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Serotonin 2A receptor mRNA levels in the neonatal dopamine-depleted rat striatum remain upregulated following suppression of serotonin hyperinnervation
Developmental Brain Research 116 Ž1999. 111–117 www.elsevier.comrlocaterbres
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Serotonin 2A receptor mRNA levels in the neonatal dopamine-depleted rat striatum remain upregulated following suppression of serotonin hyperinnervation Gregory J. Basura a , Paul D. Walker
a,b,)
a
b
Department of Anatomy and Cell Biology, Wayne State UniÕersity School of Medicine, 9352 Gordon H. Scott Hall, 540 East Canfield AÕenue, Detroit, MI 48201, USA Department of Psychiatry and BehaÕioral Neurosciences, The Cellular and Clinical Neurobiology Program, Wayne State UniÕersity School of Medicine, 540 East Canfield AÕenue, Detroit, MI 48201, USA Accepted 20 April 1999
Abstract Sixty days after bilateral dopamine ŽDA. depletion Ž) 98%. with 6-hydroxydopamine Ž6-OHDA. in neonatal rats, serotonin Ž5-HT. content doubled and 5-HT2A receptor mRNA expression rose 54% within the rostral striatum. To determine if striatal 5-HT2A receptor mRNA upregulation is dependent on increased 5-HT levels following DA depletion, neonatal rats received dual injections of 6-OHDA and 5,7-dihydroxytryptamine Ž5,7-DHT. which suppressed 5-HT content by approximately 90%. In these 6-OHDAr5,7-DHT-treated rats, striatal 5-HT2A receptor mRNA expression was still elevated Ž87% above vehicle controls.. Comparative analysis of 5-HT2C receptor mRNA expression yielded no significant changes in any experimental group. These results demonstrate that upregulated 5-HT2A receptor biosynthesis in the DA-depleted rat is not dependent on subsequent 5-HT hyperinnervation. q 1999 Elsevier Science B.V. All rights reserved. Keywords: 6-Hydroxydopamine; 5,7-Dihydroxytryptamine; In situ hybridization; Serotonin 2C receptor; Basal ganglia; Substantia nigra; Dorsal raphe
Severe dopamine ŽDA. depletion in the neonatal rodent results in the development of serotonin Ž5-HT. hyperinnervation characterized by increased 5-HT projections and terminals within the anterior striatum ŽA-STR . w2,5,17,20,24x as well as elevated 5-HT and 5-hydroxyindoleacetic acid Ž5-HIAA. content w2,25,28x. Sources of increased striatal 5-HT content originate from midbrain raphe cells which normally provide heavy innervation to the posterior striatum ŽP-STR. but sprout to hyperinnervate the DA-depleted A-STR w2,24x. Striatal 5-HT hyperinnervation has been further characterized by increased 5-HT uptake sites and capacity for stimulated 5-HT release w11x and metabolism w19x. An additional consequence of neonatal DA depletion is the postsynaptic upregulation of 5-HT2A receptors by striatal neurons. Normally, 5-HT innervation w27x and 5-HT2 receptors w1,31x are distributed more predominantly in the
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P-STR. However, following neonatal DA depletion, 5-HT2 receptor ligand binding with DOI w16,22x and 5-HT2A receptor mRNA levels w16x are markedly increased in the A-STR, suggesting that 5-HT hyperinnervation may contribute to 5-HT2A upregulation. Interestingly, chronic DA receptor agonism w16x prevents 5-HT2A receptor binding and mRNA level increases after neonatal 6-hydroxydopamine Ž6-OHDA. injection. Although this finding suggests that DA reduction triggers 5-HT2A receptor upregulation, the influence of elevated 5-HT following neonatal DA depletion has not been ruled out as a contributing factor. The present study sought to determine if increased 5-HT2A receptor mRNA expression in the DA-depleted striatum occurs in the absence of ‘‘sprouted’’ 5-HT projections. To test this, the development of the 5-HT hyperinnervation phenomenon was suppressed by co-infusing the 5-HT neurotoxin, 5,7-dihydroxytryptamine Ž5,7-DHT., into rat neonates that also received the DA neurotoxin 6-OHDA. For comparative purposes, striatal 5-HT2C receptor mRNA expression was also examined in all experimental groups.
0165-3806r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 9 . 0 0 0 6 6 - 8
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Sprague–Dawley rat litters ŽCharles River Laboratories. were used in these studies in accordance with the NIH Guide to the Care and Use of Laboratory Animals and institutional approval by Wayne State University Animal Investigation Committee. All drugs were suspended in a vehicle solution of 0.9% saline containing 0.1% ascorbic acid. The procedure for intracerebroventricular Ži.c.v.. injections of 6-OHDA was based on a previous neonatal lesion model w18x. On postnatal day 3, rat pups received subcutaneous Žs.c.. injections of desipramine Ž25 mgrkg, Sigma. 30 min prior to anesthetization by hypothermia to protect norepinephrine neurons. Using a modified surgicalrstereotaxic platform, vehicle, 6-OHDA Ž100 mg, RBI., 5,7-DHT Ž100 mg, RBI., or both 6-OHDAr5,7-DHT Ž100 mg each. were delivered over a 30-s period into each lateral ventricle Ž5 ml per ventricle. using a 10-ml Hamilton syringe equipped with a 30-gauge needle Ž"1.0 mm lateral, y2.5 mm ventral from exterior of skull relative to bregma w18x.. Sixty days following i.c.v. injection, rats were sacrificed by decapitation and brains removed and hemisected. One hemisphere was immersion fixed for 2 weeks at 48C in 4% paraformaldehyde for 5-HT2A and 5-HT2C receptor mRNA analysis by in situ hybridization. The A-STR and P-STR from the remaining hemisphere were separated at the level of the decussation of the anterior commissure using stereomicroscopic optics. Samples were frozen in liquid nitrogen and stored at y808C for monoamine analysis by high performance liquid chromatography with electrochemical detection ŽHPLC-ED. w4,15x. 5-HT2A and 5-HT2C mRNA levels were measured via in situ hybridization performed on free-floating brain sections. Coronal brain sections Ž50 mm. were obtained on a vibratome and stored at 48C in 4% paraformaldehyde, then transferred to 24-well culture plates Ž1 sectionrwell. and washed in 0.1 M PBS. Sections were pretreated Ž0.5 mlrwell. with 0.1% Triton X-100, proteinase-K Ž10 mgrml in 0.1 M TE at 378C. and acetylated Ž0.25% acetic anhydride in 0.1 M TEA at RT. prior to incubation Ž100 mlrwell. in prehybridization buffer Ž50% deionized formamide, 5 = SSPE, 10% dextran sulfate, 5 = Denhardt’s, 100 mgrml denatured herring testes DNA, 100 mgrml Escherichia coli tRNA, 10 mM DTT. at 528C for 1 h. Prehybridization buffer was replaced with the same buffer containing 35 S-labeled 5-HT2A or 5-HT2C antisense cRNA probes hydrolyzed to lengths of approximately 150 nt. 35 S-labeled 5-HT2A probes were transcribed from a 900-bp Pst1 fragment subcloned into a bluescript KS q plasmid ŽStratagene. representing 379–1275 bp of the 5-HT2A receptor cDNA w12x, while 5-HT2C probes were transcribed from a 1165 bp HindIII fragment subcloned into a bluescript KS q plasmid ŽStratagene. representing 1735–2900 bp of the 5-HT2C receptor cDNA w13x. Sections were hybridized with excess probe Ž1 = 10 6 c.p.m.rsection. for 16 h at 528C followed by 0.5 ml washes in 4 = SSPE Žtwice at RT., 2 = SSPE Žat 378C., 2 = SSPE containing
