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Intrastriatal injection of a muscarinic receptor agonist and antagonist regulates striatal neuropeptide mRNA expression in normal and amphetamine-treated rats
Brain Research 748 Ž1997. 62–70
Research report
Intrastriatal injection of a muscarinic receptor agonist and antagonist regulates striatal neuropeptide mRNA expression in normal and amphetamine-treated rats John Q. Wang, Jacqueline F. McGinty
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Department of Anatomy and Cell Biology, East Carolina UniÕersity School of Medicine, GreenÕille, NC 27858-4354, USA Accepted 15 October 1996
Abstract Systemic administration of the muscarinic receptor antagonist, scopolamine, augments, whereas the muscarinic receptor agonist, oxotremorine, attenuates behaviors Žlocomotion and stereotypies. and preprodynorphin ŽPPD. and substance P ŽSP. gene expression in striatonigral neurons induced by the indirect dopamine receptor agonist, amphetamine ŽAMPH.. In contrast, systemic scopolamine blocks, whereas oxotremorine augments, AMPH-stimulated preproenkephalin ŽPPE. gene expression in striatopallidal neurons. This study investigated the site of action of these effects by administering scopolamine and oxotremorine directly into the striatum and assessing the expression of neuropeptide mRNAs with quantitative in situ hybridization. Unilateral injection of scopolamine into the dorsal striatum augmented, and oxotremorine attenuated, AMPH Ž2.5 mgrkg, i.p..-stimulated behaviors. Intrastriatal scopolamine at a concentration of 62 mM, but not 6.2 mM, increased basal levels of PPD and SP mRNAs in the dorsal striatum. In addition, both 6.2 and 62 mM scopolamine significantly augmented AMPH-stimulated PPD and SP mRNA levels. Intrastriatal infusion of 1.6 or 8.1 mM oxotremorine did not alter basal levels of striatal PPD and SP mRNAs. However, both concentrations of oxotremorine completely blocked AMPH-stimulated SP mRNA and oxotremorine at 8.1 mM blocked AMPH-stimulated PPD mRNA. In contrast, PPE induction by AMPH was blocked by 62, but not 6.2, mM scopolamine. Both concentrations of oxotremorine tended to augment basal and AMPH-stimulated PPE mRNA in the dorsal striatum but the trend was not significant. These data demonstrate an inhibition of striatonigral, and facilitation of striatopallidal, gene expression through activation of local striatal muscarinic receptors, which is consistent with the changes seen after systemic administration of muscarinic agents. Therefore, muscarinic cholinergic regulation of basal and stimulated expression of neuropeptide mRNA is processed within the striatum. Keywords: Dynorphin; Substance P; Enkephalin; Scopolamine; Oxotremorine; Caudoputamen; Accumbens; Behavior
1. Introduction The role of muscarinic cholinergic neurotransmission in the regulation of striatal gene expression has been extensively investigated recently in this laboratory and others. Increasing evidence supports the concept that acetylcholine generally opposes the actions of dopamine in a dynamic balance that controls gene expression in striatal neurons w10,36x. For example, stimulation of D1 dopamine receptors on striatonigral neurons by the indirect dopamine receptor agonists, cocaine or amphetamine ŽAMPH., upregulates the expression of immediate early genes and the
) Corresponding author. Fax: q1 Ž919. 816-2850; E-mail: [email protected]
neuropeptides, preprodynorphin ŽPPD. and substance P ŽSP., contained in these neurons w4,16,25,37x. In contrast to the positive regulation by D 1 receptors, muscarinic receptor stimulation suppresses L-dopa-induced expression of c-fos mRNA w7x, constitutive SP mRNA w22x and AMPH-stimulated PPD and SP mRNA w38x in striatonigral neurons. Muscarinic receptor blockade induces striatal Fos protein w2x and SP and PPD mRNA w23,38x expression and potentiates D 1-stimulated immediate early gene Žc-fos and zif r 268 . and peptide ŽPPD and SP. mRNA induction in dopamine-lesioned w26,27x and intact w38–40x rats. Furthermore, chronic scopolamine augments, and chronic oxotremorine suppresses, dynorphin-like immunoreactivity in striatonigral neurons induced by the D 1rD 2 agonist, apomorphine w9x. Thus, muscarinic receptor stimulation opposes the action of D 1 receptor agonists on striatonigral
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 2 4 4 - 9
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gene expression. In striatopallidal neurons which contain preproenkephalin ŽPPE., D 2 receptor stimulation inhibits, whereas muscarinic receptor stimulation induces, Fos w2x and PPE mRNA w22,23,38x. Moreover, cholinergic facilitation is also implicated in D 2 antagonist-induced striatopallidal gene expression as demonstrated by a partial attenuation of D 2 antagonist-induced Fos w17x and enkephalin w18x immunoreactivity and PPE mRNA w30,40x in striatopallidal neurons after scopolamine pretreatment. It has been hypothesized that cholinergic interneurons in the striatum mediate the effects of muscarinic receptor ligands on D 1-stimulated striatal gene expression w10,38– 40x. However, all the above pharmacological studies were performed using systemic drug injections. Therefore, it was necessary to investigate the effects of central administration of muscarinic agents on striatal gene expression in order to identify the site of dopaminergic and cholinergic interactions. A recent report by Keefe and Gerfen w21x shows that intrastriatal injection of the D 1 antagonist, SCH-23390, is as effective as systemic injection in blocking D 1-stimulated striatal c-fos and zif r 268 mRNA in dopamine-lesioned rats. However, to date, no attempt has been made to evaluate the effects of central administration of muscarinic receptor agents on basal and stimulated peptide mRNA expression in striatonigral and striatopallidal neurons in intact rats. By utilizing quantitative in situ hybridization histochemistry, this study examined the influence of pharmacological manipulation of intrastriatal muscarinic receptor
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activity on striatonigral PPD and SP, and striatopallidal PPE, mRNA expression in normal and AMPH-stimulated rats. Local alteration of striatal muscarinic receptor activity was accomplished by direct injection of the non-selective muscarinic receptor antagonist, scopolamine, or the muscarinic agonist, oxotremorine, into the striatum.
2. Materials and methods 2.1. Animals Adult male Wistar rats Ž225–249 g, Charles River, Raleigh, NC. were individually housed and maintained on a 12:12 h lightrdark schedule with food and water provided ad libitum. A total of 60 rats were randomly assigned to 12 different groups Ž n s 5 per group.. All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. 2.2. Drug administration and behaÕior For intrastriatal infusion, the rats were anesthetized with 4% chloral hydrate Ž400 mgrkg, i.p.. and placed in a David Kopf stereotaxic holder. A 24-gauge stainless-steel guide cannula Ž10 mm in length. was implanted at the coordinates of 1 mm anterior to bregma, 2.5 mm right to
Fig. 1. Effects of scopolamine ŽA. and oxotremorine ŽB. on AMPH-stimulated behaviors. Intrastriatal injection Ž1 m l. of scopolamine Ž6.2 and 62 mM. or oxotremorine Ž1.6 and 8.1 mM. was made 15 min prior to i.p. injection of AMPH Ž2.5 mgrkg.. The behavioral ratings are expressed as mean " S.E.M. The numbers in the top legend of each panel represent the AUC values. ) P - 0.05 as compared with aCSF q saline, q P - 0.05 as compared with aCSFq AMPH.
