Local Release of GABAergic Inhibition in the Motor Cortex Induces Immediate-Early Gene Expression in Indirect Pathway Neurons of the Striatum

Application of picrotoxin to the motor cortex evokes discrete movements and localized gene induction in the neocortex and striatum

To study the effects of cortical activity on striatal immediate-early gene expression in awake, freely moving animals, we preimplanted wells over focal craniotomies, without disturbing the dura mater overlying the region of interest to minimize trauma to the cortex. To determine the optimal location for the wells, we mapped MI with electrical stimulation in three rats (Fig.1A,B). On the basis of these maps and of those of Neafsey and coworkers (1986), we chose standard coordinates estimated to correspond to the limb region of the MI map except for a slightly medial bias imposed to favor well stability (Fig.1B).

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Fig. 1.

Focal epidural application of picrotoxin onto the MI through a chronically implanted, well induced c-Fos expression and increased metabolic activity in the underlying cortex and in the dorsolateral caudoputamen. A, An example of the microstimulation maps of MI on which stereotaxic coordinates for the implanted wells were based. The area in gray represents the size and the position of the well. F, Foot;V, vibrissae; E, elbow; W, wrist; T, trunk; N, neck;H, hand; D5F, digit 5; X, no response. Coordinates relative to bregma. B, Schematic representation (anterior = 9.7; Paxinos and Watson, 1986) of the most intense c-Fos induction observed in the cortex after picrotoxin application (gray) superimposed on points at which microstimulation elicited movements of the elbow (E) and of the elbow and vibrissae (E/V) at the same position in a different experiment. C, Serial transverse sections through the caudoputamen illustrating the longitudinally extended dorsolateral induction of c-Fos in the striatum after picrotoxin application to the motor cortex. Scale bar, 500 μm.

Approximately 15 min after the addition of 300 μmpicrotoxin to the implanted wells, rats began to exhibit localized motor tics involving either the forelimb or hindlimb or both and lasting several tens of milliseconds. The tics had a frequency of ∼26/min (∼2.3 sec intervals). The tics did not interfere with the spontaneous behavior of the animals, which in most experiments spent periods of time sleeping, grooming, drinking, and feeding. The motor tics continued for the entire duration of the treatment (2 hr and 15 min). The occurrence of such discrete tics was used as the criterion for further analysis. In five rats, the stimulation evoked motor tics that evolved into generalized seizures. These rats were excluded from the study. No behavioral response was seen in the saline-treated controls or in controls with other well sites.

Because different nuclear immediate-early genes are regulated differentially (for reviews, see Sheng and Greenberg, 1990; Hughes and Dragunow, 1995), we studied the expression of four of them, c-Fos, JunB, NGFI-A, and FosB, in an attempt to survey cortically induced gene activation in the striatum. Our attempts to evoke c-Jun expression were not successful. In every rat in which well defined motor tics were elicited, we found localized induction of all four transcription factor immunoreactivities in the motor cortex and the sensorimotor sector of the ipsilateral striatum (Fig. 2A,B). Induction in the striatum was first detectable 1 hr after picrotoxin application and reached a maximum at 2 hr (data not shown). In the neocortex, the epidural picrotoxin induced intense gene expression in a wedge-shaped zone, most sharply demarcated in sections stained for c-Fos (Figs. 1B, 2A,B) and JunB (see Fig. 4A). Outside this intense focal zone, there was weaker induction in cortical neurons throughout the hemisphere (Figs. 2A, 4A). This general pattern of a dense focus and a more diffuse extended activation zone was visible also in the neocortex of rats prepared for 2-DG autoradiography (Fig. 3A). Similarly, in the striatum, the dorsolateral zone of immediate-early gene activation was matched by a comparably situated region showing heightened metabolic activity in the case prepared for 2-DG autoradiography (Fig.3B).

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Fig. 2.

Focal epidural application of picrotoxin induces c-Fos expression in a concentration-dependent manner. A,B, Photomicrographs of sections immunostained for c-Fos from experiments in which 75 μm (A) or 300 μm (B) picrotoxin was applied to MI. A wedge-shaped area of intense c-Fos induction, corresponding to the site of picrotoxin application, is detectable. In both cases, c-Fos induction in the striatum is restricted to the dorsolateral caudoputamen. The inset (a) shows bracketed region at higher magnification. Markers above the overlying cortex indicate the approximate location of the well.C, Relationship between the number of immunodetectable c-Fos-positive nuclei in the ipsilateral caudoputamen and the concentration of picrotoxin applied to MI (mean ± SEM;N = 18; n = 3/group). Scale bar, 1 mm.

