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σ Receptor blockade by BMY 14802 affects enkephalinergic and tachykinin cells differentially in the striatum of the rat
European Journal of Pharmacology, 175 (1990) 225-228
225
Elsevier EJP 20539
Short communication
o Receptor blockade by BMY 14802 affects enkephalinergic and tachykinin cells differentially in the striatum of the rat Jesus A. Angulo, J e a n L u d C a d e t ~ a n d Bruce S. M c E w e n : Columbia University, College of Physicians and Surgeons, Laboratory of Preclinical Neurosciences, Department of Neurology, New York, NY, U.S.A. and The Rockefeller University, Laboratory of Neuroendocrinology, New York, NY, U.S.A. Received 9 November 1989, accepted 14 November 1989
We determined proenkephalin and protachykinin mRNA levels in the striatum of the rat by in situ hybridization histochemistry in controls and in animals treated for 14 days with the potential neuroleptic drug BMY 14802 (15 mg/kg i.p.). Proenkephalin mRNA levels were increased relative to control values by 107, 88 and 94% and protachykinin mRNA levels decreased by 17, 28 and 10% in the rostral, medial and caudal aspects of the caudate-putamen, respectively. The data suggest that o receptor activity, directly or indirectly, affects proenkephalin and protachykinin mRNA expression in striatal neurons. Neuroleptics; BMY 14802; Proenkephalin; Protachykinin; (In situ hybridization)
1. Introduction
Neuroleptic drugs are a heterogeneous group of compounds used in the management of psychotic behavior in schizophrenic patients. They are potent dopamine receptor blockers but also display antagonistic activities at other neurotransmitter receptor systems such as adrenoceptor, serotonin, histamine, muscarinic acetylcholine and the o receptor (Peroutka and Snyder, 1980). Chronic treatment with haloperidol causes increases of proenkephalin m R N A but decreases in protachykinin m R N A in the striatum of the rat (Tang et al., 1983; Bannon et al., 1987). These effects are thought to be secondary to blockade of D-2 receptors by these agents. BMY 14802 (a-(4-fluorophenyl)-4-(5- fluoro-2-pyrimidinyl)- 1-piperazine butanol) is a potential neuroleptic which is devoid
of anti-dopaminergic activity but which is a potent antagonist at the haloperidol-sensitive o receptor site (Taylor and Dekleva, 1987). The potential anti-psychotic properties of BMY 14802 are exemplified by its ability to block the conditional avoidance response, inhibition of apomorphine-induced stereotypy, and resemblance to the atypical neuroleptic clozapine in the Sidman avoidance paradigm (Taylor and Dekleva, 1987). The present study was carried out in order for us to evaluate the effects of chronic treatment with BMY 14802 on proenkephalin and protachykinin mRNA levels in the rostral, medial, and caudal aspects of the caudate-putamen of the rat by in situ hybridization histochemistry.
2. Materials and methods 2.1. Materials
Correspondence to: J.A. Angulo, The Rockefeller University, Laboratory of Neuroendocrinology, 1230 York Avenue, New York, NY 10021, U.S.A.
Materials used are: polyvinylpyrolidone, bovine serum albumin (Cohn's fraction V), salmon sperm
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
226 DNA, sodium chloride, sodium citrate, paraformaldehyde, sodium phosphate, citric acid, potassium cacodylate, cobalt chloride and dithiothreitol from the Sigma Company; ultra pure formamide from Bethesda Research Laboratories; BMY 14802 from Bristol-Meyers; terminal deoxynucleotidyl transferase from Boehringer/Mannheim; Sephadex G10 from Pharmacia.
an overnight hybridization, the sections were washed first in 2 × SSC buffer at room temperature for 25 min, and then for 1.5 h in 1 x SSC buffer at 45 o C. The sections were then apposed to Kodak XAR film for 24-48 h at - 7 0 ° C with intensifying screens.
