- Email: [email protected]
Amphetamine and haloperidol modulate preprotachykinin A mRNA expression in rat nucleus accumbens and caudate-putamen
Molecular Brain Research, 13 (1992) 151-154 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0169-328X/92/$05.00
151
BRESM 80113
Amphetamine and haloperidol modulate preprotachykinin A mRNA expression in rat nucleus accumbens and caudate-putamen Nils Lindefors Department of Pharmacology, Karolinska Institutet, Stockholm (Sweden)
(Accepted 29 October 1991) Key words: Nucleus accumbens; Amphetamine; Caudate-putamen; mRNA; Preprotachykinin A
In situ hybridization was used to measure the effect of repeated amphetamine (1.5 mg/kg) and haloperidol (0.5 mg/kg) treatment for 7 days on the expression of preprotachykinin A (PPT-A) mRNA in rat nucleus accumbens (Acb) and caudate-putamen (CPu). Amphetamine elevated the level of PPT-A mRNA in Acb, but not in CPu. Haloperidol decreased the levels in Acb shell and CPu, but not in Acb core. Haloperidol injected together with amphetamine, prevented the amphetamine-induced increase in PPT-A mRNA expression in both Acb core and shell. Neurokinin A (NKA) and substance P (SP) belong to the tachykinin family of neuropeptides and are encoded by the same gene 11'17. Three precursor peptides, a-, r and y-preprotachykinin A (PPT-A) are formed from this gene and while SP can be translated and processed from all three forms N K A is only derived from r - and y-PPTA 11. However, r - and 7-PPT-A comprise 95% of the total PPT-A m R N A found in the rat striatum 11. Brain tachykinins are functionally closely linked to dopaminergic transmission in the mammalian brain, particularly in caudate-putamen, which is a main projection area of midbrain dopamine neurons 3. Manipulation of dopaminergic transmission by amphetamine (indirect agonist that increase dopamine release) and neuroleptics (dopamine receptor antagonist) influences the synthesis and in vivo release of brain tachykinins in the striato-nigral system of the basal ganglia 1'2'16'21, including tachykinin containing cell bodies in the caudate-putamen (CPu) projecting to the substantia nigra. Interactions between tachykinins and dopaminergic transmission is less well studied in the nucleus accumbens (Acb), a limbic forebrain region adjacent to the CPu that also contain a high density of dopaminergic terminals 3. However, ultrastructural analysis show that tachykinin-containing and dopamine-containing nerve terminals are closely related in Acb 2°, indicating extensive tachykinin/dopamine interactions also in this region of the brain. A recent study has shown that a moderate dose of the psycho-stimulant amphetamine potentiates the in vivo release of tachykinins (i.e. NKA) in Acb without affecting release in CPu 15. The present study was undertaken
to evaluate the possible activation of PPT-A gene expression in Acb by amphetamine. In situ hybridization was used to determine the effect of repeated amphetamine treatment on PPT-A m R N A expression in ventromedial Acb (shell) and dorso-lateral Acb (core) 19 (AcbSh and AcbC, respectively) and in dorsolateral caudateputamen (dlCPu). Combined haloperidol and amphetamine treatment was used to reveal a possible dopaminergic component of amphetamine-induced effects. The results show that amphetamine stimulates tachykinin synthesis in AcbSh and AcbC at the level of PPT-A m R N A expression in a dopamine-dependent manner. Male Sprague-Dawley rats (b. wt. 240-280 g; n = 20) were equally divided into 4 groups and subcutaneously injected twice daily for 7 days with either saline, amphetamine (1.5 mg/kg; D-amphetamine sulfate, Sigma), haloperidol (0.5 mg/kg; Haldol, Janssen Pharmaceutica) or a combination of both amphetamine (1.5 mg/kg) and haloperidol (0.5 mg/kg). The rats were decapitated 4 h following the last injection, the brains were dissected out, immediately frozen on dry ice and stored at -80°C until cryostat sectioning. Coronal tissue sections (at the level of: Bregma +1.0 to 1.5) including Acb and CPu were cut on a cryostat (Leitz, Wetzlar, F.R.G.) in 16/~m sections and thawed onto poly-L-lysine-coated slides (50 mg/ml). After cryostat sectioning, the tissue sections were fixed in 10% formalin in phosphate-buffered saline for 30 min, rinsed twice for 4 min in PBS, and immersed in graded series of ethanol, including a 5 min incubation with chloroform. The sections were then air-dried. The hybridiza-
Correspondence: N. Lindefors, Department of Pharmacology, Karolinska Institutet, Box 60400, S-104 01 Stockholm, Sweden.
