMP postsynaptic staining was stronger than glial staining, whereas the localization pattern was reversed for cyclic AMP. The synaptic localization of cyclic AMP and cyclic GMP immunoreactivity adds support to the idea that these compounds have an influential role in synaptic function within the striatum.

Are neostriatal dopamine receptors co-localized?

The postsynaptic effects of dopamine in the neostriatum are mediated by five G-protein-coupled receptors. The extent to which these receptors are co-localized in neostriatal neurons has become controversial. This debate has far-reaching implications for treatment strategies in disorders of dopaminergic signaling, such as Parkinson's disease and schizophrenia. This review examines the molecular and cellular evidence for and against co-localization, including new information derived from single-cell mRNA amplification and patch-clamping of isolated neurons. It is concluded that this evidence is largely consistent with co-localization of functionally significant receptors of the D1 and D2 families in the majority of neostriatal efferent neurons. This conclusion has important implications for parallel processing models of the neostriatum.

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.

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.

Localization of D2 dopamine receptors in vertebrate retinae with anti-peptide antibodies.

Dopamine plays an important role in modulating various aspects of retinal signal processing. The morphology of dopaminergic neurons and its physiological effects are well characterized. Two classes of receptor molecules (D1 and D2) were shown pharmacologically to mediate specific actions, with differences between individual groups of vertebrates. In an attempt to better understand dopaminergic mechanisms at the cellular level, we used antisera against D2 receptors and investigated the localization of the dopamine D2 receptor in the retinae of rat, rabbit, cow, chick, turtle, frog, and two fish species with immunofluorescence techniques. Antisera were raised in rabbits to two oligopeptides predicted from rat D2 receptor cDNA; one specific for the splice-variant insertion in the third cytoplasmic loop and the other directed towards the extracellular amino terminal region shared by both short and long isoforms. Preadsorption with the synthetic peptide resulted in a significant reduction of label, indicating the presence of specific binding in all species except turtle and goldfish. The pattern of labelling produced by the two antisera was essentially identical; however, the staining obtained with antiserum to the extracellular motif was always more intense. Specific staining was present in photoreceptor inner and outer segments, and in the outer and inner plexiform layers of all species. In mammals and chick, strongly fluorescent perikarya were observed in the ganglion cell layer and at the proximal margin of the inner nuclear layer. Label may be present in the pigment epithelium but could not be established beyond doubt. This pattern of labelling is in accordance with previous observations on D2 receptor localization by means of radioactive ligand binding and in situ hybridization techniques. It suggests that retinal dopamine acts as a neuromodulator as well as a transmitter. In the distal retina, it may reach its targets via diffusion over considerable distances, even crossing the outer limiting membrane; in the inner and outer plexiform layers, conventional synaptic transmission seems to coexist with paracrine addressing of more distant targets, and D2 receptors are expressed by both amacrine and ganglion cells.

Distribution of components of the guanosine 3',5'-phosphate system in rat caudate-putamen.

The cellular distribution of guanylate cyclase (EC 4.6.1.2), guanosine 3',5'-phosphate (cyclic GMP), cyclic GMP-dependent protein kinase (EC 2.7.1.38), and cyclic GMP phosphodiesterase (EC 3.1.4.17) have been examined in the rostral rat caudate-putamen complex. Immunofluorescent staining for guanylate cyclase, cyclic GMP, and cyclic GMP-dependent protein kinase in fresh frozen caudate-putamen tissues is analogous to the immunoperoxidase localization in perfusion-fixed striatal slices. Homologous immunoreactivity in the cytoplasm and processes of ovoid and rounded neurons, 15-20 microns in diameter can be seen for these three components of the cyclic GMP system. Immunoreactive neurons are uniformly distributed throughout the caudate-putamen complex of all experimental tissue examined. Occasional large neurons, greater than 25 microns in diameter, in the ventral region of the striatum show immunoreactivity. Enzyme histochemical determination of the activities of guanylate cyclase and cyclic GMP phosphodiesterase show the medium-sized neuronal population (15-20 microns) contain hydrolytic activity for these proteins. Large- to medium-sized capillaries demonstrate guanylate cyclase synthetic activity, but the endothelial cells do not exhibit immunohistochemical staining. This suggests that physiological activity of an enzyme cannot be completely discerned through application of immunohistochemical procedures. Additionally, enzymatically detected guanylate cyclase histochemical activity was not uniformly distributed throughout the striatal neuropil. Enzyme histochemical detection of cyclic GMP phosphodiesterase demonstrates homologous cellular staining to guanylate cyclase enzymatic reactivity. The activity of the phosphodiesterase hydrolytic enzyme could be detected evenly distributed throughout the neuropil within cells 15-20 microns in diameter, analogous in cytoarchitecture to immunohistochemically visualized guanylate cyclase, cyclic GMP, and protein kinase elements. Ultrastructural examination of rat caudate-putamen demonstrates that the immunoreactivity for the components of the cyclic GMP system is predominantly distributed within the medium-spiny neuron subtype of this structure. Occasional aspiny neurons demonstrate peroxidase immunoreactivity for the cyclase, cyclic GMP, and the protein kinase, as does the luminal surface of capillary endothelial cells. The subcellular distribution of the antigenic determinants for these three elements and the hydrolytic activity of the phosphodiesterase enzyme show proximity to one another and are confined to the postsynaptic region of asymmetrical, but not symmetrical, terminal boutons. The asymmetrical terminal population of the caudate-putamen is derived from striatal afferents from the neocortex, intralaminar thalamus, and substantia nigra, and to a lesser extent the intrinsic striatal circuitry.(ABSTRACT TRUNCATED AT 400 WORDS)

