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Proc. Natl. Acad. Sci. USA Vol. 83, pp. 9827-9831, December 1986 Neurobiology

Mesencephalic regulate the expression of neuropeptide mRNAs in the rat forebrain (substance P/enkephalin/dynorphin/in situ hybridization/) W. SCOTT YOUNG III*, TOM I. BONNER, AND MARK R. BRANNt Laboratory of Cell Biology, Building 36, Room 3A17, National Institute of Mental Health, Bethesda, MD 20892 Communicated by Julius Axelrod, September 11, 1986

ABSTRACT We used in situ hybridization histochemistry Changes in striatal neuropeptide biosynthesis result from with synthetic oligodeoxyribonucleotide probes to identify cells lesioning mesencephalic dopamine neurons with 6-hydroxy- that synthesize mRNAs encoding tyrosine hydroxylase in the dopamine or chronic administration of neuroleptics. SP and mesencephalon and substance P, enkephalin, and dynorphin in SP mRNA levels in the are depressed by neurolept- the rat forebrain. Dopaminergic cells in the mesencephalon ics (22-24). Conversely, Enk levels, Enk synthesis, and Enk project to the forebrain and influence neuropeptide levels. We mRNA levels are all increased by 6-hydroxydopamine le- examined the effect of unilateral 6-hydroxydopamine lesions sions or neuroleptics (25-30). The effects on Dyn biosynthe- (which eliminated tyrosine hydroxylase mRNA-containing cells sis are uncertain (31, 32). These studies were performed in in the mesencephalon) on substance P, enkephalin, and dynor- homogenate preparations that lack sufficient anatomical phin mRNA levels. Substance P mRNA levels were depressed, resolution to show whether all dopamine-innervated areas whereas enkephalin mRNA levels were elevated in consecutive are effected similarly. We sought to gain a more detailed sections from striatal areas in all animals. The effects of the understanding of the responses of neuropeptide systems to lesions on dynorphin mRNA levels were less robust, and loss of dopamine innervation after 6-hydroxydopamine le- considerable variation between animals was observed. Changes sions by using in situ hybridization histochemistry (ISHH). were evident in the levels of message in individual cells but not This technique, using synthetic 35S-labeled oligodeoxyribo- in the numbers of labeled cells. These effects were not uniform nucleotide probes for SP, Enk, and Dyn mRNAs, allowed us throughout the dopamine-innervated areas, suggesting degrees to see regional and cellular changes in these mRNA levels. of control not apparent with RNA blot-hybridization or dot- We also visualized the cells in the mesencephalon that blot analyses. synthesize dopamine with a probe for tyrosine hydroxylase (TyrOHase), the rate-limiting enzyme in dopamine synthesis Dopamine is a principal transmitter of the mesostriatal and (33). mesocortical (or mesolimbic) components of the mesence- phalic pathways to the forebrain (1), which exert major MATERIALS AND METHODS influences on an animal's behavior. In rats, lesions of these Male, Sprague-Dawley rats (200-300 g, National Institutes of pathways may lead to behavioral deficits, such as akinesia, Health) were anesthetized with Equithesin (1 ml/kg of body adipsia, and aphagia, while stimulation leads to increased weight) and 6-hydroxydopamine (8 ,ug) or vehicle (4 ,ul of motor activity and stereotyped behaviors such as sniffing, 0.9% NaCl/0.1% ascorbic acid) were injected (1 ,ul/min) into licking, and gnawing (2, 3). Side effects of neuroleptics such the left SN of each animal (AP2.6;L1.9;V8.6 mm from the as acute dystonia and tardive dyskinesia and neurological bregma and dural surface; ref. 34). The animals were fed and diseases such as Parkinson's disease, in which a deficiency in watered ad libitum and housed on a 12-hr-on/12-hr-off mesostriatal dopamine is accompanied by movement disor- lighting schedule. Two weeks after the injections, animals ders such as bradykinesia and tremor, demonstrate the were decapitated, and