Neocortical Projections Regulate the Neostriatal Proenkephalin Gene Expression

L. Just, C. Olenik and D.K. Meyer

Department of Pharmacology, Albert-Ludwigs-University, D-79104 Freiburg, Germany


    Abstract
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 Abstract
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 Materials and Methods
 Results
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There is controversial evidence that neocortical projections to the neostriatum may regulate the neostriatal expression of the proenkephalin (PEnk) gene. Therefore, we have studied PEnk gene expression in organotypic neocortico-neostriatal co-cultures as well as cultures of isolated neostriata. PEnk mRNA was determined with in situ hybridization. Removal of the neocortex caused a time-dependent reduction in the number of neostriatal cells which showed expression of the PEnk gene. A maximal decrease was seen after 3 days. Within 2 days after blockade of glutamate receptors of the NMDA type significantly fewer neostriatal cells expressed the PEnk gene, indicating that NMDA receptors mediated the expression of the gene. In isolated neostriatal slices in which the expression of the PEnk gene had been down-regulated, NMDA increased the number of cells which expressed the gene in a time-dependent manner. Maximal expression was observed after 3 days. This induction was reduced by nimodipine, which blocks L-type Ca2+-channels. The slow increase in PEnk gene expression caused by NMDA resembled a recruiting process. It seems to be specific for the neostriatum and may be due to the latter's neuronal organization.


    Introduction
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In rat neostriatum, the proenkephalin (PEnk) gene is expressed in medium-sized spiny neurons which project to the globus pallidus (Pickel et al., 1980Go; DiFiglia et al., 1982Go; Somogyi et al., 1982Go; Beckstead and Kersey, 1985Go; Izzo et al., 1987Go). These neurons are the first stage of a projection system by which the basal ganglia communicate with the thalamus and the substantia nigra (for reviews see Sandyk, 1985Go; Albin et al., 1991Go; Angulo and McEwen, 1994Go; Chesselet and Delfs, 1996Go).

The regulation of the expression of the PEnk gene in the neostriatal neurons by neocortical afferents has been studied in numerous in vivo experiments, the results of which are quite equivocal. Thus, partial ablation of the neocortex decreases the content of PEnk mRNA in the ipsilateral neostriatum by maximally 50% (Uhl et al., 1988Go; Somers and Beckstead, 1990Go; Jolkkonen et al., 1995Go) or prevents the enhancing effects of dopaminergic denervation (Campbell and Bjorklund, 1994Go). However, neocortical ischemic lesions have been reported to elevate neostriatal PEnk mRNA levels (Salin and Chesselet, 1992Go). Controversial findings were also obtained when antagonists at NMDA receptors were used. In some studies, the antagonist MK-801 decreased the neostriatal PEnk mRNA content after several days of application (Hajji et al., 1996Go; Noailles et al., 1996Go; Wang and McGinty, 1996Go; Zhang et al., 1996Go). In other investigations, short or long-term applications of MK-801 were ineffective or caused dose-dependent elevations in neostriatal PEnk mRNA content (Somers and Beckstead, 1992Go; Angulo et al., 1993Go, 1995Go; Lannes et al., 1995Go). To explain these varying effects of MK-801 one may speculate that the agent acts not only within the neostriatum but also in the neocortex, thalamus or substantia nigra, all of which send projections to the neostriatum and modulate its activity.

In the present series of experiments, we have again addressed the question as to whether projections from the neocortex to the neostriatum can regulate the neostriatal expression of the PEnk gene. To avoid the complexity of the in vivo situation, we have used organotypic cultures of rat neostriatum. Since their first use (Gähwiler, 1981Go), such cultures prepared from neonatal neocortex and neostriatum have proved to be good models for the investigation of postnatal neuronal development (Bolz et al., 1990Bolz et al., 1993; Østergaard et al., 1990Go, 1991Go, 1995Go; Götz and Bolz, 1992Go; Yamamoto et al., 1992Go; Distler and Robertson, 1993Go; Østergaard, 1993Go; Obst and Wahle, 1995Go; Just et al., 1996Go, 1998Go). Recent studies with slice cultures have shown that the neostriatal tissue has many features in common with the tissue in vivo, such as a similar striosome-matrix architecture, neuronal distribution and neuronal activity. Also neocortico-neostriatal projections are present in the slices (Liu et al., 1995Go; Plenz and Aertsen, 1996aGo). Therefore, slice cultures have been used to study the regulation of the immediate early gene c-fos and of the transcription factor CREB (Liu et al., 1995Go; Liu and Graybiel, 1996Go), as well as for electrophysiological investigations (Plenz and Aertsen, 1996aGo,bGo).