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midline and 3 mm below the surface of the skull. The guide cannula was sealed by a stainless steel wire of the same length Ž10 mm.. Rats were allowed at least 5 days for recovery from surgery. One day prior to intrastriatal injection, the inner steel wire was replaced by a longer one Ž12.5 mm in length. that protruded 2.5 mm beyond the guide cannula. This procedure created a track for the injection cannula thereby minimizing acute induction of neuronal activity around the injection cannula on the day of the experiment w34x. The next day, this inner wire was replaced by a 30-gauge stainless-steel injection cannula with the same length Ž12.5 mm., through which artificial cerebrospinal fluid ŽaCSF. or 1.14 or 5.7 m g Ž1.6 or 8.1 mM, respectively. oxotremorine or 3 or 30 m g Ž6.2 or 62 mM, respectively. scopolamine were infused into the central part of the dorsal striatum in a volume of 1 m l at a rate of 0.2 m lrmin in freely moving animals. The progress of injection was monitored by observing the movement of a small air bubble through a length of precalibrated PE-10 tubing inserted between the injection cannula and a 5 m l Hamilton microsyringe. After completing each injection, the cannula was left in place for an additional 3 min to
reduce any possible backflow of the solution along the injection track. AMPH or saline was injected i.p. 15 min after the intrastriatal injection in a volume of 1.2 mlrkg. The behavior of the rats was rated by two trained raters, who were unaware of the treatment, 5 min before the intrastriatal injection and every 5 min after the i.p. injection for 1 h, using a 9-point scale as described w33x. 2.3. In situ hybridization histochemistry Three hours after the i.p. injection, the rats were anesthetized with Equithesin Ž5 mlrkg, i.p.. and decapitated. The brains were removed and frozen in isopentane at y408C and stored at y708C. Quantitative in situ hybridization histochemistry to measure PPD, SP and PPE mRNA expression in striatal neurons was performed according to standard procedures in this laboratory w35,41x. Quantitation of the mRNA hybridization signals on X-ray films was performed using NIH Image 1.44 ŽW. Rasband, NIMH. and a Macintosh IIci as described w35x. Briefly, the 14 C standards were measured, plotted against
Fig. 2. Dark-field photomicrographs from emulsion-dipped slides illustrating the effect of intrastriatal scopolamine and oxotremorine on constitutive and AMPH-stimulated PPD mRNA expression in the dorsal striatum. A: aCSFq saline; B: scopolamine Ž62 mM. q saline; C: oxotremorine Ž8.1 mM. q saline; D: aCSFq AMPH Ž2.5 mgrkg.; E: scopolamine Ž62 mM. q AMPH Ž2.5 mgrkg.; F: oxotremorine Ž8.1 mM. q AMPH Ž2.5 mgrkg.. Arrows mark intrastriatal injection tracks. Scale bar s 1 mm.
J.Q. Wang, J.F. McGintyr Brain Research 748 (1997) 62–70
known dpmrmg, and converted to 35 S equivalents to generate a calibration curve. Film background was measured and saved as a ‘blank field’ to correct uneven illumination. Under the density slice option, the hybridization signal in the entire dorsal striatum on the injected side was measured using a circle Ždiameters 200 pixels.. The dorsal striatal area corresponding to the cannula track was not subtracted from the measurement of the entire dorsal striatum because there was little difference among rats in the extent of damage in sections that were taken at the level of the cannula track. In several rats, artificial signals due to blood clots in the cannula track were subtracted to eliminate these non-specific signals. The signals in the nucleus accumbens ŽNAc. were measured using a manual drawing which outlined the shell and core areas. Quantitative changes were expressed as Ž1. the number of labeled pixels per area Žarea., Ž2. mean density of tissue in dpmrmg, and Ž3. integrated density which is the product of area times mean density as described w33,35x. The mean " S.E.M. of each of these measures was calculated for each rat by averaging the values in three adjacent sections. 2.4. Drugs D-Amphetamine sulfate, Žy.-scopolamine hydrobromide and oxotremorine sesquifumarate were purchased from Sigma Chemical Co. ŽSt. Louis, MO.. Scopolamine and oxotremorine were dissolved in aCSF ŽmM: NaCl 154, KCl 2.68, MgCl 2 0.9 and CaCl 2 1.22, pH 7.4.. Doses of scopolamine and oxotremorine were changed by altering concentration but not volume. AMPH was dissolved in 0.9% saline. The dose of AMPH was calculated as the salt. The mM concentrations of scopolamine and oxotremorine refer to the free bases. All drugs were freshly prepared.
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AMPH Ž2.5 mgrkg. significantly increased behavioral activity which included sniffing, exploring, and rearing ŽFig. 1A.. The two concentrations of scopolamine significantly augmented the behavioral responses to AMPH ŽFig. 1A.. The augmentation was concentration-dependent as the higher concentration of scopolamine Ž62 mM. caused sustained maximal stereotypy Žcontinuous sniffing and rearing in one location of cage. 25 min after AMPH ŽFig. 1A.. Although systemic injection of oxotremorine causes typical cholinergic symptoms which include tremor, salivation and lacrimation w38x, these symptoms were not displayed after unilateral intrastriatal injection of oxotremorine at either concentration Ž1.6 and 8.1 mM.. Neither was there a significant effect of oxotremorine on spontaneous behavioral activity ŽFig. 1B.. However, intrastriatal oxotremorine significantly attenuated AMPH-stimulated behaviors in a concentration-dependent manner ŽFig. 1B.. 3.2. Effects of intrastriatal scopolamine and oxotremorine on constitutiÕe and AMPH-stimulated PPD mRNA Intrastriatal scopolamine at the lower concentration Ž6.2 mM. had no significant effect on basal PPD expression in the dorsal striatum in the injected side Ždata not shown.. However, the higher concentration of scopolamine Ž62 mM. appeared to increase the PPD hybridization signal in patch-like clusters and, to a lesser extent, in the surrounding matrix, of the dorsal striatum ŽFig. 2, B vs. A.. Neither
2.5. Statistics A 2-way analysis of variance ŽANOVA. followed by a Bonferroni ŽDunn. comparison of groups using least squares-adjusted means was performed on the area under curve ŽAUC. values calculated from plotting behavioral ratings against time w33x. Significance in area, mean density and integrated density between groups was determined by a nested two-way ANOVA followed by a Bonferroni ŽDunn. comparison of groups using least squares-adjusted means.
3. Results 3.1. BehaÕioral obserÕations Unilateral intrastriatal injection of scopolamine at two concentrations Ž6.2 and 62 mM. did not significantly increase spontaneous behaviors ŽFig. 1A.. Acute injection of
Fig. 3. Quantitiative image analysis of effects of intrastriatal injection of scopolamine ŽA. and oxotremorine ŽB. on basal and AMPH-stimulated PPD mRNA in the striatum in terms of the intrgrated density measured over the entire dorsal striatum at the level of the cannula track. ) P - 0.05 as compared with aCSFqsaline, q P - 0.05 as compared with aCSFq AMPH Ž2.5 mgrkg..