the were removed, frozen on importance of dopamine in human motor control (4). dry ice, and stored at -80°C until sectioned. Undoubtedly, dopamine, in part, exerts its influence and is Frozen 12-,um sections were thaw-mounted onto twice- influenced in turn by neuropeptide transmitter systems. gelatin-coated slides, fixed with 4% formaldehyde in 0.12 M Extensive evidence exists for complex topographical rela- sodium phosphate-buffered saline (PBS, pH 7.4) for 5 min at tionships between these systems in the basal ganglia (5-9). room temperature, and rinsed twice in PBS. Hybridizations Broadly, the caudate- (CP) system receives input were performed as decribed (35). Briefly, the sections were from the (SN) and placed in 0.25% acetic anhydride in 0.1 M triethanolamine/ 0.9% NaCl, pH 8, for 10 min at room temperature, followed (VTA), while the accumbens (NA) and olfactory by delipidation in a graded series of ethanol (70, 80, 95, and tubercle (OT) are primarily innervated by the VTA. Howev- 100%) and chloroform. They were then hybridized with er, there is some overlap in the innervations of these striatal 0.6-2.0 x 101 dpm of one of the four probes (see below) at areas by the SN and VTA. Also, the dopamine cell group A8 37°C for 22-24 hr in a buffer containing 4x NaCl/Cit (lx projects to the NA and ventral CP. These dopamine-inner- NaCl/Cit = 0.15 M NaCl/0.015 M sodium citrate, pH 7.2), vated areas contain substance P (SP) (10), enkephalin (Enk) 50% (vol/vol) formamide, 10% (wt/vol) dextran sulfate, and (ref. 11 and references within), and dynorphin (Dyn) (12, 13) cell bodies, which project in characteristic ways out of the Abbreviations: CP, caudate-putamen; SN, substantia nigra; VTA, striatum. In the rat, SP (14-16) and Dyn (17) cells project ventral tegmental area; NA, accumbens nucleus; SP, substance P; principally to the SN, especially the , whereas Enk, enkephalin; Dyn, dynorphin; OT, ; Enk cells project to the globus pallidus (18-21). TyrOHase, tyrosine hydroxylase; ISHH, in situ hybridization his- tochemistry. *To whom reprints should be addressed. The publication costs of this article were defrayed in part by page charge tPresent address: Metabolic Diseases Branch, Building 10, Room payment. This article must therefore be hereby marked "advertisement" 9C-101, National Institute of Diabetes and Digestive Disorders, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Bethesda, MD 20892. 9827 9828 Neurobiology: Young et al. Proc. Natl. Acad. Sci. USA 83 (1986) 10 mM dithiothreitol (35). Then they were washed in four England Nuclear) and terminal deoxynucleotidyltransferase 15-min rinses of 2x NaCl/Cit containing 50o formamide at and were hybridized 18 hr at 37°C as described by Bannon et 40'C followed by two 1-hr rinses of 2x NaCl/Cit at room al. (24). They then were washed with five 15-min rinses of0.3 temperature. In later experiments, sections were washed in M NaCl/0.02 M sodium phosphate/0.02 M EDTA/50% four 15-min rinses of 1 x NaCl/Cit at 550C, followed by two formamide/0.2% NaDodSO4, pH 7.4, at 40°C. The blots were 1-hr rinses in 1x NaCl/Cit at room temperature. Both wash apposed to x-ray film, which revealed single bands with the protocols were performed at about 20'C below the calculated SP, Enk, Dyn, and TyrOHase probes of about 1000, 1500, melting temperatures for the probes (36, 37). The sections 2400, and 1800 bases, respectively (data not shown), in were next dipped into water, blown dry, and examined agreement with published reports (24, 38-40). autoradiographically-first by apposition to x-ray film (X- Omat, Kodak) and then by dipping into NTB3 nuclear emulsion (1:1 with water, Kodak). The exposures were 1-14 RESULTS days with film and 1-6 weeks with nuclear emulsion. The Cells containing SP mRNA were prominent in the CP, sections were subsequently stained with cresyl violet to especially in the dorsolateral region. Labeled cells were also counterstain the cell bodies. Some