Crk activation of JNK via C3G and R-Ras.

v-crk is an oncogene identified originally in CT10 chicken tumor virus. C3G, a guanine nucleotide exchange factor (GEF) for Rap1 and R-Ras, is postulated to transduce the oncogenic signal of v-Crk to c-Jun kinase (JNK). We have found that R-Ras, but not Rap1, mediates JNK activation by v-Crk in 293T and NIH 3T3 cells. Constitutively activated R-Ras, R-Ras(Val-38), but not Rap1(Val-12), activated JNK, as did the constitutively active H-Ras(Val-12) or Rac1(Val-12). v-Crk activation of JNK was inhibited by a dominant-negative mutant of R-Ras, R-Ras(Asn-43). JNK activation by R-Ras(Val-38) was inhibited by a dominant-negative mutant of mixed lineage kinase 3. Among six GEFs for Ras-family G proteins, mSos1, Ras-GRF, C3G, CalDAG-GEFI, Ras-GRP/CalDAG-GEFII, and Epac/cAMP-GEFI, GEFs for either H-Ras or R-Ras activated JNK and c-Jun-dependent transcription. CalDAG-GEFI and Epac/cAMP-GEFI, both of which are GEFs specific for Rap1, did not activate JNK or c-Jun-dependent transcription. These results demonstrate that R-Ras, but not Rap1, is the downstream effector of C3G to stimulate JNK. Finally, we found that expression of the dominant-negative R-Ras mutant induced flat reversion of NIH 3T3 cells transformed by v-Crk, suggesting that R-Ras-dependent JNK activation is critical for the transformation by v-Crk.

Interaction between the serotoninergic and dopaminergic systems in d-fenfluramine-induced activation of c-fos and jun B genes in rat striatal neurons.

To test for the relative contributions of the dopaminergic and serotoninergic systems in the striatum to the effects of d-fenfluramine, an indirect serotonin receptor agonist, we assessed the expression of Fos/Jun proteins induced by d-fenfluramine given alone or in the presence of dopaminergic or serotoninergic agents. To determine the neuronal targets of d-fenfluramine in the striatum, we identified the phenotypes of striatal neurons in which d-fenfluramine induced Fos expression. Our results demonstrated that d-fenfluramine evokes nuclear expression of Fos/Jun B proteins in the striatum, and that the Fos expression was dose-dependent and accompanied by transient induction of c-fos mRNA. Fos expression was blocked by p-chloroamphetamine, a serotoninergic neurotoxin. Pretreatment with SCH 23390, a D1-dopamine receptor antagonist, led to a marked decrease in Fos/Jun B expression in the caudoputamen, but not in the cortex, whereas pretreatment with methiothepin, a nonselective serotonin 5-HT1 receptor antagonist, blocked Fos expression completely in the cortex and only partially in the caudoputamen. The expression of Fos/Jun B in the striatum occurred mainly in dynorphin-containing neurons and in a subpopulation of striatal interneurons that exhibited NADPH-diaphorase activity. Most of the enkephalin-containing neurons of the striatum did not show Fos/Jun B staining. These results suggest that the mechanism by which d-fenfluramine induces c-fos and jun B expression in the rat caudoputamen depends at least in part on activation of the dopaminergic system by serotonin.

A measure of striatal function predicts motor stereotypy.

To identify basal ganglia circuit dysfunctions that might produce repetitive behaviors known as motor stereotypies, we applied psychomotor stimulants and a direct dopamine receptor agonist to induce different levels of stereotypy in rats. We then used a gene induction assay to measure the functional activation of neurons in the neurochemically distinct compartments of the striatum, the striosomes and the extrastriosomal matrix. The amount by which activation in the striosomes exceeded activation in the matrix predicted the degree of motor stereotypy induced by the drug treatments. These results suggest that imbalance between compartmentally organized basal ganglia circuits may represent a neural correlate of motor stereotypy.