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concentration of scopolamine affected PPD expression in the nucleus accumbens. Acute systemic AMPH produced typical patchy increases in PPD mRNA in the dorsal striatum ŽFig. 2D.. Intrastriatal pretreatment with 62 mM scopolamine followed by AMPH caused a greater induction of PPD mRNA in the dorsal striatum ŽFig. 2E. than after either 62 mM scopolamine or AMPH alone. Furthermore, scopolamine at 6.2 mM, which was ineffective in modifying basal PPD expression Žsee above., also enhanced AMPH-stimulated PPD mRNA signal in the dorsal striatum Ždata not shown.. The augmentation by the two concentrations of scopolamine occurred in patchy areas and in the surrounding matrix of the dorsal striatum. Intrastriatal oxotremorine at 1.6 and 8.1 ŽFig. 2C. mM had no visible effect on basal levels of PPD mRNA in the dorsal striatum. However, less induction of PPD mRNA hybridization signal in the dorsal striatum after AMPH was observed following intrastriatal pretreatment with oxotremorine at 8.1 mM ŽFig. 2F., but not after 1.6 mM, than after pretreatment with aCSF.
Fig. 3 illustrates quantitation of the PPD mRNA hybridization signal in the dorsal striatum. Alterations in integrated density reflect a change in the mean density of hybridization signal and, to a larger extent, in the number of labeled particles per area. Scopolamine at 62 mM significantly increased basal levels of PPD mRNA ŽFig. 3A.. Scopolamine at both 6.2 and 62 mM significantly augmented AMPH-stimulated PPD expression ŽFig. 3A.. In contrast, 1.6 and 8.1 mM oxotremorine had no effect on basal PPD expression ŽFig. 3B.. Although 1.6 mM oxotremorine did not significantly attenuate AMPH-stimulated PPD expression, 8.1 mM oxotremorine completely blocked it ŽFig. 3B.. 3.3. Effects of intrastriatal scopolamine and oxotremorine on constitutiÕe and AMPH-stimulated SP mRNA Alterations in SP mRNA in the dorsal striatum paralleled those of PPD mRNA in response to scopolamine alone or scopolamineq AMPH, i.e., the higher concentration of scopolamine Ž62 mM. increased basal levels of SP
Fig. 4. Dark-field photomicrographs from emulsion-dipped slides illustrating the effect of intrastriatal scopolamine and oxotremorine on constitutive and AMPH-stimulated SP mRNA expression in the dorsal striatum. A: aCSF q saline; B: scopolamine Ž62 mM. q saline; C: oxotremorine Ž8.1 mM. q saline; D: aCSFq AMPH Ž2.5 mgrkg.; E: scopolamine Ž62 mM. q AMPH Ž2.5 mgrkg.; F: oxotremorine Ž8.1 mM. q AMPH Ž2.5 mgrkg.. Arrows mark intrastriatal injection tracks. Scale bar s 1 mm.
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Oxotremorine at 1.6 and 8.1 mM ŽFig. 4C. did not affect the constitutive expression of SP mRNA in the dorsal striatum ŽFig. 5B.. However, both 1.6 and 8.1 mM oxotremorine ŽFig. 4F. abolished increases in striatal SP mRNA induced by systemic AMPH administration ŽFig. 5B.. 3.4. Effects of intrastriatal scopolamine and oxotremorine on constitutiÕe and AMPH-stimulated PPE mRNA
Fig. 5. Quantitiative image analysis of effects of intrastriatal injection of scopolamine ŽA. and oxotremorine ŽB. on basal and AMPH-stimulated SP mRNA in the striatum in terms of the intrgrated density measured over the entire dorsal striatum at the level of the cannula track. ) P - 0.05 as compared with aCSFqsaline, q P - 0.05 as compared with aCSFq AMPH Ž2.5 mgrkg..
Intrastriatal scopolamine Ž6.2 and 62 mM. had no detectable effect on basal levels of PPE mRNA in the dorsal striatum ŽFig. 6A.. Systemic AMPH produced a significant increase in PPE mRNA in the dorsal striatum as described previously w37x. Intrastriatal scopolamine at 6.2 mM did not affect AMPH-stimulated PPE expression ŽFig. 6A.. In contrast, 62 mM scopolamine totally prevented AMPHstimulated PPE expression in the dorsal striatum ŽFig. 6A.. Although oxotremorine at both concentrations Ž1.6 and 8.1 mM. tended to increase basal levels of, and augment AMPH-stimulated, PPE mRNA in the dorsal striatum, these effects did not reach statistical significance ŽFig. 6B..
4. Discussion mRNA ŽFig. 4B vs. 4A and Fig. 5A., and both 6.2 and 62 ŽFig. 4E. mM scopolamine augmented AMPH-stimulated SP mRNA ŽFig. 5A..
Fig. 6. Quantitiative image analysis of effects of intrastriatal injection of scopolamine ŽA. and oxotremorine ŽB. on basal and AMPH-stimulated PPE mRNA in the striatum in terms of the integrated density measured over the entire dorsal striatum at the level of the cannula track. ) P - 0.05 as compared with aCSFqsaline, q P - 0.05 as compared with aCSFq AMPH Ž2.5 mgrkg..
Our previous study established a regulatory role for muscarinic cholinergic transmission in the basal and dopamine agonist-stimulated expression of striatal neuropeptide genes following systemic administration of the muscarinic receptor agonist, oxotremorine, and the antagonist, scopolamine w38x. This study demonstrates that direct injection of these muscarinic agents into the striatum was as effective as systemic administration in regulating striatal peptide gene expression in normal and AMPH-treated rats. For instance, intrastriatal scopolamine increased constitutive, and augmented AMPH-stimulated, PPD and SP mRNA to the same extent as that observed after systemic scopolamine. Furthermore, intrastriatal scopolamine blocked AMPH-stimulated PPE mRNA expression. In contrast, intrastriatal, like systemic, injection of oxotremorine attenuated AMPH-stimulated PPD and SP mRNA, but did not significantly increase AMPH-stimulated PPE expression. Therefore, the muscarinic receptors regulating dopamine-stimulated striatonigral and striatopallidal peptide gene expression are most likely located in the striatum. Di Chiara and colleagues w10x have proposed a detailed model of intrastriatal cell-to-cell interactions between striatonigral and striatopallidal projection neurons and cholinergic interneurons in response to endogenous dopamine receptor stimulation, which is strongly supported by this study and others w38–40x. Briefly, stimulation of D 1 receptors located on striatonigral neurons results in PPD and SP mRNA expression as well as local SP release. SP then stimulates NK 1 receptors on cholinergic neurons, the pri-