For the present investigation, neostriatal slices as well as neocortico-neostriatal co-cultures were prepared from brains of 2-day-old rats. The expression of the PEnk gene was determined with in situ and Northern blot hybridization.


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 Materials and Methods
 Results
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Preparation and Cultivation of Slice Cultures

Cultures were prepared from 2-day-old rats. Coronal slices (250 µm thick) of frontoparietal neocortex with the adjacent neostriatum were cut with a McIllwain tissue chopper. They were cultivated on translucent membranes (Millipore, Eschborn, Germany) at 37°C in a humidified atmosphere (5% CO2) (Stoppini et al., 1991Go; Heimrich and Frotscher, 1994Go). Culturing lasted for 5–11 days; the medium [HEPES buffered minimum essential medium (50%), Hank's balanced salt solution (25%) and heat-inactivated horse serum (25%)] was renewed three times a week. AP5, dizocilpine maleate (MK-801), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), KN62, (±)-{alpha}-methyl-4-carboxyphenyl-glycine (MCPG), and nimodipine were bought from RBI (Biotrend, Köln, Germany). N-Methyl-D-aspartate (NMDA) and 8Br.cAMP were purchased from Sigma (Deisenhofen, Germany).

Extraction and Filter Hybridization of RNA

Ten slice cultures were pooled per sample. Total RNA was extracted with 7.6 M guanidine hydrochloride as described previously (Cheley and Anderson, 1984Go). Total RNA was separated over a denaturing agarose/ formaldehyde gel (1.25% agarose, 6.6% formaldehyde). The running buffer contained 0.02 M MOPS, 8 mM NaOAc and 1 mM EDTA. The 28S and 18S ribosomal RNAs were visualized with ethidium bromide fluorescence and photographed. The RNA bands were electroblotted onto a nylon membrane (Boehringer, Mannheim, Germany). The membranes were then co-hybridized with antisense probes for PEnk and cyclophilin mRNA at 65°C overnight and washed with 0.2x SSC at 65°C thereafter (Höltke and Kessler, 1990Go). The digoxigenin-labeled cRNAs were made by in vitro transcription (Höltke and Kessler, 1990Go; Theodoridu et al., 1994Go). The PEnk cDNA used was a gift from Dr Steven Sabol (NIH, Bethesda, MD). It was restricted with SacI and SmaI, and cloned into the pT7T3 18U vector (Pharmacia, Freiburg, Germany). It was 935 nucleotides in length and complementary to the coding region of PEnk mRNA (Yoshikawa et al., 1984Go). The cyclophilin cDNA was obtained from Dr Mi (University Freiburg, Freiburg, Germany). It was cloned into the EcoRI/BamHI site of the pT7T3 transcription vector (Boehringer). The transcribed cRNA contained 510 bp and was complementary to the complete coding region of the cylophilin mRNA (Haendler et al., 1987). Digoxigenin-labeled mRNA hybrids were detected and quantified according to the Applications Manual `DIG DNA Labelling and Detection' (Boehringer). In short, after reacting the hybrids with the antibody–conjugate (anti-digoxigenin–alkaline phosphatase), the enzymatic reaction was started by addition of the chemiluminescent substrate disodium 3-(4-methoxyspiro(1,2-dioxetane–3,2'-(5'-chloro)-tricyclo(3.3.1.1) decan)-4-yl)phenylphosphate for 5 min. The filter was exposed to X-ray film.

In Situ Hybridization of Slice Cultures

Whole mounts were fixed with 4% paraformaldehyde in 0.1 M PBS (pH 7.4) for 30 min and washed with 0.1 M PBS. After prehybridization for 4 h, the whole mounts were hybridized overnight at 6°C with the sense or antisense probe (concentration 200 ng/ml) and then washed in 0.2x SSC at 65°C. For detection of PEnk mRNA, the antisense probe described above was used. To enhance penetration and reduce non-specific background, the probe was alkali hydrolyzed to an average length of 100–200 bases. This antisense probe detected a single specific band of 1.4 kb in filter hybridization under identical conditions (Theodoridu et al., 1994Go) and was tested by in situ hybridization of adult rat caudate putamen. Digoxigenin-labeled mRNA-hybrids were detected as described above. The color development was performed overnight in the dark with freshly prepared substrate solution of nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate (Boehringer). For hybridization controls the corresponding messenger sense probe was used.