Cortically driven Fos induction in the striatum is amplified by local dopamine D2-class receptor blockade.

Dopamine D2-class receptors have been shown to control the excitability of striatal neurons in response to cortical activation. It has been unclear, however, whether such receptors could regulate the number of striatal neurons activated by cortical stimulation, and thus affect the population response of the striatum to its cortical inputs. We used Fos induction as a readout to measure the ensemble response of striatal neurons to localized stimulation of the frontal cortex and tested for the effects of D2-class dopamine receptor blockade on this response. In freely moving rats, we stimulated the frontal cortex by local epidural application of a dose of a GABAA receptor antagonist (picrotoxin) just threshold for inducing Fos in the striatum. We combined this treatment with D2-class dopamine receptor antagonist treatments at dose levels also just threshold for inducing Fos, using either (i) systemic haloperidol or (ii) intrastriatal (-)sulpiride. Both systemic and intrastriatal blockade of D2-class receptors sharply increased the numbers of striatal neurons exhibiting cortically evoked Fos induction. These findings suggest that local activation of intrastriatal D2-class dopamine receptors can regulate the number of striatal neurons responsive to cortical inputs, thus dynamically shaping the flow of information through the striatum.

A family of cAMP-binding proteins that directly activate Rap1.

cAMP (3',5' cyclic adenosine monophosphate) is a second messenger that in eukaryotic cells induces physiological responses ranging from growth, differentiation, and gene expression to secretion and neurotransmission. Most of these effects have been attributed to the binding of cAMP to cAMP-dependent protein kinase A (PKA). Here, a family of cAMP-binding proteins that are differentially distributed in the mammalian brain and body organs and that exhibit both cAMP-binding and guanine nucleotide exchange factor (GEF) domains is reported. These cAMP-regulated GEFs (cAMP-GEFs) bind cAMP and selectively activate the Ras superfamily guanine nucleotide binding protein Rap1A in a cAMP-dependent but PKA-independent manner. Our findings suggest the need to reformulate concepts of cAMP-mediated signaling to include direct coupling to Ras superfamily signaling.

A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia.

Ras proteins, key regulators of growth, differentiation, and malignant transformation, recently have been implicated in synaptic function and region-specific learning and memory functions in the brain. Rap proteins, members of the Ras small G protein superfamily, can inhibit Ras signaling through the Ras/Raf-1/mitogen-activated protein (MAP) kinase pathway or, through B-Raf, can activate MAP kinase. Rap and Ras proteins both can be activated through guanine nucleotide exchange factors (GEFs). Many Ras GEFs, but to date only one Rap GEF, have been identified. We now report the cloning of a brain-enriched gene, CalDAG-GEFI, which has substrate specificity for Rap1A, dual binding domains for calcium (Ca2+) and diacylglycerol (DAG), and enriched expression in brain basal ganglia pathways and their axon-terminal regions. Expression of CalDAG-GEFI activates Rap1A and inhibits Ras-dependent activation of the Erk/MAP kinase cascade in 293T cells. Ca2+ ionophore and phorbol ester strongly and additively enhance this Rap1A activation. By contrast, CalDAG-GEFII, a second CalDAG-GEF family member that we cloned and found identical to RasGRP [Ebinu, J. O., Bottorff, D. A., Chan, E. Y. W., Stang, S. L., Dunn, R. J. & Stone, J. C. (1998) Science 280, 1082-1088], exhibits a different brain expression pattern and fails to activate Rap1A, but activates H-Ras, R-Ras, and the Erk/MAP kinase cascade under Ca2+ and DAG modulation. We propose that CalDAG-GEF proteins have a critical neuronal function in determining the relative activation of Ras and Rap1 signaling induced by Ca2+ and DAG mobilization. The expression of CalDAG-GEFI and CalDAG-GEFII in hematopoietic organs suggests that such control may have broad significance in Ras/Rap regulation of normal and malignant states.