152 tion cocktail contained 50% formamide, 4 × SSC (1 x SSC is 0.15 M NaC1, 0.015 M sodium citrate pH 7,0), 1 × Denhardt's solution, 1% Sarcosyl, 0.02 M Na3PO 4 (pH 7.0), 10% dextransulphate, 0.5 mg/ml yeast tRNA, 0.06 M DT-I" and 0.1 mg/ml sheared salmon sperm DNA. For rat PPT-A m R N A hybridization, a 48-mer oligonucleotide coding for amino acids 49-64 of the preprotachykinin protein (thus complementary to a-, fl- and 7-PPT-A mRNA) 11, was used. The oligonucleotides were 3'-end labeled with [a-aSS]dATP using terminal deoxyribonucleotidyl transferase (International Biotech. Inc., New Haven, CT) to a specific activity of approximately 6 x 108 cpm//~g. The labelled probes were purified on a Nensorb column (DuPont, Wilmington, DE) prior to use. Following hybridization for 16 h at 42°C, the sections were rinsed 5 times for 15 min in 1 × SSC at 55°C. Finally, the sections were rinsed in autoclaved water for 2 min, and then dehydrated through a series of graded alcohol and airdried. The sections were exposed to X-ray film (Amersham fl-max) for 20 days. The specificity of the hybridization was examined by adding a 100-fold molar excess of unlabeled PPT-A m R N A oligonucleotide probe to separate control slides thus eliminating the specific hybridization signal. To minimize the variation all in situ hybridizations used for measurements in the present study were performed simultaneously. The autoradiograms from in situ hybridizations were analyzed with Microcomputer Imaging Device (Imaging Research Inc., Canada). Optical density values were converted to the level of isotop labeled oligonucleotide probe in the tissue using external isotop standards exposed to the same film. Measured changes in optical density and hybridization signal (isotop level) are linear to relative changes in PPT-A m R N A levels in the tissue section (Bren6, Lindefors and Persson, unpublished findings) in an interval including all measurements in this study. Thus measured changes in hybridization signal correspond to underlying changes in m R N A levels of similar size. A similar linear relation between optical density values and relative levels of PPT-A m R N A measured by in situ hybridization has also been reported by Gerfen and collaborators 7. In the Acb of control brains, the expression of PPT-A m R N A was found to be approximately 2-fold stronger in AcbSh compared with AcbC, whereas in the CPu there is a medial to lateral gradient with higher levels of PPT-A m R N A in the lateral part (Fig. 1). In agreement with earlier reports an intense labeling of PPT-A m R N A expression in the normal forebrain of control animals was found in the CPu, Acb and olfactory tubercules at the coronal level 4A2'24. Repeated treatment with amphetamine (1.5 mg/kg) increased the level of PPT-A m R N A with 25% in AcbSh
and 41% in AcbC, while no change was observed in the dlCPu (Figs. 2 and 3). Conversely, repeated haloperidol (0.5 mg/kg) treatment reduced the level of PPT-A in AcbSh by 20% and by 32% in dlCPu, while no decrease was observed in AcbC (Figs. 2 and 3). Combined amphetamine and haloperidol treatment prevented the amphetamine induced increase in the PPT-A m R N A level in both AcbSh and AcbC, while in dlCPu the PPT-A m R N A level was reduced as with haloperidol only (Figs. 2 and 3). The basal ganglia and the limbic forebrain contain among the highest levels of the tachykinins SP and NKA in the brain 1°'13. Tachykinin biosynthesis in the mammalian forebrain is concentrated to subcortical regions, e.g., CPu and Acb as shown by RNA blot analysis and in situ hybridization 4'1s. Moreover, the expression of PPT-A m R N A in CPu has been shown to be dependent on intact dopaminergic transmission 2A2'24. In CPu there is evidence of potentiation of PPT-A m R N A expression by amphetamine (i.e. methamphetamine) at a high dose which could be reversed by dopamine antagonists L. The methamphetamine-induced increase in PPT-A m R N A levels in CPu has been shown to be due to an increased PPT-A gene transcription 8. The present study shows that a moderate dose of the psychostimulant amphetamine, known to release dopamine in Acb more effectively than in dorsal CPu 5, preferentially activate PPT-A m R N A expression in this brain region compared with CPu. Behavioural and biochemical analysis have shown that tachykinins (i.e. SP) facilitate dopaminergic transmission in the Acb 6'9. Furthermore, a moderate dose of amphetamine is known to induce locomotion which correlate predominantly with dopamine release in Acb rather than
Fig. 1. Autoradiograms of in situ hybridization of PPT-A mRNA in tissue sections of rat forebrain including Acb and CPu. Arrow tips indicate areas where measurements were performed, dlCPu, dorso-lateral caudate-putamen; AcbC. nucleus accumbens core; AcbSh, nucleus accumbens shell.