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Fig. 4.

Picrotoxin-induced excitation of MI elicits different patterns of c-Fos, JunB, FosB, and NGFI-A induction in the cerebral cortex (A) and striatum (B). Immediate-early gene induction in the striatum of saline-treated controls (C) was found only along the medial edge of the caudoputamen. Scale bar, 500 μm.

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Fig. 3.

Autoradiograms of two parasagittal sections demonstrating the effects of picrotoxin application to MI on the uptake of [¹⁴C] 2-DG. In A, thearrowhead indicates a wedge-shaped region of high uptake approximating the site of picrotoxin application (lateral = 2.4;Paxinos and Watson, 1986). In B, thearrowhead indicates the corresponding region of heightened labeling in the dorsolateral caudoputamen (lateral = 4.6; Paxinos and Watson, 1986). Scale bar, 1 mm.

In the neocortex, for all four transcription factors, there was widespread induction in the supragranular and infragranular layers, whereas induction was weak or absent in layer 4. Details of the distribution patterns for the protein immunoreactivities differed. This was most obvious for JunB, which lacked the differentially strong induction in layers 3 and 5 shown by c-Fos, FosB, and NGFI-A immunoreactivities (Fig. 4A). We found little or no induction of any of the four protein classes in the contralateral neocortex.

No gene induction was seen in the saline-treated controls. Furthermore, in Nissl stains, we found no evidence of obvious cellular damage in the neocortex or in the underlying striatum after picrotoxin application (also see Collins and Olney, 1982). In control rats in which picrotoxin was applied over cortical regions other than the motor cortex, the induction of Fos–Jun proteins occurred in the absence of evoked movement with different distributions and intensities. The possibility that the striatal gene induction was produced by leakage of picrotoxin from the cortex to the striatum was excluded by the facts that the gene induction in the striatum was not directly underneath the site of application and that intrastriatal injections of picrotoxin (n = 3) did not lead to local expression of the genes. Finally, the one rat with a localized hemorrhage directly under the well did not show induction of the proteins in the striatum.

Induction of c-Fos expression in the sensorimotor striatum by MI picrotoxin application is dose-dependent

In 18 rats (3 rats/group), we performed a dose–effect study by applying concentrations of picrotoxin varying from 0 to 300 μm to MI and subsequently counting the numbers of c-Fos-positive neurons in representative sections through the caudoputamen (Fig. 2C). We found an orderly dose–effect relationship with a threshold for induction at picrotoxin concentrations between 50 μm (9.3 nuclei per section ± 16.2 SEM) and 100 μm (171.0 nuclei per section ± 69.0 SEM). Strong induction (500 nuclei per section ± 184.8 SEM) was found almost invariably at 300 μm. At this dose, which was used for all subsequent experiments, there was a consistent, somatotopically localized behavioral response to the picrotoxin and a consistent core of intense c-Fos induction in the sensorimotor zone accompanied by a margin of weaker induction.

Stimulation of the motor cortex induces immediate-early genes in a topographically ordered pattern in the striatum

Each of the four transcription factors showed a main zone of induction in the dorsolateral caudoputamen (Figs. 2A,4B) in the region corresponding to the sensorimotor sector, which receives direct input from MI (McGeorge and Faull, 1989). Within the principal zone of induction, the activation of gene expression was ordered in a roughly topographic manner, judging from the movement elicited during stimulation. In rats with hindlimb motor tics, the fields of induction were generally dorsomedial to those in rats with forelimb motor tics. In cases in which the location of the well was shifted rostrally or caudally with respect to the standard site (n = 6), gene induction in the striatum occurred with different distributions.

The results for two forelimb cases are shown in Figures1C and 4B. The zones of induction were elongated anteroposteriorly (Fig. 1C). At some levels, especially posteriorly, immunopositive nuclei tended to form clusters. Anteriorly, the distribution of labeled nuclei was more diffuse. Immunostaining for c-Fos, JunB, FosB, and NGFI-A showed roughly comparable major zones of induction (Fig. 4B), but the levels of induction in the sensorimotor striatum in any given animal were generally stronger for JunB and NGFI-A than for c-Fos, and they were least for FosB (Fig. 3B). FosB was also exceptional in showing considerable induction in the ventral foot of the caudoputamen and adjoining nucleus accumbens (Fig.4B). As judged by comparisons with expression levels in saline-treated controls (Fig. 4C), picrotoxin induced only weak striatal expression of the other protein species outside the dorsolateral zone.