Tracing of Nerve Fibers with Biocytin

For the tracing of neocortical projections, small biocytin crystals were injected into the neocortical part of the neocortico-neostriatal co-culture. After 2 h the cultures were washed twice with fresh medium to remove the soluble tracer. Then they were incubated for additional 48 h and fixed with 4% paraformaldehyde in 0.1 M PBS for 1 h. After rinsing in PBS, the cultures were incubated for 2 h in ABC-Elite reagent (dilution 1:50, Vector Laboratories, Burlingame, CA) and washed again in PBS. For visualization of labeled fibers a diaminobenzidine-nickel/cobalt procedure was used (Adams, 1981Go).

Data Analysis

Cells containing PEnk mRNA were quantified in a frame the area of which is given in the figure legends. For statistical evaluation, the Kruskal–Wallis test and the Mann–Whitney U-test were used. Means ± SEM are shown.


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 Materials and Methods
 Results
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 References
 
Projections from the Neocortex Maintain the PEnk Gene Expression in the Neostriatum

When neocortico-neostriatal slices were prepared from brains of 2-day-old rats and brought into culture for 7 days, numerous neurons strongly expressed the gene in the neostriatum. In contrast, only a few labeled cells were found in the neocortex (Fig. 1Go). Thus, the expression of the PEnk gene in slices resembled that found in vivo (Olenik and Meyer, 1997Go). When the neocortex was removed during preparation of the slices, markedly fewer cells expressed the PEnk gene in the neostriatal slices (Fig. 1Go). The adherent neocortex thus appeared to be necessary for the expression of the PEnk gene in the neostriatal neurons.



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Figure 1.  Effect of removal of neocortex on expression of the PEnk gene in neostriatum: in situ hybridization for PEnk mRNA of whole mounts. Slices were prepared from brains of 2-day-old rats and cultured for 7 days. (A) neocorticoneostriatal co-culture; (B) neostriatal culture where the neocortex was removed during preparation. Scale bar = 400 µm.

 
To study the time-course of the reduction in PEnk gene expression in the neostriatum, we removed the neocortex at different days in vitro. The neostriata of neocortico-neostriatal cultures served as controls (Fig. 2Go). There was no change 24 h after removal of the neocortex. After 48 h, however, PEnk mRNA could only be detected in a reduced number of cells. Only a few cells detectably expressed the PEnk gene 72 h after removal of the neocortex. This finding confirmed that the neostriatal expression of the PEnk gene depended on the adjacent neocortex. Moreover, it showed that the decline in the expression of the PEnk gene was not immediate but took ~3 days. However, it provided no information on the nature of neocortico-neostriatal interactions. Were neuronal projections or humoral factors from the neocortex essential for the maintenance of the neostriatal expression?



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Figure 2.  Time-course of the decrease in PEnk gene expression in neostriatum after removal of neocortex: in situ hybridization for PEnk mRNA of whole mounts. Neocortico-neostriatal slices were prepared from brains of 2-day-old rats and cultured for 11 days. (A) Control; (B) the neocortex was removed at day 10 in vitro, i.e. 24 h before histological processing of the slices; (C) the neocortex was removed at day 9 in vitro, i.e. 48 h before histological processing of the slices; (D) the neocortex was removed at day 8 in vitro, i.e. 72 h before histological processing of the slices. Scale bar = 50 µm.

 
To study this question, we prepared neocortico-neostriatal co-cultures and severed the connections between both areas without removing the neocortex. Nine days afterwards, the number of cells which visibly expressed the PEnk gene in the neostriatum was not reduced compared with that in untreated co-cultures (Fig. 3Go). Two alternative explanations were possible for this surprising finding: either humoral factors were released from the neocortex or reinnervation of the neostriatum by neocortical projections occurred. To study the possiblity of reinnervation, the neocortico-neostriatal connections were severed during slice preparation and again after 4 and 7 days in vitro. As a result, the number of cells with detectable PEnk gene expression was diminished to 31% compared with controls. This reduction corresponded to that found in isolated neostriata (27% of controls) (Fig. 3Go).