153
Fig. 2. Autoradiograms of in situ hybridization of PPT-A mRNA in tissue sections of rat forebrain including Acb and CPu. Upper left panel shows hybridization in a control brain, upper right panel in the brain of an amphetamine treated animal, lower left panel in a haloperidol treated and lower right panel in the brain of an amphetamine and haloperidol treated animal. Note increase in PPT-A mRNA levels in AcbSh and AcbC after amphetamine treatment and attenuation after combined amphetamine and haloperidol treatment, amph, amphetamine; halo, haloperidol; dlCPu, dorso-lateral caudate-putamen; AcbC, nucleus accumbens core; AcbSh, nucleus accumbens shell. AcbSh
150
AcbC
dlCPu in the CPu 22 and to potentiate tachykinin release in Acb rather than in CPu 15. The present findings demonstrate
100
"1-
T
t
< z 50
IN
t Fig. 3. Hybridization signal from autoradiograms of PPT-A mRNA is situ hybridizations. Measurements were made over a total of 1 mm2 on three consecutive sections from each brain. The mean value measured in the medial Acb of the control brains was given the value of 100%. The statistical analysis used ANOVA with repeated measurements and Scheffe's post-comparative test. Values are mean + S.E.M. (n = 5). Abbreviations: see legend to Fig. 2. *Significant difference compared to saline-treated control animals (P < 0.05); *significant difference compared to amphetaminetreated animals (P < 0.05).
that the selective stimulatory effect by a moderate dose of a m p h e t a m i n e in the Acb occurs also at the level of P P T - A m R N A expression. In line with earlier data 1, haloperidol treatment was shown to inhibit P P T - A m R N A expression in the CPu. In addition, I show that haloperidol decreases the level of P P T - A m R N A levels in AcbSh to a similar extent which is in line with previous findings of a haloperidolinduced decrease in tachykinins levels in this structure as measured by radioimmunoassay including whole tissue extracts of Acb TM. However, a recent study using R N A protection analysis did not show any change in the tissue level of P P T - A m R N A in Acb following repeated haloperidol treatment (1 mg/kg) 23. This is in contrast to the findings of the present study. There are two obvious differences between the two studies that may explain the
154 difference. First the whole tissue levels measured in the previous study by dissection using a 1 m m punch may
expression in AcbSh is reversed by dopamine receptor
not have included edge parts of AcbSh which are impor-
blockade. Thus the stimulatory action of amphetamine on tachykinin synthesis and release in Acb appears to be
tant, since the present results showed changes in P P T - A m R N A levels only in AcbSh and not in the AcbC. Secondly, Shibata and collaborators 23 measured P P T - A
mediated by a dopaminergic mechanism. This give further support to the close functional interaction by tachykinins and dopamine in this part of the limbic forebrain.
m R N A content in A c b C and AcbSh together which
A substantial body of research has characterized the interaction between dopaminergic and tachykinergic
should be expected to attenuate the possibility to measure any drug effect. The fact that in situ hybridization allow a more detailed analysis further suggest that this technique should be used to study specific regulation in subpopulations of neurons within brain regions like Acb. Further experiments are needed to explain the mechanism of the differential effect by haloperidol on P P T - A m R N A expression in AcbSh and AcbC. However, combined a m p h e t a m i n e and haloperidol treatment shows that amphetamine-induced activation of P P T - A m R N A
1 Bannon, M.J., Elliot, P.J. and Bunney, E.B., Striatal tachykinin biosynthesis: regulation of mRNA and peptide levels by dopamine agonists and antagonists, Mol. Brain Res., 3 (1987) 31-37. 2 Bannon, M.J., Lee, J.-M., Giraus, P., Young, A., Affolter, H.U. and Bonner, T.I., Dopamine antagonist haloperidol decreases substance P, substance K, and preprotachykinin mRNAs in rat striatonigral neurons, J. Biol. Chem., 261 (1986) 66406642. 3 BjSrklund, A. and Lindvall, O., Dopamine-containing systems in the CNS. In A. Bj~rklund and T. HSkfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 2, Classical Transmitters in the CNS, Part L Elsevier, Amsterdam, 1984, pp. 55-122.