We consistently found weak induction of c-Fos and JunB in the sensorimotor sector of the contralateral caudoputamen in a roughly mirror-image position to the main ipsilateral field. Because of the higher levels of basal expression of NGFI-A and FosB, such low levels of induction, if present, would have been virtually impossible to detect.

Stimulation of the motor cortex with picrotoxin selectively activates subclasses of striatal projection neurons and interneurons

To identify the phenotypes of sriatal neurons activated by the cortical stimulation to express immediate-early genes, we used dual-antiserum immunohistochemistry with standard neurochemical markers for different classes of striatal neurons. The two main classes of striatal projection neurons coexpress different neuropeptides along with GABA. Most direct pathway neurons (and most neurons of striosomes, which project to the substantia nigra) express dynorphin. Most indirect pathway neurons express enkephalin (Graybiel, 1990; Gerfen, 1992). We therefore were able to identify these neurons as distinct classes. We also identified three classes of striatal interneurons with antiserum against parvalbumin to label the fast-spiking GABAergic interneurons, antiserum against ChAT to label the large aspiny cholinergic striatal interneurons, and enzyme histochemistry for NADPH diaphorase to detect the striatal interneurons that express this enzyme (putatively identified as nitric oxide synthase) along with neuropeptide Y and somatostatin (Kawaguchi et al., 1995; Figueredo-Cardenas et al., 1996). We restricted the analysis to double-labeling for c-Fos and JunB because of their relatively low levels of constitutive expression in the striatum.

We found remarkable selectivity in the patterns of gene induction in both the projection neurons and the interneurons (Figs.5, 6; Table 1). Projection neurons make up >90–95% of all neurons in the rat striatum and are equally divided into dynorphin-containing (direct pathway) and enkephalin-containing (indirect pathway) types. After the MI stimulation, fully 70–80% of all the striatal neurons expressing c-Fos were enkephalin-containing neurons (Fig. 5B, Table 1). Within the region of most intense induction, 35–40% of enkephalin-positive neurons expressed c-Fos. Only 7% of c-Fos-positive neurons expressed dynorphin (Figs. 5A, 6A; Table 1). Thus nearly all of the striatal projection neurons activated by cortical stimulation to express c-Fos-like protein were neurons of the indirect pathway. These double-labeled projection neurons were concentrated in the region of densest c-Fos induction, and the small numbers of dynorphin-containing neurons excited to express c-Fos-like protein were always found near the center of this region (Fig.6A).

Nearly all the remaining c-Fos-positive neurons were parvalbumin-containing interneurons (Figs. 5C,6A; Table 1). Up to 22% of c-Fos-positive nuclei were in parvalbumin-positive neurons, a remarkably high percentage given that the parvalbumin-containing neurons are thought to make up only 3–5% of all neurons in the rat’s caudoputamen (Kawaguchi et al., 1995). The activated parvalbumin-positive neurons were distributed both within the dense core of the field of induction and in its periphery (Fig. 6A). To estimate how fully this interneuronal population was activated to express c-Fos immunoreactivity, we counted all the neurons immunostained for parvalbumin and all of those that in addition were c-Fos-positive. In the region of densest induction, the percentage of all parvalbumin-positive neurons that were c-Fos-labeled was 88 ± 2.3 SEM. The percentage of parvalbumin-positive neurons that were c-Fos-positive was as high as 78 ± 4.6 SEM when the surrounding zone of less intense induction was also included.

Of the remaining c-Fos-positive neurons, 3–4% expressed NADPH diaphorase and thus corresponded mainly to the somatostatin-containing neurons of the striatum (Figueredo-Cardenas et al., 1996) (Fig.6A, Table 1). They had a clearly patterned distribution that was different from those of any other of the c-Fos-labeled neurons (Fig. 6A), and they were least well represented in the central field of induction, increased in number in the more weakly labeled marginal zone around this field, and extended into a broad zone including much of the lateral part of the striatum unoccupied by other c-Fos-labeled cells. Within this entire region, the percentage of NADPH diaphorase-positive neurons that expressed c-Fos was 44 ± 2.8 SEM. In the central region, this percentage dropped to 32 ± 5.1 SEM. We found no ChAT-positive neurons double-labeled for c-Fos. We did not stain for calretinin, a marker for another class of striatal interneurons, but the labeling we did find must collectively have accounted for most striatal interneurons.