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mary striatal neurons which express NK 1 receptors w1,13,15x, to increase striatal acetylcholine release. The released acetylcholine would in turn Ž1. inhibit PPDrSP gene expression in striatonigral neurons, and Ž2. facilitate PPE gene expression in striatopallidal neurons. These sequentially-occurring intercellular events suggest that cholinergic neurons are in a strategic position to play a buffer-like role in normalizing responses of their neighboring projection neurons to changes in dopaminergic activity w10,36x. However, until this study, demonstration that the striatum is the site of these acetylcholinerdopamine interactions was lacking. Nisenbaum et al. w29x reported that seven daily injections of systemic scopolamine prevented the 6-hydroxydopamine-induced elevation of PPE mRNA and reduction of SP mRNA in the striatum Žbut see w23x.. However, seven daily intrastriatal injections of scopolamine in concentrations ranging from 0.5 to 50 mM were not able to mimic this effect. They concluded that extrastriatal cholinergic receptors regulate striatal peptide gene expression in dopamine-depleted rats. This conclusion is different from the intrastriatal cholinergic regulation of striatal peptide gene expression under normal and dopamine-stimulated conditions demonstrated in this study. The possibility exists that intrastriatal cholinergic regulation in intact rats is altered so that extrastriatal cholinergic regulation dominates in dopamine-depleted rats. However, it is also possible that these authors did not use a high enough concentration of scopolamine to test their hypothesis because in the present study, the lower concentration of scopolamine Ž6.2 mM. did not affect AMPH-stimulated PPE mRNA whereas it did affect SP mRNA. Nonetheless, both intra- and extra-striatal cholinergic transmission is likely to contribute to the regulation of striatonigral and striatopallidal peptide gene expression. The subtype of muscarinic receptors mediating inhibition of striatonigral PPDrSP expression or facilitation of striatopallidal PPE expression remains to be determined. Since M 4 receptors are contained in most PPDrSP neurons w3,43x and are coupled to inhibition of cAMP formation w12,19x, which opposes D 1-stimulated cAMP formation, it is likely that this subtype mediates inhibition of striatonigral gene expression. The subtype most likely to mediate facilitation of striatopallidal gene expression is M 1 because this subtype is expressed by all striatopallidal neurons w3,43x and is coupled to stimulation of phosphoinositide turnover w19x. Interestingly, oxotremorine is a poor stimulator of phosphoinositide hydrolysis w14x which may explain its weak effects on striatopallidal neurons Žbut see below for an alternative explanation.. Unavailability of pharmacological agents with satisfactory subtype specificity hinders the ability to investigate the muscarinic subtypes involved in striatal gene expression. However, recently available muscarinic toxins which have much higher subtype selectivity than any other pharmacological compounds to date w20,31x provide an alternative avenue to
evaluate the relevant subtypes. Recently in this laboratory, muscarinic toxin 3 ŽMT3., a peptide toxin isolated from green mamba venom, which has a high selectivity for cloned m4 receptors w20x, was used in vivo to evaluate the contribution of M 4 receptors to cholinergic inhibition of PPDrSP gene expression. It was found that intrastriatal MT3 administration elevated basal, and augmented AMPH-stimulated, PPD and SP mRNA expression w42x similar to the effect of scopolamine. Thus, the M 4 receptor appears to be a subtype which mediates cholinergic inhibition of striatonigral PPDrSP gene expression. Two main striatal compartments, the patch Žstriosomes. and matrix, are distinguished by a number of biochemical markers, afferent and efferent connections, as well as functional differences. However, it is unclear whether the cholinergic regulation of peptide mRNA expression in striatal medium spiny neurons is compartment-specific because our analysis did not extend to this level. However, scopolamine has been found to induce c-fos immediate early gene expression in both striosomal and matrix neurons whereas oxotremorine induced c-fos predominantly in striosomal neurons w2x. If the effects of these drugs on PPDrSP and PPE expression in this study were similar, oxotremorine-induced PPE expression in striatopallidal neurons may be occurring in striosomes, which consist of only about 15% of striatal neurons. In this case, changes in PPE mRNA would be less detectable than scopolamine-induced PPDrSP expression which occurs in both compartments. The concentrations of the muscarinic agents injected intrastriatally fall within the dose range shown previously to be effective in modifying muscarinic receptor-mediated functions w6,8x. In fact, because the striatum encompasses a large area, actual tissue concentration of injected drugs should have been reduced substantially by diffusion. Moreover, both scopolamine and oxotremorine affected gene expression only in the injected dorsal striatum, but not in the ipsilateral nucleus accumbens, indicating that the diffusion of drugs used in this study was limited to the ipsilateral caudoputamen. It was somewhat unexpected that unilateral intrastriatal injection of muscarinic agents, which was anticipated to affect striatal function only on the injected side, altered the behavioral activity of AMPH-stimulated rats in a manner similar to systemic injections. Behavioral effects also have been seen after an AMPH injection into one side of the striatum although the unilateral injection produced oral stereotypy half as intense as that observed after bilateral injections w11x. Furthermore, in many other experimental conditions, bilateral effects have been frequently seen after unilateral manipulations of striatal functions w5,23,28x. The mechanism for this phenomenon is unclear. However, it is not likely because of diffusion of drugs to the uninjected side of the striatum because muscarinic agents had no significant effect on basal or AMPH-stimulated peptide gene expression in the contralateral side. Alternatively,
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unilateral modification of striatal function may affect motor activity by activating bilateral and commissural projections in sensorimotor cortical areas w24,32x. In summary, this study demonstrates that local blockade of striatal muscarinic receptors by intrastriatal injection of scopolamine augmented constitutive and AMPH-stimulated striatonigral PPD and SP gene expression in the dorsal striatum. Local activation of striatal muscarinic receptors by intrastriatal injection of oxotremorine blocked AMPH-stimulated PPD and SP gene expression. In addition, intrastriatal scopolamine blocked AMPH-stimulated striatopallidal PPE expression. These data are in good accordance with those observed after systemic injection of scopolamine and oxotremorine w38x. Thus, muscarinic cholinergic regulation of striatonigral and striatopallidal gene expression is most likely mediated by intrastriatal cholinergic interneurons in intact rats.
Acknowledgements This research was supported by DA03982. The authors wish to thank Denise C. Mayer, Jennifer S. Starling and William T. Bohler for their technical assistance.
