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 RNA synthesis in isolated brain nuclei was ABSTRACT analyzed 2.5 hr after the intravenous administration of d-lysergic acid diethylamide (LSD) to young rabbits. The drug stimulated transcription by 54% in brain stem nuclei and by 13% in cerebral hemisphere nuclei expressed over saline controls. Both nucleoplasmic and nucleolar RNA synthesis were increased. The main activity, in the isolated nuclei assay was due to nucleoplasmic RNA polymerase, since a-amanitin reduced synthesis by over 70% in either drug or control treatments.
The complexity of transcription in neural tissue has previously been analyzed by RNA excess hybridization techniques. Our studies (1, 2) and those of others (3-5) indicate that RNA isolated from the mammalian brain hybridizes to a greater fraction of nonrepeated DNA as compared to RNA from other organs. Change in the transcription of nonrepeated DNA occurs during neural development. In this report we analyze whether a drug, which is known to affect the brain at the psychological and physiological level, influences transcription in the brain. Recently we reported that d-lysergic acid diethylamide (LSD) affects the brain at the level of covalent modification of chromosomal proteins (6). Moderate dosages of the drug were found to increase the acetylation of specific histones in the rabbit brain 30 min after intravenous drug administration. Since modification of chromosomal proteins may be associated with regulation of gene activity (7), we now examine RNA synthesis at a period shortly after the acetylation change in the LSD-treated rabbit brain. To circumvent the nucleotide pool size difficulties often inherent with determinations of RNA synthesis in vivo, we use an isolated nuclei system. In this report cerebral hemisphere and brain stem nuclei are shown to demonstrate increased ability to synthesize RNA after drug administration in vivo. MATERIALS d-Lysergic acid diethylamide (LSD), a product of Sandoz Pharmaceuticals, Switzerland, was obtained through the Department of National Health and Welfare, Ottawa, Canada. [3H]UTP was purchased from New England Nuclear, and other nucleotides and RNase-free sucrose from Schwarz/Mann. a-Amanitin from Boehringer, Germany, was donated by Dr. I. A. Menon, University of Toronto. METHODS Drug Administration. Young male New Zealand white rabbits weighing 1 kg were used throughout. LSD dissolved in Abbreviation: LSD, d-lysergic acid diethylamide. 837
0.9% (w/v) NaCl at 1 mg/ml was injected into the ear vein at 100 Ag/kg of body weight. Control rabbits received an appropriate volume of saline. Injections were always carried out at the same hour of the day. The animals were left undisturbed for 2.5 hr, then dispatched by cervical dislocation of the neck. Isolation of Nuclei. Brains were dissected into cerebral hemispheres and brain stem (total brain minus cerebral hemispheres and cerebellum), then homogenized with a Teflonglass homogenizer in 4 volumes of solution A [0.32 M sucrose, 50 mM sodium phosphate (pH 6.5), 50 mM KCl, 1 mM dithiothreitol, 2 mM MgC12, and 0.1% Triton X-100]. The homogenate volume was increased to 15 ml per brain region, filtered through one layer of cheesecloth, and centrifuged for 10 min at 1000 X g. The crude nuclear pellet was then homogenized again in solution A followed by centrifugation. The resultant pellet was suspended in 28 ml of solution B [2.2 M sucrose, 50 mM sodium phosphate (pH 6.5), 1 mM dithiothreitol, 2 mM Mg C12], layered over 10 ml of fresh solution B, and centrifuged for 45 min at 22,000 rpm in an SW 27 rotor. The purified nuclear pellet recovered after the centrifugation through dense sucrose was suspended in 2 ml of solution C [25% glycerol, 40 mM Tris HCl (pH 8.0), 1 mM dithiothreitol, 10 mM MgC12, 0.1 mM EDTA] and centrifuged for 10 minutes at 1000 X g. The washing procedure was repeated to remove sucrose prior to the determination of nuclear DNA concentration by the diphenylamine reaction. The final nuclear suspension was adjusted to 0.1 mg of DNA per ml with solution C.