Local release of GABAergic inhibition in the motor cortex induces immediate-early gene expression in indirect pathway neurons of the striatum.

The neocortex is thought to exert a powerful influence over the functions of the basal ganglia via its projection to the striatum. It is not known, however, whether corticostriatal effects are similar across different types of striatal projection neurons and interneurons or are unique for cells having different functions within striatal networks. To examine this question, we developed a method for focal synchronous activation of the primary motor cortex (MI) of freely moving rats by local release of GABAergic inhibition. With this method, we monitored cortically evoked activation of two immediate-early gene protein products, c-Fos and JunB, in phenotypically identified striatal neurons. We further studied the influence of glutamate receptor antagonists on the stimulated expression of c-Fos, JunB, FosB, and NGFI-A. Local disinhibition of MI elicited remarkably selective induction of c-Fos and JunB in enkephalinergic projection neurons. These indirect pathway neurons, through their projections to the globus pallidus, can inhibit thalamocortical motor circuits. The dynorphin-containing projection neurons of the direct pathway, with opposite effects on the thalamocortical circuits, showed very little induction of c-Fos or JunB. The gene response of striatal interneurons was also highly selective, affecting principally parvalbumin- and NADPH diaphorase-expressing interneurons. The glutamate NMDA receptor antagonist MK-801 strongly reduced the cortically evoked striatal gene expression in all cell types for each gene examined. Because the gene induction that we found followed known corticostriatal somatotopy, was dose-dependent, and was selectively sensitive to glutamate receptor antagonists, we suggest that the differential activation patterns reflect functional specialization of cortical inputs to the direct and indirect pathways of the basal ganglia and functional plasticity within these circuits.

Cortically driven immediate-early gene expression reflects modular influence of sensorimotor cortex on identified striatal neurons in the squirrel monkey.

Current understanding of basal ganglia function emphasizes their involvement in the focal, context-dependent release of motor and cognitive circuits in the brainstem and frontal lobes. How such selective action can arise despite the existence of massively convergent inputs from the cerebral cortex is unknown. However, anatomical work has suggested that specificity could be achieved in corticostriatal circuits by modular patterns of convergent and divergent cortical inputs to striatal projection neurons. To test for such modular activation of striatal neurons, we electrically microstimulated physiologically identified sites in the primary somatosensory (SI) and primary motor (MI) cortex of the squirrel monkey. We compared the efferent fiber distributions anterogradely traced from these sites to the distributions of striatal neurons activated by microstimulation to express Fos- and Jun B-like immediate-early gene proteins. We show that the microstimulation of sensorimotor cortex induces Fos and Jun B expression in localized cell clusters in the putamen and that these clusters match the anatomical input fiber clusters (matrisomes). The modular activation of striatal neurons by sensorimotor cortex seems likely. Unexpectedly, >75% of the Fos-positive nuclei in densely labeled cell clusters were in enkephalin-immunoreactive neurons. This expression pattern suggests that the primate sensorimotor cortex exerts a differential influence on the enkephalinergic (indirect pathway) as opposed to the substance P/dynorphin (direct pathway) projection neurons of the putamen. The densely labeled clusters of Fos-labeled enkephalinergic neurons occurred within larger zones containing sparsely distributed Fos-labeled parvalbumin neurons. Moreover, when the cortical stimulation induced expression of Fos-like protein only in sparsely distributed neurons, almost every putamenal neuron expressing Fos was a parvalbumin-containing (GABAergic) interneuron. These patterns suggest a model in which the primate sensorimotor cortex can target parvalbumin-containing inhibitory interneurons, which in turn depress the remaining neuronal activity within and around matrisomes in a feed-forward manner until sufficient coherent cortical input can overcome the inhibition to influence selectively enkephalinergic projection neurons in the activated matrisomes. Tuning of cortical input by striatal interneurons thus may be an important mechanism by which broader anatomical connections are dynamically adjusted to achieve selective flow of information through the basal ganglia.