Long-term changes in striatal D1 dopamine receptor distribution after dopaminergic deafferentation.

The morphochemical disposition of the adenylate cyclase-linked dopamine receptor (D1 type) in the rat striatum has been assessed at various time points after a neurotoxic lesion of the dopaminergic afferent pathway to the caudate nucleus. D1 receptor binding sites in the caudate nucleus were determined by in vitro autoradiography of the substituted benzazepine D1 antagonists, [3H]SCH 23390 or [125I]SCH 23982, and contrasted to the pattern of striatal immunohistochemical reactivity of the second messenger compound, cyclic 3',5'-adenosine monophosphate. The results demonstrate that the specific association of this dopamine receptor type with cyclic 3',5'-adenosine monophosphate-stained neurons is abolished at 7 days following chemical interruption of the nigrostriatal pathway, and the receptor disruption is persistent for durations as long as 20 weeks. This investigation suggests that once the postsynaptic receptor pathology is produced by deafferentation, it does not recover the selective morphochemical relationship normally established with the target cell containing the second messenger. This is in contrast to modest biochemical recuperation in D1 dopamine receptor binding seen using this experimental paradigm. This change in D1 dopamine receptor morphochemistry is discussed in relation to the neurochemical deficits produced by dopaminergic denervation and in Parkinson's disease.

Cyclic nucleotide distribution within rat striatonigral neurons.

Adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate have differential immunohistochemical distributions within retrogradely-labeled striatonigral neurons of the rat. Adenosine cyclic 3',5'-monophosphate is localized within more than half of the striatonigral projection neurons. It is also within the cytoplasm of other neurons and oligodendroglia. Guanosine cyclic 3'-5'-monophosphate is localized within 80% of the identified striatonigral neurons. These large percentages of cyclic nucleotide immunoreactivity within the striatonigral neurons suggest some of these efferent cells must contain both cyclic nucleotides. The immunofluorescent staining for guanosine cyclic 3',5'-monophosphate is almost identical to that reported for efferent neurotransmitter-containing neurons of the caudate nucleus. However, the large proportion of striatonigral neurons demonstrating guanosine cyclic 3',5'-monophosphate immunoreactivity precludes the association of the cyclic nucleotide with a selective neurotransmitter agent. Adenosine cyclic 3',5'-monophosphate-reactive elements are very different in staining appearance from guanosine cyclic 3',5'-monophosphate and neurotransmitter-identified somata. The number of striatonigral cells exhibiting reaction for this cyclic nucleotide does not eliminate the possibility that adenosine cyclic 3',5'-monophosphate might be preferentially co-localized with a specific neurotransmitter, such as gamma-aminobutyrate, as has been previously suggested through biochemical experimentation.

Behavioral effects of fetal substantia nigra tissue grafted into the dopamine-denervated striatum: responses to selective D1 and D2 dopamine receptor agonists.