10 mgrml RNAse-A Žat 378C., and 1 = SSPE Žat 608C.. Sections were mounted out of 0.01 M PBS onto glass slides. Once air-dried, slides were exposed to b-max film ŽAmersham. so that all sections were represented on a single film. For each mRNA, three film exposures were obtained spanning 3–5 days. For presentation purposes, representative sections of the A-STR from each experimental group were pseudocolored ŽPaintshop Pro. to visualize 5-HT2A or 5-HT2C mRNA expression signal ŽFig. 1.. This involved assigning one of 16 colors to each of the 16 levels of gray within four sections taken from the same scanned b-Max film. This method prevented subjective coloring of any section since all sections on a given film would be equally affected. The resultant image demonstrates a qualitative color gradient reflective of the 5-HT2A and 5-HT2C mRNA expression density within one animal from each group. This procedure had no influence on subsequent quantitative analysis. b-Max films containing raw 5-HT2A or 5-HT2C receptor mRNA hybridization signals were scanned on a Molecular Dynamics Personal Densitometer and analyzed with ImageQuant software. Separate films for 5-HT2A or 5-HT2C receptor mRNAs from multiple exposures Ž3–5 days. were scanned individually and the data presented ŽFig. 2. for each respective receptor were derived from sections from the same film exposure. Data obtained from subsequent scans of different exposures replicated the results of Fig. 2. For 5-HT2A and 5-HT2C receptor mRNA analysis, the striatum was selected and outlined in each section from the same film with the experimenter blind to which group the section belonged. Once background was subtracted Žvalue taken from the corpus callosum which does not express 5-HT2A or 5-HT2C mRNA., a volume report was generated that contained optical density values per unit area. Values obtained from the volume report were analyzed using one-way ANOVA with Tukey–Kramer post-hoc test Žsignificant at p - 0.05. and data were then expressed as a percentage of vehicle-injected controls ŽFig. 2.. Sixty days following i.c.v. injection of 6-OHDA, striatal DA and DOPAC levels were significantly reduced while 5-HT and 5-HIAA levels were significantly elevated ŽTable 1.. Injection of 5,7-DHT reduced striatal 5-HT and 5-HIAA levels with no alterations in DA or DOPAC, while co-infusion of 6-OHDAr5,7-DHT significantly reduced striatal DA and DOPAC content as well as 5-HT and 5-HIAA levels ŽTable 1.. These results were consistent in both A-STR and P-STR. Following 6-OHDA injection, 5-HT2A mRNA levels were upregulated to 154.5% "16.07 of vehicle controls Ž p - 0.05. in the A-STR ŽFigs. 1 and 2A. while no changes Ž115.4% "10.21. occurred in the P-STR ŽFig. 2B.. Injection of 5,7-DHT did not alter 5-HT2A mRNA expression in either striata ŽFigs. 1 and 2A, B.. Interestingly, 5-HT2A mRNA levels remained elevated to 186.9% "10.63 Ž p - 0.05. in the A-STR ŽFigs. 1 and 2A. following co-infusion of both 6-OHDAr5,7-DHT, while no al-
G.J. Basura, P.D. Walkerr DeÕelopmental Brain Research 116 (1999) 111–117 Fig. 1. Pseudocolored images of brain sections, representative of A-STR at level of the nucleus accumbens. Colors demonstrate varying densities of 5-HT2A vs. 5-HT2C receptor mRNA expression following neonatal i.c.v. injection of vehicle, 6-OHDA, 5,7-DHT, or the co-infusion of 6-OHDAr5,7-DHT. Color gradient in order of decreasing optical density: black ) red ) orange) yellow ) green) blue ŽSTR sstriatum, CTX s cortex.. For each mRNA species, representative sections from each group were chosen from the same film. 113
114 G.J. Basura, P.D. Walkerr DeÕelopmental Brain Research 116 (1999) 111–117 Fig. 2. 5-HT2A and 5-HT2C receptor mRNA levels in the A-STR ŽA. and ŽC. and P-STR ŽB. and ŽD. respectively, 60 days following i.c.v. injection of vehicle Ž ns8., 6-OHDA Ž ns 7., 5,7-DHT Ž ns8., or co-infusion of 6-OHDAr5,7-DHT Ž ns8.. Values are based on O.D. volume reports compared between groups by One-way ANOVA and analyzed by Tukey’s post-hoc test with significance at p- 0.05 ŽU significant as compared to vehicle; c significant as compared to 5,7-DHT; ns s not significant compared to 6-OHDA.. All data for each respective mRNA species were derived from the same film exposure and expressed in bar graph form as a percentage of respective vehicle controls.
G.J. Basura, P.D. Walkerr DeÕelopmental Brain Research 116 (1999) 111–117
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Table 1 HPLC-ED quantitative levels of DA, DOPAC, 5-HT, and 5-HIAA levels in the A-STR vs. P-STR 60 days following neonatal i.c.v. injection of vehicle, 6-OHDA, 5,7-DHT, or the co-infusion of 6-OHDAr5,7-DHT. Values are nanogram monoamine or metabolite per milligram protein Žmean " S.E.M.. with percent of vehicle or 6-OHDA in parentheses. Differences between individual group means were determined using One-way ANOVA with Tukey–Kramer post-hoc comparisons Ž p - 0.05 compared to vehicleU or 6-OHDAc groups.
A-STR
P-STR
Group
DA
DOPAC
5-HT
5-HIAA
Vehicle % Veh 6-OHDA % Veh 5,7-DHT % Veh 6-OHDAr 5,7-DHT % Veh % 6-OHDA Vehicle % Veh 6-OHDA % Veh 5,7-DHT % Veh 6-OHDAr 5,7-DHT % Veh % 6-OHDA
173.0 " 13.4 Ž100.0 " 7.7. 2.32 " 0.60U Ž1.34 " 0.35. 193.9 " 19.9 Ž112.1 " 11.5. 3.65 " 0.99U Ž2.11 " 0.57. Ž156.9 " 44.1. 26.9 " 2.68 Ž100.0 " 9.93. 0.21 " 0.04U Ž0.77 " 0.16. 32.8 " 5.97 Ž121.5 " 22.1. 0.50 " 0.17U Ž1.85 " 0.63. Ž241.5 " 82.8.
22.9 " 2.3 Ž100.0 " 10.0. 0.66 " 0.11U Ž2.86 " 0.46. 22.2 " 1.88 Ž96.8 " 8.18. 0.95 " 0.32U Ž4.13 " 1.41. Ž144.5 " 52.3. 1.87 " 0.22 Ž100.0 " 11.6. 0.03 " 0.01U Ž1.86 " 0.69. 2.09 " 0.30 Ž111.7 " 15.8. 0.05 " 0.02U Ž2.74 " 1.28. Ž147.0 " 68.7.
5.95 " 0.50 Ž100.0 " 8.43. 13.46 " 0.92U Ž226.3 " 15.5. 0.62 " 0.24U Ž10.4 " 4.10. 1.42 " 0.41U Ž23.9 " 6.84. Ž10.6 " 3.03.c 2.43 " 0.39 Ž100.0 " 15.9. 4.43 " 0.55U Ž182.5 " 22.6. 0.10 " 0.02U Ž4.01 " 0.69. 0.40 " 0.19U c Ž16.5 " 7.65. Ž9.05 " 4.19.