The induction of JunB-like protein by stimulation of MI was even more selective for enkephalin-containing projection neurons than that for c-Fos (Fig. 6B). Of all JunB-positive neurons, 85–90% were immunoreactive for enkephalin (Figs. 5E,6B; Table 1). Approximately 50% of the enkephalinergic neurons within the region of most intense induction were activated to express JunB. Only 4–6% of the JunB-positive neurons expressed dynorphin (Figs. 5D, 6B; Table 1). These few dynorphin-positive, JunB-positive neurons were distributed within the core of the field of induction. In contrast to c-Fos, JunB was not detectably activated in large numbers of striatal interneurons (Figs. 5F, 6B; Table 1). Only 0.7% of JunB-positive nuclei were in parvalbumin-containing interneurons. These made up only 17 ± 9% SEM of the parvalbumin-positive neurons within the total region of induction that expressed JunB. They were located mainly within the central field of induction. Approximately 1–2% of the entire JunB-positive population expressed NADPH diaphorase (Table 1). These neurons accounted for ∼29% of all NADPH diaphorase-containing cells and were widely scattered within the striatum (Fig. 6B). We found no ChAT-immunoreactive neurons that expressed JunB.

Taken together, these results suggest marked selectivity in the pattern of gene expression induced in the striatum in response to stimulation of the motor cortex, including coordinate and selective induction of c-Fos and JunB in enkephalinergic indirect pathway projection neurons and, for interneurons, selective induction of c-Fos in parvalbumin-containing and NADPH diaphorase-containing neurons.

Cortically evoked expression of c-Fos and Jun expression in the striatum requires glutamate NMDA receptors and is modulated by glutamate AMPA/kainate receptors

Corticostriatal fibers release glutamate or a closely related excitatory amino acid as neurotransmitter (Fonnum et al., 1981), and both NMDA and non-NMDA glutamate receptors are strongly expressed in the caudoputamen (Albin et al., 1992; Petralia and Wenthold, 1992; Tallaksen-Greene and Albin, 1994; Testa et al., 1994;Landwehrmeyer et al., 1995). To test for the relative requirements of these glutamate receptor subtypes in mediating the effects of cortical excitation on striatal gene induction, we combined the local epidural picrotoxin treatment with pretreatment with either a noncompetitive antagonist of the NMDA receptor, MK-801, or an AMPA/kainate receptor antagonist, GYKI 52466.

Injections of MK-801 (1 mg/kg, i.p; n = 6) and GYKI 52466 (10 mg/kg, i.p.; n = 6) by themselves did not induce the expression of any of the transcription factor immunoreactivities we monitored nor did the vehicles for MK-801 (saline; n = 5) or GYKI 52446 (CREMOPHOR EL plus saline; n = 5) affect the levels of picrotoxin-induced gene expression when given as control pretreatments before the standard application of 300 μm picrotoxin to the cortex (data not shown). Pretreatment with each antagonist, however, did affect the cortically induced gene expression observed in striatal neurons (Fig.7).

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Fig. 7.

Effects of glutamate receptor antagonists on cortically driven gene expression in the striatum. A, The glutamate NMDA receptor antagonist MK-801 injected systemically before picrotoxin application (n = 10) significantly reduced picrotoxin-stimulated c-Fos, JunB, NGFI-A, and FosB induction in the caudoputamen relative to the levels of induction found in control rats that received vehicle injection before picrotoxin application (n = 10; p ≤ 0.025). B, The glutamate AMPA receptor antagonist GYKI 52466 increased JunB induction in the caudoputamen but did not have detectable effects on c-Fos, NGFI-A, or FosB induction relative to control levels in rats treated with vehicle before picrotoxin (n = 10; p ≤ 0.025).Asterisks indicate values significant by Kruskal–Wallis testing followed by comparisons of treatments versus control;circled asterisks indicate differences by ANOVA testing followed by Scheffé’s post hoc test.

