- Email: [email protected]
Cocaine-induced expression of striatal c-fos in the rat is inhibited by NMDA receptor antagonists
0361-9230/93 $6.00 + .OO Copyright 0 1992 Pergamon Press Ltd.
Brarn Research Bulhin, Vol. 30, pp. 173-176, 1993 Printed in the USA. All rights reserved.
Cocaine-Induced Expression of Striatal c-fos in the Rat is Inhibited by NMDA Receptor Antagonists GERMAN
TORRES’
AND CATHERINE
RIVIER
The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037 Received 29 June 1992; Accepted 28 July 1992 TORRES, G. AND C. RIVIER. Cocaine-induced expression of striatal c-fos in the rut is inhibited by NMDA receptor antagonists. BRAIN RES BULL 30( l/2) 173-176, 1993.-To assess the possible involvement of NMDA receptors in mediating the expression of striatal c-fos by cocaine injection, we investigated the effects of the noncompetitive NMDA receptor antagonists, ketamine and (+)-S-methyl-IO,1 1-dibydro-5%dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) as well as the competitive NMDA receptor antagonist, 3-(2-carboxypiperazin-4-yl)-propyl- I-phosphonic acid (CPP), in the perikarya of cocaine-treated rat brains. As previously shown by our group, administration of 20 mg/kg cocaine (IP) resulted in the immunocytochemical expression of the protooncogene in numerous cells of the caudate putamen (dorsal/sensorimotor striatum). A ketamine mixture anesthetic (2 mg/kg), however, administered 30 min prior to cocaine exposure completely blocked such genomic expression. Pretreatment with MK-80 1 (I mg/kg) or CPP (5 mg/kg) also interfered, albeit to a lesser extent, with the expression of c-fos by cocaine in awake animals. These results indicate that cocaine induction of cellular c-fos in the caudate putamen is mediated at least in part by NMDA-sensitive receptors.
Rat
Cocaine
c-fos
Ketamine mixture
MK 801
COCAINE administration to rats has been reported to induce the expression of the immediate early gene, c-fos, in striatal neurons (5,14,17). FOS (a product of c-fos) is a nuclear protein that binds with high affinity to the DNA transcription promotor element, AP-1 (9). Because AP-1 is essential for the transcription of several genes, induction of c-fos expression by cocaine could represent some of the initial genomic mechanisms by which this drug exerts its stimulatory effects in the mammalian brain. Cocaine-induced expression of c-fos appears to be primarily mediated through the postsynaptic activation of D, receptors localized on striatonigral neurons (2). Evidence for this hypothesis comes from observations that D, receptor antagonists such as SCH-23390 effectively block the expression of the immediate early gene in cocaine-treated rats (5,17). However, there is also evidence that striatal dopamine release is mediated by glutamate receptors sensitive to N-methyl-D-asperate (NMDA) (12). Thus, the expression of c-fos by cocaine could also be mediated by receptors other than D, receptors. Excitatory amino acids such as L-glutamate and L-aspartate are likely to represent the major excitatory neurotransmitters in the brain. The actions of these molecules are mediated by at least three classes of membrane receptors, including NMDA, quisqualate, and kainate (3,8,16). The most investigated of these
CPP
and perhaps the most abundant and widely distributed in parenchyma tissue is the NMDA receptor. This ionotropic receptor is linked to membrane cation channels and can be selectively blocked noncompetitively by ketamine and (+)-5-methyl10,l I -dihydro-5H-dibenzo[a,&yclohepten-5, IO-imine hydrogen maleate (MK-801) or competitively by 3-(2-carboxypiperazin-4-yl)-propyl- 1-phosphonic acid (CPP) (8,16). It is significant that high levels of NMDA receptors are found in the rat striatum (caudate putamen and nucleus accumbens) because perikarya within these brain regions selectively express c-fos in response to cocaine exposure. We report here, as suggested by preliminary studies (15) that the expression of striatal c-fos by cocaine is markedly inhibited by systemic pretreatment of the above NMDA receptor antagonists. METHOD
Male, albino Sprague-Dawley rats (Haltzman, Madison, WI) weighing between 230-280 g were used for all experiments. Rats were housed in groups of three to four under a 12L: 12D cycle and standard ambient temperature; food and water were available ad lib. All experiments were conducted between 1400 and 1500 h of the light cycle. For the ketamine experiments, rats
’ To whom requests for reprints should be addressed.