9.40 " 0.73 Ž100.0 " 7.76. 14.58 " 0.84U Ž155.1 " 8.92. 0.78 " 0.31U Ž8.32 " 3.30. 1.91 " 0.58U Ž20.34 " 6.22. Ž13.1 " 3.98.c 2.66 " 0.22 Ž100.0 " 8.30. 3.89 " 0.38U Ž146.2 " 14.4. 0.18 " 0.06U Ž6.92 " 2.10. 0.52 " 0.18U c Ž19.5 " 6.87. Ž13.3 " 4.69.
terations Ž120.2% "9.08. were observed in the P-STR ŽFig. 2B.. Furthermore, no significant changes in striatal 5-HT2C mRNA levels were detected in any experimental group ŽFigs. 1 and 2C, D.. Although slight qualitative increases in 5-HT2A receptor mRNA levels in the 6OHDAr5,7-DHT group above that of the 6-OHDA lesion group seem apparent in some sections ŽFig. 1., subsequent quantitative analysis revealed no significant differences between 6-OHDA and 6-OHDAr5,7-DHT groups ŽFig. 2A.. Reductions in striatal DA and DOPAC, accompanied by increases in 5-HT and 5-HIAA levels in the present study, confirmed published data w2,25x on 5-HT hyperinnervation following neonatal DA depletion. Co-infusion of 5,7DHTr6-OHDA prevented the development of 5-HT ‘‘sprouting’’ by destroying 5-HT afferent projection neurons w3x. While this treatment did not completely deplete striatal 5-HT content, it effectively inhibited elevated 5-HT levels normally seen after neonatal 6-OHDA lesion, thereby suppressing the hyperinnervation phenomenon. In fact, when compared to the 6-OHDA group alone, 5-HT levels were reduced by approximately 90% similar to the effects of 5,7-DHT alone as compared to vehicle controls. Following neonatal DA depletion, increases in 5-HT2A receptor mRNA levels were restricted to the A-STR, confirming previously reported data w16x. In addition, striatal 5-HT2C receptor mRNA expression levels were unaltered in any group confirming prior observations in an adult 6-OHDA lesion model w21x. Interestingly, the increases in 5-HT2A mRNA levels were maintained in the absence of 5-HT hyperinnervation, indicating that upregulated 5-HT2A receptors are not the result of the ‘‘sprouting’’ phenomenon. This conclusion is supported by infusion of
5,7-DHT alone, which reduced striatal 5-HT levels, yet did not alter striatal 5-HT2A mRNA levels in the present study, or whole brain 5-HT2 receptor binding w6x. Moreover, despite increased terminals w5,20x and 5-HT content w2,25x, basal extracellular levels of 5-HT are not significantly elevated following 5-HT hyperinnervation w11x, an observation attributed to increased 5-HT reuptake sites w19x and elevated 5-HT1B autoreceptors w22x. This suggests that 5-HT2A receptors located on striatal neurons are not exposed to increasing amounts of 5-HT in the DA-depleted rat under basal conditions. Therefore, anatomically and biochemically, striatal 5-HT hyperinnervation does not seem to influence 5-HT2A receptors as compared to normal 5-HT innervation patterns. This idea is further supported by increased striatal 5-HT2 mRNA levels following 6OHDA lesion of adult rodent nigrostriatal DA neurons w21x where raphestriatal 5-HT sprouting is less consistent w14,24–26,32,33x. Maintenance of 5-HT2A mRNA upregulation following suppression of 5-HT hyperinnervation suggests that DA normally inhibits these receptors, which become upregulated following 6-OHDA lesion. This is supported by previously reported increases in striatal 5-HT2A mRNA levels and DOI binding following neonatal DA depletion that were prevented after the chronic application of the general DA receptor agonist apomorphine or the D 1 receptor agonist, SKF-38393 w16x. Furthermore, this effect was blocked with the D 1 receptor antagonist, SCH-23390, suggesting a D 1 receptor-mediated regulation of 5-HT2A receptors w16x. Maintenance of 5-HT2A mRNA upregulation following co-infusion of 6-OHDAr5,7-DHT indicates that a majority of 5-HT2A receptors are not located within DA- or 5-HT-
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G.J. Basura, P.D. Walkerr DeÕelopmental Brain Research 116 (1999) 111–117
containing neurons. Rather, 5-HT2A as well as 5-HT2C receptor mRNAs have been co-localized with preprotachykinin ŽPPT. and preproenkephalin ŽPPE. mRNA expressing cells comprising direct and indirect striatal output pathways, respectively w31x. Interestingly, following neonatal DA depletion, upregulated 5-HT2A receptor mRNA was found primarily in non PPE-containing neurons w16x, suggesting localization in PPT neurons which express D 1 receptors w7x. Therefore, upregulated 5-HT2A receptors following neonatal DA depletion may result from removal of D 1-mediated inhibition of 5-HT2A receptors in PPT neurons and not from 5-HT hyperinnervation. The presence of increased 5-HT2A receptors in the A-STR following removal of DA transmission may provide a new experimental avenue to manipulate striatal PPT mRNA levels which decline after DA depletion w23x. Although 5-HT2 receptors appear positively coupled to striatal PPT mRNA regulation w8,10,29,30x, effects are largely observed in the P-STR w29x where 5-HT2A receptors are predominant w1,31x. Interestingly, normally unresponsive PPT mRNA levels in the A-STR become sensitive to 5-HT2 receptor agonism in the DA-depleted adult rat w10x. In addition, a synergistic interaction between 5-HT2 and D 1 receptors has been observed in the regulation of PPT mRNA levels in the dorsomedial striatum in the adult model of DA depletion w9x. The absence of 5-HT2C receptor mRNA level alterations following neonatal or adult w21x DA depletion further highlights a strengthened 5-HT2A receptor-mediated linkage to striatal neuropeptide gene regulation when DA is lost. Additional studies are needed to determine the significance of 5-HT2A receptor stimulation on neuropeptide gene regulation in the DA-depleted A-STR.
Acknowledgements The authors thank Dr. Bryan Roth, Case Western Reserve University, for providing the 5-HT2A and 5-HT2C receptor cDNAs and Dr. Leon Carlock and Ms. Christine Perry, Wayne State University School of Medicine, for technical assistance. Supported by NS30550 ŽP.D.W.. and WSU research grants.
References w1x C.A. Altar, W.C. Boyar, M.R. Marien, 125 I-LSD autoradiography confirms the preferential localization of caudate–putamen S 2 receptors to the caudal Žperipallidal. region, Brain Res. 372 Ž1986. 130–136. w2x T.W. Berger, S. Kaul, E.M. Stricker, M.J. Zigmond, Hyperinnervation of the striatum by dorsal raphe afferents after dopamine-depleting brain lesions in neonatal rats, Brain Res. 336 Ž1985. 354–358. w3x G.R. Breese, B.R. Cooper, Behavioral and biochemical interactions of 5,7-dihydroxytryptamine with various drugs when administered intracisternally to adult and developing rats, Brain Res. 98 Ž1975. 517–527.