Systemic injection of MK-801 reduced expression of each of the four protein classes in the striatum (Fig. 7A). The nonparametric Kruskal–Wallis test, followed by comparisons of treatment and control conditions, showed that MK-801 significantly (p ≤ 0.025) decreased cortically driven induction of c-Fos, JunB, NGFI-A, and FosB in the striatum. Testing with ANOVA followed by Scheffé post hoc test showed significance only for c-Fos (p < 0.0001), probably because of the large variability within the groups.

In the cerebral cortex, the MK-801 completely abolished the induction of c-Fos in regions not directly under the picrotoxin well, so that only the activated wedge of induction remained (data not shown). The induction of JunB and NGFI-A in the cortex was also reduced but not completely abolished. Analysis of the phenotypes of the striatal neurons that still expressed c-Fos after MK-801 pretreatment showed that there was a comparable decrease in each of the neuronal subclasses: no phenotype was particularly affected.

In contrast to MK-801, GYKI 52446 did not change the cortically driven striatal induction of c-Fos, NGFI-A, and FosB, and it increased the induction of JunB in the caudoputamen (ANOVA followed by Scheffépost hoc test, p = 0.009; Kruskal–Wallis followed by comparisons of treatments vs control, p ≤ 0.025) (Fig. 7B). As observed for MK-801, all striatal phenotypes showed a comparable response to the treatment with picrotoxin and GYKI 52466. Neither antagonist had a detectable effect on the motor tics evoked by the picrotoxin treatment.

Picrotoxin-induced excitation of motor cortex induces gene expression in other subcortical sites

We did a partial screening of cortically evoked gene induction in other subcortical regions of the forebrain, including other sites in basal ganglia circuitry and in the midbrain (Table2).

For the thalamus, the results were surprising. There was little, if any, induction in the nucleus ventralis anterior–nucleus ventralis lateralis (VA–VL) complex, the main thalamic region interconnected with MI. By contrast, there was consistent induction of Fos–Jun proteins in neurons of the nucleus reticularis of the thalamus. In the center median (but not in the nucleus parafascicularis), there was activation of c-Fos but little activation of the other protein classes. This nucleus-specific pattern of activation is interesting given that each of these thalamic regions receives a direct projection from the stimulated MI.

In other nuclei of the basal ganglia circuit, the subthalamic nucleus, which receives a direct input from the MI, showed consistent induction of c-Fos and NGFI-A, as did the entopeduncular nucleus (internal pallidum) and the substantia nigra pars reticulata. There was more modest induction in the globus pallidus (external pallidum).

Of other regions analyzed, the amygdaloid complex (central and basolateral amygdaloid nuclei) showed marked activation, especially of c-Fos. The cortical stimulation also induced c-Fos, but not the other proteins, at immunodetectable levels in the pontine nuclei, which receive direct MI input. Altogether, c-Fos and NGFI-A showed the greatest responsivity across the structures analyzed.

Effects of MI picrotoxin application on striatal neurons

Because medium spiny striatal neurons receive numerous but only weakly effective synaptic inputs and are dominated by an inward rectifier that shunts depolarization, coherent cortical excitation is required to induce their firing (Wilson, 1995). We attempted to produce locally coherent activation by releasing a small region of MI from the inhibitory network of GABAergic interneurons that controls the spread of cortical excitation (Chagnac-Amitai and Connors, 1989a; Jacobs and Donoghue, 1991; Thomson and Deuchars, 1994). By using picrotoxin to lift cortical inhibition locally, rather than electrical stimulation to excite local regions, we avoided choosing an arbitrary pattern of cortical activity. We also avoided the activation of fibers of passage within the cortical region of interest. The observed well localized motor tics evoked by picrotoxin treatment and the subsequent clearly demarcated local regions of intense gene induction and 2-DG activation at the site of picrotoxin application and in the sensorimotor sector of the striatum are consistent with effects evoked from a discrete region of the motor cortex. We cannot directly relate, however, the gene induction patterns to specific patterns of neuronal firing in the striatum. Indeed, our results show that the selectivity of the gene induction for different classes of striatal neurons differs for different genes, as does the sensitivity of the induction to pretreatment with glutamate antagonists.