173
TORRES
174
were injected (SC) with an anesthetic mixture of ketamine:acepromozine:xylazine ( 160 pl/ 100 g body weight) containing 2.5 ml ketamine (Vetelar, 100 mg/ml solution; Aveco Co.) 1 ml acepromazine maleate (10 mg/ml solution; Fermenta Animal Health Co.) 2.5 ml xylazine (Rompun, 20 mg/ml solution; Miles Laboratories), and 4 ml distilled water. Animals were then left undisturbed after administration of the anesthetic for 30 min. After this time delay, these rats (n = 5) were injected (IP) with cocaine HCI (Mallincodt, St Louis, MO) that had been dissolved in sterile saline at a dose of 20 mg/kg (calculated as free base). Thirty minutes later, animals (still under anesthesia) were perfused transcardially with heparinized saline followed by cold 4% paraformaldehyde in 0.1 M sodium phosphate (pH 7.2) buffer. To evaluate the effects of the above ketamine mixture on nuclear c-fos already induced by cocaine in the striatum, rats (n = 3) were injected with cocaine and 0.5 h later anesthetized with the aforementioned ketamine mixture. Following another 30-min delay, these rats were perfused as previously described. Another group of rats were injected (IP) with MK-801 (n = 4) or CPP (n = 5) (Research Biochemicals Inc., Natick, MA) dissolved in sterile saline at a dose of 1 or 5 mg/kg, respectively. Thirty minutes later, rats were injected with cocaine (20 mg/ kg). In all experiments, control rats were always injected with sterile saline. Following a 30-min delay, animals were anesthetized with sodium pentobarbital(50 mg/kg) and perfused as previously described. Thus, different rats received the following experimental drug designs: (a) ketamine mixture + cocaine; (b) MK-801 + cocaine; (c) CPP + cocaine; (d) saline + cocaine (n = 4); (e) saline + saline (n = 3). In addition, other rats was injected with only the ketamine mixture (n = 3) MK-801 (n = 5) or CPP (n = 2). Specifics of c-fos determination by immunocytochemistry are reported elsewhere (14). Briefly, free-floating brain sections (40 pm) were incubated for 48 h with a c-fos antibody [rabbit polyclonal immunoglobulin (1g)G; 1: 1,000 dilution; specificity: rodent P62c-fos; Oncogene Science, NY]. The sections were reacted with DAB, I % HzOz, and Ni04. 6Hz0. After several rinses in 50 mM KPBS, brains sections were mounted onto gelatin-chrome alum-coated slides and coverslipped with DPX. To determine the specificity of c-fos IgG, random brain sections were treated in the same manner as described above but were incubated without the c-fos antibody. Alternate brain sections were counterstained with neutral red to subsequently identify striatal regions expressing c-fos. The number of perikarya expressing the immediate early gene was counted using a grid reticle on a microscope. Rat brain sections corresponding to plate I8 of Paxinos and Watson (11) were chosen for counting purposes. The medial aspect of the caudate putamen was selected as the counting region because it was this area where a significant blockade of c-fos expression by the NMDA receptor antagonists was most often discernible. The dorsal (adjacent to the corpus callosum) and lateral (adjacent to the lateral ventricle) portions of the caudate putamen served as primary margins for the aforementioned medial region. The data were analyzed by analysis of variance (ANOVA) followed by posthoc (t test) comparisons for each drug treatment. RESULTS
When c-fos immunoreactivity was assessed in the caudate putamen of rats injected with saline alone, the phosphoprotein was not measurably detected in such forebrain structure (Fig. 1). However, nonspecific, scattered fos-immunopositive cells were observed in other brain regions including the piriform cortex, septal region, and lateral hypothalamus. This pattern of nuclear reactivity was observed in brains of both anesthetized and
AND
RIVIER
awake animals. The injection of 20 mg/kg cocaine to awake rats caused a robust and wide expression of c-fos relative to salinetreated rats (Fig. 2; p < 0.0 I). This genomic induction was characterized by intense nuclear labeling of numerous neurons within the caudate putamen, in particular its dorsal-medial-lateral subdivisions; axons and dendrites were not labeled (Fig. 1). This pattern of nuclear reactivity, however, was completely abolished in rats that had been anesthetized with a ketamine mixture prior to administration ofcocaine (Fig. 1). Interestingly, the ketamine mixture did not inhibit the endogenous expression of c-fos in other cytoarchitectonic regions of the rat brain, including those in the piriform cortex, septal region, or paraventricular thalamic nucleus. When rats were injected with cocaine and 30 min later anesthetized with the ketamine mixture, c-fos expression in the caudate putamen was still fully present in terms of its topographical distribution and nuclear intense labeling (data not shown). Thus, blockade of c-fos expression by the ketamine mixture occurs only when the NMDA receptor antagonist is administered prior to cocaine exposure. Pretreatment with the NMDA receptor antagonist MK-801 30 min before cocaine injections markedly reduced the number of striatal perikarya expressing c-fos relative to saline + cocainetreated rats (Fig. 2; p < 0.01). The reduction of this immediate early gene was more discernible in the medial subdivision of the caudate putamen than its dorsal or lateral regions. Brains from rats injected with MK-801 with or without cocaine posttreatment also exhibited