4 Bren6, S., Lindefors, N., Friedman, W. and Persson, H., Developmental changes of preprotachykinin A mRNA expression in rat forebrain, Dev. Brain Res., 57 (1990) 151-162. 5 Carboni, E., Imperato, A., Perezzani, L. and Di Chiara, G., Amphetamine, cocaine, phencycidine and nomifenesine increase extracellular dopamine concentrations preferentially in the nucleus accumbens in freely moving rats, Neuroscience, 28 (1989) 653-661. 6 Elliott, P.J., Nemeroff, C.B. and Kilts, C.D., Evidence for a tonic facilitatory influence of substance P on dopamine release in the nucleus accumbens, Brain Res., 385 (1986) 379-382. 7 Gerfen, C.R., McGinty, J.E and Young III, S., Dopamine differentially regulates dynorphin, substance P, and enkephalin expression in striatal neurons: in situ hybridization histochemical analysis, J. Neurosci. 11 (1991) 1016-1031. 8 Haverstick, D.M. and Bannon, M.J., Evidence for dual mechanisms involved in metamphetamine-induced increases in striatal preprotachykinin mRNA, J. Biol. Chem., 264 (1989) 1314013144. 9 Kalivas, P.W. and Miller, J.S., Substance P modulation of dopamine in the nucleus accumbens, Neurosci. Lett., 48 (1984) 55-59. 10 Kanazawa, 1., Ogawa, T., Kimura, S. and Munekata, E., Regional distribution of substance P, neurokinin A and neurokinin B in rat central nervous system, Neurosci. Res., 2 (1984) 111120. 11 Krause, J.E., Chirgwin, J.M., Carter, M.S., Xu, Z.S. and Hershey, A.D., Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A, Proc. Natl. Acad. Sci. U.S.A., 79 (1987) 881-885. 12 Lindefors, N., Bren6, S., Herrera-Marschitz, M. and Persson, H., Neuropeptide gene expression in brain is differentially reg-
transmission in the m a m m a l i a n brain. The present study adds to this literature in demonstrating a dopamine-dep e n d e n t regulation of P P T - A gene expression in both core and shell of Acb by repeated a m p h e t a m i n e and of a differential effect of neuroleptic treatment on these subregions of Acb. This study was supported by The Swedish Medical Research Council (no. 8653) and Ake Wibergs Stiftelse.
13
14
15 16 17
18 19 20
21
22
23 24
ulated by midbrain dopamine neurons, Exp. Brain Res., 80 (1990) 489-500. Lindefors, N., Brodin, E., Theodorsson-Norheim, E. and Ungerstedt, U., Regional distribution and in vivo release of tachykinin-like immunoreactivities in rat brain: evidence for regional differences in relative propotions of tachykinins, Regul. Pept., 10 (1985) 217-230. Lindefors, N., Brodin, E. and Ungerstedt, U., Subchronic neuroleptic treatment induces region-specific changes in levels of neurokinin A and substance P in rat brain, Neuropeptides, 7 (1986) 265-280. Lindefors, N., Brodin, E. and Ungerstedt, U., Amphetamine facilitates the in vivo release of neurokinin A in the nucleus accumbens of the rat, Eur. J. Pharmacol., 160 (1989) 417-420. Lindefors, N., Brodin, E. and Ungerstedt, U., Subchronic haloperidol treatment decreases in vivo release of tachykinins in rat substantia nigra, Eur. J. Pharmacol., 161 (1989) 95-98. Nawa, H., Hirose, T., Takashima, H., Inayama, S. and Nakanishi, S., Nucleotide sequence of cloned cDNAs for two types of bovine brain substance P precursor, Nature, 306 (1983) 3236. Nawa, H., Kotani, H. and Nakanishi, S., Tissue-specificgeneration of two preprotachykinin mRNAs from one gene by alternative RNA splicing, Nature, 312 (1984) 729-734. Paxinos, S. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, 1986. Pickel, V.M., Joh, T.H. and Chan, J., Substance P in the rat nucleus accumbens: ultrastrucural localization in axon terminals and their relation to dopaminergic afferents, Brain Res., 444 (1988) 247-264. Ritter, J.K., Schmidt, C.J., Gibb, J.W. and Hanson, G.R., Increases of substance P-like immunoreactivity within striatal-nigral structures after subacute metamphetamine treatment, J. Pharmacol. Exp. Ther., 229 (1984) 487-492. Sharp, T., Zetterstr6m, T., Ljungberg, T. and Ungerstedt, U., A direct comparison of amphetamine induced behaviours and regional brain dopamine release in the rat using intracerebral dialysis, Brain Res., 401 (1987) 322-330. Shibata, K., Haverstick, D.M. and Bannon, M.J., Tachykinin gene expression in rat limbic nuclei: modulation by dopamine antagonists, J. Pharmacol. Exp. Ther., 255 (1990) 388-392. Young, W.S., Bonner, T.I. and Brann, M.R., Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 9827-9831.