Spatiotemporal dynamics of CREB phosphorylation: transient versus sustained phosphorylation in the developing striatum.

The cAMP response element-binding protein (CREB) is a plasticity-associated transcription factor that can potentially integrate cAMP and calcium signals at the gene activation level. We tested for convergent Ser-133 phosphorylation of CREB via dopamine D1/D5 receptors and L-type calcium channels in organotypic cultures of neonatal striatum. We found such convergence only transiently. Sustained CREB phosphorylation by D1/D5 receptor and L-type channel agonists was targeted to opposite (striosome and matrix) cellular phenotypes. Subsequent expression of the CRE-containing gene, c-fos, matched the divergent patterns of sustained CREB phosphorylation, and both divergent patterns could be switched by inhibition of phosphatases, including calcineurin. Control of the duration of CREB phosphorylation may be a critical regulator of CRE-mediated gene expression by dopamine and calcium.

D1-class dopamine receptors influence cocaine-induced persistent expression of Fos-related proteins in striatum.

Chronic intermittent exposure to psychomotor stimulants induces in the striatum the expression of Fos-related proteins (Fras) that persist after the end of drug treatment. We carried out experiments to determine whether such Fras ("chronic Fras') require dopamine D1-class receptor function for their persistent expression in the striatum. We chronically administered cocaine to rats in a behavioral sensitization protocol and blocked D1-class receptors with SCH23390 before a final cocaine challenge. Western blotting and immunohistochemical analyses indicate that Fras persistently expressed in response to chronic treatment include proteins of two types: those that have become independent of D1-class dopamine receptor activation and those that remain dependent on D1-class receptors for their expression following drug challenge.

Atypical and typical neuroleptic treatments induce distinct programs of transcription factor expression in the striatum.

Atypical and typical neuroleptics, when administered chronically, can bring about profound but contrasting changes in schizophrenic symptoms and motor activation and dramatically modulate brain neurochemistry. To explore the transcriptional events that might be involved in this neurochemical regulation, we used immunohistochemistry and immunoblotting to examine the expression patterns of two bZip transcription factors, c-Fos and FosB, in the striatum of rats treated acutely and chronically with neuroleptic drugs of different classes. Typical and atypical neuroleptic drugs produced contrasting regulatory effects on a FosB-like protein of ca. 36-39 kDa, the molecular weight of truncated FosB (delta FosB). Chronic treatments with two typical neuroleptics, haloperidol and metoclopramide, but not with the atypical neuroleptic clozapine, led to markedly enhanced FosB-like immunoreactivity in the caudoputamen. Further, c-Fos-like protein in the striatum, considered a marker for the induction of antipsychotic actions by neuroleptic treatments, was downregulated by chronic treatment with the two potent antipsychotic drugs tested, but not by chronic treatment with metoclopramide, which has low antipsychotic efficacy but induces extrapyramidal side effects. These results suggest that chronic treatments with neuroleptics having different effects on cognitive and motor behavior induce different long-term changes in transcription factor expression in the striatum. Nevertheless, we found that neuroleptics of both classes regulated transcription factor expression in overlapping populations of striatal neurons expressing enkephalin or DARPP-32. Contrasting patterns of transcriptional regulation in these neurons may thus contribute to the distinct neurochemical and behavioral effects that characterize neuroleptics of different classes.

Network-level changes in expression of inducible Fos-Jun proteins in the striatum during chronic cocaine treatment and withdrawal.

Repeated exposure to psychomotor stimulants produce long-term changes in behavior ranging from addiction to behavioral sensitization. Many of these behaviors depend on the nigrostriatal system of the basal ganglia. We show here that chronic cocaine exposure not only leads to time-varying alterations in the inducibility of bZIP transcription factors in individual striatal neurons, but also to long-lasting network changes in which ensembles of striatal neurons express these proteins. These network-level adaptations suggest that the behavioral sensitization induced by repeated psychomotor stimulant exposure may reflect an enduring functional reorganization of basal ganglia circuits.