intense and consistent immunoreactivity cells in the thalamus and the parvocellular subdivision of the paraventricular nucleus of the hypothalamus (data not shown). This latter effect may be a reflection of increased activity in the hypothalamic-pituitary-adrenal axis because systemic injection of MK-80 1 stimulates corticotropin and corticosterone secretion ( 15). When animals were pretreated with CPP, cocaine exposure elicited a modest expression of c-fos as compared to rats injected with the saline + cocaine experimental drug design (Fig. 2; p < 0.01). The inhibition of the immediate early gene was more prominent in the medial cellular subdivision of the caudate putamen. Thus, the aforementioned blockade of c-fos by CPP followed a similar pattern of topographical inhibition to that seen for MK-801 (Fig. 1). Rat brains injected with saline followed by CPP showed a modest but not significant expression of c-fos in striatal or hypothalamic tissue. DISCUSSION
These studies were undertaken to assess the possible participation of NMDA receptors in the expression of c-fos by cocaine. We report here that three NMDA receptor antagonists administered prior to drug exposure blocked, to various degrees, such nuclear induction. These results suggest that NMDA receptors mediate at least in part the expression of striatal c-fos by cocaine. We and others have shown that cocaine administration to rats leads to rapid protooncogene expression in the perikarya of the caudate putamen and nucleus accumbens (5,14,17). These forebrain structures have received ample experimental attention because projecting dopamine terminal plexuses to these brain regions have been implicated in the reinforcing and behavioralactivating properties of cocaine. These psychostimulant properties are thought to have a neurochemical basis via enhanced dopamine neurotransmission and a preferential activation of striatal D, as opposed to Dz receptors (5). Concomitantly, the D, receptor subtype has been hypothesized to modulate the induction of nuclear c-fos by cocaine and other dopamine agonists (4,5, IO). However, our present results also support the hypothesis that striatal NMDA receptors are involved in the molecular
COCAINE AND STRIATAL
175
C-FOS EXPRESSION
KET + COC
SAL + SAL
SAL + COC
MK-801 + COC
CPP + COC
FIG. 1. Representative brightfield photomicrographs of coronal sections through the medial portion of the caudate putamen. Rats were pretreated with saline (Sal), a ketamine mixture (Ket; 2 mg/kg), MK-801 (1 mg/kg), or CPP (5 mg/kg) followed 30 min later by Sal or cocaine (Cot; 20 mg/ kg) IP injections. (A). Forebrain section illustrating the region of the caudate putamen depicted in subsequent photomicrographs. Blacks arrows point to unreactive myelinated bundles of corticofugal fibers coursing through the caudate putamen. Original magnification 10X.
changes that invariably occur following cocaine exposure. These data are consistent with evidence showing that Lglutamate evokes the release of dopamine in the striatum and this release is primarily mediated by NMDA-sensitive receptors proximal to or on dopamine terminal plexuses ( 12). The NMDA receptor is linked to a Na+ ionic channel with an appreciable Ca*+ permeability as well. The aforementioned postsynaptic
5
Sal + Sal
Sal + Cot
MK-801 + Cot CPP + Cot
FIG. 2. Effects of NMDA receptor antagonists on the induction of c-fos expression by cocaine in the rat caudate putamen. Saline (Sal), MK 801 (I mg/kg), or CPP (5 mg/kg) were administered 30 min prior to cocaine (Cot; 20 mg/kg) IP injections. Each bar represents the means f SEM of immunopositive perikarya in 10 mm2 sections of the medial caudate putamen (one side). Pretreatment with the ketamine mixture completely abolished the expression of c-fos by cocaine. **p -c0.01 relative to MK80 1 + Cot- or CPP + Cot-treated rats.
receptor is blocked by a number of highly specific noncompetitive and competitive antagonist compounds, such as ketamine, MK801, and CPP. Ketamine, a dissociative anesthetic, has been shown to bind with high affinity to the cation channel, thus antagonizing the intracellular regulation of the receptor by (perhaps) altering its voltage-gated dependency to a nonconductive state (8,16). Although in the present experiments we employed a ketamine mixture instead of ketamine alone, our results are in agreement with related findings showing that ketamine alone effectively blocks the expression of hypothalamic c-fos by photic stimulation ( 1). Moreover, we have ascertained that this ketamine mixture exhibits no measurable adverse effects on striatal dopamine metabolism as demonstrated by in vivo intracerebral microdialysis ( 15). Thus, the use of a ketamine mixture appears to be an appropriate method by which the expression of c-fos by cocaine in the brain parenchyma can be effectively antagonized at the receptor level. MK-801, one of the most potent NMDA antagonists yet described, also blocked the expression of c-fos by cocaine exposure. It is important to note, however, that although MK-801 blocked such expression the degree of blockade was greater for the ketamine mixture. The basis for this potency differential between the ketamine mixture and MK-80 1 is unknown. One possibility may be related to the mixture of xylazine and acepromozine in our ketamine prep aration. Xylazine, a nonnarcotic, sedative analgesic, could have potentiated the antagonistic action of ketamine by further decreasing the nonconductive state of the cation channel. Further research is obviously needed to validate this hypothesis. CPP, a competitive NMDA receptor antagonist, also blocked the expression of c-fos by cocaine. CPP readily penetrates the blood-brain barrier and is thought to inhibit the aforementioned receptor by binding to the