151
BRESM 80113
Amphetamine and haloperidol modulate preprotachykinin A mRNA expression in rat nucleus accumbens and caudate-putamen Nils Lindefors Department of Pharmacology, Karolinska Institutet, Stockholm (Sweden)
(Accepted 29 October 1991) Key words: Nucleus accumbens; Amphetamine; Caudate-putamen; mRNA; Preprotachykinin A
In situ hybridization was used to measure the effect of repeated amphetamine (1.5 mg/kg) and haloperidol (0.5 mg/kg) treatment for 7 days on the expression of preprotachykinin A (PPT-A) mRNA in rat nucleus accumbens (Acb) and caudate-putamen (CPu). Amphetamine elevated the level of PPT-A mRNA in Acb, but not in CPu. Haloperidol decreased the levels in Acb shell and CPu, but not in Acb core. Haloperidol injected together with amphetamine, prevented the amphetamine-induced increase in PPT-A mRNA expression in both Acb core and shell. Neurokinin A (NKA) and substance P (SP) belong to the tachykinin family of neuropeptides and are encoded by the same gene 11'17. Three precursor peptides, a-, r and y-preprotachykinin A (PPT-A) are formed from this gene and while SP can be translated and processed from all three forms N K A is only derived from r - and y-PPTA 11. However, r - and 7-PPT-A comprise 95% of the total PPT-A m R N A found in the rat striatum 11. Brain tachykinins are functionally closely linked to dopaminergic transmission in the mammalian brain, particularly in caudate-putamen, which is a main projection area of midbrain dopamine neurons 3. Manipulation of dopaminergic transmission by amphetamine (indirect agonist that increase dopamine release) and neuroleptics (dopamine receptor antagonist) influences the synthesis and in vivo release of brain tachykinins in the striato-nigral system of the basal ganglia 1'2'16'21, including tachykinin containing cell bodies in the caudate-putamen (CPu) projecting to the substantia nigra. Interactions between tachykinins and dopaminergic transmission is less well studied in the nucleus accumbens (Acb), a limbic forebrain region adjacent to the CPu that also contain a high density of dopaminergic terminals 3. However, ultrastructural analysis show that tachykinin-containing and dopamine-containing nerve terminals are closely related in Acb 2°, indicating extensive tachykinin/dopamine interactions also in this region of the brain. A recent study has shown that a moderate dose of the psycho-stimulant amphetamine potentiates the in vivo release of tachykinins (i.e. NKA) in Acb without affecting release in CPu 15. The present study was undertaken
to evaluate the possible activation of PPT-A gene expression in Acb by amphetamine. In situ hybridization was used to determine the effect of repeated amphetamine treatment on PPT-A m R N A expression in ventromedial Acb (shell) and dorso-lateral Acb (core) 19 (AcbSh and AcbC, respectively) and in dorsolateral caudateputamen (dlCPu). Combined haloperidol and amphetamine treatment was used to reveal a possible dopaminergic component of amphetamine-induced effects. The results show that amphetamine stimulates tachykinin synthesis in AcbSh and AcbC at the level of PPT-A m R N A expression in a dopamine-dependent manner. Male Sprague-Dawley rats (b. wt. 240-280 g; n = 20) were equally divided into 4 groups and subcutaneously injected twice daily for 7 days with either saline, amphetamine (1.5 mg/kg; D-amphetamine sulfate, Sigma), haloperidol (0.5 mg/kg; Haldol, Janssen Pharmaceutica) or a combination of both amphetamine (1.5 mg/kg) and haloperidol (0.5 mg/kg). The rats were decapitated 4 h following the last injection, the brains were dissected out, immediately frozen on dry ice and stored at -80°C until cryostat sectioning. Coronal tissue sections (at the level of: Bregma +1.0 to 1.5) including Acb and CPu were cut on a cryostat (Leitz, Wetzlar, F.R.G.) in 16/~m sections and thawed onto poly-L-lysine-coated slides (50 mg/ml). After cryostat sectioning, the tissue sections were fixed in 10% formalin in phosphate-buffered saline for 30 min, rinsed twice for 4 min in PBS, and immersed in graded series of ethanol, including a 5 min incubation with chloroform. The sections were then air-dried. The hybridiza-
Correspondence: N. Lindefors, Department of Pharmacology, Karolinska Institutet, Box 60400, S-104 01 Stockholm, Sweden.