Dopaminergic regulation of transcription factor expression in organotypic cultures of developing striatum.

Dopamine is a major neurotransmitter in neural systems innervating the striatum, and dopamine receptors are expressed during early pattern formation in the developing striatum. To test for the functional responsiveness of developing striatal neurons to dopaminergic stimulation, we established an organotypic slice culture of newborn rat striatum. We analyzed the effects of dopamine receptor agonists and of adenylate cyclase and protein kinase activation on striatal neurons by measuring the induction of Fos-like and Fra-like proteins in the cultured striatum. Fos-like and Fra-like proteins were induced in striatal neurons by activation of D1-like dopamine receptors but not by activation of D2-like receptors. The induction of Fos-like protein was mainly in striosomes and a medial compartment next to the ventricular zone, whereas Fra-like protein was induced in the striatal matrix as well. cAMP analogs and forskolin induced widespread expression of both Fos-like and Fra-like proteins. Our findings thus suggest that neurons of developing striosome and matrix compartments not only have different functional coupling of D1-like receptors to adenylate cyclase, but also have distinct maturational programs for dopaminergic regulation of individual transcription factors. Finally, despite evidence that protein kinase was involved in the induction of Fos-like protein, experiments with kinase inhibitors suggested that the induction of Fos-like protein had unusual pharmacological characteristics and raised the possibility that a novel protein kinase A-like molecule may have been involved in the induction. The cultured striatal slice preparation should provide a valuable tool for analyzing the molecular determinants of striatal development and function.

Coordinate expression of c-fos and jun B is induced in the rat striatum by cocaine.

In cells in culture, specific stimuli induce selective patterns of immediate-early gene induction. In the present study, we tested for such selectivity of stimulated gene expression by monitoring the expression of fos/jun gene mRNAs in the striatum in rats treated in vivo with the indirect dopamine agonist cocaine. We found by Northern blot and in situ hybridization analysis that cocaine induces the coordinate expression of c-fos and jun B mRNAs in neurons of the rat's striatum. By contrast, another immediate-early gene of the leucine-zipper family, c-jun, was not induced in striatal neurons by cocaine at any time tested from 1 to 24 hr after treatment. With the same probe, we could detect the induction of c-jun mRNA (as well as that of c-fos and jun B mRNAs) in the hippocampus following administration of pentylenetetrazol. The induction of expression of c-fos and jun B was rapid and transient, with peak expression occurring at approximately 1 hr after cocaine administration, and the induction of the two genes was in similar striatal sites. These results establish that differential patterns of expression of fos/jun genes occur in striatal neurons following exposure to cocaine, a potent psychomotor stimulant. We suggest that these tissue-specific patterns of gene expression may contribute to the response specificity of striatal neurons to stimulation by monoamines including dopamine.

Influence of mesostriatal afferents on the development and transmitter regulation of intrastriatal grafts derived from embryonic striatal primordia.