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Figure 3.  In slice cultures, projections from neocortex can reinnervate the neostriatum. Neocortico-neostriatal slices were prepared from brains of 2-day-old rats and cultured for 11 days. Connections between the neocortex and the neostriatum were severed during the dissection. In some cultures, this procedure was repeated after 4 and 7 days in vitro. Untreated co-cultures and striatal cultures served as references. After in situ hybridization for PEnk mRNA, the positive cells were counted in a frame of 0.3 mm2. Data are expressed as % ± SEM (n = 5). a, P < 0.05 compared with neocortico-neostriatal co-cultures; b, P < 0.05 compared with neostriatal cultures.

 
Reinnervation of the neostriatum was also directly demonstrated. For this purpose, co-cultures were prepared and the connections between the neocortex and neostriatum were severed. Seven days later, biocytin was injected into the neocortex of the co-cultures (Fig. 4Go), which were then cultivated for another 2 days. In these slices, biocytin-labeled neocortical cells with pyramidal morphology were found at the injection site. Within the neostriatum, a dense network of fibers was stained by the dye, indicating a profuse innervation from the neocortex (Fig. 4Go). Thus, neocortical neurons had indeed reinnervated the neostriatum of the co-cultures.



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Figure 4.  In neocortico-neostriatal slices neocortical neurons project to the neostriatum. The slices were prepared from brains of 2-day-old rats and cultured for 9 days. Biocytin was injected into the neocortical part of the slice to label the adjacent neurons and to visualize their projections. (A) A biocytin labeled neuron in the neocortex; (B) biocytin labeled axonal network in the neostriatum. Scale bar = 25 µm.

 
In the final experiment of this series, we tested whether the projections which maintained the neostriatal expression of the PEnk gene originated in the upper or lower half of the neocortex. When the upper half of the neocortex was removed, the number of neostriatal cells which expressed the PEnk gene (111 ± 7%; n = 3) remained unchanged compared with controls (100 ± 6%; n = 4).

Taken together, these results showed that projections from the lower half of the neocortex maintained the PEnk gene transcription in the neostriatum.

Endogenous Glutamate Maintains the PEnk Gene Expression in Neocortico-neostriatal Slices via NMDA Receptors

Neocortical projections to the neostriatum are glutamatergic. Therefore, we used receptor antagonists to determine whether glutamate indeed regulated neostriatal PEnk gene expression in our slice cultures. The blockers were added to the incubation medium of neocortico-neostriatal cultures for 24 and 48 h to study the time-course of their effects. MK-801 blocks the cation channel of the NMDA-receptor (Wong et al., 1986Go). When applied for 24 h at a concentration of 1 µmol/l, MK-801 diminished by 41% the number of cells with a detectable gene expression. After 48 h, this reduction was ~55% (Fig. 5Go). AP5 blocks the glutamate binding site of the receptor (Monaghan et al., 1984Go, 1985Go; Olverman et al., 1984Go). At a concentration of 100 µmol/l, AP5 had no effect when applied for 24 h but reduced the number of positive cells by 70% after 48 h (Fig. 5Go).



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Figure 5.  NMDA receptor antagonists reduce the neostriatal expression of the PEnk gene in neocortical-neostriatal co-cultures. Slices were prepared from brains of 2-day-old rats and cultured for 7 days. During the last 24 or 48 h of incubation the cultures were treated with MK-801 (1 µmol/l; panel A) or AP5 (100 µmol/l; panel B). After in situ hybridization for PEnk mRNA, the number of positive cells in the neostriatum in a 0.15 mm2 frame was counted. Data are expressed as % ± SEM (n = 5). Asterisk indicates P < 0.05 compared with respective controls.

 
To test the possible involvement of other glutamate receptors, we blocked those of the AMPA/kainate type with CNQX (Blake et al., 1988Go; Honore et al., 1988Go) and metabotropic glutamate receptors with MCPG (Recasens et al., 1987Go; Sugiyama et al., 1987Go). Both antagonists did not affect the neostriatal expression of the PEnk gene. In co-cultures treated with 50 µmol/l CNQX, the number of cells which showed expression was 111 ± 5% (n = 3) and 110 ± 7% (n = 6) of controls (100 ± 5%; n = 6) 24 and 48 h after application respectively. In co-cultures treated with 1 mmol/l MCPG for 24 or 48 h the number of positive cells was 90 ± 4% (n = 4) and 96 ± 28% (n = 3) of controls (100 ± 5%; n = 6). Taken together, these results indicated that glutamate receptors of the NMDA type were involved in the maintenance of the expression of the PEnk gene in neostriatal neurons.