152 tion cocktail contained 50% formamide, 4 × SSC (1 x SSC is 0.15 M NaC1, 0.015 M sodium citrate pH 7,0), 1 × Denhardt's solution, 1% Sarcosyl, 0.02 M Na3PO 4 (pH 7.0), 10% dextransulphate, 0.5 mg/ml yeast tRNA, 0.06 M DT-I" and 0.1 mg/ml sheared salmon sperm DNA. For rat PPT-A m R N A hybridization, a 48-mer oligonucleotide coding for amino acids 49-64 of the preprotachykinin protein (thus complementary to a-, fl- and 7-PPT-A mRNA) 11, was used. The oligonucleotides were 3'-end labeled with [a-aSS]dATP using terminal deoxyribonucleotidyl transferase (International Biotech. Inc., New Haven, CT) to a specific activity of approximately 6 x 108 cpm//~g. The labelled probes were purified on a Nensorb column (DuPont, Wilmington, DE) prior to use. Following hybridization for 16 h at 42°C, the sections were rinsed 5 times for 15 min in 1 × SSC at 55°C. Finally, the sections were rinsed in autoclaved water for 2 min, and then dehydrated through a series of graded alcohol and airdried. The sections were exposed to X-ray film (Amersham fl-max) for 20 days. The specificity of the hybridization was examined by adding a 100-fold molar excess of unlabeled PPT-A m R N A oligonucleotide probe to separate control slides thus eliminating the specific hybridization signal. To minimize the variation all in situ hybridizations used for measurements in the present study were performed simultaneously. The autoradiograms from in situ hybridizations were analyzed with Microcomputer Imaging Device (Imaging Research Inc., Canada). Optical density values were converted to the level of isotop labeled oligonucleotide probe in the tissue using external isotop standards exposed to the same film. Measured changes in optical density and hybridization signal (isotop level) are linear to relative changes in PPT-A m R N A levels in the tissue section (Bren6, Lindefors and Persson, unpublished findings) in an interval including all measurements in this study. Thus measured changes in hybridization signal correspond to underlying changes in m R N A levels of similar size. A similar linear relation between optical density values and relative levels of PPT-A m R N A measured by in situ hybridization has also been reported by Gerfen and collaborators 7. In the Acb of control brains, the expression of PPT-A m R N A was found to be approximately 2-fold stronger in AcbSh compared with AcbC, whereas in the CPu there is a medial to lateral gradient with higher levels of PPT-A m R N A in the lateral part (Fig. 1). In agreement with earlier reports an intense labeling of PPT-A m R N A expression in the normal forebrain of control animals was found in the CPu, Acb and olfactory tubercules at the coronal level 4A2'24. Repeated treatment with amphetamine (1.5 mg/kg) increased the level of PPT-A m R N A with 25% in AcbSh
and 41% in AcbC, while no change was observed in the dlCPu (Figs. 2 and 3). Conversely, repeated haloperidol (0.5 mg/kg) treatment reduced the level of PPT-A in AcbSh by 20% and by 32% in dlCPu, while no decrease was observed in AcbC (Figs. 2 and 3). Combined amphetamine and haloperidol treatment prevented the amphetamine induced increase in the PPT-A m R N A level in both AcbSh and AcbC, while in dlCPu the PPT-A m R N A level was reduced as with haloperidol only (Figs. 2 and 3). The basal ganglia and the limbic forebrain contain among the highest levels of the tachykinins SP and NKA in the brain 1°'13. Tachykinin biosynthesis in the mammalian forebrain is concentrated to subcortical regions, e.g., CPu and Acb as shown by RNA blot analysis and in situ hybridization 4'1s. Moreover, the expression of PPT-A m R N A in CPu has been shown to be dependent on intact dopaminergic transmission 2A2'24. In CPu there is evidence of potentiation of PPT-A m R N A expression by amphetamine (i.e. methamphetamine) at a high dose which could be reversed by dopamine antagonists L. The methamphetamine-induced increase in PPT-A m R N A levels in CPu has been shown to be due to an increased PPT-A gene transcription 8. The present study shows that a moderate dose of the psychostimulant amphetamine, known to release dopamine in Acb more effectively than in dorsal CPu 5, preferentially activate PPT-A m R N A expression in this brain region compared with CPu. Behavioural and biochemical analysis have shown that tachykinins (i.e. SP) facilitate dopaminergic transmission in the Acb 6'9. Furthermore, a moderate dose of amphetamine is known to induce locomotion which correlate predominantly with dopamine release in Acb rather than
Fig. 1. Autoradiograms of in situ hybridization of PPT-A mRNA in tissue sections of rat forebrain including Acb and CPu. Arrow tips indicate areas where measurements were performed, dlCPu, dorso-lateral caudate-putamen; AcbC. nucleus accumbens core; AcbSh, nucleus accumbens shell.