w4x T.J. Cummings, P.D. Walker, Serotonin depletion exacerbates changes in striatal gene expression following quinolinic acid injection, Brain Res. 743 Ž1996. 240–248. w5x L. Descarries, J.-J. Soghomonian, S. Garcia, G. Doucet, J.P. Bruno, Ultrastructural analysis of the serotonin hyperinnervation in adult rat neostriatum following neonatal dopamine denervation with 6-hydroxydopamine, Brain Res. 569 Ž1992. 1–13. w6x C.T. Fischette, B. Nock, K. Renner, Effects of 5,7-dihydroxytryptamine on serotonin 1 and serotonin 2 receptors throughout the rat central nervous system using quantitative autoradiography, Brain Res. 421 Ž1987. 263–279. w7x C.R. Gerfen, T.M. Engber, L.C. Mahan, Z. Susel, T.N. Chase, F.J. Monsma, D.R. Sibley, D1 and D 2 dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons, Science 250 Ž1990. 1429–1432. w8x P.J. Gresch, P.D. Walker, Acute p-chloroamphetamine increases striatal preprotachykinin mRNA: role of the serotonin 2Ar2C receptor, Mol. Brain Res. 67 Ž1999. 190–193. w9x P.J. Gresch, P.D. Walker, Synergistic interaction between serotonin-2 receptor and dopamine D1 receptor stimulation on striatal preprotachykinin mRNA expression in the 6-hydroxydopamine lesioned rat, Mol. Brain Res. Ž1999. in press. w10x P.J. Gresch, P.D. Walker, Serotonin-2 receptor stimulation normalizes striatal preprotachykinin mRNA in an animal model of Parkinson’s disease, Neuroscience Ž1999. in press. w11x D. Jackson, E.D. Abercrombie, In vivo neurochemical evaluation of striatal serotonergic hyperinnervation in rats depleted of dopamine at infancy, J. Neurochem. 58 Ž1992. 890–897. w12x D. Julius, K.N. Huang, T.J. Livelle, R. Axel, T.M. Jessell, The 5-HT2 receptor defines a family of structurally distinct but functionally conserved serotonin receptors, Proc. Natl. Acad. Sci. U.S.A. 87 Ž1990. 928–932. w13x D. Julius, A.B. MacDermott, R. Axel, T.M. Jessell, Molecular characterization of a functional cDNA encoding the serotonin 1c receptor, Science 241 Ž1988. 558–564. w14x P.J. Karstaedt, H. Kerasidis, J.H. Pincus, R. Meloni, J. Graham, K. Gale, Unilateral destruction of dopamine pathways increases ipsilateral striatal serotonin turnover in rats, Exp. Neurol. 126 Ž1994. 25–30. w15x I.C. Kilpatrick, M.W. Jones, O.T. Phillipson, A semiautomated analysis method for catecholamines, indolamines, and some prominent metabolites in microdissected regions of the nervous system: an isocratic HPLC technique employing coulometric detection and minimal sample preparation, J. Neurochem. 46 Ž1986. 1865–1876. w16x N. Laprade, F. Radja, T.A. Reader, J.-J. Soghomonian, Dopamine receptor agonists regulate levels of the serotonin 5-HT2A receptor and its mRNA in a subpopulation of rat striatal neurons, J. Neurosci. 16 Ž1996. 3727–3736. w17x J. Luthman, B. Bolioli, T. Tsutsumi, A. Verhofstad, G. Jonsson, Sprouting of striatal serotonin nerve terminals following selective lesions of nigrostriatal dopamine neurons in neonatal rats, Brain Res. Bull. 19 Ž1987. 269–274. w18x J. Luthman, E. Brodin, E. Sundstrom, B. Wiehager, Studies on brain monoamine and neuropeptide systems after neonatal intracerebroventricular 6-hydroxydopamine treatment, Int. J. Dev. Neurosci. 8 Ž1990. 549–560. w19x E. Molina-Holgado, K.M. Dewar, L. Descarries, T.A. Reader, Altered dopamine and serotonin metabolism in the dopamine-denervated and serotonin-hyperinnervated neostriatum of adult rat after neonatal 6-hydroxydopamine, J. Pharmacol. Exp. Ther. 270 Ž1994. 713–721. w20x A. Mrini, J.P. Soucy, F. Lafaille, P. Lemoine, L. Descarries, Quantification of the serotonin hyperinnervation in adult rat neostriatum after neonatal 6-hydroxydopamine lesion of nigral dopamine neurons, Brain Res. 669 Ž1995. 303–308. w21x S. Numan, K.H. Lundgren, D.E. Wright, J.P. Herman, K.B. Seroogy, Increased expression of 5-HT2 receptor mRNA in rat striatum
G.J. Basura, P.D. Walkerr DeÕelopmental Brain Research 116 (1999) 111–117
w22x
w23x
w24x
w25x
w26x
w27x
following 6-OHDA lesions of the adult nigrostriatal pathway, Mol. Brain. Res. 29 Ž1995. 391–396. F. Radja, L. Descarries, K. Dewar, T. Reader, Serotonin 5-HT1 and 5-HT2 receptors in adult rat brain after neonatal destruction of nigrostriatal dopamine neurons: a quantitative autoradiographic study, Brain Res. 606 Ž1993. 273–285. S.P. Sivam, G.R. Breese, J.E. Krause, T.C. Napier, R.A. Mueller, J.-S. Hong, Neonatal and adult 6-hydroxydopamine-induced lesions differentially alter tachykinin and enkephalin gene expression, J. Neurochem. 49 Ž1987. 1623–1633. A.M. Snyder, M.J. Zigmond, R. Lund, Sprouting of serotonergic afferents into striatum after dopamine-depleting lesions in infant rats: a retrograde transport and immunocytochemical study, J. Comp. Neurol. 245 Ž1986. 274–281. M.K. Stachowiak, J.P. Bruno, A.M. Snyder, E.M. Stricker, M.J. Zigmond, Apparent sprouting of striatal serotonergic terminals after dopamine-depleting lesions in neonatal rats, Brain Res. 291 Ž1984. 164–167. Y. Takeuchi, T. Sawada, S. Blunt, P. Jenner, C.D. Marsden, Effects of 6-hydroxydopamine lesions of the nigrostriatal pathway on striatal serotonin innervation in adult rats, Brain Res. 562 Ž1991. 301–305. J.P. Ternaux, F. Hery, S. Bourgoin, J. Adrien, J. Glowinski, M. Hamon, The topographical distribution of serotonergic terminals in
w28x
w29x
w30x
w31x
w32x
w33x
117
the neostriatum of the rat and caudate nucleus of the cat, Brain Res. 121 Ž1977. 311–326. A.C. Towle, H.E. Criswell, E.H. Maynard, J.M. Lauder, T.H. Joh, R.A. Mueller, G.R. Breese, Serotonergic innervation of the rat caudate following a neonatal 6-hydroxydopamine lesion: an anatomical, biochemical and pharmacological study, Pharmacol. Biochem. Behav. 34 Ž1989. 367–374. P.D. Walker, J.J. Capodilupo, W.A. Wolf, L.R. Carlock, Preprotachykinin and preproenkephalin mRNA expression within striatal subregions in response to altered serotonin transmission, Brain Res. 732 Ž1996. 25–35. P.D. Walker, L.A. Riley, R.P. Hart, G.M. Jonakait, Serotonin regulation of tachykinin biosynthesis in the rat neostriatum, Brain Res. 546 Ž1991. 33–39. R.W. Ward, D.M. Dorsa, Colocalization of serotonin receptor subtypes 5-HT2A , 5-HT2C , and 5-HT6 with neuropeptide mRNAs in the rat forebrain, J. Comp. Neurol. 370 Ž1996. 405–414. P.T.-H. Wong, W.L. Teo, T. Lee, A microdialysis study on striatal dopamine, 5-HT and metabolites in conscious rats after various treatments: evidence for extravesicular release of dopamine, NeuroReport 5 Ž1993. 53–56. F.C. Zhou, S. Bledsoe, J. Murphy, Serotonergic sprouting is induced by dopamine lesion in substantia nigra of adult rat brain, Brain Res. 556 Ž1991. 108–116.