TORRES AND RIVIER
176
agonist site of the receptor rather than its ionic channel (8,16). As observed for MK-80 1, the blockade of c-fos by CPP was of a lesser magnitude than that seen for the ketamine mixture but equally effective relative to that of MK-80 1. It remains also unclear the differential blockade susceptibility for c-fos between the medial versus dorsal or lateral portions of the caudate putamen to the antagonistic effects of MK 80 1 and CPP. The rat striatum, which includes both the caudate putamen and the nucleus accumbens, is structurally complex, displaying a mosaic organization of neurochemical systems (4). Relating these systems to the postsynaptic effects of cocaine has proven difficult because we do not know for certain the phenotypic coding of most neurons expressing c-fos in the aforementioned forebrain region. In preliminary studies, Graybiel(4) has shown that the induction of the immediate early gene by cocaine occurs in numerous dopamine and cyclic adenosine monophosphate (CAMP)-regulated phosphoprotein (DARPP-32) neurons. This neuronal protein is phosphorylated by dopamine (6) and its regulation appears to be orchestrated by the interactions between dopamine, acting through CAMP, and L-glutamate, acting
through Ca”. Thus, the above topographical differences may be related to a preferential activation of c-fos within a striatal subset of medium-size spiny cells that biochemically interact at the level of protein phosphorylation (6). More research is needed to expand this hypothesis. In summary, our results clearly suggest the involvement of NMDA-sensitive receptors in the postsynaptic induction of cfos by cocaine. These observations are substantiated by recent articles showing that NMDA receptor blockade considerably modifies cocaine-induced convulsions ( 13) and cocaine-induced behavioral sensitization (7,15). Thus, D, as well as NMDA receptors mediate some of the neurochemical and behavioral effects of cocaine. ACKNOWLEDGEMENTS
This work was supported by the Foundation for Medical Research, Inc. and NIDA Grant DA05602. G.T. is the recipient of a Minority Research NIDA grant. C.R. is a Foundation for Medical Research Investigator.
REFERENCES 1. Abe, H.; Rusak, B.; Robertson, H. A. NMDA and nonNMDA receptor antagonists inhibit photic induction of fos protein in the hamster suprachiasmatic nucleus. Brain Res. Bull. 2883 l-835; 1992. 2. Cenci, A. M.; Campbell, K.; Wictorin, K.; Bjorklund, A. Striatal cfos induction by cocaine or apomorphine occurs preferentially in output neurons projecting to the substantia nigra in the rat. Eur. J. Neurosci. 4:376-380; 1992. 3. Cotman, C. W.; Monaghan, D. T.; Ottersen, 0. P.; Math&n-Storm, J. Anatomical organization of excitatory amino acid receptors and their pathways. Trends Neurosci. 10(7):273-280; 1987. 4. Graybiel, A. M. Immediate early gene activation in the striatum: New clue to function and plasticity in the basal ganglia. IBRO News 19:7; 1991. 5. Graybiel, A. M.; Moratalla, R.; Robertson, H. A. Amphetamine and cocaine induce drug-specific activation of the c-fos gene in striasomematrix compartments and limbic subdivisions of the striatum. Proc. Natl. Acad. Sci. USA 87:69 12-69 16; 1990. 6. Hemmings, H. C.; Walaas, S. 1.; Ouimet, C. C.; Greengard, P. Dopaminergic regulation of protein phosphorylation in the striatum: DARPPI32. Trends Neurosci. 10(9):377-382; 1987. 7. Karler. R.: Calder. L. D.: Chaudhrv. I. A.: Turkanis. S. A. Blockade of “reverse tolerance” to cocaine’and amphetamine by MK-801. Life Sci. 45599-606; 1989. 8. Kemp, J. A.; Foster, A. C.; Wong, H. F. Noncompetitive antagonists of excitatory amino acid receptors. Trends Neurosci. 10 (7):294298; 1987.
9. Morgan, J. I.; Curran, T. Proto-oncogene transcription factors and epilepsy. Trends Pharmacol. I2:343-349; 199 1. 10. Paul, M. L.; Graybiel. A. M.; Robertson, H. A. Synergistic activation of the immediate early gene c-fos in striasomes by D, and D2 dopamine agonists. Sot. Neurosci Abstr. 16:954; 1990. 1I. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. New York: Academic Press; 1986. 12. Roberts, P. J.; Anderson, S. D. Stimulatory effect of t_-glutamate and related amino acids on [‘Hldopamine release from rat striatum: An in vitro model of glutamate actions. J. Neurochem. 32:15391545; 1979. 13. Rockhold, R. W.; Oden, G.; Ho, I. K.; Andrew, M.; Farley, J. M. Glutamate receptor antagonists block cocaine-induced convulsions and death. Brain Res. Bull. 27:721-723; 1991. 14. Torres, G.; Rivier, C. Differential effects of intermittent or continuous exposure to cocaine on the hypothalamic-pituitary-adrenal axis and c-fos expression. Brain Res. 57 1:204-211; 1992. 15. Torres, G.; Rivier, C.; Weiss, F. Effects of cocaine on ACTH secretion, striatal c-fos expression, and locomotor sensitization in awake and anesthetized rats. Sot. Neurosci. Abstr. 18:670; 1992. 16. Watkins, J. C.; Olverman, H. J. Agonists and antagonists for excitatory amino acid receptors. Trends Neurosci. 10(7):265-272; 1987. 17. Young, S. T.; Porrino, L. J.; ladarola, M. J. Cocaine induces striatal c-fos-immunoreactive proteins via dopaminergic D, receptors. Proc. Nat]. Acad. Sci. USA 88:1291-1295; 1991.