NMDA Increases the Expression of the PEnk Gene in Isolated Neostriatal Slices

To further test this hypothesis, we studied the effect of NMDA on the expression of the PEnk gene in slice cultures of isolated neostriata, which had been precultured for at least 4 days to diminish the expression of the PEnk gene. In previous studies on the expression of the PEnk gene in neocortical slice cultures, a concentration of 10 µmol/l NMDA proved to result in a maximal stimulation (Just et al., 1998Go). Therefore, this concentration was also used in the present study. It enhanced the number of positive cells in a time-dependent manner, i.e. by 101% after 24 h, by 172% after 48 h and by 253% after 72 h (Fig. 6Go). To corroborate this finding, Northern blot analysis for PEnk mRNA was used. Again, neostriatal cultures were treated with 10 µmol/l NMDA for 24 or 72 h and then extracted for total RNA. Northern blot analysis showed that NMDA increased the PEnk mRNA band by 76% after 24 h and by 197% after 72 h compared with the controls (Fig. 7Go). In additional experiments, we tested whether lower concentrations of NMDA were effective. However, 1 mmol/l NMDA added for 72 h did not increase the number of neurons expressing the PEnk gene in isolated neostriata (controls 100 ± 7% versus 1 µmol/l NMDA 112 ± 5%; mean ± SEM; n = 6).



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Figure 6.  NMDA increases the expression of the PEnk gene in neostriatal slices. Original photographs are shown in upper panel; quantification by counting of positive cells is shown in lower panel. Slices were prepared from brains of 2-day-old rats and cultured for 7 days. Upper panel: (A) control; (B) NMDA (10 µmol/l) was present in the incubation medium during the last 72h. Scale bar = 400 µm. Lower panel: cultures were treated with 10 µmol/l NMDA during the last 72, 48 or 24 h. After in situ hybridization for PEnk mRNA, the positive cells were counted in a frame of 0.15 mm2. Data are expressed as % ± SEM (n = 6). Asterisks indicate P < 0.05 compared with controls.

 


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Figure 7.  In neostriatal slices, NMDA enhances the expression of the PEnk gene (as determined by Northern blot hybridization). Slices were prepared from brains of 2-day-old rats and cultured for 7 days. During the last 72 or 24 h, cultures were treated with 10 µmol/l NMDA. Slices were then extracted for total RNA. Ten cultures were pooled for each sample. Bands for PEnk and cyclophilin (Cyp) mRNA are shown.

 
While the time-course experiment showed that a strong effect of NMDA was present after about 72 h, it did not indicate whether NMDA initiated a process which required 3 days to develop, or whether the continous presence of NMDA was necessary. To clarify this, we treated isolated neostriata with NMDA for 24 h. After a further 48 h in the absence of NMDA, the number of positive cells increased by 90%, compared with controls (Fig. 8Go). However, in slices which had been continuously treated with NMDA for 72 h the number of positive cells was significantly increased (by ~230%; P < 0.05; Fig. 8Go). Taken together, these results showed that continuous stimulation of NMDA receptors was necessary to cause the maximal activation of the PEnk gene in isolated neostriata.



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Figure 8.  Continuous stimulation of NMDA receptors is necessary to enhance the neostriatal expression of the PEnk gene. Neostriatal slices were prepared from brains of 2-day-old rats and cultured for 7 days. In some cultures, 10 µmol/l NMDA was added to the incubation after 4 days in vitro and remained throughout the last 72 h of incubation. In other cultures, 10 µmol/l NMDA was added after 4 days in vitro but removed 24 h later. These cultures were kept in control medium for the next 48 h. After in situ hybridization for PEnk mRNA, the number of positive cells in a 0.15 mm2 frame was counted. Data are expressed as % ± SEM (n = 6). Asterisks indicate P < 0.05, compared with controls.