typical x-ray film autoradi- seen in the NA, pyramidal and polymorph (not the large cells) ograms were analyzed by using a Loats image analysis cell layers of the OT, , bed nucleus of the system (Loats, Westminster, MD). "S-impregnated , , lateral and medial septal nuclei, paste standards were concurrently exposed and used to various regions of the hypothalamus, medial , and determine the relationship between optical density and tissue . No cells were seen in the rostral globus radioactivity (35). pallidus. The oligodeoxyribonucleotides were 48 bases long. The SP Cells containing Enk mRNA were also prominent in the oligomer was complementary to the base sequence encoding CP, NA, and OT. In the OT, they were located in the the first 16 amino acids of exon 3 of the rat SP precursor polymorph and in the pyramidal cell layer adjacent to the mRNA (5' GAG GCA ATG CCG GAG CCC TTT GAG CAT islands of Calleja. Unlike the cells containing SP mRNA, CTT CTT CAG AGA ATC GCC CGA 3'; T.I.B., A. C. Enk-labeled cells were not observed in the most ventral Young, and H.-U. Affolter, unpublished data). The Enk, portions below the islands. Enk cells were also seen in the Dyn, and TyrOHase oligomers were complementary to bases primary olfactory cortex, neocortex (especially layers 2, 5, 388-435, 862-909, and 1441-1488 of the preproenkephalin A and 6), amygdala, diagonal bands, bed nucleus of the stria (38), prodynorphin (39), and TyrOHase (40) mRNAs, respec- terminalis, and various hypothalamic areas and were scat- tively. The oligomers were made on an Applied Biosystems tered throughout all layers of the . DNA synthesizer (Applied Biosystems, Foster City, CA) Scattered, lightly labeled Enk cells also were seen in the (courtesy of M. J. Brownstein, National Institute of Mental globus pallidus. Health) and purified on a 8% polyacrylamide/8 M urea Cells containing Dyn mRNA were also seen in the CP, NA, preparative sequencing gel. They were labeled by using and OT. In the OT, the cells were distributed in the same deoxyadenosine [a-thioltriphosphate labeled at the a posi- pattern as Enk. Other labeled cells were seen in the neocor- tion with 35S (>1000 Ci/mmol, New England Nuclear; 1 Ci = tex, lateral septum, hypothalamus (especially the magnocell- 37 GBq) and terminal deoxynucleotidyltransferase (Bethesda ular cells of the paraventricular and supraoptic nuclei), bed Research Laboratories) to specific activities of 4-10 x 103 nucleus of the stria terminalis, various regions of the hypo- Ci/mmol. In order to verify the specificity of the probes, we , and cerebral cortex. Dyn cells were prominent in performed blot-hybridization analyses of total RNA in stri- the granule cell layer of the as well. atal (with SP, Enk, and Dyn probes) and ventral mesence- The TyrOHase probe labeled cells in the SN compacta phalic (with the TyrOHase probe) tissue extracts. The probes (Fig. 1) and in the VTA. Cells in All and dorsal extension of were labeled by using [a-32P]dATP (>3000 Ci/mmol, New the VTA were the only other cells labeled at this level (5.0 mm

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FIG. 1. TyrOHase mRNA-containing cells of the SN from the control side of a lesioned animal. (Bar = 25 am.) Neurobiology: Young et al. Proc. Natl. Acad. Sci. USA 83 (1986) 9829 behind the bregma; ref. 41). 6-Hydroxydopamine-treated mRNA levels in the OT were also decreased. The changes in animals had no TyrOHase mRNA-containing cells in the SN Dyn mRNA levels were less robust than those of SP and Enk. and VTA on the lesioned side of the brain. The unlesioned In four of nine brains examined, the levels decreased in the side showed labeling identical to that seen on either side of striatum (Fig. 3). However, the levels increased in one and the vehicle-injected animals. The forebrains of the animals were unchanged in four. The average change in Dyn mRNA with complete lesions, as well as several from vehicle- in the striatum at the level