RNA Synthesis Assay. The conditions for assaying RNA synthesis were a modification of the method of Reeder and Roeder (8). The standard reaction mixture contained in a final volume of 0.5 ml: 12.5% glycerol, 40 mM Tris HCl (pH 8.0), 200 mM KCl, 1 mM dithiothreitol, 5 mM MgCl2, 2.5 M MnClg, 0.05 mM EDTA, 5 mM creatine phosphate, 25 1Ag of creatine phosphokinase, 1.25 ,Ci of [3H]UTP, 0.15 mM each of ATP, GTP, and CTP, and 25 J.g of nuclear DNA. The complete mixture was mixed on a Vortex mixer and incubated at 370 for 30 min. Reactions were terminated by the addition of 0.5 mg of bovine serum albumin as carrier followed by an equal volume of 10% trichloroacetic acid. The acid-insoluble pellet was washed twice with 5% trichloroacetic acid and dissolved overnight in 0.5 ml of 0.3 M NaOH. Radioactivity of samples was determined by liquid scintillation in 10 ml of a Beckman Bio-Solv-toluene cocktail. All assays were run in duplicate.
838
Proc. Nat. Acad. Sci. USA 72 (1976)
Biochemistry: Brown 20
6A 0.
12
o
x 2
x
8
Ea. cL
I
10
1.
.1
I
40 30 20 DNA (ug/assay)
I
50
FIG. 1. Effect of DNA concentration on RNA synthesis in isolated brain nuclei. Nuclei purified from cerebral hemispheres were incubated at various concentrations under standard assay conditions as described in Methods.
RESULTS
Isolated Brain Nuclei. The nuclear isolation procedure received critical attention in order to achieve pure brain nuclei while retaining the ability for active RNA synthesis. The pH of the initial homogenization medium and concentration of magnesium ions were found to be particularly important. Clumping of brain nuclei occurred at pH greater than 6.5. Divalent ion concentrations less than 2,-mM caused nuclear breakage, while higher levels produced condensed nuclei with adherent cytoplasm. Inclusion of Triton X-100 in the initial medium improved the nuclear yield and markedly decreased cytoplasmic contamination. The 2.2 M sucrose gradient was required for complete removal of myelin fragments. Phase-contrast and electron microscopy of the final nuclear pellet revealed that the brain nuclei were free of cytoplasmic contamination. Recovery of DNA was 40% of the initial homogenate, and yield per brain was 800-900,ug for the cerebral hemispheres and 400-500.ug for the brain stem. In 25% glycerol solution C, nuclei were stable on ice for at least 2 hr. Nuclei could be quickly frozen in a dry ice/ethanol mixture, stored at - 500 for up to a month, and assayed immediately after thawing without significant loss of activity. RNA Synthesis. Purified brain nuclei actively synthesized RNA in an in vitro assay which included ['H]UTP and three unlabeled ribonucleotide triphosphates. The amount of isotope incorporated by brain nuclei was linearly dependent on
ocamanitin (ug/assay) FIG. 3. Effect of a-amanitin on RNA synthesis in brain nuclei. a-Amanitin was incubated with cerebral hemisphere nuclei under standard assay conditions. the concentration of nuclear DNA in the reaction mixture for the range 10-50 ,ug per assay (Fig. 1). Incorporation was dependent on the inclusion of all four ribonucleotides. In the presence of 5 mM magnesium and under high salt conditions, RNA synthesis was found to be stimulated by manganese ions. A 3-fold increase was observed when nuclei were incubated with 2.5 mM MnCl2 (Fig. 2). a-Amanitin reduced RNA synthesis by 73% under the standard assay conditions of 5 mM MgCl2 plus 2.5 mM MnCl2, indicating that a major portion of the activity was due to nucleoplasmic RNA polymerase (Fig. 3). Inhibitor concentrations of 0.1 ug/0.5 ml of assay were sufficient to produce maximal inhibition. Experiments in Figs. 1-3 used cerebral hemisphere nuclei; however, similar results were obtained with brain stem nuclei. Effect of Administration of LSD In Vivo. The injection of LSD into young rabbits produced a marked stimulation in the ability of isolated brain stem nuclei to synthesize RNA in the in vitro assay (Fig. 4). The increase was 54.3% ± 1.2 after 30 min of nuclear incubation expressed over parallel controls which received saline injections (average of four trials). Brain stem is the total brain minus cerebral hemispheres and cerebellum. Cerebral hemisphere nuclei also showed an enhancement in their ability to synthesize RNA (Fig. 5). The stimulation was 13.2% i 0.2 (average of four trials). In these experiments LSD was injected intravenously into the rabbits 2.5 hr prior to removal of the brain for nuclear isolation. No
x4 2
x/ -6
2
4
10
20
30
Time (min)
2
0
1
2
3
4
MnCl(mM)
FIG. 2. Effect of MnCl2 concentration on RNA synthesis in brain nuclei. Cerebral hemisphere nuclei were incubated under standard assay conditions with the exception of variation in concentration of MnCl2.
