Proc. Nat. Acad. Sci. USA Vol. 72, No. 3, pp. 837-839, March 1975

RNA Synthesis in Isolated Brain Nuclei after Administration of d-Lysergic Acid Diethylamide (LSD) In Vivo (rabbit brain regions/intravenous LSD/a-amanitin) IAN R. BROWN Department of Zoology, Scarborough College, University of Toronto, West Hill, Ontario M1C 1A4, Canada Communicated by Richard B. Roberts, December 12, 1974

ABSTRACT RNA synthesis in isolated brain nuclei was 0.9% (w/v) NaCl at 1 mg/ml was injected into the ear vein analyzed 2.5 hr after the intravenous administration of at 100 of body weight. Control rabbits received an d-lysergic acid diethylamide (LSD) to young rabbits. The Ag/kg drug stimulated transcription by 54% in brain stem nuclei appropriate volume of saline. Injections were always carried and by 13% in cerebral hemisphere nuclei expressed over out at the same hour of the day. The animals were left un- saline controls. Both nucleoplasmic and nucleolar RNA disturbed for 2.5 hr, then dispatched by cervical dislocation of synthesis were increased. The main activity, in the isolated the neck. nuclei assay was due to nucleoplasmic RNA polymerase, since a-amanitin reduced synthesis by over 70% in either Isolation of Nuclei. Brains were dissected into cerebral drug or control treatments. hemispheres and brain stem (total brain minus cerebral hemi- The complexity of transcription in neural tissue has pre- spheres and cerebellum), then homogenized with a Teflon- viously been analyzed by RNA excess hybridization tech- glass homogenizer in 4 volumes of solution A [0.32 M sucrose, niques. Our studies (1, 2) and those of others (3-5) indicate 50mM sodium phosphate (pH 6.5), 50 mM KCl, 1 mM dithio- that RNA isolated from the mammalian brain hybridizes to a threitol, 2 mM MgC12, and 0.1% Triton X-100]. The homog- greater fraction of nonrepeated DNA as compared to RNA enate volume was increased to 15 ml per brain region, fil- from other organs. Change in the transcription of nonrepeated tered through one layer of cheesecloth, and centrifuged for 10 DNA occurs during neural development. In this report we min at 1000 X g. The crude nuclear pellet was then homog- analyze whether a drug, which is known to affect the brain at enized again in solution A followed by centrifugation. The the psychological and physiological level, influences transcrip- resultant pellet was suspended in 28 ml of solution B [2.2 M tion in the brain. sucrose, 50 mM sodium phosphate (pH 6.5), 1 mM dithio- Recently we reported that d-lysergic acid diethylamide threitol, 2 mM Mg C12], layered over 10 ml of fresh solution (LSD) affects the brain at the level of covalent modification of B, and centrifuged for 45 min at 22,000 rpm in an SW 27 rotor. chromosomal proteins (6). Moderate dosages of the drug The purified nuclear pellet recovered after the centrifugation were found to increase the acetylation of specific histones in through dense sucrose was suspended in 2 ml of solution C the rabbit brain 30 min after intravenous drug administration. [25% glycerol, 40 mM Tris HCl (pH 8.0), 1 mM dithio- Since modification of chromosomal proteins may be associated threitol, 10 mM MgC12, 0.1 mM EDTA] and centrifuged for with regulation of gene activity (7), we now examine RNA 10 minutes at 1000 X g. The washing procedure was repeated synthesis at a period shortly after the acetylation change in to remove sucrose prior to the determination of nuclear DNA the LSD-treated rabbit brain. To circumvent the nucleotide concentration by the diphenylamine reaction. The final nu- pool size difficulties often inherent with determinations of clear suspension was adjusted to 0.1 mg of DNA per ml with RNA synthesis in vivo, we use an isolated nuclei system. In solution C. this report cerebral hemisphere and brain stem nuclei are RNA Synthesis Assay. The conditions for assaying RNA shown to demonstrate increased ability to synthesize RNA synthesis were a modification of the method of Reeder and after drug administration in vivo. Roeder (8). The standard reaction mixture contained in a MATERIALS final volume of 0.5 ml: 12.5% glycerol, 40 mM Tris HCl (pH d-Lysergic acid diethylamide (LSD), a product of Sandoz 8.0), 200 mM KCl, 1 mM dithiothreitol, 5 mM MgCl2, 2.5 M Pharmaceuticals, Switzerland, was obtained through the MnClg, 0.05 mM EDTA, 5 mM creatine phosphate, 25 1Ag of Department of National Health and Welfare, Ottawa, creatine phosphokinase, 1.25 ,Ci of [3H]UTP, 0.15 mM each Canada. [3H]UTP was purchased from New England Nu- of ATP, GTP, and CTP, and 25 J.g of nuclear DNA. The clear, and other nucleotides and RNase-free sucrose from complete mixture was