Brarn Research Bulhin, Vol. 30, pp. 173-176, 1993 Printed in the USA. All rights reserved.
Cocaine-Induced Expression of Striatal c-fos in the Rat is Inhibited by NMDA Receptor Antagonists GERMAN
TORRES’
AND CATHERINE
RIVIER
The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037 Received 29 June 1992; Accepted 28 July 1992 TORRES, G. AND C. RIVIER. Cocaine-induced expression of striatal c-fos in the rut is inhibited by NMDA receptor antagonists. BRAIN RES BULL 30( l/2) 173-176, 1993.-To assess the possible involvement of NMDA receptors in mediating the expression of striatal c-fos by cocaine injection, we investigated the effects of the noncompetitive NMDA receptor antagonists, ketamine and (+)-S-methyl-IO,1 1-dibydro-5%dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) as well as the competitive NMDA receptor antagonist, 3-(2-carboxypiperazin-4-yl)-propyl- I-phosphonic acid (CPP), in the perikarya of cocaine-treated rat brains. As previously shown by our group, administration of 20 mg/kg cocaine (IP) resulted in the immunocytochemical expression of the protooncogene in numerous cells of the caudate putamen (dorsal/sensorimotor striatum). A ketamine mixture anesthetic (2 mg/kg), however, administered 30 min prior to cocaine exposure completely blocked such genomic expression. Pretreatment with MK-80 1 (I mg/kg) or CPP (5 mg/kg) also interfered, albeit to a lesser extent, with the expression of c-fos by cocaine in awake animals. These results indicate that cocaine induction of cellular c-fos in the caudate putamen is mediated at least in part by NMDA-sensitive receptors.
Rat
Cocaine
c-fos
Ketamine mixture
MK 801
COCAINE administration to rats has been reported to induce the expression of the immediate early gene, c-fos, in striatal neurons (5,14,17). FOS (a product of c-fos) is a nuclear protein that binds with high affinity to the DNA transcription promotor element, AP-1 (9). Because AP-1 is essential for the transcription of several genes, induction of c-fos expression by cocaine could represent some of the initial genomic mechanisms by which this drug exerts its stimulatory effects in the mammalian brain. Cocaine-induced expression of c-fos appears to be primarily mediated through the postsynaptic activation of D, receptors localized on striatonigral neurons (2). Evidence for this hypothesis comes from observations that D, receptor antagonists such as SCH-23390 effectively block the expression of the immediate early gene in cocaine-treated rats (5,17). However, there is also evidence that striatal dopamine release is mediated by glutamate receptors sensitive to N-methyl-D-asperate (NMDA) (12). Thus, the expression of c-fos by cocaine could also be mediated by receptors other than D, receptors. Excitatory amino acids such as L-glutamate and L-aspartate are likely to represent the major excitatory neurotransmitters in the brain. The actions of these molecules are mediated by at least three classes of membrane receptors, including NMDA, quisqualate, and kainate (3,8,16). The most investigated of these
CPP
and perhaps the most abundant and widely distributed in parenchyma tissue is the NMDA receptor. This ionotropic receptor is linked to membrane cation channels and can be selectively blocked noncompetitively by ketamine and (+)-5-methyl10,l I -dihydro-5H-dibenzo[a,&yclohepten-5, IO-imine hydrogen maleate (MK-801) or competitively by 3-(2-carboxypiperazin-4-yl)-propyl- 1-phosphonic acid (CPP) (8,16). It is significant that high levels of NMDA receptors are found in the rat striatum (caudate putamen and nucleus accumbens) because perikarya within these brain regions selectively express c-fos in response to cocaine exposure. We report here, as suggested by preliminary studies (15) that the expression of striatal c-fos by cocaine is markedly inhibited by systemic pretreatment of the above NMDA receptor antagonists. METHOD
Male, albino Sprague-Dawley rats (Haltzman, Madison, WI) weighing between 230-280 g were used for all experiments. Rats were housed in groups of three to four under a 12L: 12D cycle and standard ambient temperature; food and water were available ad lib. All experiments were conducted between 1400 and 1500 h of the light cycle. For the ketamine experiments, rats
’ To whom requests for reprints should be addressed.