shown in Fig. 2 was +9 ± 9% (n injected animals, were examined for changes in SP, Enk, and = 8). Dyn mRNA levels. In all six animals with complete lesions, the CP on the side DISCUSSION of the lesion had less SP mRNA than did the other side (Fig. 2). Conversely, adjacent sections from the six rats, plus The results and conclusions of this paper rely on the speci- sections from four additional lesioned rats, had increased ficity of the probes used for ISHH. Several factors argue for Enk mRNA levels on the side of the lesion compared to the the specificity of our probes. First, the probes were used for other side (Fig. 2). To gain a perspective on the extent of blot-hybridizations of total mRNA preparations under con- these changes, we measured the levels of probe radioactivity ditions of comparable stringency as used for ISHH, and (35) in the sections shown in Fig. 2. The SP probe levels single bands of appropriate length were observed. Second, decreased by 40% on the lesion side in both the CP and NA. the different patterns of hybridizations argue against nonspe- The levels of Enk probe radioactivity increased by 100% and cific binding. And, third, the distributions of SP, Enk, Dyn, 30% in the CP and NA, respectively. The average changes in and TyrOHase mRNAs observed with ISHH are in excellent SP and Enk mRNA levels in the striatum at the level shown agreement with previous immunocytochemical reports. in Fig. 2 were -55 ± 26% (±SEM, n = 4, 2 sections per SP, Enk, and Dyn mRNAs were detected in cells in the CP, animal) and +52 ± 24% (n = 8), respectively. When exam- NA, and OT, where their cell bodies had been described ined at higher resolution, these changes were evident in the immunocytochemically (refs. 7 and 10-13 and references amount of label per cell and not in the number of cells within). SP and Enk cell bodies in the rat globus pallidus have expressing the particular mRNA species (Fig. 2). For exam- been described by some authors (14, 42, 43), but we only ple, in the lateral striatum shown in Fig. 2, there were 43 and observed scattered, lightly labeled Enk mRNA-containing 39 SP cells per 0.1 mm2 on the control and lesioned sides, cells there. Our observation of SP cells in the islands of respectively. Similarly, there were 43 and 47 Enk cells per 0.1 Calleja has not been reported previously in the rat, but it has mm2 on the control and lesioned sides, respectively. SP been reported in the cat (44). The islands of Calleja resemble

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FIG. 2. ISHH localization of SP (Upper) and Enk (Lower) mRNAs in consecutive sections from a rat forebrain after unilateral 6-hydroxydopamine lesion of the SN and VTA. The darkfield photomicrographs, in which the silver grains appear white, show that SP mRNA levels are reduced in the CP and NA on the side of the lesion (left), whereas Enk mRNA levels are increased. The four brightfield photomicrographs show labeled cells in the same medial areas of the CP ipsilateral (I) and contralateral (C) to the lesion. Note, respectively, the decreased and increased cell labeling in the SP and Enk cells ipsilaterally. Solid arrows point to the major islands of Calleja, and the hollowed arrow points to the pyramidal cell layer of the OT. (Darkfield bar = 1.5 mm; brightfield bar = 25 um.) 9830 Neurobiology: Young et al. Proc. Natl. Acad. Sci. USA 83 (1986)