153
Fig. 2. Autoradiograms of in situ hybridization of PPT-A mRNA in tissue sections of rat forebrain including Acb and CPu. Upper left panel shows hybridization in a control brain, upper right panel in the brain of an amphetamine treated animal, lower left panel in a haloperidol treated and lower right panel in the brain of an amphetamine and haloperidol treated animal. Note increase in PPT-A mRNA levels in AcbSh and AcbC after amphetamine treatment and attenuation after combined amphetamine and haloperidol treatment, amph, amphetamine; halo, haloperidol; dlCPu, dorso-lateral caudate-putamen; AcbC, nucleus accumbens core; AcbSh, nucleus accumbens shell. AcbSh
150
AcbC
dlCPu in the CPu 22 and to potentiate tachykinin release in Acb rather than in CPu 15. The present findings demonstrate
100
"1-
T
t
< z 50
IN
t Fig. 3. Hybridization signal from autoradiograms of PPT-A mRNA is situ hybridizations. Measurements were made over a total of 1 mm2 on three consecutive sections from each brain. The mean value measured in the medial Acb of the control brains was given the value of 100%. The statistical analysis used ANOVA with repeated measurements and Scheffe's post-comparative test. Values are mean + S.E.M. (n = 5). Abbreviations: see legend to Fig. 2. *Significant difference compared to saline-treated control animals (P < 0.05); *significant difference compared to amphetaminetreated animals (P < 0.05).
that the selective stimulatory effect by a moderate dose of a m p h e t a m i n e in the Acb occurs also at the level of P P T - A m R N A expression. In line with earlier data 1, haloperidol treatment was shown to inhibit P P T - A m R N A expression in the CPu. In addition, I show that haloperidol decreases the level of P P T - A m R N A levels in AcbSh to a similar extent which is in line with previous findings of a haloperidolinduced decrease in tachykinins levels in this structure as measured by radioimmunoassay including whole tissue extracts of Acb TM. However, a recent study using R N A protection analysis did not show any change in the tissue level of P P T - A m R N A in Acb following repeated haloperidol treatment (1 mg/kg) 23. This is in contrast to the findings of the present study. There are two obvious differences between the two studies that may explain the
154 difference. First the whole tissue levels measured in the previous study by dissection using a 1 m m punch may
expression in AcbSh is reversed by dopamine receptor
not have included edge parts of AcbSh which are impor-
blockade. Thus the stimulatory action of amphetamine on tachykinin synthesis and release in Acb appears to be
tant, since the present results showed changes in P P T - A m R N A levels only in AcbSh and not in the AcbC. Secondly, Shibata and collaborators 23 measured P P T - A
mediated by a dopaminergic mechanism. This give further support to the close functional interaction by tachykinins and dopamine in this part of the limbic forebrain.
m R N A content in A c b C and AcbSh together which
A substantial body of research has characterized the interaction between dopaminergic and tachykinergic
should be expected to attenuate the possibility to measure any drug effect. The fact that in situ hybridization allow a more detailed analysis further suggest that this technique should be used to study specific regulation in subpopulations of neurons within brain regions like Acb. Further experiments are needed to explain the mechanism of the differential effect by haloperidol on P P T - A m R N A expression in AcbSh and AcbC. However, combined a m p h e t a m i n e and haloperidol treatment shows that amphetamine-induced activation of P P T - A m R N A
1 Bannon, M.J., Elliot, P.J. and Bunney, E.B., Striatal tachykinin biosynthesis: regulation of mRNA and peptide levels by dopamine agonists and antagonists, Mol. Brain Res., 3 (1987) 31-37. 2 Bannon, M.J., Lee, J.-M., Giraus, P., Young, A., Affolter, H.U. and Bonner, T.I., Dopamine antagonist haloperidol decreases substance P, substance K, and preprotachykinin mRNAs in rat striatonigral neurons, J. Biol. Chem., 261 (1986) 66406642. 3 BjSrklund, A. and Lindvall, O., Dopamine-containing systems in the CNS. In A. Bj~rklund and T. HSkfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 2, Classical Transmitters in the CNS, Part L Elsevier, Amsterdam, 1984, pp. 55-122.