173
TORRES
174
were injected (SC) with an anesthetic mixture of ketamine:acepromozine:xylazine ( 160 pl/ 100 g body weight) containing 2.5 ml ketamine (Vetelar, 100 mg/ml solution; Aveco Co.) 1 ml acepromazine maleate (10 mg/ml solution; Fermenta Animal Health Co.) 2.5 ml xylazine (Rompun, 20 mg/ml solution; Miles Laboratories), and 4 ml distilled water. Animals were then left undisturbed after administration of the anesthetic for 30 min. After this time delay, these rats (n = 5) were injected (IP) with cocaine HCI (Mallincodt, St Louis, MO) that had been dissolved in sterile saline at a dose of 20 mg/kg (calculated as free base). Thirty minutes later, animals (still under anesthesia) were perfused transcardially with heparinized saline followed by cold 4% paraformaldehyde in 0.1 M sodium phosphate (pH 7.2) buffer. To evaluate the effects of the above ketamine mixture on nuclear c-fos already induced by cocaine in the striatum, rats (n = 3) were injected with cocaine and 0.5 h later anesthetized with the aforementioned ketamine mixture. Following another 30-min delay, these rats were perfused as previously described. Another group of rats were injected (IP) with MK-801 (n = 4) or CPP (n = 5) (Research Biochemicals Inc., Natick, MA) dissolved in sterile saline at a dose of 1 or 5 mg/kg, respectively. Thirty minutes later, rats were injected with cocaine (20 mg/ kg). In all experiments, control rats were always injected with sterile saline. Following a 30-min delay, animals were anesthetized with sodium pentobarbital(50 mg/kg) and perfused as previously described. Thus, different rats received the following experimental drug designs: (a) ketamine mixture + cocaine; (b) MK-801 + cocaine; (c) CPP + cocaine; (d) saline + cocaine (n = 4); (e) saline + saline (n = 3). In addition, other rats was injected with only the ketamine mixture (n = 3) MK-801 (n = 5) or CPP (n = 2). Specifics of c-fos determination by immunocytochemistry are reported elsewhere (14). Briefly, free-floating brain sections (40 pm) were incubated for 48 h with a c-fos antibody [rabbit polyclonal immunoglobulin (1g)G; 1: 1,000 dilution; specificity: rodent P62c-fos; Oncogene Science, NY]. The sections were reacted with DAB, I % HzOz, and Ni04. 6Hz0. After several rinses in 50 mM KPBS, brains sections were mounted onto gelatin-chrome alum-coated slides and coverslipped with DPX. To determine the specificity of c-fos IgG, random brain sections were treated in the same manner as described above but were incubated without the c-fos antibody. Alternate brain sections were counterstained with neutral red to subsequently identify striatal regions expressing c-fos. The number of perikarya expressing the immediate early gene was counted using a grid reticle on a microscope. Rat brain sections corresponding to plate I8 of Paxinos and Watson (11) were chosen for counting purposes. The medial aspect of the caudate putamen was selected as the counting region because it was this area where a significant blockade of c-fos expression by the NMDA receptor antagonists was most often discernible. The dorsal (adjacent to the corpus callosum) and lateral (adjacent to the lateral ventricle) portions of the caudate putamen served as primary margins for the aforementioned medial region. The data were analyzed by analysis of variance (ANOVA) followed by posthoc (t test) comparisons for each drug treatment. RESULTS
When c-fos immunoreactivity was assessed in the caudate putamen of rats injected with saline alone, the phosphoprotein was not measurably detected in such forebrain structure (Fig. 1). However, nonspecific, scattered fos-immunopositive cells were observed in other brain regions including the piriform cortex, septal region, and lateral hypothalamus. This pattern of nuclear reactivity was observed in brains of both anesthetized and
AND
RIVIER
awake animals. The injection of 20 mg/kg cocaine to awake rats caused a robust and wide expression of c-fos relative to salinetreated rats (Fig. 2; p < 0.0 I). This genomic induction was characterized by intense nuclear labeling of numerous neurons within the caudate putamen, in particular its dorsal-medial-lateral subdivisions; axons and dendrites were not labeled (Fig. 1). This pattern of nuclear reactivity, however, was completely abolished in rats that had been anesthetized with a ketamine mixture prior to administration ofcocaine (Fig. 1). Interestingly, the ketamine mixture did not inhibit the endogenous expression of c-fos in other cytoarchitectonic regions of the rat brain, including those in the piriform cortex, septal region, or paraventricular thalamic nucleus. When rats were injected with cocaine and 30 min later anesthetized with the ketamine mixture, c-fos expression in the caudate putamen was still fully present in terms of its topographical distribution and nuclear intense labeling (data not shown). Thus, blockade of c-fos expression by the ketamine mixture occurs only when the NMDA receptor antagonist is administered prior to cocaine exposure. Pretreatment with the NMDA receptor antagonist MK-801 30 min before cocaine injections markedly reduced the number of striatal perikarya expressing c-fos relative to saline + cocainetreated rats (Fig. 2; p < 0.01). The reduction of this immediate early gene was more discernible in the medial subdivision of the caudate putamen than its dorsal or lateral regions. Brains from rats injected with MK-801 with or without cocaine posttreatment also exhibited intense and consistent immunoreactivity cells in the thalamus and the parvocellular subdivision of the paraventricular nucleus of the hypothalamus (data not shown). This latter effect may be a reflection of increased activity in the hypothalamic-pituitary-adrenal axis because systemic injection of MK-80 1 stimulates corticotropin and corticosterone secretion ( 15). When animals were pretreated with CPP, cocaine exposure elicited a modest expression of c-fos as compared to rats injected with the saline + cocaine experimental drug design (Fig. 2; p < 0.01). The inhibition of the immediate early gene was more prominent in the medial cellular subdivision of the caudate putamen. Thus, the aforementioned blockade of c-fos by CPP followed a similar pattern of topographical inhibition to that seen for MK-801 (Fig. 1). Rat brains injected with saline followed by CPP showed a modest but not significant expression of c-fos in striatal or hypothalamic tissue. DISCUSSION