2.2. Animals
Proenkephalin m R N A was detected with two synthetic oligonucleotides ( 5 ' d C A A G T C G T C C T C A T C C T G T T T G C T G C T G C T and 5'dCTCCACGGGGTAAAGCTCATCCATCTTCTT) complementary to the nucleotide sequence coding for amino acids 86-95 and 112-121, respectively, of the rat proenkephalin (Rosen et al., 1985). The synthetic oligonucleotides used to detect protachykinin mRNA (5'dCATTAATCCAAAGAAC T G C T G A G G C T T G G G T C T and 5'dGCCCATTAGTCCAACAAAGGAATCTGTTTTATG) correspond to the nucleotide sequence coding for amino acids 58-68 and 83-93, respectively, of the rat protachykinin (Krause et al., 1987). Northern gel analysis revealed one band of the appropriate molecular size for proenkephalin and protachykinin mRNAs, respectively (Rosen et al., 1985; Krause et al., 1987). Addition of unlabeled probe at 15-fold molar excess blocked the in situ hybridization signal for proenkephalin and protachykinin mRNAs, respectively, demonstrating a high degree of specificity by the oligonucleotide probes. The in situ hybridization signal on the autoradiogram was quantitated using the Drexel DUMAS computerized image analysis system. Standards were used to calibrate the optical system from 0.05 to 1.0 optical density units. Measurements were made in the linear range of the X-ray film. The values obtained represent mRNA level for the entire plane of the caudate-putamen in coronal section (dark area in fig. 1). The data were then analyzed using Student's t-test since there were only two groups.
Male CD rats weighing approximately 200 g at the start of the experiment were exposed to a 12 h light/dark cycle at constant temperature and humidity with free access to food and water. The rats were housed in plastic cages at a density of three animals per cage.
2.3. Drug treatment The rats were injected i.p. daily for 14 days with BMY 14802 (15 mg/kg) dissolved in saline. Control rats received saline injections. The animals were killed 24 h after the last injection.
2.4. Detection of mRNA by in situ hybridization histochemistry The animals were killed by decapitation and the brains frozen on powdered dry ice. Coronal sections (16 t~m in thickness) were cut in the cryostat at - 1 5 ° C and thermally mounted onto gelatin-coated glass slides pretreated with diethylpyrocarbonate. Rostral, medial, and caudal aspects of the caudate-putamen correspond to plates 14, 22 and 31, respectively, of the Stereotaxic Atlas of the Rat Brain (Pellegrino et al., 1979). The tissue was fixed for 6 rain at 4 ° C in 2% paraformaldehyde/0.1 M sodium phosphate buffer (pH 7.2), and then washed in ice-cold 0.5 × SSC buffer for 1-2 rain (1 × SSC = 0.15 M sodium chloride/0.015 M sodium citrate buffer, pH 7.0). The sections were air-dried at room temperature and hybridized at 37°C in a humidified environment with a solution consisting of 0.2% (w/v) of bovine serum albumin, 0.1% (w/v) polyvinylpyrolidone, 4 × SSC buffer, 50% formamide, 100 t~g/ml of salmon sperm DNA, and 10 6 c p m / m l of 32p_ labeled synthetic oligonucleotide probe. Following
2.5. Specificity of hybridization probes
3. Results
The photomicrographs in fig. 1 show the hybridization signal for proenkephalin (A and B)
227
A
6
Fig. 1. In situ hybridization histochemistry for proenkephalin and protachykinin mRNAs in coronal brain sections from control and BMY 14802-treated animals. The autoradiograms were photographed in bright field. A and B represent proenkephalin mRNA level in saline-injected and BMY 14802-treated animals, respectively,for the medial aspect of the caudate-putamen. C and D represent the level of protachykinin mRNA in control and BMY 14802-treated animals.
and protachykinin (C and D) m R N A s in coronal sections for the medial aspect of the caudate-putamen. The hybridization signal on the X-ray film was quantitated by optical density scanning. The area quantitated included the entire caudate nucleus in coronal section. Optical density readings were taken bilaterally at each level. A total of 8 sections (16 readings) were quantitated per level and the values obtained taken as representative of the m R N A level. Chronic treatment with the non-dopamine o receptor blocker BMY 14802 caused increases in proenkephalin m R N A levels in the rostral, medial, and caudal aspects of the caudate-putamen by 107, 88 and 94% above controls (fig. 2A). In contrast, protachykinin m R N A levels were decreased in the same animals by 17, 28 and 10% below controls in rostral, medial and caudal aspects, respectively (fig. 2B).