 
The NMDA-induced PEnk Gene Expression is Mediated by a Ca2+-dependent Mechanism

Serine/threonine protein kinases, some of which are Ca2+-activated, can increase the transcription of the PEnk gene. Since NMDA receptor activation raises intracellular Ca2+ concentrations, we tested whether the NMDA-induced expression of the PEnk gene was Ca2+-dependent. Firstly, we used KN62 (10 µmol/l), which inhibits Ca2+/calmodulin kinase II as well as voltage-dependent Ca2+-channels (Sihra and Pearson, 1995Go). In neostriatal slices, the effect of KN62 was time-dependent. KN62 had no effect when added only during the last 24 h of the 3 day treatment period with NMDA (10 µmol/l) (Fig. 9Go). However, when KN62 was present together with NMDA for 3 days, it almost abolished the effect of NMDA (Fig. 9Go). Also, in neocortico-neostriatal co-cultures, KN62 (10 mmol/l) reduced the expression of the PEnk gene in a time-dependent manner (Fig. 10Go). Next, we tested the effects of the specific L-type Ca2+-channel blocker nimodipine. Nimodipine (10 µmol/l) also attenuated the increase in the number of cells with detectable expression induced by NMDA, when added to isolated neostriata for 72 h (Fig. 9Go). In neocortico-neostriatal co-cultures treated with nimodipine (10 µmol/l) for 2 days, the number of positive cells was significantly decreased compared with controls (Fig. 10Go). Taken together, these data indicated that NMDA receptor stimulation increased the expression of the PEnk gene via Ca2+-dependent mechanisms.



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Figure 9.  KN62 and nimodipin reduce the increase in neostriatal PEnk gene expression caused by NMDA. Slices were prepared from brains of 2-day-old rats and cultured for 7 days. NMDA (10 µmol/l) was added to the incubation medium after 4 days in vitro and remained throughout the last 72 h of incubation. KN62 (10 µmol/l; panel A) and nimodipin (Nimo; 10 µmol/l; panel B) were added during the last 24 or 72 h of the incubation. After in situ hybridization for PEnk mRNA, the number of positive cells in a 0.15 mm2 frame was counted. Data are expressed as % ± SEM (n = 6). a, P 100 < 0.05 compared with controls; b, P < 0.05 compared with cultures treated with NMDA.

 


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Figure 10.  KN62 and nimodipin diminish neostriatal PEnk gene expression in neocortico-neostriatal co-cultures. Slices were prepared from brains of 2-day-old ratsand cultured for 7 days. KN62 (10 µmol/l; panel A) was present in the incubation medium during the last 24 or 48 h of incubation, while nimodipin (Nimo; 10 µmol/l; panel B) was added 48 h before the end of the incubation. After in situ hybridization for PEnk mRNA, the number of positive cells in a 0.15 mm2 frame was counted. Data are expressed as % ± SEM (n = 6). Asterisks indicate P < 0.05 compared with controls.

 
8Br.cAMP Does not Increase the PEnk Gene Expression in Isolated Neostriatal Slices

In dissociated neostriatal neurons, activation of protein kinase A induces a threefold increase in PEnk gene transcription within 6 h (Giraud et al., 1991Go). Therefore, we used 8Br.cAMP to compare its effect with that of NMDA on the expression of the gene in the neostriatal slices. Surprisingly, 8Br.cAMP did not affect the number of cells which detectably expressed the gene, when added to the incubation medium for 24 h [controls: 100 ± 14 (n = 6) versus 8Br.cAMP: 110 ± 17 (n = 5)].


    Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, we have used neocortico-neostriatal co-cultures to investigate whether neocortical projections maintain PEnk gene expression in neostriatal neurons. Our data showed that these afferents indeed do so via NMDA receptors in a Ca2+-dependent manner. The changes in PEnk gene expression had characteristic kinetic features: the decrease induced by deafferentiation as well as the increase after NMDA receptor stimulation took at least 48 h to become fully established.

Neocortical Projections Mediate the Neostriatal PEnk Gene Expression

Complete removal of the neocortex reduced neostriatal PEnk gene expression in our slices by at least 70%, while partial ablation of the neocortex in vivo causes a reduction of ~50% (Uhl et al., 1988Go; Campbell and Bjorklund, 1994Go). The kinetics of the reductions were also quite similar in vivo and in vitro. In vivo, a maximal reduction in PEnk gene expression takes up to 4 days (Uhl et al., 1988Go; Campbell and Bjorklund, 1994Go), while in our cultures it was found after 3 days. Taken together, these findings suggest that similar mechanisms may induce the reductions in PEnk gene expression in vivo and in slice cultures.