These studies were undertaken to assess the possible participation of NMDA receptors in the expression of c-fos by cocaine. We report here that three NMDA receptor antagonists administered prior to drug exposure blocked, to various degrees, such nuclear induction. These results suggest that NMDA receptors mediate at least in part the expression of striatal c-fos by cocaine. We and others have shown that cocaine administration to rats leads to rapid protooncogene expression in the perikarya of the caudate putamen and nucleus accumbens (5,14,17). These forebrain structures have received ample experimental attention because projecting dopamine terminal plexuses to these brain regions have been implicated in the reinforcing and behavioralactivating properties of cocaine. These psychostimulant properties are thought to have a neurochemical basis via enhanced dopamine neurotransmission and a preferential activation of striatal D, as opposed to Dz receptors (5). Concomitantly, the D, receptor subtype has been hypothesized to modulate the induction of nuclear c-fos by cocaine and other dopamine agonists (4,5, IO). However, our present results also support the hypothesis that striatal NMDA receptors are involved in the molecular
COCAINE AND STRIATAL
175
C-FOS EXPRESSION
KET + COC
SAL + SAL
SAL + COC
MK-801 + COC
CPP + COC
FIG. 1. Representative brightfield photomicrographs of coronal sections through the medial portion of the caudate putamen. Rats were pretreated with saline (Sal), a ketamine mixture (Ket; 2 mg/kg), MK-801 (1 mg/kg), or CPP (5 mg/kg) followed 30 min later by Sal or cocaine (Cot; 20 mg/ kg) IP injections. (A). Forebrain section illustrating the region of the caudate putamen depicted in subsequent photomicrographs. Blacks arrows point to unreactive myelinated bundles of corticofugal fibers coursing through the caudate putamen. Original magnification 10X.
changes that invariably occur following cocaine exposure. These data are consistent with evidence showing that Lglutamate evokes the release of dopamine in the striatum and this release is primarily mediated by NMDA-sensitive receptors proximal to or on dopamine terminal plexuses ( 12). The NMDA receptor is linked to a Na+ ionic channel with an appreciable Ca*+ permeability as well. The aforementioned postsynaptic
5
Sal + Sal
Sal + Cot
MK-801 + Cot CPP + Cot
FIG. 2. Effects of NMDA receptor antagonists on the induction of c-fos expression by cocaine in the rat caudate putamen. Saline (Sal), MK 801 (I mg/kg), or CPP (5 mg/kg) were administered 30 min prior to cocaine (Cot; 20 mg/kg) IP injections. Each bar represents the means f SEM of immunopositive perikarya in 10 mm2 sections of the medial caudate putamen (one side). Pretreatment with the ketamine mixture completely abolished the expression of c-fos by cocaine. **p -c0.01 relative to MK80 1 + Cot- or CPP + Cot-treated rats.
receptor is blocked by a number of highly specific noncompetitive and competitive antagonist compounds, such as ketamine, MK801, and CPP. Ketamine, a dissociative anesthetic, has been shown to bind with high affinity to the cation channel, thus antagonizing the intracellular regulation of the receptor by (perhaps) altering its voltage-gated dependency to a nonconductive state (8,16). Although in the present experiments we employed a ketamine mixture instead of ketamine alone, our results are in agreement with related findings showing that ketamine alone effectively blocks the expression of hypothalamic c-fos by photic stimulation ( 1). Moreover, we have ascertained that this ketamine mixture exhibits no measurable adverse effects on striatal dopamine metabolism as demonstrated by in vivo intracerebral microdialysis ( 15). Thus, the use of a ketamine mixture appears to be an appropriate method by which the expression of c-fos by cocaine in the brain parenchyma can be effectively antagonized at the receptor level. MK-801, one of the most potent NMDA antagonists yet described, also blocked the expression of c-fos by cocaine exposure. It is important to note, however, that although MK-801 blocked such expression the degree of blockade was greater for the ketamine mixture. The basis for this potency differential between the ketamine mixture and MK-80 1 is unknown. One possibility may be related to the mixture of xylazine and acepromozine in our ketamine prep aration. Xylazine, a nonnarcotic, sedative analgesic, could have potentiated the antagonistic action of ketamine by further decreasing the nonconductive state of the cation channel. Further research is obviously needed to validate this hypothesis. CPP, a competitive NMDA receptor antagonist, also blocked the expression of c-fos by cocaine. CPP readily penetrates the blood-brain barrier and is thought to inhibit the aforementioned receptor by binding to the
TORRES AND RIVIER
176
agonist site of the receptor rather than its ionic channel (8,16). As observed for MK-80 1, the blockade of c-fos by CPP was of a lesser magnitude than that seen for the ketamine mixture but equally effective relative to that of MK-80 1. It remains also unclear the differential blockade susceptibility for c-fos between the medial versus dorsal or lateral portions of the caudate putamen to the antagonistic effects of MK 80 1 and CPP. The rat striatum, which includes both the caudate putamen and the nucleus accumbens, is structurally complex, displaying a mosaic organization of neurochemical systems (4). Relating these systems to the postsynaptic effects of cocaine has proven difficult because we do not know for certain the phenotypic coding of most neurons expressing c-fos in the aforementioned forebrain region. In preliminary studies, Graybiel(4) has shown that the induction of the immediate early gene by cocaine occurs in numerous dopamine and cyclic adenosine monophosphate (CAMP)-regulated phosphoprotein (DARPP-32) neurons. This neuronal protein is phosphorylated by dopamine (6) and its regulation appears to be orchestrated by the interactions between dopamine, acting through CAMP, and L-glutamate, acting
through Ca”. Thus, the above topographical differences may be related to a preferential activation of c-fos within a striatal subset of medium-size spiny cells that biochemically interact at the level of protein phosphorylation (6). More research is needed to expand this hypothesis. In summary, our results clearly suggest the involvement of NMDA-sensitive receptors in the postsynaptic induction of cfos by cocaine. These observations are substantiated by recent articles showing that NMDA receptor blockade considerably modifies cocaine-induced convulsions ( 13) and cocaine-induced behavioral sensitization (7,15). Thus, D, as well as NMDA receptors mediate some of the neurochemical and behavioral effects of cocaine. ACKNOWLEDGEMENTS
This work was supported by the Foundation for Medical Research, Inc. and NIDA Grant DA05602. G.T. is the recipient of a Minority Research NIDA grant. C.R. is a Foundation for Medical Research Investigator.