4. Discussion
Blockade of the o receptor by the potential antipsychotic drug BMY 14802 caused changes in striatal proenkephalin and protachykinin m R N A
that parallel those observed after classical neuroleptic treatment. Since all neuroleptics block dopamine receptors at clinically effective doses (Peroutka and Snyder, 1980), the effect on striatal neuropeptides was believed to occur solely through the dopamine receptor. Moreover, deafferentation studies in which the dopaminergic cells in the substantia nigra were ablated by the chemical neurotoxin 6-hydroxydopamine affected proenkephalin and protachykinin m R N A s in the same direction and magnitude as chronic neuroleptic treatment (Young et al., 1986). These observations had suggested that dopaminergic neurotransmission might cause direct modulation of biosynthesis in rat brain. Nevertheless, in addition to their dopamine D-2 blocking activity, neuroleptics can also antagonize the a receptor (Tam and Cook, 1984). For example, haloperidol competes with the specific o receptor agonist N-allylnormetazocine with an apparent K i value of 4 × 10 -9 M for binding sites in membrane preparations from guinea pig brain (Tam and Cook, 1984). Moreover, o receptor sites in the brain of the rat are localized on intrinsic neurons in the caudate-putamen (Gundlach et al.,
228 250
effects of the o antagonist BMY 14802 support this idea and demonstrate that the enkephalin and tachykinin systems may be influenced by other neurotransmitter systems in rat brain. The present observations provide new impetus for further characterization of the effects of o receptor-active drugs on the molecular regulation of brain peptides.
200
150
§ o
t00
50
R
H
C
t25
100
75 g o ~e
50
25
R
H
Fig. 2. Effect of chronic treatment with BMY 14802 on proenkephalin (A) and protachykinin (B) m R N A levels in the rostral, medial and caudal aspects of the caudate-putamen. The values shown represent the average from three animals (n = 3). Two sets of sections were collected from each animal: one set was used to determine proenkephalin m R N A and the other for protachykinin m R N A . Criss-cross bars represent controls and solid bars BMY 14802-treated animals. Values over the bars represent mean S.E.M. R, rostral; M, medial; and C, caudal aspects of the caudate-putamen. * P < 0.05, * * P < 0.01, and • ** P < 0.0001 in comparison to control values (Student's t-test).
1986). When taken together, these data had raised the possibility that o receptor antagonists or agonists might modulate the biosynthesis of certain peptidergic systems whose cell bodies are localized in the striatum. The present data on the
References Bannon, M., P. Elliott and E. Bunney, 1987, Striatal tachykinin biosynthesis: regulation of m R N A and peptide levels by dopamine agonists and antagonists, Mol. Brain Res. 3, 31. Gundlach, A., B. Largent and S. Snyder, 1986, Autoradiographic localization of sigma receptor binding sites in guinea pig and rat central nervous system with (+)3H-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine, J. Neurosci. 6, 1757. Krause, J., J. Chirgwin, M. Carter, Z. Xu and A. Hershey, 1987, Three rat preprotachykinin m R N A s encode the neuropeptides substance P and neurokinin A, Proc. Natl. Acad. Sci. U.S.A. 84, 881. Pellegrino, L., A. Pellegrino and A. Cushman, 1979, A stereotaxic atlas of the rat brain, 2nd edn. (Plenum, New York). Peroutka, S. and S. Snyder, 1980, Relationship of neuroleptic drug effects at brain dopamine, serotonin, alpha-adrenergic, and histamine receptors to clinical potency, Am. J. Psychiat. 137, 1518. Rosen, H., J. Douglass and E. Herbert, 1984, Isolation and characterization of the rat proenkephalin gene, J. Biol. Chem. 259, 14309. Tam, W. and L. Cook, 1984, Sigma opiates and certain antipsychotic drugs mutually inhibit ( + ) - [ 3 H ] S K F 10047 and [3H]haloperidol binding in guinea pig brain membranes, Proc. Natl. Acad. Sci. U.S.A. 81, 5618. Tang, F., E. Costa and J. Schwartz, 1983, Increase of proenkephalin m R N A and enkephalin content of rat striatum after daily injection of haloperidol for 2 to 3 weeks, Proc. Natl, Acad. Sci. U.S.A. 80, 3841. Taylor, D. and J. Dekleva, 1987, Potential antipsychotic BMY 14802 selectively binds to sigma sites, Drug Devel. Res. 11, 65. Young, S., T. Bonner and M. Brann, 1986, Mesencephalic dopamine neurons regulate the expression of neuropeptide m R N A in the rat forebrain, Proc. Natl. Acad. Sci. U.S.A. 83, 9827.