Short communication
Serotonin 2A receptor mRNA levels in the neonatal dopamine-depleted rat striatum remain upregulated following suppression of serotonin hyperinnervation Gregory J. Basura a , Paul D. Walker
a,b,)
a
b
Department of Anatomy and Cell Biology, Wayne State UniÕersity School of Medicine, 9352 Gordon H. Scott Hall, 540 East Canfield AÕenue, Detroit, MI 48201, USA Department of Psychiatry and BehaÕioral Neurosciences, The Cellular and Clinical Neurobiology Program, Wayne State UniÕersity School of Medicine, 540 East Canfield AÕenue, Detroit, MI 48201, USA Accepted 20 April 1999
Abstract Sixty days after bilateral dopamine ŽDA. depletion Ž) 98%. with 6-hydroxydopamine Ž6-OHDA. in neonatal rats, serotonin Ž5-HT. content doubled and 5-HT2A receptor mRNA expression rose 54% within the rostral striatum. To determine if striatal 5-HT2A receptor mRNA upregulation is dependent on increased 5-HT levels following DA depletion, neonatal rats received dual injections of 6-OHDA and 5,7-dihydroxytryptamine Ž5,7-DHT. which suppressed 5-HT content by approximately 90%. In these 6-OHDAr5,7-DHT-treated rats, striatal 5-HT2A receptor mRNA expression was still elevated Ž87% above vehicle controls.. Comparative analysis of 5-HT2C receptor mRNA expression yielded no significant changes in any experimental group. These results demonstrate that upregulated 5-HT2A receptor biosynthesis in the DA-depleted rat is not dependent on subsequent 5-HT hyperinnervation. q 1999 Elsevier Science B.V. All rights reserved. Keywords: 6-Hydroxydopamine; 5,7-Dihydroxytryptamine; In situ hybridization; Serotonin 2C receptor; Basal ganglia; Substantia nigra; Dorsal raphe
Severe dopamine ŽDA. depletion in the neonatal rodent results in the development of serotonin Ž5-HT. hyperinnervation characterized by increased 5-HT projections and terminals within the anterior striatum ŽA-STR . w2,5,17,20,24x as well as elevated 5-HT and 5-hydroxyindoleacetic acid Ž5-HIAA. content w2,25,28x. Sources of increased striatal 5-HT content originate from midbrain raphe cells which normally provide heavy innervation to the posterior striatum ŽP-STR. but sprout to hyperinnervate the DA-depleted A-STR w2,24x. Striatal 5-HT hyperinnervation has been further characterized by increased 5-HT uptake sites and capacity for stimulated 5-HT release w11x and metabolism w19x. An additional consequence of neonatal DA depletion is the postsynaptic upregulation of 5-HT2A receptors by striatal neurons. Normally, 5-HT innervation w27x and 5-HT2 receptors w1,31x are distributed more predominantly in the
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P-STR. However, following neonatal DA depletion, 5-HT2 receptor ligand binding with DOI w16,22x and 5-HT2A receptor mRNA levels w16x are markedly increased in the A-STR, suggesting that 5-HT hyperinnervation may contribute to 5-HT2A upregulation. Interestingly, chronic DA receptor agonism w16x prevents 5-HT2A receptor binding and mRNA level increases after neonatal 6-hydroxydopamine Ž6-OHDA. injection. Although this finding suggests that DA reduction triggers 5-HT2A receptor upregulation, the influence of elevated 5-HT following neonatal DA depletion has not been ruled out as a contributing factor. The present study sought to determine if increased 5-HT2A receptor mRNA expression in the DA-depleted striatum occurs in the absence of ‘‘sprouted’’ 5-HT projections. To test this, the development of the 5-HT hyperinnervation phenomenon was suppressed by co-infusing the 5-HT neurotoxin, 5,7-dihydroxytryptamine Ž5,7-DHT., into rat neonates that also received the DA neurotoxin 6-OHDA. For comparative purposes, striatal 5-HT2C receptor mRNA expression was also examined in all experimental groups.
0165-3806r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 9 . 0 0 0 6 6 - 8
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Sprague–Dawley rat litters ŽCharles River Laboratories. were used in these studies in accordance with the NIH Guide to the Care and Use of Laboratory Animals and institutional approval by Wayne State University Animal Investigation Committee. All drugs were suspended in a vehicle solution of 0.9% saline containing 0.1% ascorbic acid. The procedure for intracerebroventricular Ži.c.v.. injections of 6-OHDA was based on a previous neonatal lesion model w18x. On postnatal day 3, rat pups received subcutaneous Žs.c.. injections of desipramine Ž25 mgrkg, Sigma. 30 min prior to anesthetization by hypothermia to protect norepinephrine neurons. Using a modified surgicalrstereotaxic platform, vehicle, 6-OHDA Ž100 mg, RBI., 5,7-DHT Ž100 mg, RBI., or both 6-OHDAr5,7-DHT Ž100 mg each. were delivered over a 30-s period into each lateral ventricle Ž5 ml per ventricle. using a 10-ml Hamilton syringe equipped with a 30-gauge needle Ž"1.0 mm lateral, y2.5 mm ventral from exterior of skull relative to bregma w18x.. Sixty days following i.c.v. injection, rats were sacrificed by decapitation and brains removed and hemisected. One hemisphere was immersion fixed for 2 weeks at 48C in 4% paraformaldehyde for 5-HT2A and 5-HT2C receptor mRNA analysis by in situ hybridization. The A-STR and P-STR from the remaining hemisphere were separated at the level of the decussation of the anterior commissure using stereomicroscopic optics. Samples were frozen in liquid nitrogen and stored at y808C for monoamine analysis by high performance liquid chromatography with electrochemical detection ŽHPLC-ED. w4,15x. 5-HT2A and 5-HT2C mRNA levels were measured via in situ hybridization performed on free-floating brain sections. Coronal brain sections Ž50 mm. were obtained on a vibratome and stored at 48C in 4% paraformaldehyde, then transferred to 24-well culture plates Ž1 sectionrwell. and washed in 0.1 M PBS. Sections were pretreated Ž0.5 mlrwell. with 0.1% Triton X-100, proteinase-K Ž10 mgrml in 0.1 M TE at 378C. and acetylated Ž0.25% acetic anhydride in 0.1 M TEA at RT. prior to incubation Ž100 mlrwell. in prehybridization buffer Ž50% deionized formamide, 5 = SSPE, 10% dextran sulfate, 5 = Denhardt’s, 100 mgrml denatured herring testes DNA, 100 mgrml Escherichia coli tRNA, 10 mM DTT. at 528C for 1 h. Prehybridization buffer was replaced with the same buffer containing 35 S-labeled 5-HT2A or 5-HT2C antisense cRNA probes hydrolyzed to lengths of approximately 150 nt. 35 S-labeled 5-HT2A probes were transcribed from a 900-bp Pst1 fragment subcloned into a bluescript KS q plasmid ŽStratagene. representing 379–1275 bp of the 5-HT2A receptor cDNA w12x, while 5-HT2C probes were transcribed from a 1165 bp HindIII fragment subcloned into a bluescript KS q plasmid ŽStratagene. representing 1735–2900 bp of the 5-HT2C receptor cDNA w13x. Sections were hybridized with excess probe Ž1 = 10 6 c.p.m.rsection. for 16 h at 528C followed by 0.5 ml washes in 4 = SSPE Žtwice at RT., 2 = SSPE Žat 378C., 2 = SSPE containing