Embryonic striatal grafts develop a modular organization in which patches of tissue enriched in many transmitter substances characteristic of striatum (P regions) are embedded in surrounds (NP regions) expressing only low levels of these substances. Catecholaminergic fibers from the host brain, identified by their expression of tyrosine hydroxylase (TH), grow into such grafts and selectively terminate in the striatum-like P regions. This terminal pattern suggests that cell-cell affinities between neurons of the substantia nigra and striatum may play a role either in the aggregation of the striatal cells into P regions, or in the targeting of the TH-positive fibers to the cell clusters. In the present study, we tested the first of these possibilities. Striatal grafts derived from embryonic day 15 striatal primordia were implanted into the ibotenate-damaged host striatum of rats previously treated with 6-hydroxydopamine (6-OHDA) to destroy TH-containing dopaminergic nigrostriatal afferents. The 6-OHDA lesions that eliminated nearly all TH-like immunostaining in the host striatum also resulted in disappearance of nearly all TH-positive fibers in the grafts. In this dopamine-depleted environment, the grafts nevertheless developed a clear modular organization. They contained striatum-like patches with neurons expressing many of the neurochemicals characteristic of striatum (ACh, ChAT, calbindin-D28KD, met-enkephalin, and dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein-32,000 or DARPP-32), and these patches were surrounded by graft tissue expressing few of these striatal markers. These observations suggest that the ingrowth of TH-positive fibers from the host is not obligatory for the sorting out of striatal from nonstriatal cells during the formation of P regions in embryonic striatal grafts. Despite the fact that dopaminergic denervation of the host striatum did not disrupt either the aggregation of grafted cells into P regions or the acquisition of striatal neurochemical phenotypes by cells in the P regions, there were clear differences between the staining patterns of these grafts and grafts placed into dopamine-innervated striatum. Most striking was a sharp increase of met-enkephalin-like immunostaining in the P zones of the denervated grafts. Upregulation of met-enkephalin is known to occur in the dopamine-depleted mature striatum, and was observed in the parts of host striatum surrounding the grafts on the side ipsilateral to the 6-OHDA lesions. This result suggests that functional interactions between dopaminergic and enkephalinergic systems can occur in the striatal circuits reconstructed by embryonic striatal grafting. More generally, our results suggest that TH-containing afferents from the host striatum, though not required for induction and maintenance of striatal phenotypy in striatal grafts, can chronically regulate neurotransmitter/neuromodulator expression in neurons of the striatum-like P zones in a manner similar to that found for the normal striatum.

D1-like and D2-like dopamine receptors synergistically activate rotation and c-fos expression in the dopamine-depleted striatum in a rat model of Parkinson's disease.

Selective agonists for D1-like and D2-like dopamine receptors can interact synergistically to enhance each other's actions on locomotion and behavior in experimental animals. Clinically, the combination of the D2 agonist bromocriptine with L-dopa (which has pronounced D1 effects) is a highly effective treatment for Parkinson's disease. The mechanisms underlying this important receptor interaction are poorly understood and are the subject of intense study in vitro. In rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway, D1-selective (but not D2-selective) dopamine agonists produce a marked increase in expression of the immediate-early gene c-fos in the striatum ipsilateral to the 6-OHDA lesion. In the experiments reported here, we have used this in vivo model to explore the possibility that combinations of D1-selective and D2-selective agonists might have effects on c-fos transcription that are different from those exhibited by D1 or D2 agonists administered alone. We examined the effects of the D1-selective agonist SKF-38393 and the D2-selective agonist quinpirole (LY 171555) on the expression of Fos-like protein and c-fos mRNA in the caudoputamen and made parallel behavioral observations in the same animals. A low dose of SKF-38393 produced little contraversive rotation and little induction of Fos-like immunoreactivity in the striatum. A low dose of quinpirole elicited contralateral rotation but little or no induction of Fos-like immunoreactivity in the caudoputamen; there was, however, induction of Fos in the globus pallidus ipsilateral to the 6-OHDA lesion. Combination of the low dose of SKF-38393 and quinpirole produced a synergistic effect on rotation and elicited, in the dopamine-depleted caudoputamen, a striking pattern of Fos-like protein expression in which Fos-positive neurons were concentrated in striosomes and in the dorsolateral caudoputamen. Northern blot analysis showed that c-fos mRNA was expressed following combined agonist treatment but was not detectable after the single-agonist treatments. Both the contraversive rotation and the induction of Fos-like immunoreactivity were blocked by the preadministration of the D1-preferring antagonist SCH-23390 and the D2-selective antagonist raclopride in combination. Pretreatment with the glutamate NMDA receptor antagonist MK-801 also blocked the induction of Fos-like immunoreactivity, and it reversed the rotation. These findings suggest a D1/D2 synergistic mechanism that involves the participation of D1-responsive striatonigral and D2-responsive striatopallidal output pathways, and that is sensitive to glutamatergic modulation.