NMDA receptor antagonists reduce, but AMPA/kainate receptor antagonists enhance, cortically evoked gene expression in the striatum

Membrane depolarization of striatal neurons is known to be mediated by both NMDA and non-NMDA glutamate receptors (Kita, 1996, and references therein). Our experiments with MK-801 clearly implicate glutamate NMDA receptors in the cortically driven immediate-early gene induction in striatal neurons. Thus activation of NMDA receptors by corticostriatal fibers may have increased intracellular Ca²⁺ in the responsive striatal neurons, triggering the activation of intracellular cascades with the final result of gene induction (Bito et al., 1996; Liu and Graybiel, 1996; Xia et al., 1996). MK-801 also reduced gene induction in the neocortex, and it could have acted intracortically to limit excitation of corticostriatal neurons, even though it did not eliminate motor tics.

Surprisingly, blockade of AMPA receptors with GYKI 52466 did not block the cortically evoked gene induction in the striatum. The GYKI 52466 pretreatment actually increased the induction of JunB. Kita (1996) has demonstrated AMPA/kainate responses in GABAergic interneurons, which in turn elicit a GABA response in nearby projection neurons. AMPA blockade could thus increase the response of projection neurons to cortical stimulation. We were unable to test for the effects of kainate receptor blockade because of the lack of selective antagonists for use in vivo (Lerma et al., 1997), and this was true also for metabotropic glutamate receptors.

Non-NMDA receptors have been implicated in cortically induced LTD, and NMDA receptors have been implicated in LTP in the striatum (Calabresi et al., 1992). Under conditions of subthreshold depolarization, the glutamate responses of striatal projection neurons show NMDA components, which are larger and slower than the AMPA/kainate responses of these cells, favoring summation of excitatory responses (Kita, 1996). We do not claim strict parallels between these electrical events and immediate-early gene induction, but note that the differences between NMDA and non-NMDA responses in the corticostriatal system are reflected also in long-lasting events involving gene induction and protein synthesis.

Differential control of the direct and indirect pathways by motor cortex

The most striking finding in our experiments is that activation of MI differentially affects gene expression in the direct and indirect projection neurons of the striatum. Stimulation of MI activated c-Fos and JunB expression overwhelmingly (with 300 μmpicrotoxin) or exclusively (with 75 μm picrotoxin) in enkephalin-containing indirect pathway neurons. By contrast, very few dynorphin-containing direct pathway neurons showed a gene response to motor cortex excitation (Fig. 8). In the squirrel monkey, electrical stimulation of motor or somatosensory cortex also predominantly induces c-Fos in the enkephalinergic projection neurons of the putamen (Parthasarathy and Graybiel, 1997). Our results, in a reproducible paradigm devoid of anesthesia, corroborate and substantially extend the results obtained in the primate.

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Fig. 8.

Top. Model of MI activation of the sensorimotor striatum. Local application of picrotoxin to MI relieves projection neurons of inhibitory control by intrinsic GABAergic interneurons. Corticostriatal neurons (Ctx;red) become activated as a consequence and in turn stimulate a restricted population of striatal neurons (CPu; red) to express the immediate-early gene products c-Fos and JunB. Most of the activated striatal neurons are enkephalinergic (e) and thus are part of the indirect basal ganglia pathway projecting to the external pallidal segment (GPe). Dynorphin-positive projection neurons (d), part of the direct pathway projecting to the internal pallidum (EN/SN), show very little cortically driven induction of c-Fos and JunB after MI stimulation. Differential cortical effects on these two striatal projection neuron subtypes potentially amplify the specificity with which MI controls the indirect and direct pathways of the basal ganglia. GPe, External pallidum; EN, entopeduncular nucleus;SN, substantia nigra; CPu, caudoputamen;Ctx, motor cortex.

The most extreme interpretation of our findings would be that MI projects almost exclusively to indirect pathway neurons rather than to direct pathway neurons. From the available anatomical evidence, we do not think that complete segregation of inputs is likely (Dubé et al., 1988; Hersch et al., 1995; Kincaid and Wilson, 1996); however, differences in the innervation densities or placement of cortical inputs to the two cell types could account for the results, as could different levels of expression of glutamate receptor subtypes, Ca²⁺ channels, or kinase or phosphatase cascades controlling nuclear events (Malenka and Nicoll, 1993; Tallaksen-Greene and Albin, 1994; Ghosh and Greenberg, 1995; Landwehrmeyer et al., 1995;Bito et al., 1996; Liu and Graybiel, 1996; Wilson and Kawaguchi, 1996). Specialization in the intrinsic connectivity of the two cell types or in noncortical extrinsic connections could also be critical (Dubéet al., 1988; Lapper and Bolam, 1992; Kawaguchi et al., 1990;Sidibé and Smith, 1996). Particularly interesting is the report by Sidibé and Smith (1996) that thalamostriatal afferents preferentially innervate direct pathway neurons. This suggests that a cortico-thalamo-striatal activation pathway is not likely to account for the gene activation we found in enkephalinergic neurons, although differences in thalamic or other connectivity might contribute to the difference in direct and indirect neuron responsivity that we found. We emphasize that in any given experiment, on the order of 35% (c-Fos) to 50% (JunB) of the enkephalin-immunoreactive neurons in the field of induction showed immunodetectable levels of immediate-early gene induction. Conceivably, this could reflect the presence of subtypes of enkephalinergic neurons.