225
Elsevier EJP 20539
Short communication
o Receptor blockade by BMY 14802 affects enkephalinergic and tachykinin cells differentially in the striatum of the rat Jesus A. Angulo, J e a n L u d C a d e t ~ a n d Bruce S. M c E w e n : Columbia University, College of Physicians and Surgeons, Laboratory of Preclinical Neurosciences, Department of Neurology, New York, NY, U.S.A. and The Rockefeller University, Laboratory of Neuroendocrinology, New York, NY, U.S.A. Received 9 November 1989, accepted 14 November 1989
We determined proenkephalin and protachykinin mRNA levels in the striatum of the rat by in situ hybridization histochemistry in controls and in animals treated for 14 days with the potential neuroleptic drug BMY 14802 (15 mg/kg i.p.). Proenkephalin mRNA levels were increased relative to control values by 107, 88 and 94% and protachykinin mRNA levels decreased by 17, 28 and 10% in the rostral, medial and caudal aspects of the caudate-putamen, respectively. The data suggest that o receptor activity, directly or indirectly, affects proenkephalin and protachykinin mRNA expression in striatal neurons. Neuroleptics; BMY 14802; Proenkephalin; Protachykinin; (In situ hybridization)
1. Introduction
Neuroleptic drugs are a heterogeneous group of compounds used in the management of psychotic behavior in schizophrenic patients. They are potent dopamine receptor blockers but also display antagonistic activities at other neurotransmitter receptor systems such as adrenoceptor, serotonin, histamine, muscarinic acetylcholine and the o receptor (Peroutka and Snyder, 1980). Chronic treatment with haloperidol causes increases of proenkephalin m R N A but decreases in protachykinin m R N A in the striatum of the rat (Tang et al., 1983; Bannon et al., 1987). These effects are thought to be secondary to blockade of D-2 receptors by these agents. BMY 14802 (a-(4-fluorophenyl)-4-(5- fluoro-2-pyrimidinyl)- 1-piperazine butanol) is a potential neuroleptic which is devoid
of anti-dopaminergic activity but which is a potent antagonist at the haloperidol-sensitive o receptor site (Taylor and Dekleva, 1987). The potential anti-psychotic properties of BMY 14802 are exemplified by its ability to block the conditional avoidance response, inhibition of apomorphine-induced stereotypy, and resemblance to the atypical neuroleptic clozapine in the Sidman avoidance paradigm (Taylor and Dekleva, 1987). The present study was carried out in order for us to evaluate the effects of chronic treatment with BMY 14802 on proenkephalin and protachykinin mRNA levels in the rostral, medial, and caudal aspects of the caudate-putamen of the rat by in situ hybridization histochemistry.
2. Materials and methods 2.1. Materials
Correspondence to: J.A. Angulo, The Rockefeller University, Laboratory of Neuroendocrinology, 1230 York Avenue, New York, NY 10021, U.S.A.
Materials used are: polyvinylpyrolidone, bovine serum albumin (Cohn's fraction V), salmon sperm
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
226 DNA, sodium chloride, sodium citrate, paraformaldehyde, sodium phosphate, citric acid, potassium cacodylate, cobalt chloride and dithiothreitol from the Sigma Company; ultra pure formamide from Bethesda Research Laboratories; BMY 14802 from Bristol-Meyers; terminal deoxynucleotidyl transferase from Boehringer/Mannheim; Sephadex G10 from Pharmacia.
an overnight hybridization, the sections were washed first in 2 × SSC buffer at room temperature for 25 min, and then for 1.5 h in 1 x SSC buffer at 45 o C. The sections were then apposed to Kodak XAR film for 24-48 h at - 7 0 ° C with intensifying screens.