FIG. 4. Time course of RNA synthesis in isolated brain stem nuclei after LSD administration in vivo. LSD (100 ,Ag/kg) was injected intravenously into young rabbits, which were killed 2.5 hr later. Nuclei purified from the brain stem (total brain minus cerebral hemispheres and cerebellum) were incubated under standard assay conditions, as described in Methods. *, LSD treatment; 0, saline control.
Proc. Nat. Acad. Sci. UASA 72
RNA Synthesis in Isolated Brain Nuclei after LSD
(1975)
TABLE 1. a-Amanitin-resistant RNA synthesis in isolated brain nuclei
839
165 >1 co
% a-Amanitin-resistant RNA
synthesis Brain region
Cerebral hemisphere nuclei Brain stem nuclei
Saline control LSD treatment 27.3 4 1.0 28. 1 4+ 1. 3 26.9 i 1.2 27.9 i 1.3
Brain nuclei were incubated under standard assay conditions with or without a-amanitin at 0.5 .g per assay. Each value is the mean of five experiments i SEM.
stimulation was observed in either brain region when nuclei were isolated 30 min after drug administration. To determine whether the LSD effect was due solely to increased ribosomal RNA synthesis, I analyzed the percentage of a-amanitin-resistant synthesis. As shown in Table 1, percentage a-amanitin-resistant RNA synthesis was the same in nuclei from control and LSD-treated animals. This result suggested that the drug-induced stimulation was due to proportionate increases in both nucleoplasmic and nucleolar RNA synthesis. DISCUSSION Isolated nuclei systems have been useful for the study of transcription in neural tissue: maturation-dependent changes in template ability of brain nuclei have been demonstrated (9), the presence of poly(A)-containing RNA suggested (10), and a report made of multiple RNA polymerases (11). We have utilized an isolated nuclei system to test whether LSD affects transcription in regions of thewrabbit brain. Under the conditions of our in vitro assay, the major synthetic activity is due to nucleoplasmic RNA polymerase. The drug was found to stimulate the ability of nuclei from two brain regions to synthesize RNA. The effect was pronounced in nuclei from the brain stem while less increase was observed in cerebral hemisphere nuclei. Both nucleoplasmic and nucleolar RNA synthesis were stimulated. The location of the maximal effect correlates with our previous findings on LSD stimulation of brain histone acetylation in which the midbrain demonstrated the greatest increase (6). The change in RNA synthesis at 2.5 hr after intravenous LSD administration is subsequent to the histone acetylation alteration, which occurs at 30 min. Modification of chromosomal proteins has been associated with regulation of transcription (7). At the
4A 4A
m
122
IM6
x
E3
E
0.
0
a
I
I 11
11%
20
30
10
if%
Time (min)
FIG. 5. Time course of RNA synthesis in isolated cerebral hemisphere nuclei after LSD administration in vivo. Procedure was as given in legend of Fig. 4. 0, ISD treatment; 0, saline control.
physiological level, LSD has been reported to specifically affect neuron firing and serotonin metabolism in the raphe nucleus region (12). This area is included in our brain stem fraction. Transcription in the brain is known to be complex. More transcripts of nonrepeated DNA are present in neural tissue than in other organs (1-5). LSD may provide a convenient method for inducing a change in RNA synthesis which can be used to advantage in studies that attempt to analyze basic mechanisms of neural transcription. The excellent technical assistance of Miss S. W. Lee is gratefully acknowledged. This project was supported under the program of Research on Drug Abuse administered by the NonMedical Use of Drugs Directorate, Health and Welfare, Canada. 1. Brown, I. R. & Church, R. B. (1971) Biochem. Biophys. Res. Commun. 42, 850-856. 2. Brown, I. R. & Church, R. B. (1972) Develop. Biol. 29, 7384. 3. Hahn, W. E. & Laird, C. D. (1971) Science 173, 158-161. 4. Grouse, L., Chilton, M. D. & McCarthy, B. J. (1972) Biochemistry 11, 798-805. 5. Grouse, L., Omenn, G. A. & McCarthy, B. J. (1973) J. Neurochem. 20, 1063-1073. 6. Brown, I. R. & Liew, C. C. (1975) Science, in press. 7. Allfrey, V. G. (1971) in Histones and Nucleohistones, ed. Phillip, D. P. (Plenum Press, New York), pp. 241-294. 8. Reeder, R. H. & Roeder, R. G. (1972) J. Mol. Biol. 67, 433441. 9. Banks, S. P. & Johnson, T. C. (1972) Brain Res. 41, 155169. 10. Banks, S. P. & Johnson, T. C. (1973) Science 181, 10641065. 11. Thompson, R. J. (1973) J. Neurochem. 21, 19-40. 12. Sourkes, T. L. (1972) in Basic Neurochemistry, eds. Albers, R. W. et al. (Little, Brown & Co., Boston), pp. 581-606.