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J.Q. Wang, J.F. McGintyr Brain Research 748 (1997) 62–70 muscarinic regulation of striatal enkephalin and substace P messenger RNAs following striatal dopamine denervation: effects of systemic and central administration of quinpirole and scopolamine, Neuroscience, 63 Ž1994. 435–449. Pollack, A.E. and Wooten, G.F., D 2 dopaminergic regulation of striatal preproenkephalin mRNA levels in mediated at least in part through cholinergic interneurons, Mol. Brain Res., 13 Ž1992. 35–41. Potter, L.T., Hanchett-Valentine, H., Liang, J.S., Max, S.I., Purkerson, S.L., Silberberg, H.A. and Strauss, W.L., m1-toxin, Life Sci., 52 Ž1993. 433–440. Savaki, H.E., Raos, V.C. and Dermon, C.R., Bilateral cerebral metabolic effects of pharmacological manipulations of the substantia nigra in the rat: unilateral intranigral application of the inhibitory GABA A receptor against muscimol, Neuroscience, 50 Ž1992. 781– 794. Smith, A.J.W. and McGinty, J.F., Acute amphetamine or methamphetamine alters opioid peptide mRNA expression in rat striatum, Mol. Brain Res., 21 Ž1994. 359–362. Steiner, H. and Gerfen, C.R., Dynorphin opioid inhibition of cocaine-induced, D1 dopamine receptor-mediated immediate-early gene expression in the striatum, J. Comp. Neurol., 353 Ž1995. 200–212. Wang, J.Q. and McGinty, J.F., Dose-dependent alteration in zif r 268 and preprodynorphin mRNA expression induced by amphetamine or methamphetamine in rat forebrain, J. Pharmacol. Exp. Ther., 273 Ž1995. 909–917. Wang, J.Q. and McGinty, J.F., Glutamatergic and cholinergic regulation of immediate early gene and neuropeptide gene expression in the striatum. In K.M. Merchant ŽEd.., Pharmacological Regulation
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of Gene Expression in the CNS, CRC Press, Boca Raton, 1996, pp. 81–113. Wang, J.Q. and McGinty, J.F., D1 and D 2 receptor regulation of preproenkephalin and preprodynorphin mRNA in rat striatum following acute injection of amphetamine or methamphetamine, Synapse, 22 Ž1996. 114–122. Wang, J.Q. and McGinty, J.F., Muscarinic receptors regulates striatal neuropeptide gene expression in normal and amphetamine-treated rats, Neuroscience, 75 Ž1996. 43–56. Wang, J.Q. and McGinty, J.F., Scopolamine augments c-fos and zif r 268 mRNA expression induced by the full D 1 dopamine receptor agonist SKF-82958 in the intact rat striatum, Neuroscience, 72 Ž1996. 601–616. Wang, J.Q. and McGinty, J.F., The full D1 dopamine receptor agonist SKF-82958 induces neuropeptide mRNA in the normosensitive striatum of rats: regulation of D1 rD 2 interactions by muscarinic receptors, submitted Ž1996.. Wang, J.Q., Smith, A.J.W. and McGinty, J.F., A single injection of amphetamine or methamphetamine induces dynamic alterations in c-fos, zif r 268 and preprodynorphin mRNA expression in rat forebrain, Neuroscience, 68 Ž1995. 83–95. Wang, J.Q. and McGinty, J.F., The M 4 muscarinic toxin MT3 augments neuropeptide mRNA in rat striatum in vivo, NeuroReport, Ž1996. submitted. Weiner, D.M., Levey, A. and Brann, M.R., Expression of muscarinic acetylcholine and dopamine receptor mRNAs in rat basal ganglia, Proc. Natl. Acad. Sci. USA, 87 Ž1990. 7050–7054.
Research report
Intrastriatal injection of a muscarinic receptor agonist and antagonist regulates striatal neuropeptide mRNA expression in normal and amphetamine-treated rats John Q. Wang, Jacqueline F. McGinty
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Department of Anatomy and Cell Biology, East Carolina UniÕersity School of Medicine, GreenÕille, NC 27858-4354, USA Accepted 15 October 1996
Abstract Systemic administration of the muscarinic receptor antagonist, scopolamine, augments, whereas the muscarinic receptor agonist, oxotremorine, attenuates behaviors Žlocomotion and stereotypies. and preprodynorphin ŽPPD. and substance P ŽSP. gene expression in striatonigral neurons induced by the indirect dopamine receptor agonist, amphetamine ŽAMPH.. In contrast, systemic scopolamine blocks, whereas oxotremorine augments, AMPH-stimulated preproenkephalin ŽPPE. gene expression in striatopallidal neurons. This study investigated the site of action of these effects by administering scopolamine and oxotremorine directly into the striatum and assessing the expression of neuropeptide mRNAs with quantitative in situ hybridization. Unilateral injection of scopolamine into the dorsal striatum augmented, and oxotremorine attenuated, AMPH Ž2.5 mgrkg, i.p..-stimulated behaviors. Intrastriatal scopolamine at a concentration of 62 mM, but not 6.2 mM, increased basal levels of PPD and SP mRNAs in the dorsal striatum. In addition, both 6.2 and 62 mM scopolamine significantly augmented AMPH-stimulated PPD and SP mRNA levels. Intrastriatal infusion of 1.6 or 8.1 mM oxotremorine did not alter basal levels of striatal PPD and SP mRNAs. However, both concentrations of oxotremorine completely blocked AMPH-stimulated SP mRNA and oxotremorine at 8.1 mM blocked AMPH-stimulated PPD mRNA. In contrast, PPE induction by AMPH was blocked by 62, but not 6.2, mM scopolamine. Both concentrations of oxotremorine tended to augment basal and AMPH-stimulated PPE mRNA in the dorsal striatum but the trend was not significant. These data demonstrate an inhibition of striatonigral, and facilitation of striatopallidal, gene expression through activation of local striatal muscarinic receptors, which is consistent with the changes seen after systemic administration of muscarinic agents. Therefore, muscarinic cholinergic regulation of basal and stimulated expression of neuropeptide mRNA is processed within the striatum. Keywords: Dynorphin; Substance P; Enkephalin; Scopolamine; Oxotremorine; Caudoputamen; Accumbens; Behavior
1. Introduction The role of muscarinic cholinergic neurotransmission in the regulation of striatal gene expression has been extensively investigated recently in this laboratory and others. Increasing evidence supports the concept that acetylcholine generally opposes the actions of dopamine in a dynamic balance that controls gene expression in striatal neurons w10,36x. For example, stimulation of D1 dopamine receptors on striatonigral neurons by the indirect dopamine receptor agonists, cocaine or amphetamine ŽAMPH., upregulates the expression of immediate early genes and the
) Corresponding author. Fax: q1 Ž919. 816-2850; E-mail: [email protected]
neuropeptides, preprodynorphin ŽPPD. and substance P ŽSP., contained in these neurons w4,16,25,37x. In contrast to the positive regulation by D 1 receptors, muscarinic receptor stimulation suppresses L-dopa-induced expression of c-fos mRNA w7x, constitutive SP mRNA w22x and AMPH-stimulated PPD and SP mRNA w38x in striatonigral neurons. Muscarinic receptor blockade induces striatal Fos protein w2x and SP and PPD mRNA w23,38x expression and potentiates D 1-stimulated immediate early gene Žc-fos and zif r 268 . and peptide ŽPPD and SP. mRNA induction in dopamine-lesioned w26,27x and intact w38–40x rats. Furthermore, chronic scopolamine augments, and chronic oxotremorine suppresses, dynorphin-like immunoreactivity in striatonigral neurons induced by the D 1rD 2 agonist, apomorphine w9x. Thus, muscarinic receptor stimulation opposes the action of D 1 receptor agonists on striatonigral
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 2 4 4 - 9
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gene expression. In striatopallidal neurons which contain preproenkephalin ŽPPE., D 2 receptor stimulation inhibits, whereas muscarinic receptor stimulation induces, Fos w2x and PPE mRNA w22,23,38x. Moreover, cholinergic facilitation is also implicated in D 2 antagonist-induced striatopallidal gene expression as demonstrated by a partial attenuation of D 2 antagonist-induced Fos w17x and enkephalin w18x immunoreactivity and PPE mRNA w30,40x in striatopallidal neurons after scopolamine pretreatment. It has been hypothesized that cholinergic interneurons in the striatum mediate the effects of muscarinic receptor ligands on D 1-stimulated striatal gene expression w10,38– 40x. However, all the above pharmacological studies were performed using systemic drug injections. Therefore, it was necessary to investigate the effects of central administration of muscarinic agents on striatal gene expression in order to identify the site of dopaminergic and cholinergic interactions. A recent report by Keefe and Gerfen w21x shows that intrastriatal injection of the D 1 antagonist, SCH-23390, is as effective as systemic injection in blocking D 1-stimulated striatal c-fos and zif r 268 mRNA in dopamine-lesioned rats. However, to date, no attempt has been made to evaluate the effects of central administration of muscarinic receptor agents on basal and stimulated peptide mRNA expression in striatonigral and striatopallidal neurons in intact rats. By utilizing quantitative in situ hybridization histochemistry, this study examined the influence of pharmacological manipulation of intrastriatal muscarinic receptor
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activity on striatonigral PPD and SP, and striatopallidal PPE, mRNA expression in normal and AMPH-stimulated rats. Local alteration of striatal muscarinic receptor activity was accomplished by direct injection of the non-selective muscarinic receptor antagonist, scopolamine, or the muscarinic agonist, oxotremorine, into the striatum.