2.2. Animals
Proenkephalin m R N A was detected with two synthetic oligonucleotides ( 5 ' d C A A G T C G T C C T C A T C C T G T T T G C T G C T G C T and 5'dCTCCACGGGGTAAAGCTCATCCATCTTCTT) complementary to the nucleotide sequence coding for amino acids 86-95 and 112-121, respectively, of the rat proenkephalin (Rosen et al., 1985). The synthetic oligonucleotides used to detect protachykinin mRNA (5'dCATTAATCCAAAGAAC T G C T G A G G C T T G G G T C T and 5'dGCCCATTAGTCCAACAAAGGAATCTGTTTTATG) correspond to the nucleotide sequence coding for amino acids 58-68 and 83-93, respectively, of the rat protachykinin (Krause et al., 1987). Northern gel analysis revealed one band of the appropriate molecular size for proenkephalin and protachykinin mRNAs, respectively (Rosen et al., 1985; Krause et al., 1987). Addition of unlabeled probe at 15-fold molar excess blocked the in situ hybridization signal for proenkephalin and protachykinin mRNAs, respectively, demonstrating a high degree of specificity by the oligonucleotide probes. The in situ hybridization signal on the autoradiogram was quantitated using the Drexel DUMAS computerized image analysis system. Standards were used to calibrate the optical system from 0.05 to 1.0 optical density units. Measurements were made in the linear range of the X-ray film. The values obtained represent mRNA level for the entire plane of the caudate-putamen in coronal section (dark area in fig. 1). The data were then analyzed using Student's t-test since there were only two groups.
Male CD rats weighing approximately 200 g at the start of the experiment were exposed to a 12 h light/dark cycle at constant temperature and humidity with free access to food and water. The rats were housed in plastic cages at a density of three animals per cage.
2.3. Drug treatment The rats were injected i.p. daily for 14 days with BMY 14802 (15 mg/kg) dissolved in saline. Control rats received saline injections. The animals were killed 24 h after the last injection.
2.4. Detection of mRNA by in situ hybridization histochemistry The animals were killed by decapitation and the brains frozen on powdered dry ice. Coronal sections (16 t~m in thickness) were cut in the cryostat at - 1 5 ° C and thermally mounted onto gelatin-coated glass slides pretreated with diethylpyrocarbonate. Rostral, medial, and caudal aspects of the caudate-putamen correspond to plates 14, 22 and 31, respectively, of the Stereotaxic Atlas of the Rat Brain (Pellegrino et al., 1979). The tissue was fixed for 6 rain at 4 ° C in 2% paraformaldehyde/0.1 M sodium phosphate buffer (pH 7.2), and then washed in ice-cold 0.5 × SSC buffer for 1-2 rain (1 × SSC = 0.15 M sodium chloride/0.015 M sodium citrate buffer, pH 7.0). The sections were air-dried at room temperature and hybridized at 37°C in a humidified environment with a solution consisting of 0.2% (w/v) of bovine serum albumin, 0.1% (w/v) polyvinylpyrolidone, 4 × SSC buffer, 50% formamide, 100 t~g/ml of salmon sperm DNA, and 10 6 c p m / m l of 32p_ labeled synthetic oligonucleotide probe. Following
2.5. Specificity of hybridization probes
3. Results
The photomicrographs in fig. 1 show the hybridization signal for proenkephalin (A and B)
227
A
6
Fig. 1. In situ hybridization histochemistry for proenkephalin and protachykinin mRNAs in coronal brain sections from control and BMY 14802-treated animals. The autoradiograms were photographed in bright field. A and B represent proenkephalin mRNA level in saline-injected and BMY 14802-treated animals, respectively,for the medial aspect of the caudate-putamen. C and D represent the level of protachykinin mRNA in control and BMY 14802-treated animals.
and protachykinin (C and D) m R N A s in coronal sections for the medial aspect of the caudate-putamen. The hybridization signal on the X-ray film was quantitated by optical density scanning. The area quantitated included the entire caudate nucleus in coronal section. Optical density readings were taken bilaterally at each level. A total of 8 sections (16 readings) were quantitated per level and the values obtained taken as representative of the m R N A level. Chronic treatment with the non-dopamine o receptor blocker BMY 14802 caused increases in proenkephalin m R N A levels in the rostral, medial, and caudal aspects of the caudate-putamen by 107, 88 and 94% above controls (fig. 2A). In contrast, protachykinin m R N A levels were decreased in the same animals by 17, 28 and 10% below controls in rostral, medial and caudal aspects, respectively (fig. 2B).