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 RNA synthesis in isolated brain nuclei was ABSTRACT analyzed 2.5 hr after the intravenous administration of d-lysergic acid diethylamide (LSD) to young rabbits. The drug stimulated transcription by 54% in brain stem nuclei and by 13% in cerebral hemisphere nuclei expressed over saline controls. Both nucleoplasmic and nucleolar RNA synthesis were increased. The main activity, in the isolated nuclei assay was due to nucleoplasmic RNA polymerase, since a-amanitin reduced synthesis by over 70% in either drug or control treatments.
The complexity of transcription in neural tissue has previously been analyzed by RNA excess hybridization techniques. Our studies (1, 2) and those of others (3-5) indicate that RNA isolated from the mammalian brain hybridizes to a greater fraction of nonrepeated DNA as compared to RNA from other organs. Change in the transcription of nonrepeated DNA occurs during neural development. In this report we analyze whether a drug, which is known to affect the brain at the psychological and physiological level, influences transcription in the brain. Recently we reported that d-lysergic acid diethylamide (LSD) affects the brain at the level of covalent modification of chromosomal proteins (6). Moderate dosages of the drug were found to increase the acetylation of specific histones in the rabbit brain 30 min after intravenous drug administration. Since modification of chromosomal proteins may be associated with regulation of gene activity (7), we now examine RNA synthesis at a period shortly after the acetylation change in the LSD-treated rabbit brain. To circumvent the nucleotide pool size difficulties often inherent with determinations of RNA synthesis in vivo, we use an isolated nuclei system. In this report cerebral hemisphere and brain stem nuclei are shown to demonstrate increased ability to synthesize RNA after drug administration in vivo. MATERIALS d-Lysergic acid diethylamide (LSD), a product of Sandoz Pharmaceuticals, Switzerland, was obtained through the Department of National Health and Welfare, Ottawa, Canada. [3H]UTP was purchased from New England Nuclear, and other nucleotides and RNase-free sucrose from Schwarz/Mann. a-Amanitin from Boehringer, Germany, was donated by Dr. I. A. Menon, University of Toronto. METHODS Drug Administration. Young male New Zealand white rabbits weighing 1 kg were used throughout. LSD dissolved in Abbreviation: LSD, d-lysergic acid diethylamide. 837
0.9% (w/v) NaCl at 1 mg/ml was injected into the ear vein at 100 Ag/kg of body weight. Control rabbits received an appropriate volume of saline. Injections were always carried out at the same hour of the day. The animals were left undisturbed for 2.5 hr, then dispatched by cervical dislocation of the neck. Isolation of Nuclei. Brains were dissected into cerebral hemispheres and brain stem (total brain minus cerebral hemispheres and cerebellum), then homogenized with a Teflonglass homogenizer in 4 volumes of solution A [0.32 M sucrose, 50 mM sodium phosphate (pH 6.5), 50 mM KCl, 1 mM dithiothreitol, 2 mM MgC12, and 0.1% Triton X-100]. The homogenate volume was increased to 15 ml per brain region, filtered through one layer of cheesecloth, and centrifuged for 10 min at 1000 X g. The crude nuclear pellet was then homogenized again in solution A followed by centrifugation. The resultant pellet was suspended in 28 ml of solution B [2.2 M sucrose, 50 mM sodium phosphate (pH 6.5), 1 mM dithiothreitol, 2 mM Mg C12], layered over 10 ml of fresh solution B, and centrifuged for 45 min at 22,000 rpm in an SW 27 rotor. The purified nuclear pellet recovered after the centrifugation through dense sucrose was suspended in 2 ml of solution C [25% glycerol, 40 mM Tris HCl (pH 8.0), 1 mM dithiothreitol, 10 mM MgC12, 0.1 mM EDTA] and centrifuged for 10 minutes at 1000 X g. The washing procedure was repeated to remove sucrose prior to the determination of nuclear DNA concentration by the diphenylamine reaction. The final nuclear suspension was adjusted to 0.1 mg of DNA per ml with solution C.