10 mgrml RNAse-A Žat 378C., and 1 = SSPE Žat 608C.. Sections were mounted out of 0.01 M PBS onto glass slides. Once air-dried, slides were exposed to b-max film ŽAmersham. so that all sections were represented on a single film. For each mRNA, three film exposures were obtained spanning 3–5 days. For presentation purposes, representative sections of the A-STR from each experimental group were pseudocolored ŽPaintshop Pro. to visualize 5-HT2A or 5-HT2C mRNA expression signal ŽFig. 1.. This involved assigning one of 16 colors to each of the 16 levels of gray within four sections taken from the same scanned b-Max film. This method prevented subjective coloring of any section since all sections on a given film would be equally affected. The resultant image demonstrates a qualitative color gradient reflective of the 5-HT2A and 5-HT2C mRNA expression density within one animal from each group. This procedure had no influence on subsequent quantitative analysis. b-Max films containing raw 5-HT2A or 5-HT2C receptor mRNA hybridization signals were scanned on a Molecular Dynamics Personal Densitometer and analyzed with ImageQuant software. Separate films for 5-HT2A or 5-HT2C receptor mRNAs from multiple exposures Ž3–5 days. were scanned individually and the data presented ŽFig. 2. for each respective receptor were derived from sections from the same film exposure. Data obtained from subsequent scans of different exposures replicated the results of Fig. 2. For 5-HT2A and 5-HT2C receptor mRNA analysis, the striatum was selected and outlined in each section from the same film with the experimenter blind to which group the section belonged. Once background was subtracted Žvalue taken from the corpus callosum which does not express 5-HT2A or 5-HT2C mRNA., a volume report was generated that contained optical density values per unit area. Values obtained from the volume report were analyzed using one-way ANOVA with Tukey–Kramer post-hoc test Žsignificant at p - 0.05. and data were then expressed as a percentage of vehicle-injected controls ŽFig. 2.. Sixty days following i.c.v. injection of 6-OHDA, striatal DA and DOPAC levels were significantly reduced while 5-HT and 5-HIAA levels were significantly elevated ŽTable 1.. Injection of 5,7-DHT reduced striatal 5-HT and 5-HIAA levels with no alterations in DA or DOPAC, while co-infusion of 6-OHDAr5,7-DHT significantly reduced striatal DA and DOPAC content as well as 5-HT and 5-HIAA levels ŽTable 1.. These results were consistent in both A-STR and P-STR. Following 6-OHDA injection, 5-HT2A mRNA levels were upregulated to 154.5% "16.07 of vehicle controls Ž p - 0.05. in the A-STR ŽFigs. 1 and 2A. while no changes Ž115.4% "10.21. occurred in the P-STR ŽFig. 2B.. Injection of 5,7-DHT did not alter 5-HT2A mRNA expression in either striata ŽFigs. 1 and 2A, B.. Interestingly, 5-HT2A mRNA levels remained elevated to 186.9% "10.63 Ž p - 0.05. in the A-STR ŽFigs. 1 and 2A. following co-infusion of both 6-OHDAr5,7-DHT, while no al-
G.J. Basura, P.D. Walkerr DeÕelopmental Brain Research 116 (1999) 111–117 Fig. 1. Pseudocolored images of brain sections, representative of A-STR at level of the nucleus accumbens. Colors demonstrate varying densities of 5-HT2A vs. 5-HT2C receptor mRNA expression following neonatal i.c.v. injection of vehicle, 6-OHDA, 5,7-DHT, or the co-infusion of 6-OHDAr5,7-DHT. Color gradient in order of decreasing optical density: black ) red ) orange) yellow ) green) blue ŽSTR sstriatum, CTX s cortex.. For each mRNA species, representative sections from each group were chosen from the same film. 113
114 G.J. Basura, P.D. Walkerr DeÕelopmental Brain Research 116 (1999) 111–117 Fig. 2. 5-HT2A and 5-HT2C receptor mRNA levels in the A-STR ŽA. and ŽC. and P-STR ŽB. and ŽD. respectively, 60 days following i.c.v. injection of vehicle Ž ns8., 6-OHDA Ž ns 7., 5,7-DHT Ž ns8., or co-infusion of 6-OHDAr5,7-DHT Ž ns8.. Values are based on O.D. volume reports compared between groups by One-way ANOVA and analyzed by Tukey’s post-hoc test with significance at p- 0.05 ŽU significant as compared to vehicle; c significant as compared to 5,7-DHT; ns s not significant compared to 6-OHDA.. All data for each respective mRNA species were derived from the same film exposure and expressed in bar graph form as a percentage of respective vehicle controls.
G.J. Basura, P.D. Walkerr DeÕelopmental Brain Research 116 (1999) 111–117
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Table 1 HPLC-ED quantitative levels of DA, DOPAC, 5-HT, and 5-HIAA levels in the A-STR vs. P-STR 60 days following neonatal i.c.v. injection of vehicle, 6-OHDA, 5,7-DHT, or the co-infusion of 6-OHDAr5,7-DHT. Values are nanogram monoamine or metabolite per milligram protein Žmean " S.E.M.. with percent of vehicle or 6-OHDA in parentheses. Differences between individual group means were determined using One-way ANOVA with Tukey–Kramer post-hoc comparisons Ž p - 0.05 compared to vehicleU or 6-OHDAc groups.
A-STR
P-STR
Group
DA
DOPAC
5-HT
5-HIAA
Vehicle % Veh 6-OHDA % Veh 5,7-DHT % Veh 6-OHDAr 5,7-DHT % Veh % 6-OHDA Vehicle % Veh 6-OHDA % Veh 5,7-DHT % Veh 6-OHDAr 5,7-DHT % Veh % 6-OHDA
173.0 " 13.4 Ž100.0 " 7.7. 2.32 " 0.60U Ž1.34 " 0.35. 193.9 " 19.9 Ž112.1 " 11.5. 3.65 " 0.99U Ž2.11 " 0.57. Ž156.9 " 44.1. 26.9 " 2.68 Ž100.0 " 9.93. 0.21 " 0.04U Ž0.77 " 0.16. 32.8 " 5.97 Ž121.5 " 22.1. 0.50 " 0.17U Ž1.85 " 0.63. Ž241.5 " 82.8.
22.9 " 2.3 Ž100.0 " 10.0. 0.66 " 0.11U Ž2.86 " 0.46. 22.2 " 1.88 Ž96.8 " 8.18. 0.95 " 0.32U Ž4.13 " 1.41. Ž144.5 " 52.3. 1.87 " 0.22 Ž100.0 " 11.6. 0.03 " 0.01U Ž1.86 " 0.69. 2.09 " 0.30 Ž111.7 " 15.8. 0.05 " 0.02U Ž2.74 " 1.28. Ž147.0 " 68.7.
5.95 " 0.50 Ž100.0 " 8.43. 13.46 " 0.92U Ž226.3 " 15.5. 0.62 " 0.24U Ž10.4 " 4.10. 1.42 " 0.41U Ž23.9 " 6.84. Ž10.6 " 3.03.c 2.43 " 0.39 Ž100.0 " 15.9. 4.43 " 0.55U Ž182.5 " 22.6. 0.10 " 0.02U Ž4.01 " 0.69. 0.40 " 0.19U c Ž16.5 " 7.65. Ž9.05 " 4.19.