Such differential control by the motor cortex over indirect pathway striatal neurons is of great potential clinical interest, because many therapeutic approaches to basal ganglia disorders involve selective manipulation of the direct and indirect pathways (Chesselet and Delfs, 1996; Graybiel, 1996, and references therein). Our results and those of Parthasarathy and Graybiel (1997) provide the first evidence for a functional difference in cortical control of the direct and indirect pathways. It will be of great interest to learn whether the distributions we show here hold for inputs from other areas of cortex and for other transcriptional events.

We looked for, but did not find, different levels of immediate-early gene induction in the two segments of the globus pallidus. In the primate, however, our group has found that electrical stimulation of the primary somatosensory and motor cortex induces transneuronal c-Fos expression exclusively in the external segment of the globus pallidus, the target of indirect pathway neurons (Parthasarathy and Graybiel, 1997). Our different findings in the rat may reflect the greater arborization in the basal ganglia pathways of the rodent (Chang et al., 1981; Kawaguchi et al., 1990).

Differential cortical control of striatal interneurons

Interneurons constitute a minority of all striatal cells but can critically modulate basal ganglia function (Aosaki et al., 1995;Kawaguchi et al., 1995). We found a remarkably selective induction of c-Fos in parvalbumin-containing interneurons in and around the focus of activated projection neurons, and an even wider field of induction of NADPH diaphorase-containing interneurons, principally around rather than within the central zone of activation. These patterns of induction, taken together, suggest that the striatal response to a local excitation of MI includes (1) a local focus of c-Fos and JunB induction in which as many as 80–90% of the immediate-early gene-positive nuclei are expressed in indirect pathway neurons and parvalbumin interneurons and (2) a surrounding region in which almost no projection neurons are excited to express c-Fos, but in which many parvalbumin and somatostatin neurons are. Differences in the relative threshold excitability of the different neuronal types and activation of the intrinsic networks formed by the interneurons may determine these core and surround distributions (Fig. 9).

c-Fos and JunB were induced in nearly identical patterns in enkephalin-containing neurons but not in parvalbumin-positive neurons after cortical stimulation. We do not know the source of the differential regulation in the parvalbumin-containing neurons, but our findings do demonstrate that the same pattern of cortical activity can lead to changes in gene expression that are specific for different classes of neurons. These selective responses, in turn, should have different consequences on protein synthesis because of the heteromeric combinatorial control of transactivational ability (for review, seeHughes and Dragunow, 1995).

Plasticity in corticostriatal circuits

Cortical interneurons powerfully shape cortical activity (Connors et al., 1988; Kawaguchi and Kubota, 1995), and the network formed by inhibitory interneurons is crucial to dynamic cortical processes and to the reorganization of cortical maps (e.g., Jacobs and Donoghue, 1991). The application of picrotoxin in our experiments could mimic, in part, such cortical remapping. Given the strong and functionally important connections between cortex and striatum, such stimulation could induce plastic changes in the cortex and in turn generate medium- or long-term events in the striatum by affecting protein synthesis. The patterns of cortically evoked gene expression in our experiments point to such changes as being highly differentiated according to the different neuronal populations in the affected striatum. The regulatory machinery governing the expression of c-Fos and JunB is highly responsive to cortical stimulation in striatal neurons that give rise to the indirect pathway, which decreases activation of the motor thalamus, but it is almost unresponsive in striatal neurons that give rise to the direct pathway, which inhibits the motor thalamus. By differentially affecting gene expression and protein synthesis in indirect pathway neurons, the motor cortex could selectively affect their responsivity and activity patterns and therefore induce plastic changes with repercussions on the entire circuit.