2. Materials and methods 2.1. Animals Adult male Wistar rats Ž225–249 g, Charles River, Raleigh, NC. were individually housed and maintained on a 12:12 h lightrdark schedule with food and water provided ad libitum. A total of 60 rats were randomly assigned to 12 different groups Ž n s 5 per group.. All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. 2.2. Drug administration and behaÕior For intrastriatal infusion, the rats were anesthetized with 4% chloral hydrate Ž400 mgrkg, i.p.. and placed in a David Kopf stereotaxic holder. A 24-gauge stainless-steel guide cannula Ž10 mm in length. was implanted at the coordinates of 1 mm anterior to bregma, 2.5 mm right to
Fig. 1. Effects of scopolamine ŽA. and oxotremorine ŽB. on AMPH-stimulated behaviors. Intrastriatal injection Ž1 m l. of scopolamine Ž6.2 and 62 mM. or oxotremorine Ž1.6 and 8.1 mM. was made 15 min prior to i.p. injection of AMPH Ž2.5 mgrkg.. The behavioral ratings are expressed as mean " S.E.M. The numbers in the top legend of each panel represent the AUC values. ) P - 0.05 as compared with aCSF q saline, q P - 0.05 as compared with aCSFq AMPH.
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midline and 3 mm below the surface of the skull. The guide cannula was sealed by a stainless steel wire of the same length Ž10 mm.. Rats were allowed at least 5 days for recovery from surgery. One day prior to intrastriatal injection, the inner steel wire was replaced by a longer one Ž12.5 mm in length. that protruded 2.5 mm beyond the guide cannula. This procedure created a track for the injection cannula thereby minimizing acute induction of neuronal activity around the injection cannula on the day of the experiment w34x. The next day, this inner wire was replaced by a 30-gauge stainless-steel injection cannula with the same length Ž12.5 mm., through which artificial cerebrospinal fluid ŽaCSF. or 1.14 or 5.7 m g Ž1.6 or 8.1 mM, respectively. oxotremorine or 3 or 30 m g Ž6.2 or 62 mM, respectively. scopolamine were infused into the central part of the dorsal striatum in a volume of 1 m l at a rate of 0.2 m lrmin in freely moving animals. The progress of injection was monitored by observing the movement of a small air bubble through a length of precalibrated PE-10 tubing inserted between the injection cannula and a 5 m l Hamilton microsyringe. After completing each injection, the cannula was left in place for an additional 3 min to
reduce any possible backflow of the solution along the injection track. AMPH or saline was injected i.p. 15 min after the intrastriatal injection in a volume of 1.2 mlrkg. The behavior of the rats was rated by two trained raters, who were unaware of the treatment, 5 min before the intrastriatal injection and every 5 min after the i.p. injection for 1 h, using a 9-point scale as described w33x. 2.3. In situ hybridization histochemistry Three hours after the i.p. injection, the rats were anesthetized with Equithesin Ž5 mlrkg, i.p.. and decapitated. The brains were removed and frozen in isopentane at y408C and stored at y708C. Quantitative in situ hybridization histochemistry to measure PPD, SP and PPE mRNA expression in striatal neurons was performed according to standard procedures in this laboratory w35,41x. Quantitation of the mRNA hybridization signals on X-ray films was performed using NIH Image 1.44 ŽW. Rasband, NIMH. and a Macintosh IIci as described w35x. Briefly, the 14 C standards were measured, plotted against
Fig. 2. Dark-field photomicrographs from emulsion-dipped slides illustrating the effect of intrastriatal scopolamine and oxotremorine on constitutive and AMPH-stimulated PPD mRNA expression in the dorsal striatum. A: aCSFq saline; B: scopolamine Ž62 mM. q saline; C: oxotremorine Ž8.1 mM. q saline; D: aCSFq AMPH Ž2.5 mgrkg.; E: scopolamine Ž62 mM. q AMPH Ž2.5 mgrkg.; F: oxotremorine Ž8.1 mM. q AMPH Ž2.5 mgrkg.. Arrows mark intrastriatal injection tracks. Scale bar s 1 mm.
J.Q. Wang, J.F. McGintyr Brain Research 748 (1997) 62–70
known dpmrmg, and converted to 35 S equivalents to generate a calibration curve. Film background was measured and saved as a ‘blank field’ to correct uneven illumination. Under the density slice option, the hybridization signal in the entire dorsal striatum on the injected side was measured using a circle Ždiameters 200 pixels.. The dorsal striatal area corresponding to the cannula track was not subtracted from the measurement of the entire dorsal striatum because there was little difference among rats in the extent of damage in sections that were taken at the level of the cannula track. In several rats, artificial signals due to blood clots in the cannula track were subtracted to eliminate these non-specific signals. The signals in the nucleus accumbens ŽNAc. were measured using a manual drawing which outlined the shell and core areas. Quantitative changes were expressed as Ž1. the number of labeled pixels per area Žarea., Ž2. mean density of tissue in dpmrmg, and Ž3. integrated density which is the product of area times mean density as described w33,35x. The mean " S.E.M. of each of these measures was calculated for each rat by averaging the values in three adjacent sections. 2.4. Drugs D-Amphetamine sulfate, Žy.-scopolamine hydrobromide and oxotremorine sesquifumarate were purchased from Sigma Chemical Co. ŽSt. Louis, MO.. Scopolamine and oxotremorine were dissolved in aCSF ŽmM: NaCl 154, KCl 2.68, MgCl 2 0.9 and CaCl 2 1.22, pH 7.4.. Doses of scopolamine and oxotremorine were changed by altering concentration but not volume. AMPH was dissolved in 0.9% saline. The dose of AMPH was calculated as the salt. The mM concentrations of scopolamine and oxotremorine refer to the free bases. All drugs were freshly prepared.
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AMPH Ž2.5 mgrkg. significantly increased behavioral activity which included sniffing, exploring, and rearing ŽFig. 1A.. The two concentrations of scopolamine significantly augmented the behavioral responses to AMPH ŽFig. 1A.. The augmentation was concentration-dependent as the higher concentration of scopolamine Ž62 mM. caused sustained maximal stereotypy Žcontinuous sniffing and rearing in one location of cage. 25 min after AMPH ŽFig. 1A.. Although systemic injection of oxotremorine causes typical cholinergic symptoms which include tremor, salivation and lacrimation w38x, these symptoms were not displayed after unilateral intrastriatal injection of oxotremorine at either concentration Ž1.6 and 8.1 mM.. Neither was there a significant effect of oxotremorine on spontaneous behavioral activity ŽFig. 1B.. However, intrastriatal oxotremorine significantly attenuated AMPH-stimulated behaviors in a concentration-dependent manner ŽFig. 1B.. 3.2. Effects of intrastriatal scopolamine and oxotremorine on constitutiÕe and AMPH-stimulated PPD mRNA Intrastriatal scopolamine at the lower concentration Ž6.2 mM. had no significant effect on basal PPD expression in the dorsal striatum in the injected side Ždata not shown.. However, the higher concentration of scopolamine Ž62 mM. appeared to increase the PPD hybridization signal in patch-like clusters and, to a lesser extent, in the surrounding matrix, of the dorsal striatum ŽFig. 2, B vs. A.. Neither
2.5. Statistics A 2-way analysis of variance ŽANOVA. followed by a Bonferroni ŽDunn. comparison of groups using least squares-adjusted means was performed on the area under curve ŽAUC. values calculated from plotting behavioral ratings against time w33x. Significance in area, mean density and integrated density between groups was determined by a nested two-way ANOVA followed by a Bonferroni ŽDunn. comparison of groups using least squares-adjusted means.