When compared with the in vivo situation, the slices have a distinct advantage as they represent a reduced system which can be easily manipulated. Since all projections from the thalamus or the substantia nigra to the neostriatum could be removed, it was possible to analyze the effects of the neocortical projections without any subcortical interference. Under these conditions, we obtained clear evidence that neocortical projections can induce the expression of the PEnk gene in neostriatal neurons. This finding is of importance in view of the controversial in vivo data which deal with this question (Uhl et al., 1988Go; Salin and Chesselet, 1992Go; Campbell and Bjorklund, 1994Go).

The neocortico-neostriatal co-cultures also enabled us to study the effects of reinnervation after previous severance of the neocortical projections. This experiment showed that neostriatal PEnk gene expression was dependent on the neocortical innervation. In such co-cultures, labeling of the neocortical projection neurons with biocytin showed a dense network of stained axons in the neostriatum. Thus, the neocortical neurons were still able to reinnervate the neostriatum several days after birth. Obviously, this model can be used for studies on axonal outgrowth and pathfinding of neocortical projection neurons.

Neocortical neurons from the somatosensory or prelimbic areas which project to subcortical regions originate in layer V. They give off collaterals to the striatum on their way to the brain stem or spinal cord (Levesque et al., 1996Go; Levesque and Parent, 1998Go). The neocortical neurons which induce neostriatal PEnk gene expression are also situated in deeper neocortical layers, since removal of the upper half of the neocortex did not reduce PEnk gene expression in co-cultures.

NMDA Receptors Mediate the Induction of PEnk Gene Expression

In our neocortico-neostriatal co-cultures, antagonists at metabotropic glutamate receptors or at kainate/AMPA receptors did not affect the expression of the PEnk gene. In contrast, MK-801 and AP5, which block NMDA receptors via different mechanisms, reduced the expression within 48 h by 55 and 77% respectively. Taken together, these results showed that endogenous glutamate released from neostriatal projections enhanced the neostriatal expression of the PEnk gene solely via NMDA receptors. Also this finding is important in view of controversial effects of MK-801 which have been reported in in vivo studies (Somers and Beckstead, 1992Go; Angulo et al., 1993Go, 1995Go; Lannes et al., 1995Go; Hajji et al., 1996Go; Noailles et al., 1996Go; Wang and McGinty, 1996Go; Zhang et al., 1996Go). To explain this discrepancy it is tempting to speculate that in vivo MK-801 exerts actions not only in the neostriatum but also in other brain areas. However, the different time-schedules and routes of administration used may also have contributed to the varying effects of the antagonist.

NMDA was tested in neostriata which had been previously depleted of PEnk mRNA by removing the neocorte x. As postulated, NMDA produced a concentration and time-dependent increase in PEnk gene expression. In addition, this series of experiments allowed us to evaluate two important aspects of our methodology. Firstly, it showed that removal of the neocortex did not cause a loss of the cells which expressed the PEnk gene, but reversibly decreased their expression of the gene. Secondly, it showed that NMDA had similar effects on PEnk mRNA levels when quantified by both Northern blot analysis and by counting of cells which contained visible amounts of PEnk mRNA.

Counting the positive cells provided important information on the time-course of the increase in PEnk gene expression induced by NMDA. Surprisingly, NMDA did not stimulate a maximal number of positive cells within 24 h; instead, the number of positive cells increased by 70 to 100% (as compared with controls) per day during the 3 day treatment period with NMDA. When slices were treated with NMDA for 1 day only and then incubated in drug-free medium for the following 2 days, the resulting increase in the number of positive cells was 90%, compared with 230% after continuous treatment with NMDA for 3 days. Thus, the continuous presence of NMDA was necessary for the linear increase in the number of cells with a detectable expression of the PEnk gene.

Is the Gradual Effect of NMDA on PEnk Gene Expression Due to the Neuronal Organization of the Neostriatum?