4 Bren6, S., Lindefors, N., Friedman, W. and Persson, H., Developmental changes of preprotachykinin A mRNA expression in rat forebrain, Dev. Brain Res., 57 (1990) 151-162. 5 Carboni, E., Imperato, A., Perezzani, L. and Di Chiara, G., Amphetamine, cocaine, phencycidine and nomifenesine increase extracellular dopamine concentrations preferentially in the nucleus accumbens in freely moving rats, Neuroscience, 28 (1989) 653-661. 6 Elliott, P.J., Nemeroff, C.B. and Kilts, C.D., Evidence for a tonic facilitatory influence of substance P on dopamine release in the nucleus accumbens, Brain Res., 385 (1986) 379-382. 7 Gerfen, C.R., McGinty, J.E and Young III, S., Dopamine differentially regulates dynorphin, substance P, and enkephalin expression in striatal neurons: in situ hybridization histochemical analysis, J. Neurosci. 11 (1991) 1016-1031. 8 Haverstick, D.M. and Bannon, M.J., Evidence for dual mechanisms involved in metamphetamine-induced increases in striatal preprotachykinin mRNA, J. Biol. Chem., 264 (1989) 1314013144. 9 Kalivas, P.W. and Miller, J.S., Substance P modulation of dopamine in the nucleus accumbens, Neurosci. Lett., 48 (1984) 55-59. 10 Kanazawa, 1., Ogawa, T., Kimura, S. and Munekata, E., Regional distribution of substance P, neurokinin A and neurokinin B in rat central nervous system, Neurosci. Res., 2 (1984) 111120. 11 Krause, J.E., Chirgwin, J.M., Carter, M.S., Xu, Z.S. and Hershey, A.D., Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A, Proc. Natl. Acad. Sci. U.S.A., 79 (1987) 881-885. 12 Lindefors, N., Bren6, S., Herrera-Marschitz, M. and Persson, H., Neuropeptide gene expression in brain is differentially reg-
transmission in the m a m m a l i a n brain. The present study adds to this literature in demonstrating a dopamine-dep e n d e n t regulation of P P T - A gene expression in both core and shell of Acb by repeated a m p h e t a m i n e and of a differential effect of neuroleptic treatment on these subregions of Acb. This study was supported by The Swedish Medical Research Council (no. 8653) and Ake Wibergs Stiftelse.
13
14
15 16 17
18 19 20
21
22
23 24
ulated by midbrain dopamine neurons, Exp. Brain Res., 80 (1990) 489-500. Lindefors, N., Brodin, E., Theodorsson-Norheim, E. and Ungerstedt, U., Regional distribution and in vivo release of tachykinin-like immunoreactivities in rat brain: evidence for regional differences in relative propotions of tachykinins, Regul. Pept., 10 (1985) 217-230. Lindefors, N., Brodin, E. and Ungerstedt, U., Subchronic neuroleptic treatment induces region-specific changes in levels of neurokinin A and substance P in rat brain, Neuropeptides, 7 (1986) 265-280. Lindefors, N., Brodin, E. and Ungerstedt, U., Amphetamine facilitates the in vivo release of neurokinin A in the nucleus accumbens of the rat, Eur. J. Pharmacol., 160 (1989) 417-420. Lindefors, N., Brodin, E. and Ungerstedt, U., Subchronic haloperidol treatment decreases in vivo release of tachykinins in rat substantia nigra, Eur. J. Pharmacol., 161 (1989) 95-98. Nawa, H., Hirose, T., Takashima, H., Inayama, S. and Nakanishi, S., Nucleotide sequence of cloned cDNAs for two types of bovine brain substance P precursor, Nature, 306 (1983) 3236. Nawa, H., Kotani, H. and Nakanishi, S., Tissue-specificgeneration of two preprotachykinin mRNAs from one gene by alternative RNA splicing, Nature, 312 (1984) 729-734. Paxinos, S. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, San Diego, 1986. Pickel, V.M., Joh, T.H. and Chan, J., Substance P in the rat nucleus accumbens: ultrastrucural localization in axon terminals and their relation to dopaminergic afferents, Brain Res., 444 (1988) 247-264. Ritter, J.K., Schmidt, C.J., Gibb, J.W. and Hanson, G.R., Increases of substance P-like immunoreactivity within striatal-nigral structures after subacute metamphetamine treatment, J. Pharmacol. Exp. Ther., 229 (1984) 487-492. Sharp, T., Zetterstr6m, T., Ljungberg, T. and Ungerstedt, U., A direct comparison of amphetamine induced behaviours and regional brain dopamine release in the rat using intracerebral dialysis, Brain Res., 401 (1987) 322-330. Shibata, K., Haverstick, D.M. and Bannon, M.J., Tachykinin gene expression in rat limbic nuclei: modulation by dopamine antagonists, J. Pharmacol. Exp. Ther., 255 (1990) 388-392. Young, W.S., Bonner, T.I. and Brann, M.R., Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 9827-9831.