mixed on a Vortex mixer and incubated Schwarz/Mann. a-Amanitin from Boehringer, Germany, was at 370 for 30 min. Reactions were terminated by the addition donated by Dr. I. A. Menon, University of Toronto. of 0.5 mg of bovine serum albumin as carrier followed by an equal volume of 10% trichloroacetic acid. The acid-insoluble METHODS pellet was washed twice with 5% trichloroacetic acid and dis- Drug Administration. Young male New Zealand white solved overnight in 0.5 ml of 0.3 M NaOH. Radioactivity of rabbits weighing 1 kg were used throughout. LSD dissolved in samples was determined by liquid scintillation in 10 ml of a Beckman Bio-Solv-toluene cocktail. All assays were run in Abbreviation: LSD, d-lysergic acid diethylamide. duplicate. 837 Downloaded by guest on September 27, 2021 838 Biochemistry: Brown Proc. Nat. Acad. Sci. USA 72 (1976)

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I 1. .1 I I 10 20 30 40 50 DNA (ug/assay) ocamanitin (ug/assay) FIG. 1. Effect of DNA concentration on RNA synthesis in FIG. 3. Effect of a-amanitin on RNA synthesis in brain isolated brain nuclei. Nuclei purified from cerebral hemispheres nuclei. a-Amanitin was incubated with cerebral hemisphere were incubated at various concentrations under standard assay nuclei under standard assay conditions. conditions as described in Methods. the concentration of nuclear DNA in the reaction mixture for RESULTS the range 10-50 ,ug per assay (Fig. 1). Incorporation was de- Isolated Brain Nuclei. The nuclear isolation procedure re- pendent on the inclusion of all four ribonucleotides. In the ceived critical attention in order to achieve pure brain nuclei presence of 5 mM magnesium and under high salt conditions, while retaining the ability for active RNA synthesis. The pH RNA synthesis was found to be stimulated by manganese ions. of the initial homogenization medium and concentration of A 3-fold increase was observed when nuclei were incubated magnesium ions were found to be particularly important. with 2.5 mM MnCl2 (Fig. 2). a-Amanitin reduced RNA syn- Clumping of brain nuclei occurred at pH greater than 6.5. thesis by 73% under the standard assay conditions of 5 mM Divalent ion concentrations less than 2,-mM caused nuclear MgCl2 plus 2.5 mM MnCl2, indicating that a major portion of breakage, while higher levels produced condensed nuclei with the activity was due to nucleoplasmic RNA polymerase (Fig. adherent cytoplasm. Inclusion of Triton X-100 in the initial 3). Inhibitor concentrations of 0.1 ug/0.5 ml of assay were medium improved the nuclear yield and markedly decreased sufficient to produce maximal inhibition. Experiments in cytoplasmic contamination. The 2.2 M sucrose gradient was Figs. 1-3 used cerebral hemisphere nuclei; however, similar required for complete removal of myelin fragments. results were obtained with brain stem nuclei. Phase-contrast and electron microscopy of the final nuclear The injection of were of Effect of Administration of LSD In Vivo. pellet revealed that the brain nuclei free cytoplasmic a marked stimulation in the DNA was of the initial LSD into young rabbits produced contamination. Recovery of 40% stem nuclei to synthesize RNA in the yield per brain was 800-900,ug for the cere- ability of isolated brain homogenate, and in vitro assay (Fig. 4). The increase was 54.3% ± 1.2 after 30 bral hemispheres and 400-500.ug for the brain stem. In 25% 2 hr. min of nuclear incubation expressed over parallel controls glycerol solution C, nuclei were stable on ice for at least (average of four trials). frozen in a dry ice/ethanol mixture, which received saline injections Nuclei could be quickly Brain stem is the total brain minus cerebral hemispheres and stored at - 500 for up to a month, and assayed immediately showed an en- loss of cerebellum. Cerebral hemisphere nuclei also after thawing without significant activity. hancement in their ability to synthesize RNA (Fig. 5). The RNA Synthesis. Purified brain nuclei actively synthesized stimulation was 13.2% i 0.2 (average of four trials). In these RNA in an in vitro assay which included ['H]UTP and three experiments LSD was injected intravenously into the rabbits unlabeled ribonucleotide triphosphates. The amount of iso- 2.5 hr prior to removal of the brain for nuclear isolation. No tope incorporated by brain nuclei was linearly dependent on