The present knowledge on the regulation of the PEnk gene does not provide an explanation for an induction process which takes up to 3 days. In cultures of dissociated neurons and glial cells, protein kinases A and C as well as Ca2+-dependent kinases activate transcription factors such as CREB or members of the c-fos and c-jun families which enhance the expression (Comb et al., 1986Go, 1988Go; Sonnenberg et al., 1989Go; Nguyen et al., 1990Go; Kobierski et al., 1991Go; Konradi et al., 1993Go, 1994Go, 1996Go; Bacher et al., 1996Go). Recently, the involvement of tyrosine kinases has been suggested (Puchacz et al., 1993Go; Tan et al., 1994Go). All of these stimuli induce a maximal PEnk gene expression in <24 h (for references see above). This also applies to dissociated neostriatal neurons (Giraud et al., 1991Go; Konradi et al., 1995Go).

Was the gradual effect of NMDA due to its slow entry into the slices? This explanation is unlikely for several reasons. Firstly, the explanted slices are known to flatten during the incubation (Liu et al., 1995Go). Thus, they were probably less than 200 µm thick at the time of drug incubation. Secondly, NMDA increased more than fivefold the number of neurons with detectable PEnk mRNA within 24 h in neocortical slice cultures of similar width (Just et al., 1998Go). Thirdly, microscopic analysis showed that the positive cells found after 24 h of NMDA treatment were not only located close to the surface of a given slice but were found throughout its width. However, the complete development of the expression took 3 days.

Did the specific organization of the neostriatal neurons cause the gradual increase in the expression of the PEnk gene? There is some evidence for this hypothesis. In contrast to its slowly developing effect in neostriatal slices, NMDA induced within 24 h a fivefold increase in the expression of the PEnk gene in neocortical slices (Just et al., 1998Go). Moreover, the neostriatal neurons within the slices did not respond to 1 mmol/l 8Br.cAMP with an enhanced expression of the PEnk gene within a period of 24 h, while in dissociated neostriatal neurons activation of protein kinase A strongly enhances the expression of PEnk gene responses within 6 h (Giraud et al., 1991Go; Konradi et al., 1995Go). Since 8Br.cAMP also strongly increases the expression of the PEnk gene within 4 h in slice cultures of the subventricular zone of the neocortex (Just et al., 1998Go), it is unlikely that the agent cannot penetrate into slices. Instead, one must assume that those events which induce PEnk gene transcription in a variety of cells are not present in the slice cultures. Finally, morphological studies have shown extensive connections via axon collaterals of the neostriatal projection neurons, some of which express the PEnk gene which seem to form a lateral inhibitory network (Preston et al., 1980Go; Wilson and Groves, 1980Go; Bishop et al., 1982Go; Bolam and Izzo, 1988Go). Therefore, it is tempting to speculate that NMDA initiated interactions in the neostriatal network which gradually recruited neurons to express the PEnk gene. Increased intracellular concentrations of Ca2+ induced by NMDA seem to be necessary for this recruitment, since blockade of L-type Ca2+-channels with nimodipine reduced the effect of NMDA by 72%. This result also confirmed the recent finding that NMDA receptors in the neostriatum can be functionally coupled to L-type Ca2+-channels in postsynaptic densities (Liu and Graybiel, 1996Go).

Prospectus

There is strong evidence that neurons in organotypic cultures have many properties in common with those in vivo (Liu et al., 1995Go; Plenz and Aertsen, 1996aGo). Is it thus likely that glutamate released from neocortical projections in vivo also induces a gradual increase in neostriatal PEnk gene expression? There is some evidence for this conclusion. In our cultures, the removal of the glutamatergic innervation by surgical interruption of the neocortical projections or by receptor blockade caused a gradual decrease in the expression of the gene within 2–3 days. The changes of neostriatal PEnk gene expression in vivo which are brought about by such procedures also take several days to occur. This similarity may indicate that the same mechanisms are involved. Organotypic co-cultures would be suitable models with which to investigate this recruitment phenomenon.


    Notes
 
We thank Ms A Uhl for excellent technical assistance. The editorial help of Dr S. Cox is appreciated. Supported by Deutsche Forschungsgemeinschaft (Me541/6-2).

Address correspondence to D.K. Meyer, Department of Pharmacology, Albert-Ludwigs-University, Hermann-Herderstraße 5, D-79104 Freiburg, Germany. Email: meyerdk{at}uni-freiburg.de.


    References
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