3. Results 3.1. BehaÕioral obserÕations Unilateral intrastriatal injection of scopolamine at two concentrations Ž6.2 and 62 mM. did not significantly increase spontaneous behaviors ŽFig. 1A.. Acute injection of
Fig. 3. Quantitiative image analysis of effects of intrastriatal injection of scopolamine ŽA. and oxotremorine ŽB. on basal and AMPH-stimulated PPD mRNA in the striatum in terms of the intrgrated density measured over the entire dorsal striatum at the level of the cannula track. ) P - 0.05 as compared with aCSFqsaline, q P - 0.05 as compared with aCSFq AMPH Ž2.5 mgrkg..
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concentration of scopolamine affected PPD expression in the nucleus accumbens. Acute systemic AMPH produced typical patchy increases in PPD mRNA in the dorsal striatum ŽFig. 2D.. Intrastriatal pretreatment with 62 mM scopolamine followed by AMPH caused a greater induction of PPD mRNA in the dorsal striatum ŽFig. 2E. than after either 62 mM scopolamine or AMPH alone. Furthermore, scopolamine at 6.2 mM, which was ineffective in modifying basal PPD expression Žsee above., also enhanced AMPH-stimulated PPD mRNA signal in the dorsal striatum Ždata not shown.. The augmentation by the two concentrations of scopolamine occurred in patchy areas and in the surrounding matrix of the dorsal striatum. Intrastriatal oxotremorine at 1.6 and 8.1 ŽFig. 2C. mM had no visible effect on basal levels of PPD mRNA in the dorsal striatum. However, less induction of PPD mRNA hybridization signal in the dorsal striatum after AMPH was observed following intrastriatal pretreatment with oxotremorine at 8.1 mM ŽFig. 2F., but not after 1.6 mM, than after pretreatment with aCSF.
Fig. 3 illustrates quantitation of the PPD mRNA hybridization signal in the dorsal striatum. Alterations in integrated density reflect a change in the mean density of hybridization signal and, to a larger extent, in the number of labeled particles per area. Scopolamine at 62 mM significantly increased basal levels of PPD mRNA ŽFig. 3A.. Scopolamine at both 6.2 and 62 mM significantly augmented AMPH-stimulated PPD expression ŽFig. 3A.. In contrast, 1.6 and 8.1 mM oxotremorine had no effect on basal PPD expression ŽFig. 3B.. Although 1.6 mM oxotremorine did not significantly attenuate AMPH-stimulated PPD expression, 8.1 mM oxotremorine completely blocked it ŽFig. 3B.. 3.3. Effects of intrastriatal scopolamine and oxotremorine on constitutiÕe and AMPH-stimulated SP mRNA Alterations in SP mRNA in the dorsal striatum paralleled those of PPD mRNA in response to scopolamine alone or scopolamineq AMPH, i.e., the higher concentration of scopolamine Ž62 mM. increased basal levels of SP
Fig. 4. Dark-field photomicrographs from emulsion-dipped slides illustrating the effect of intrastriatal scopolamine and oxotremorine on constitutive and AMPH-stimulated SP mRNA expression in the dorsal striatum. A: aCSF q saline; B: scopolamine Ž62 mM. q saline; C: oxotremorine Ž8.1 mM. q saline; D: aCSFq AMPH Ž2.5 mgrkg.; E: scopolamine Ž62 mM. q AMPH Ž2.5 mgrkg.; F: oxotremorine Ž8.1 mM. q AMPH Ž2.5 mgrkg.. Arrows mark intrastriatal injection tracks. Scale bar s 1 mm.
J.Q. Wang, J.F. McGintyr Brain Research 748 (1997) 62–70
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Oxotremorine at 1.6 and 8.1 mM ŽFig. 4C. did not affect the constitutive expression of SP mRNA in the dorsal striatum ŽFig. 5B.. However, both 1.6 and 8.1 mM oxotremorine ŽFig. 4F. abolished increases in striatal SP mRNA induced by systemic AMPH administration ŽFig. 5B.. 3.4. Effects of intrastriatal scopolamine and oxotremorine on constitutiÕe and AMPH-stimulated PPE mRNA
Fig. 5. Quantitiative image analysis of effects of intrastriatal injection of scopolamine ŽA. and oxotremorine ŽB. on basal and AMPH-stimulated SP mRNA in the striatum in terms of the intrgrated density measured over the entire dorsal striatum at the level of the cannula track. ) P - 0.05 as compared with aCSFqsaline, q P - 0.05 as compared with aCSFq AMPH Ž2.5 mgrkg..
Intrastriatal scopolamine Ž6.2 and 62 mM. had no detectable effect on basal levels of PPE mRNA in the dorsal striatum ŽFig. 6A.. Systemic AMPH produced a significant increase in PPE mRNA in the dorsal striatum as described previously w37x. Intrastriatal scopolamine at 6.2 mM did not affect AMPH-stimulated PPE expression ŽFig. 6A.. In contrast, 62 mM scopolamine totally prevented AMPHstimulated PPE expression in the dorsal striatum ŽFig. 6A.. Although oxotremorine at both concentrations Ž1.6 and 8.1 mM. tended to increase basal levels of, and augment AMPH-stimulated, PPE mRNA in the dorsal striatum, these effects did not reach statistical significance ŽFig. 6B..
4. Discussion mRNA ŽFig. 4B vs. 4A and Fig. 5A., and both 6.2 and 62 ŽFig. 4E. mM scopolamine augmented AMPH-stimulated SP mRNA ŽFig. 5A..
Fig. 6. Quantitiative image analysis of effects of intrastriatal injection of scopolamine ŽA. and oxotremorine ŽB. on basal and AMPH-stimulated PPE mRNA in the striatum in terms of the integrated density measured over the entire dorsal striatum at the level of the cannula track. ) P - 0.05 as compared with aCSFqsaline, q P - 0.05 as compared with aCSFq AMPH Ž2.5 mgrkg..