x4 2 -6 x/ 2 4 10 20 30 2 Time (min) FIG. 4. Time course of RNA synthesis in isolated brain stem 0 1 2 3 4 nuclei after LSD administration in vivo. LSD (100 ,Ag/kg) was MnCl(mM) injected intravenously into young rabbits, which were killed 2.5 on RNA in hr later. Nuclei purified from the brain stem (total brain minus FIG. 2. Effect of MnCl2 concentration synthesis under brain nuclei. Cerebral hemisphere nuclei were incubated under cerebral hemispheres and cerebellum) were incubated as described in Methods. LSD standard assay conditions with the exception of variation in con- standard assay conditions, *, centration of MnCl2. treatment; 0, saline control. Downloaded by guest on September 27, 2021 Proc. Nat. Acad. Sci. UASA 72 (1975) RNA Synthesis in Isolated Brain Nuclei after LSD 839 TABLE 1. a-Amanitin-resistant RNA synthesis in isolated brain nuclei 165 >1 co 4A 4A % a-Amanitin-resistant RNA m 122 IM- synthesis 6 x E3 Brain region Saline control LSD treatment E 0. Cerebral hemisphere nuclei 27.3 4 1.0 28. 1 4+ 1. 3 Brain stem nuclei 26.9 i 1.2 27.9 i 1.3 0 a I I1011 11%20 30if% Brain nuclei were incubated under standard assay conditions Time (min) with or without a-amanitin at 0.5 .g per assay. Each value is the FIG. 5. Time course of RNA synthesis in isolated cerebral mean of five experiments i SEM. hemisphere nuclei after LSD administration in vivo. Procedure was as given in legend of Fig. 4. 0, ISD treatment; 0, saline con- stimulation was observed in either brain region when nuclei trol. were isolated 30 min after drug administration. To determine whether the LSD effect was due solely to in- physiological level, LSD has been reported to specifically affect creased ribosomal RNA synthesis, I analyzed the percentage neuron firing and serotonin metabolism in the raphe nucleus of a-amanitin-resistant synthesis. As shown in Table 1, per- region (12). This area is included in our brain stem fraction. centage a-amanitin-resistant RNA synthesis was the same in Transcription in the brain is known to be complex. More nuclei from control and LSD-treated animals. This result sug- transcripts of nonrepeated DNA are present in neural tissue gested that the drug-induced stimulation was due to propor- than in other organs (1-5). LSD may provide a convenient tionate increases in both nucleoplasmic and nucleolar RNA method for inducing a change in RNA synthesis which can be synthesis. used to advantage in studies that attempt to analyze basic DISCUSSION mechanisms of neural transcription. Isolated nuclei systems have been useful for the study of The excellent technical assistance of Miss S. W. Lee is grate- transcription in neural tissue: maturation-dependent changes fully acknowledged. This project was supported under the pro- gram of Research on Drug Abuse administered by the Non- in template ability of brain nuclei have been demonstrated Medical Use of Drugs Directorate, Health and Welfare, Canada. (9), the presence of poly(A)-containing RNA suggested (10), and a report made of multiple RNA polymerases (11). We 1. Brown, I. R. & Church, R. B. (1971) Biochem. Biophys. Res. Commun. 42, 850-856. have utilized an isolated nuclei system to test whether LSD 2. Brown, I. R. & Church, R. B. (1972) Develop. Biol. 29, 73- affects transcription in regions of thewrabbit brain. Under the 84. conditions of our in vitro assay, the major synthetic activity is 3. Hahn, W. E. & Laird, C. D. (1971) Science 173, 158-161. due to nucleoplasmic RNA polymerase. 4. Grouse, L., Chilton, M. D. & McCarthy, B. J. (1972) Bio- The drug was found to stimulate the ability of from chemistry 11, 798-805. nuclei 5. Grouse, L., Omenn, G. A. & McCarthy, B. J. (1973) J. two brain regions to synthesize RNA. The effect was pro- Neurochem. 20, 1063-1073. nounced in nuclei from the brain stem while less increase was 6. Brown, I. R. & Liew, C. C. (1975) Science, in press. observed in cerebral hemisphere nuclei. Both nucleoplasmic 7. Allfrey, V. G. (1971) in Histones and Nucleohistones, ed. and nucleolar RNA synthesis were stimulated. The location Phillip, D. P. (Plenum Press, New York), pp. 241-294. 8. Reeder, R. H. & Roeder, R. G. (1972) J. Mol. Biol. 67, 433- of the maximal effect correlates with our previous findings on 441. LSD stimulation of brain histone acetylation in which the mid- 9. Banks, S. P. & Johnson, T. C. (1972) Brain Res. 41, 155- brain demonstrated the greatest increase (6). The change in 169. RNA synthesis at 2.5 hr after intravenous LSD administration 10. Banks, S. P. & Johnson, T. C. (1973) Science 181, 1064- is subsequent to the histone acetylation alteration, 1065. which oc- 11. Thompson, R. J. (1973) J. Neurochem. 21, 19-40. curs at 30 min. Modification of chromosomal proteins has 12. Sourkes, T. L. (1972) in Basic Neurochemistry, eds. Albers, been associated with regulation of transcription (7). At the R. W. et al. (Little, Brown & Co., Boston), pp. 581-606. Downloaded by guest on September 27, 2021