9.40 " 0.73 Ž100.0 " 7.76. 14.58 " 0.84U Ž155.1 " 8.92. 0.78 " 0.31U Ž8.32 " 3.30. 1.91 " 0.58U Ž20.34 " 6.22. Ž13.1 " 3.98.c 2.66 " 0.22 Ž100.0 " 8.30. 3.89 " 0.38U Ž146.2 " 14.4. 0.18 " 0.06U Ž6.92 " 2.10. 0.52 " 0.18U c Ž19.5 " 6.87. Ž13.3 " 4.69.
terations Ž120.2% "9.08. were observed in the P-STR ŽFig. 2B.. Furthermore, no significant changes in striatal 5-HT2C mRNA levels were detected in any experimental group ŽFigs. 1 and 2C, D.. Although slight qualitative increases in 5-HT2A receptor mRNA levels in the 6OHDAr5,7-DHT group above that of the 6-OHDA lesion group seem apparent in some sections ŽFig. 1., subsequent quantitative analysis revealed no significant differences between 6-OHDA and 6-OHDAr5,7-DHT groups ŽFig. 2A.. Reductions in striatal DA and DOPAC, accompanied by increases in 5-HT and 5-HIAA levels in the present study, confirmed published data w2,25x on 5-HT hyperinnervation following neonatal DA depletion. Co-infusion of 5,7DHTr6-OHDA prevented the development of 5-HT ‘‘sprouting’’ by destroying 5-HT afferent projection neurons w3x. While this treatment did not completely deplete striatal 5-HT content, it effectively inhibited elevated 5-HT levels normally seen after neonatal 6-OHDA lesion, thereby suppressing the hyperinnervation phenomenon. In fact, when compared to the 6-OHDA group alone, 5-HT levels were reduced by approximately 90% similar to the effects of 5,7-DHT alone as compared to vehicle controls. Following neonatal DA depletion, increases in 5-HT2A receptor mRNA levels were restricted to the A-STR, confirming previously reported data w16x. In addition, striatal 5-HT2C receptor mRNA expression levels were unaltered in any group confirming prior observations in an adult 6-OHDA lesion model w21x. Interestingly, the increases in 5-HT2A mRNA levels were maintained in the absence of 5-HT hyperinnervation, indicating that upregulated 5-HT2A receptors are not the result of the ‘‘sprouting’’ phenomenon. This conclusion is supported by infusion of
5,7-DHT alone, which reduced striatal 5-HT levels, yet did not alter striatal 5-HT2A mRNA levels in the present study, or whole brain 5-HT2 receptor binding w6x. Moreover, despite increased terminals w5,20x and 5-HT content w2,25x, basal extracellular levels of 5-HT are not significantly elevated following 5-HT hyperinnervation w11x, an observation attributed to increased 5-HT reuptake sites w19x and elevated 5-HT1B autoreceptors w22x. This suggests that 5-HT2A receptors located on striatal neurons are not exposed to increasing amounts of 5-HT in the DA-depleted rat under basal conditions. Therefore, anatomically and biochemically, striatal 5-HT hyperinnervation does not seem to influence 5-HT2A receptors as compared to normal 5-HT innervation patterns. This idea is further supported by increased striatal 5-HT2 mRNA levels following 6OHDA lesion of adult rodent nigrostriatal DA neurons w21x where raphestriatal 5-HT sprouting is less consistent w14,24–26,32,33x. Maintenance of 5-HT2A mRNA upregulation following suppression of 5-HT hyperinnervation suggests that DA normally inhibits these receptors, which become upregulated following 6-OHDA lesion. This is supported by previously reported increases in striatal 5-HT2A mRNA levels and DOI binding following neonatal DA depletion that were prevented after the chronic application of the general DA receptor agonist apomorphine or the D 1 receptor agonist, SKF-38393 w16x. Furthermore, this effect was blocked with the D 1 receptor antagonist, SCH-23390, suggesting a D 1 receptor-mediated regulation of 5-HT2A receptors w16x. Maintenance of 5-HT2A mRNA upregulation following co-infusion of 6-OHDAr5,7-DHT indicates that a majority of 5-HT2A receptors are not located within DA- or 5-HT-
116
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containing neurons. Rather, 5-HT2A as well as 5-HT2C receptor mRNAs have been co-localized with preprotachykinin ŽPPT. and preproenkephalin ŽPPE. mRNA expressing cells comprising direct and indirect striatal output pathways, respectively w31x. Interestingly, following neonatal DA depletion, upregulated 5-HT2A receptor mRNA was found primarily in non PPE-containing neurons w16x, suggesting localization in PPT neurons which express D 1 receptors w7x. Therefore, upregulated 5-HT2A receptors following neonatal DA depletion may result from removal of D 1-mediated inhibition of 5-HT2A receptors in PPT neurons and not from 5-HT hyperinnervation. The presence of increased 5-HT2A receptors in the A-STR following removal of DA transmission may provide a new experimental avenue to manipulate striatal PPT mRNA levels which decline after DA depletion w23x. Although 5-HT2 receptors appear positively coupled to striatal PPT mRNA regulation w8,10,29,30x, effects are largely observed in the P-STR w29x where 5-HT2A receptors are predominant w1,31x. Interestingly, normally unresponsive PPT mRNA levels in the A-STR become sensitive to 5-HT2 receptor agonism in the DA-depleted adult rat w10x. In addition, a synergistic interaction between 5-HT2 and D 1 receptors has been observed in the regulation of PPT mRNA levels in the dorsomedial striatum in the adult model of DA depletion w9x. The absence of 5-HT2C receptor mRNA level alterations following neonatal or adult w21x DA depletion further highlights a strengthened 5-HT2A receptor-mediated linkage to striatal neuropeptide gene regulation when DA is lost. Additional studies are needed to determine the significance of 5-HT2A receptor stimulation on neuropeptide gene regulation in the DA-depleted A-STR.
Acknowledgements The authors thank Dr. Bryan Roth, Case Western Reserve University, for providing the 5-HT2A and 5-HT2C receptor cDNAs and Dr. Leon Carlock and Ms. Christine Perry, Wayne State University School of Medicine, for technical assistance. Supported by NS30550 ŽP.D.W.. and WSU research grants.
References w1x C.A. Altar, W.C. Boyar, M.R. Marien, 125 I-LSD autoradiography confirms the preferential localization of caudate–putamen S 2 receptors to the caudal Žperipallidal. region, Brain Res. 372 Ž1986. 130–136. w2x T.W. Berger, S. Kaul, E.M. Stricker, M.J. Zigmond, Hyperinnervation of the striatum by dorsal raphe afferents after dopamine-depleting brain lesions in neonatal rats, Brain Res. 336 Ž1985. 354–358. w3x G.R. Breese, B.R. Cooper, Behavioral and biochemical interactions of 5,7-dihydroxytryptamine with various drugs when administered intracisternally to adult and developing rats, Brain Res. 98 Ž1975. 517–527.