REFERENCES 1. Abe, H.; Rusak, B.; Robertson, H. A. NMDA and nonNMDA receptor antagonists inhibit photic induction of fos protein in the hamster suprachiasmatic nucleus. Brain Res. Bull. 2883 l-835; 1992. 2. Cenci, A. M.; Campbell, K.; Wictorin, K.; Bjorklund, A. Striatal cfos induction by cocaine or apomorphine occurs preferentially in output neurons projecting to the substantia nigra in the rat. Eur. J. Neurosci. 4:376-380; 1992. 3. Cotman, C. W.; Monaghan, D. T.; Ottersen, 0. P.; Math&n-Storm, J. Anatomical organization of excitatory amino acid receptors and their pathways. Trends Neurosci. 10(7):273-280; 1987. 4. Graybiel, A. M. Immediate early gene activation in the striatum: New clue to function and plasticity in the basal ganglia. IBRO News 19:7; 1991. 5. Graybiel, A. M.; Moratalla, R.; Robertson, H. A. Amphetamine and cocaine induce drug-specific activation of the c-fos gene in striasomematrix compartments and limbic subdivisions of the striatum. Proc. Natl. Acad. Sci. USA 87:69 12-69 16; 1990. 6. Hemmings, H. C.; Walaas, S. 1.; Ouimet, C. C.; Greengard, P. Dopaminergic regulation of protein phosphorylation in the striatum: DARPPI32. Trends Neurosci. 10(9):377-382; 1987. 7. Karler. R.: Calder. L. D.: Chaudhrv. I. A.: Turkanis. S. A. Blockade of “reverse tolerance” to cocaine’and amphetamine by MK-801. Life Sci. 45599-606; 1989. 8. Kemp, J. A.; Foster, A. C.; Wong, H. F. Noncompetitive antagonists of excitatory amino acid receptors. Trends Neurosci. 10 (7):294298; 1987.
9. Morgan, J. I.; Curran, T. Proto-oncogene transcription factors and epilepsy. Trends Pharmacol. I2:343-349; 199 1. 10. Paul, M. L.; Graybiel. A. M.; Robertson, H. A. Synergistic activation of the immediate early gene c-fos in striasomes by D, and D2 dopamine agonists. Sot. Neurosci Abstr. 16:954; 1990. 1I. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. New York: Academic Press; 1986. 12. Roberts, P. J.; Anderson, S. D. Stimulatory effect of t_-glutamate and related amino acids on [‘Hldopamine release from rat striatum: An in vitro model of glutamate actions. J. Neurochem. 32:15391545; 1979. 13. Rockhold, R. W.; Oden, G.; Ho, I. K.; Andrew, M.; Farley, J. M. Glutamate receptor antagonists block cocaine-induced convulsions and death. Brain Res. Bull. 27:721-723; 1991. 14. Torres, G.; Rivier, C. Differential effects of intermittent or continuous exposure to cocaine on the hypothalamic-pituitary-adrenal axis and c-fos expression. Brain Res. 57 1:204-211; 1992. 15. Torres, G.; Rivier, C.; Weiss, F. Effects of cocaine on ACTH secretion, striatal c-fos expression, and locomotor sensitization in awake and anesthetized rats. Sot. Neurosci. Abstr. 18:670; 1992. 16. Watkins, J. C.; Olverman, H. J. Agonists and antagonists for excitatory amino acid receptors. Trends Neurosci. 10(7):265-272; 1987. 17. Young, S. T.; Porrino, L. J.; ladarola, M. J. Cocaine induces striatal c-fos-immunoreactive proteins via dopaminergic D, receptors. Proc. Nat]. Acad. Sci. USA 88:1291-1295; 1991.