Grafts of fetal ventral mesencephalon/substantia nigra cell suspensions into the dopamine-denervated striatum have been shown to reduce many of the behavioral alterations associated with striatal dopamine depletion. In this report, the behavioral response to amphetamine, apomorphine, the D1 receptor agonist SKF82958, and the D2 receptor agonist LY171555 were tested before and after intrastriatal grafts of fetal substantia nigra, of fetal striatum or no implantation procedure in animals with unilateral dopamine denervation. Grafts of fetal substantia nigra tissue were associated with significant behavioral recovery, as indicated by decreased turning induced by amphetamine (P ≤ 0.005), SKF82958 (P < 0.005), and LY171555 (P < 0.002). These effects were significantly different from the response in animals that did not receive grafts (P < 0.05) and occurred in the absence of decreased apomorphine-induced turning. These data suggest that the response to selective D1 or D2 dopamine receptor agonists is diminished following grafts of fetal dopaminergic tissue and that this behavioral effect is dissociable from the phenomena of behavioral supersensitivity to apomorphine. In a subset of substantia nigra grafted animals, it was found that D1 or D2 dopamine receptor antagonists administered 30 min prior to apomorphine could significantly reduce apomorphine-induced turning.

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.

Neurotransmitter receptor autoradiography in immunohistochemically identified neurons.

A method for the simultaneous visualization of neurotransmitter receptor binding sites and immunohistochemically characterized cells is described using in vitro sections of rat striatum. The striatum provides a rich neurochemical environment in which to examine muscarinic acetylcholine receptor relations to peptide-reactive somata, or to assess the cellular locale of the dopamine receptor linked to adenylate cyclase activation. Different modifications of the procedure are employed to determine the localization pattern of 3H-radioligands with slow or rapid receptor dissociation times. The present technique is compatible for use with any combination of immunohistochemical antibody/radioligand probe that can be studied on fresh-frozen, slide-mounted tissue sections.

Muscarinic acetylcholine receptors are localized on striatal cyclic GMP-containing neurons.

The localization of muscarinic acetylcholine receptors has been determined using in vitro binding of radiolabeled quinuclidinyl benzilate ([3H]QNB), a specific reversible muscarinic receptor antagonist. All cyclic GMP-immunoreactive neurons in the rat striatum show clustering of [3H]QNB silver grains overlying their somata following autoradiographic analysis. The autoradiography of total binding of silver grains over the cyclic GMP-containing neurons was approximately 800 times as dense as the surrounding neuropil localization of radioligand binding sites. Incubation of striatal tissue slices in the presence of micromolar atropine, to determine non-specific binding of [3H]QNB, decreased the autoradiographic silver grain density of the neuropil about 2.5 times, and lessened the number of receptor sites detectable on cyclic GMP-positive neurons at least 5-fold. Biochemical examination of [3H]QNB binding on tissue sections demonstrated that the ligand binding is saturable and dependent on section thickness for the muscarine receptor subtype of acetylcholine.

Comparison of dopamine binding sites in the rat superior cervical ganglion and caudate nucleus.

A comparison of morphological and biochemical characteristics of the D1-type dopamine receptor has been assessed in two experimental tissues, the superior cervical ganglion (SCG) and caudate nucleus of the rat. Correlation of the distribution of this dopaminergic binding site using in vitro autoradiographic localization of [3H]SCH 23390, a selective D1-binding antagonist, demonstrated no specific association of this receptor subtype with cyclic AMP immunoreactive structures in the superior cervical ganglion. In contrast, the caudate nucleus demonstrated specific D1-binding sites associated with cyclic AMP immunoreactive elements. Biochemical analyses of the D1-dopamine binding sites showed only 20% of the amount of radioligand bound in SCG homogenates as compared to the quantity bound in homogenates of the caudate nucleus. The non-cyclase linked dopaminergic receptor, assessed using D2-type radioligand binding, was much less prevalent than the binding of the D1-subtype in either experimental tissue. Only a small amount of [3H]sulpiride binding, indicative of the D2-receptor subtype, could be measured in the SCG as compared to the caudate nucleus. This work has demonstrated differences in the amount, and the cellular association of dopamine binding sites in peripheral versus central nervous system areas with dopamine sensitive adenylate cyclase mechanisms.

Striatal D1 dopamine receptor distribution following chemical lesion of the nigrostriatal pathway.