w4x T.J. Cummings, P.D. Walker, Serotonin depletion exacerbates changes in striatal gene expression following quinolinic acid injection, Brain Res. 743 Ž1996. 240–248. w5x L. Descarries, J.-J. Soghomonian, S. Garcia, G. Doucet, J.P. Bruno, Ultrastructural analysis of the serotonin hyperinnervation in adult rat neostriatum following neonatal dopamine denervation with 6-hydroxydopamine, Brain Res. 569 Ž1992. 1–13. w6x C.T. Fischette, B. Nock, K. Renner, Effects of 5,7-dihydroxytryptamine on serotonin 1 and serotonin 2 receptors throughout the rat central nervous system using quantitative autoradiography, Brain Res. 421 Ž1987. 263–279. w7x C.R. Gerfen, T.M. Engber, L.C. Mahan, Z. Susel, T.N. Chase, F.J. Monsma, D.R. Sibley, D1 and D 2 dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons, Science 250 Ž1990. 1429–1432. w8x P.J. Gresch, P.D. Walker, Acute p-chloroamphetamine increases striatal preprotachykinin mRNA: role of the serotonin 2Ar2C receptor, Mol. Brain Res. 67 Ž1999. 190–193. w9x P.J. Gresch, P.D. Walker, Synergistic interaction between serotonin-2 receptor and dopamine D1 receptor stimulation on striatal preprotachykinin mRNA expression in the 6-hydroxydopamine lesioned rat, Mol. Brain Res. Ž1999. in press. w10x P.J. Gresch, P.D. Walker, Serotonin-2 receptor stimulation normalizes striatal preprotachykinin mRNA in an animal model of Parkinson’s disease, Neuroscience Ž1999. in press. w11x D. Jackson, E.D. Abercrombie, In vivo neurochemical evaluation of striatal serotonergic hyperinnervation in rats depleted of dopamine at infancy, J. Neurochem. 58 Ž1992. 890–897. w12x D. Julius, K.N. Huang, T.J. Livelle, R. Axel, T.M. Jessell, The 5-HT2 receptor defines a family of structurally distinct but functionally conserved serotonin receptors, Proc. Natl. Acad. Sci. U.S.A. 87 Ž1990. 928–932. w13x D. Julius, A.B. MacDermott, R. Axel, T.M. Jessell, Molecular characterization of a functional cDNA encoding the serotonin 1c receptor, Science 241 Ž1988. 558–564. w14x P.J. Karstaedt, H. Kerasidis, J.H. Pincus, R. Meloni, J. Graham, K. Gale, Unilateral destruction of dopamine pathways increases ipsilateral striatal serotonin turnover in rats, Exp. Neurol. 126 Ž1994. 25–30. w15x I.C. Kilpatrick, M.W. Jones, O.T. Phillipson, A semiautomated analysis method for catecholamines, indolamines, and some prominent metabolites in microdissected regions of the nervous system: an isocratic HPLC technique employing coulometric detection and minimal sample preparation, J. Neurochem. 46 Ž1986. 1865–1876. w16x N. Laprade, F. Radja, T.A. Reader, J.-J. Soghomonian, Dopamine receptor agonists regulate levels of the serotonin 5-HT2A receptor and its mRNA in a subpopulation of rat striatal neurons, J. Neurosci. 16 Ž1996. 3727–3736. w17x J. Luthman, B. Bolioli, T. Tsutsumi, A. Verhofstad, G. Jonsson, Sprouting of striatal serotonin nerve terminals following selective lesions of nigrostriatal dopamine neurons in neonatal rats, Brain Res. Bull. 19 Ž1987. 269–274. w18x J. Luthman, E. Brodin, E. Sundstrom, B. Wiehager, Studies on brain monoamine and neuropeptide systems after neonatal intracerebroventricular 6-hydroxydopamine treatment, Int. J. Dev. Neurosci. 8 Ž1990. 549–560. w19x E. Molina-Holgado, K.M. Dewar, L. Descarries, T.A. Reader, Altered dopamine and serotonin metabolism in the dopamine-denervated and serotonin-hyperinnervated neostriatum of adult rat after neonatal 6-hydroxydopamine, J. Pharmacol. Exp. Ther. 270 Ž1994. 713–721. w20x A. Mrini, J.P. Soucy, F. Lafaille, P. Lemoine, L. Descarries, Quantification of the serotonin hyperinnervation in adult rat neostriatum after neonatal 6-hydroxydopamine lesion of nigral dopamine neurons, Brain Res. 669 Ž1995. 303–308. w21x S. Numan, K.H. Lundgren, D.E. Wright, J.P. Herman, K.B. Seroogy, Increased expression of 5-HT2 receptor mRNA in rat striatum
G.J. Basura, P.D. Walkerr DeÕelopmental Brain Research 116 (1999) 111–117
w22x
w23x
w24x
w25x
w26x
w27x
following 6-OHDA lesions of the adult nigrostriatal pathway, Mol. Brain. Res. 29 Ž1995. 391–396. F. Radja, L. Descarries, K. Dewar, T. Reader, Serotonin 5-HT1 and 5-HT2 receptors in adult rat brain after neonatal destruction of nigrostriatal dopamine neurons: a quantitative autoradiographic study, Brain Res. 606 Ž1993. 273–285. S.P. Sivam, G.R. Breese, J.E. Krause, T.C. Napier, R.A. Mueller, J.-S. Hong, Neonatal and adult 6-hydroxydopamine-induced lesions differentially alter tachykinin and enkephalin gene expression, J. Neurochem. 49 Ž1987. 1623–1633. A.M. Snyder, M.J. Zigmond, R. Lund, Sprouting of serotonergic afferents into striatum after dopamine-depleting lesions in infant rats: a retrograde transport and immunocytochemical study, J. Comp. Neurol. 245 Ž1986. 274–281. M.K. Stachowiak, J.P. Bruno, A.M. Snyder, E.M. Stricker, M.J. Zigmond, Apparent sprouting of striatal serotonergic terminals after dopamine-depleting lesions in neonatal rats, Brain Res. 291 Ž1984. 164–167. Y. Takeuchi, T. Sawada, S. Blunt, P. Jenner, C.D. Marsden, Effects of 6-hydroxydopamine lesions of the nigrostriatal pathway on striatal serotonin innervation in adult rats, Brain Res. 562 Ž1991. 301–305. J.P. Ternaux, F. Hery, S. Bourgoin, J. Adrien, J. Glowinski, M. Hamon, The topographical distribution of serotonergic terminals in
w28x
w29x
w30x
w31x
w32x
w33x
117
the neostriatum of the rat and caudate nucleus of the cat, Brain Res. 121 Ž1977. 311–326. A.C. Towle, H.E. Criswell, E.H. Maynard, J.M. Lauder, T.H. Joh, R.A. Mueller, G.R. Breese, Serotonergic innervation of the rat caudate following a neonatal 6-hydroxydopamine lesion: an anatomical, biochemical and pharmacological study, Pharmacol. Biochem. Behav. 34 Ž1989. 367–374. P.D. Walker, J.J. Capodilupo, W.A. Wolf, L.R. Carlock, Preprotachykinin and preproenkephalin mRNA expression within striatal subregions in response to altered serotonin transmission, Brain Res. 732 Ž1996. 25–35. P.D. Walker, L.A. Riley, R.P. Hart, G.M. Jonakait, Serotonin regulation of tachykinin biosynthesis in the rat neostriatum, Brain Res. 546 Ž1991. 33–39. R.W. Ward, D.M. Dorsa, Colocalization of serotonin receptor subtypes 5-HT2A , 5-HT2C , and 5-HT6 with neuropeptide mRNAs in the rat forebrain, J. Comp. Neurol. 370 Ž1996. 405–414. P.T.-H. Wong, W.L. Teo, T. Lee, A microdialysis study on striatal dopamine, 5-HT and metabolites in conscious rats after various treatments: evidence for extravesicular release of dopamine, NeuroReport 5 Ž1993. 53–56. F.C. Zhou, S. Bledsoe, J. Murphy, Serotonergic sprouting is induced by dopamine lesion in substantia nigra of adult rat brain, Brain Res. 556 Ž1991. 108–116.