FIG. 3. Dyn mRNA ISHH in the forebrain of a rat. The left side of each section is ipsilateral to the SN and VTA 6-hydroxydopanilne lesion. Note the reduced signal in the left CP and NA (below the small arrows). The arrowhead points to labeled cells in the cortex, and the single arrow points to granule cell labeling of the dentate gyrus. (Bar = 1.5 mm.) striatopallidal complexes, and they contain SP, Enk, and Dyn of dopamine D2 receptors are seen in the CP, NA, and OT, fibers (45). Our findings suggest that the cells ofthe pyramidal including the islands ofCalleja, and, interestingly enough, we layer in the OT next to the islands contribute to Enk and Dyn only detected changes in the levels of SP and Enk mRNAs in fibers, whereas the SP fibers originate there or in the islands these three areas. The effect of the lesions on Dyn mRNA themselves. levels were generally small, suggesting a less significant Previous studies had linked dopaminergic innervation of effect of dopamine on Dyn synthesis. the CP to changes in neuropeptide synthesis. This is not The resolution of ISHH allowed us to show that striatal surprising considering the evidence for direct catechol- changes were due to changes in mRNA levels per cell, not in aminergic innervation of SP and Enk neurons in the CP (46, numbers of expressing cells. Furthermore, we were able to 47). Chronic neuroleptics have been shown to decrease SP examine the CP and other forebrain regions in detail to see if and increase Enk levels in the CP (22, 23, 25, 27). Hong et al. the dopamine were uniform or not. Our results showed that (26) and Chou et al. (28) also demonstrated that the Enk the same lesion could produce changes ofdifferent degrees in increase resulted from increased synthesis. Recent studies of different regions. For example, Enk mRNA levels were mRNAs in homogenate preparations of CP have shown that increased to a greater extent in the CP than in the NA and not chronic neuroleptic administration leads to decreased SP (24) at all in the pyramidal cell layer of the OT. These observa- and increased Enk (29, 30) mRNA levels. Although neuro- tions suggest that we can use the resolution of ISHH after leptics interact with other nondopamine receptors (48), our more selective lesions and various pharmacological manip- results suggest that the effects of the neuroleptics on striatal ulations (e.g., specific opiate or SP receptor blockade) to SP and Enk are mediated by dopamine receptors in the basal study interrelations among constituents of the basal ganglia. ganglia. Indeed, the regional distribution of dopamine D2 ISHH should prove useful in the study of human neuro- receptors in the forebrain demonstrated by receptor autoradi- logical diseases, many of which have alterations in neuro- ography (48-50) bears a striking similarity to the distribution peptide levels. Decreases in Enk and SP in the globus pallidus of mRNA level changes described in this study. High levels and SN have been found in the brains of patients with Neurobiology: Young et al. Proc. Natl. Acad. Sci. USA 83 (1986) 9831 Parkinson's disease (51-53). It is difficult to know whether W. (1981) J. Pharmacol. Exp. Ther. 218, 568-574. these changes reflect decreased synthesis, depletion due to 24. Bannon, M. J., Lee, J.-M., Giraud, P., Young, A., Affolter, 261, 6640-6642. increased synthesis, or cell death. H.-U. & Bonner, T. I. (1986) J. Biol. Chem. increased release despite 25. Hong, J., Yang, H.-Y. T., Fratta, W. & Costa, E. (1978) J. Measurement ofmRNA levels should resolve this ambiguity, Pharmacol. Exp. Ther. 205, 141-147. if proper controls for postmortem delays are used, since 26. Hong, J. S., Yang, H.-Y. T., Gillin, J. C., DiGiulio, A. M., evidence is accumulating that steady-state mRNA levels may Fratta, W. & Costa, E. (1979) Brain Res. 160, 192-195. reflect synthetic activity [e.g., Enk after dopamine denerva- 27. Thal, L. J., Sharpless, N. S., Hirschhorn, I. D., Horowitz, tion (as discussed above) and vasopressin and corticotropin- S. G. & Makman, M. H. (1983) Biochem. Pharmacol. 32, releasing factor after adrenalectomy (35, 54, 55)]. Because 3297-3301. ISHH can be used to detect neuropeptide-synthesizing cells 28. Chou, J., Tang, J., Yang, H.-Y. T. & Costa, E. (1984) J. can to detect immunocytochemically in Pharmacol. Exp. Ther. 229, 171-174. that be difficult & Biochem. prove useful in studying mRNA 29. Sabol, S. L., Yoshikawa, K. Hong, J. S. (1983) humans (56), ISHH should Biophys. Res. 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