Our previous study established a regulatory role for muscarinic cholinergic transmission in the basal and dopamine agonist-stimulated expression of striatal neuropeptide genes following systemic administration of the muscarinic receptor agonist, oxotremorine, and the antagonist, scopolamine w38x. This study demonstrates that direct injection of these muscarinic agents into the striatum was as effective as systemic administration in regulating striatal peptide gene expression in normal and AMPH-treated rats. For instance, intrastriatal scopolamine increased constitutive, and augmented AMPH-stimulated, PPD and SP mRNA to the same extent as that observed after systemic scopolamine. Furthermore, intrastriatal scopolamine blocked AMPH-stimulated PPE mRNA expression. In contrast, intrastriatal, like systemic, injection of oxotremorine attenuated AMPH-stimulated PPD and SP mRNA, but did not significantly increase AMPH-stimulated PPE expression. Therefore, the muscarinic receptors regulating dopamine-stimulated striatonigral and striatopallidal peptide gene expression are most likely located in the striatum. Di Chiara and colleagues w10x have proposed a detailed model of intrastriatal cell-to-cell interactions between striatonigral and striatopallidal projection neurons and cholinergic interneurons in response to endogenous dopamine receptor stimulation, which is strongly supported by this study and others w38–40x. Briefly, stimulation of D 1 receptors located on striatonigral neurons results in PPD and SP mRNA expression as well as local SP release. SP then stimulates NK 1 receptors on cholinergic neurons, the pri-
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mary striatal neurons which express NK 1 receptors w1,13,15x, to increase striatal acetylcholine release. The released acetylcholine would in turn Ž1. inhibit PPDrSP gene expression in striatonigral neurons, and Ž2. facilitate PPE gene expression in striatopallidal neurons. These sequentially-occurring intercellular events suggest that cholinergic neurons are in a strategic position to play a buffer-like role in normalizing responses of their neighboring projection neurons to changes in dopaminergic activity w10,36x. However, until this study, demonstration that the striatum is the site of these acetylcholinerdopamine interactions was lacking. Nisenbaum et al. w29x reported that seven daily injections of systemic scopolamine prevented the 6-hydroxydopamine-induced elevation of PPE mRNA and reduction of SP mRNA in the striatum Žbut see w23x.. However, seven daily intrastriatal injections of scopolamine in concentrations ranging from 0.5 to 50 mM were not able to mimic this effect. They concluded that extrastriatal cholinergic receptors regulate striatal peptide gene expression in dopamine-depleted rats. This conclusion is different from the intrastriatal cholinergic regulation of striatal peptide gene expression under normal and dopamine-stimulated conditions demonstrated in this study. The possibility exists that intrastriatal cholinergic regulation in intact rats is altered so that extrastriatal cholinergic regulation dominates in dopamine-depleted rats. However, it is also possible that these authors did not use a high enough concentration of scopolamine to test their hypothesis because in the present study, the lower concentration of scopolamine Ž6.2 mM. did not affect AMPH-stimulated PPE mRNA whereas it did affect SP mRNA. Nonetheless, both intra- and extra-striatal cholinergic transmission is likely to contribute to the regulation of striatonigral and striatopallidal peptide gene expression. The subtype of muscarinic receptors mediating inhibition of striatonigral PPDrSP expression or facilitation of striatopallidal PPE expression remains to be determined. Since M 4 receptors are contained in most PPDrSP neurons w3,43x and are coupled to inhibition of cAMP formation w12,19x, which opposes D 1-stimulated cAMP formation, it is likely that this subtype mediates inhibition of striatonigral gene expression. The subtype most likely to mediate facilitation of striatopallidal gene expression is M 1 because this subtype is expressed by all striatopallidal neurons w3,43x and is coupled to stimulation of phosphoinositide turnover w19x. Interestingly, oxotremorine is a poor stimulator of phosphoinositide hydrolysis w14x which may explain its weak effects on striatopallidal neurons Žbut see below for an alternative explanation.. Unavailability of pharmacological agents with satisfactory subtype specificity hinders the ability to investigate the muscarinic subtypes involved in striatal gene expression. However, recently available muscarinic toxins which have much higher subtype selectivity than any other pharmacological compounds to date w20,31x provide an alternative avenue to
evaluate the relevant subtypes. Recently in this laboratory, muscarinic toxin 3 ŽMT3., a peptide toxin isolated from green mamba venom, which has a high selectivity for cloned m4 receptors w20x, was used in vivo to evaluate the contribution of M 4 receptors to cholinergic inhibition of PPDrSP gene expression. It was found that intrastriatal MT3 administration elevated basal, and augmented AMPH-stimulated, PPD and SP mRNA expression w42x similar to the effect of scopolamine. Thus, the M 4 receptor appears to be a subtype which mediates cholinergic inhibition of striatonigral PPDrSP gene expression. Two main striatal compartments, the patch Žstriosomes. and matrix, are distinguished by a number of biochemical markers, afferent and efferent connections, as well as functional differences. However, it is unclear whether the cholinergic regulation of peptide mRNA expression in striatal medium spiny neurons is compartment-specific because our analysis did not extend to this level. However, scopolamine has been found to induce c-fos immediate early gene expression in both striosomal and matrix neurons whereas oxotremorine induced c-fos predominantly in striosomal neurons w2x. If the effects of these drugs on PPDrSP and PPE expression in this study were similar, oxotremorine-induced PPE expression in striatopallidal neurons may be occurring in striosomes, which consist of only about 15% of striatal neurons. In this case, changes in PPE mRNA would be less detectable than scopolamine-induced PPDrSP expression which occurs in both compartments. The concentrations of the muscarinic agents injected intrastriatally fall within the dose range shown previously to be effective in modifying muscarinic receptor-mediated functions w6,8x. In fact, because the striatum encompasses a large area, actual tissue concentration of injected drugs should have been reduced substantially by diffusion. Moreover, both scopolamine and oxotremorine affected gene expression only in the injected dorsal striatum, but not in the ipsilateral nucleus accumbens, indicating that the diffusion of drugs used in this study was limited to the ipsilateral caudoputamen. It was somewhat unexpected that unilateral intrastriatal injection of muscarinic agents, which was anticipated to affect striatal function only on the injected side, altered the behavioral activity of AMPH-stimulated rats in a manner similar to systemic injections. Behavioral effects also have been seen after an AMPH injection into one side of the striatum although the unilateral injection produced oral stereotypy half as intense as that observed after bilateral injections w11x. Furthermore, in many other experimental conditions, bilateral effects have been frequently seen after unilateral manipulations of striatal functions w5,23,28x. The mechanism for this phenomenon is unclear. However, it is not likely because of diffusion of drugs to the uninjected side of the striatum because muscarinic agents had no significant effect on basal or AMPH-stimulated peptide gene expression in the contralateral side. Alternatively,
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unilateral modification of striatal function may affect motor activity by activating bilateral and commissural projections in sensorimotor cortical areas w24,32x. In summary, this study demonstrates that local blockade of striatal muscarinic receptors by intrastriatal injection of scopolamine augmented constitutive and AMPH-stimulated striatonigral PPD and SP gene expression in the dorsal striatum. Local activation of striatal muscarinic receptors by intrastriatal injection of oxotremorine blocked AMPH-stimulated PPD and SP gene expression. In addition, intrastriatal scopolamine blocked AMPH-stimulated striatopallidal PPE expression. These data are in good accordance with those observed after systemic injection of scopolamine and oxotremorine w38x. Thus, muscarinic cholinergic regulation of striatonigral and striatopallidal gene expression is most likely mediated by intrastriatal cholinergic interneurons in intact rats.
Acknowledgements This research was supported by DA03982. The authors wish to thank Denise C. Mayer, Jennifer S. Starling and William T. Bohler for their technical assistance.
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