Dopamine and glutamate agonists stimulate neuron-specific expression of Fos-like protein in the striatum.

1. The monoamine dopamine and the amino acid glutamate are major neurotransmitters in the basal ganglia implicated in the normal functions of the striatum and in extrapyramidal disease states. To study the effects of these neurotransmitters on gene transcription in striatal neurons, we treated rats with dopamine (monoamine) agonists and with glutamate agonists and monitored the induction of Fos-like protein in striatal neurons. We administered the indirect monoamine agonists cocaine and amphetamine intraperitoneally and gave the glutamate agonist quinolinic acid by direct intrastriatal injection. We identified the phenotypes of the responsive neurons by immunohistochemistry and by enzyme histochemistry in double staining protocols. 2. Both the indirect monoamine agonists and the glutamate receptor agonist stimulated rapid nuclear expression of Fos-like protein in specific classes of striatal neurons. The induction by cocaine and amphetamine was blocked by pretreatment with the dopamine D1-like receptor antagonist SCH23390, and the induction by quinolinic acid was blocked by pretreatment with MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) glutamate receptor. 3. The monoamine and glutamate agonists both induced Fos-like protein exclusively in striatal neurons that constitutively expressed the protein phosphatase inhibitor DARPP-32 (dopamine and cAMP-regulated phosphoprotein). 4. The dopamine agonists failed to induce detectable Fos-like protein in striatal neurons expressing enkephalin, even though many such neurons expressed DARPP-32. By contrast, many enkephalinergic neurons did express Fos-like protein in response to glutamatergic stimulation. 5. Glutamate agonist stimulation, but not dopamine agonist stimulation, induced Fos-like protein in a subpopulation of striatal interneurons, namely, a group of neurons exhibiting NADPH-diaphorase activity. 6. These findings suggest that stimulation of dopamine D1-like receptors (or related monoamine receptors) and glutamate NMDA receptors activates neuron-specific programs of immediate-early gene expression in the striatum. Our findings further suggest that monoamine and glutamate may act cooperatively at the transcriptional level on a functionally defined subset of striatal neurons.

5'-nucleotidase: a new marker for striosomal organization in the rat caudoputamen.

The distribution of the adenosine-producing ectoenzyme 5'-nucleotidase was studied by means of a histochemical lead technique in the caudoputamen of normal adult rats and of rats in which injections either of 6-hydroxydopamine in the medial forebrain bundle or of ibotenic acid in the caudoputamen had been made 1-3 weeks previously. The patterns of striatal 5'-nucleotidase activity in these animals were compared in serial sections to the patterns of calbindin-D28k immunoreactivity and of 3H-naloxone ligand binding, which respectively mark the known matrix and striosome (patch) compartments of the caudoputamen. In the normal rats, 5'-nucleotidase activity was differentially concentrated in striosomes, where it produced a dense staining of the neuropil. The enzymatic staining followed a striosomal distribution in all but the caudal caudoputamen. Within the striatal matrix, 5'-nucleotidase staining also observed a lateromedial density gradient. Depletion of the dopamine-containing nigrostriatal innervation of the caudoputamen with 6-hydroxydopamine did not alter the striosomal selectivity of 5'-nucleotidase activity. Destruction of intrastriatal neurons by ibotenic acid led to a strongly 5'-nucleotidase-positive gliosis within the resulting necrotic region. Elsewhere in the caudoputamen, the enzyme's striosomal distribution was not detectably altered. We conclude that 5'-nucleotidase histochemistry provides an advantageous tool for detecting the striosomal architecture of the rat's caudoputamen. Moreover, 5'-nucleotidase is prominently associated with glial membranes in the central nervous system, so that the concentration of this enzyme in striosomes could mark these as sites of selective glial populations within striatum. These properties and actions of 5'-nucleotidase in purinergic neurotransmission and in neuroadhesion may contribute to the specialized functions of striosomes and matrix.