4. Discussion
Blockade of the o receptor by the potential antipsychotic drug BMY 14802 caused changes in striatal proenkephalin and protachykinin m R N A
that parallel those observed after classical neuroleptic treatment. Since all neuroleptics block dopamine receptors at clinically effective doses (Peroutka and Snyder, 1980), the effect on striatal neuropeptides was believed to occur solely through the dopamine receptor. Moreover, deafferentation studies in which the dopaminergic cells in the substantia nigra were ablated by the chemical neurotoxin 6-hydroxydopamine affected proenkephalin and protachykinin m R N A s in the same direction and magnitude as chronic neuroleptic treatment (Young et al., 1986). These observations had suggested that dopaminergic neurotransmission might cause direct modulation of biosynthesis in rat brain. Nevertheless, in addition to their dopamine D-2 blocking activity, neuroleptics can also antagonize the a receptor (Tam and Cook, 1984). For example, haloperidol competes with the specific o receptor agonist N-allylnormetazocine with an apparent K i value of 4 × 10 -9 M for binding sites in membrane preparations from guinea pig brain (Tam and Cook, 1984). Moreover, o receptor sites in the brain of the rat are localized on intrinsic neurons in the caudate-putamen (Gundlach et al.,
228 250
effects of the o antagonist BMY 14802 support this idea and demonstrate that the enkephalin and tachykinin systems may be influenced by other neurotransmitter systems in rat brain. The present observations provide new impetus for further characterization of the effects of o receptor-active drugs on the molecular regulation of brain peptides.
200
150
§ o
t00
50
R
H
C
t25
100
75 g o ~e
50
25
R
H
Fig. 2. Effect of chronic treatment with BMY 14802 on proenkephalin (A) and protachykinin (B) m R N A levels in the rostral, medial and caudal aspects of the caudate-putamen. The values shown represent the average from three animals (n = 3). Two sets of sections were collected from each animal: one set was used to determine proenkephalin m R N A and the other for protachykinin m R N A . Criss-cross bars represent controls and solid bars BMY 14802-treated animals. Values over the bars represent mean S.E.M. R, rostral; M, medial; and C, caudal aspects of the caudate-putamen. * P < 0.05, * * P < 0.01, and • ** P < 0.0001 in comparison to control values (Student's t-test).
1986). When taken together, these data had raised the possibility that o receptor antagonists or agonists might modulate the biosynthesis of certain peptidergic systems whose cell bodies are localized in the striatum. The present data on the
References Bannon, M., P. Elliott and E. Bunney, 1987, Striatal tachykinin biosynthesis: regulation of m R N A and peptide levels by dopamine agonists and antagonists, Mol. Brain Res. 3, 31. Gundlach, A., B. Largent and S. Snyder, 1986, Autoradiographic localization of sigma receptor binding sites in guinea pig and rat central nervous system with (+)3H-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine, J. Neurosci. 6, 1757. Krause, J., J. Chirgwin, M. Carter, Z. Xu and A. Hershey, 1987, Three rat preprotachykinin m R N A s encode the neuropeptides substance P and neurokinin A, Proc. Natl. Acad. Sci. U.S.A. 84, 881. Pellegrino, L., A. Pellegrino and A. Cushman, 1979, A stereotaxic atlas of the rat brain, 2nd edn. (Plenum, New York). Peroutka, S. and S. Snyder, 1980, Relationship of neuroleptic drug effects at brain dopamine, serotonin, alpha-adrenergic, and histamine receptors to clinical potency, Am. J. Psychiat. 137, 1518. Rosen, H., J. Douglass and E. Herbert, 1984, Isolation and characterization of the rat proenkephalin gene, J. Biol. Chem. 259, 14309. Tam, W. and L. Cook, 1984, Sigma opiates and certain antipsychotic drugs mutually inhibit ( + ) - [ 3 H ] S K F 10047 and [3H]haloperidol binding in guinea pig brain membranes, Proc. Natl. Acad. Sci. U.S.A. 81, 5618. Tang, F., E. Costa and J. Schwartz, 1983, Increase of proenkephalin m R N A and enkephalin content of rat striatum after daily injection of haloperidol for 2 to 3 weeks, Proc. Natl, Acad. Sci. U.S.A. 80, 3841. Taylor, D. and J. Dekleva, 1987, Potential antipsychotic BMY 14802 selectively binds to sigma sites, Drug Devel. Res. 11, 65. Young, S., T. Bonner and M. Brann, 1986, Mesencephalic dopamine neurons regulate the expression of neuropeptide m R N A in the rat forebrain, Proc. Natl. Acad. Sci. U.S.A. 83, 9827.