The morphochemical and biochemical distribution of the adenylate cyclase-linked dopamine receptor, or D1 subpopulation, has been examined in the rat striatum following a chemical lesion of the dopaminergic nigrostriatal pathway. The cellular pattern of D1 dopaminergic binding was assessed using in vitro autoradiographic localization of [3H]SCH 23390 or [125I]SCH 23982, selective D1 receptor antagonists, 1 week following unilateral infusion of the neurotoxin 6-hydroxydopamine (6-OHDA) into the substantia nigra. The specific association of the D1 binding sites with cyclic AMP-immunoreactive striatal neurons was abolished after lesion of the dopaminergic nigral afferents. The morphochemical disruption of the caudate D1 dopamine binding sites in relation to cyclic AMP-positive elements, a large proportion of which are striatonigral neurons, probably contributes to the dysfunctions in this subpopulation of dopamine receptor after depletion of the catecholamine neurotransmitter.

Rat striatal cyclic nucleotide-reactive cells and acetylcholinesterase reactive interneurons are separate populations.

The concurrent localization of cyclic nucleotide immunofluorescent cells and acetylcholinesterase-containing neurons in the rat caudate-putamen complex has been examined. Cyclic AMP and cyclic GMP stained elements do not exhibit coincident localization with the enzymatically detectable hydrolysis of acetylcholine. These data add further support for the preferential association of the cyclic nucleotides with striatal efferent projection systems, while the large cholinergic somata are part of the interneuron population of the rat caudate-putamen complex.

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.

Neurochemical differences in the superior cervical ganglion of the spontaneously hypertensive rat stroke-prone variant.

The localization and distribution of catecholamines, selected neuropeptides, and the cyclic nucleotide second messengers has been determined in the superior cervical ganglion of the stroke-prone variant of the spontaneously hypertensive rat (SHR) and its normotensive Wistar-kyoto (WKY) control. Significant alteration in the frequency of occurrence of dopaminergic small intensely fluorescent cell clusters was seen in the stroke-prone variant of the SHR. The immunofluorescent localization of cyclic AMP (cAMP) and cyclic GMP (cGMP) were also changed in the stroke-prone variant, as was the immunofluorescent staining quantity of the neuropeptides somatostatin and substance P. The morphological pattern of staining for the various compounds in the normotensive control (WKY) was equivalent to the Sprague-Dawley rat strain. The implications of the altered neurochemistry in the superior cervical ganglion on the high blood pressure, and the predisposition for stroke in this strain are discussed.

Biochemical characterization of postsynaptically localized cyclic nucleotide phosphodiesterase.

This study demonstrates the postsynaptic localization of one of the isozymes of cyclic nucleotide phosphodiesterase (PDE) activity at asymmetrical, axospinous terminals in the rat corpus striatum and neocortex. Characterization of this enzymatic activity demonstrates that the PDE form surviving aldehyde fixation for electron cytochemistry can be considered to preferentially hydrolyze cyclic 3'5'-guanosine monophosphate, and it requires calcium and a heat-stable calcium-dependent regulator protein (CDR) for full hydrolytic activity. Ion exchange chromatographic analysis of extracts of corresponding unfixed brain regions demonstrates that only one enzyme activity peak exhibits similar aldehyde resistance and calcium and regulator protein activatibility.

Co-localization of cyclic GMP in superior cervical ganglion with peptide neurotransmitters.

The immunofluorescent localization of cyclic 3',5'-guanosine monophosphate (cyclic GMP) in rat superior cervical ganglion has been compared to postganglionic neurons having immunoreactivity to neuropeptides. Cyclic GMP-positive somata are equally distributed among cell bodies fluorescent for substance P, somatostatin, and methionine-enkephalin. 40% of the total number of cells stained for each peptide demonstrate co-incident cyclic GMP localization, suggesting a large majority of the neurons employ non-cyclic GMP mechanisms.

Striatal muscarinic receptors are associated with substance P and somatostatin containing neurons.

The cellular localization of muscarinic acetylcholine binding sites (mAChr) in relation to immunohistochemically characterized cell populations within the rat caudate nucleus has been determined using in vitro autoradiography of the reversible antagonist ligand, quinuclidinyl benzilate [( 3H]QNB). The pattern of autoradiographic silver grain deposition in the striatum was contrasted with the localization of two peptide-containing neuronal populations in the striatum. Substance P-immunoreactive somata demonstrated prevalent association of mAChr binding sites, as did somatostatin-immunoreactive cells. Substantially more striatal muscarinic binding sites were aggregated over the somatostatin interneuron population of the caudate nucleus than were associated with the substance P somata in concurrently performed experiments. This data further substantiates the heterogeneity in organization of the caudate nucleus, and the results are discussed in relation to the processing of information within this basal ganglia region.