RNA Synthesis Assay. The conditions for assaying RNA synthesis were a modification of the method of Reeder and Roeder (8). The standard reaction mixture contained in a final volume of 0.5 ml: 12.5% glycerol, 40 mM Tris HCl (pH 8.0), 200 mM KCl, 1 mM dithiothreitol, 5 mM MgCl2, 2.5 M MnClg, 0.05 mM EDTA, 5 mM creatine phosphate, 25 1Ag of creatine phosphokinase, 1.25 ,Ci of [3H]UTP, 0.15 mM each of ATP, GTP, and CTP, and 25 J.g of nuclear DNA. The complete mixture was mixed on a Vortex mixer and incubated at 370 for 30 min. Reactions were terminated by the addition of 0.5 mg of bovine serum albumin as carrier followed by an equal volume of 10% trichloroacetic acid. The acid-insoluble pellet was washed twice with 5% trichloroacetic acid and dissolved overnight in 0.5 ml of 0.3 M NaOH. Radioactivity of samples was determined by liquid scintillation in 10 ml of a Beckman Bio-Solv-toluene cocktail. All assays were run in duplicate.
838
Proc. Nat. Acad. Sci. USA 72 (1976)
Biochemistry: Brown 20
6A 0.
12
o
x 2
x
8
Ea. cL
I
10
1.
.1
I
40 30 20 DNA (ug/assay)
I
50
FIG. 1. Effect of DNA concentration on RNA synthesis in isolated brain nuclei. Nuclei purified from cerebral hemispheres were incubated at various concentrations under standard assay conditions as described in Methods.
RESULTS
Isolated Brain Nuclei. The nuclear isolation procedure received critical attention in order to achieve pure brain nuclei while retaining the ability for active RNA synthesis. The pH of the initial homogenization medium and concentration of magnesium ions were found to be particularly important. Clumping of brain nuclei occurred at pH greater than 6.5. Divalent ion concentrations less than 2,-mM caused nuclear breakage, while higher levels produced condensed nuclei with adherent cytoplasm. Inclusion of Triton X-100 in the initial medium improved the nuclear yield and markedly decreased cytoplasmic contamination. The 2.2 M sucrose gradient was required for complete removal of myelin fragments. Phase-contrast and electron microscopy of the final nuclear pellet revealed that the brain nuclei were free of cytoplasmic contamination. Recovery of DNA was 40% of the initial homogenate, and yield per brain was 800-900,ug for the cerebral hemispheres and 400-500.ug for the brain stem. In 25% glycerol solution C, nuclei were stable on ice for at least 2 hr. Nuclei could be quickly frozen in a dry ice/ethanol mixture, stored at - 500 for up to a month, and assayed immediately after thawing without significant loss of activity. RNA Synthesis. Purified brain nuclei actively synthesized RNA in an in vitro assay which included ['H]UTP and three unlabeled ribonucleotide triphosphates. The amount of isotope incorporated by brain nuclei was linearly dependent on
ocamanitin (ug/assay) FIG. 3. Effect of a-amanitin on RNA synthesis in brain nuclei. a-Amanitin was incubated with cerebral hemisphere nuclei under standard assay conditions. the concentration of nuclear DNA in the reaction mixture for the range 10-50 ,ug per assay (Fig. 1). Incorporation was dependent on the inclusion of all four ribonucleotides. In the presence of 5 mM magnesium and under high salt conditions, RNA synthesis was found to be stimulated by manganese ions. A 3-fold increase was observed when nuclei were incubated with 2.5 mM MnCl2 (Fig. 2). a-Amanitin reduced RNA synthesis by 73% under the standard assay conditions of 5 mM MgCl2 plus 2.5 mM MnCl2, indicating that a major portion of the activity was due to nucleoplasmic RNA polymerase (Fig. 3). Inhibitor concentrations of 0.1 ug/0.5 ml of assay were sufficient to produce maximal inhibition. Experiments in Figs. 1-3 used cerebral hemisphere nuclei; however, similar results were obtained with brain stem nuclei. Effect of Administration of LSD In Vivo. The injection of LSD into young rabbits produced a marked stimulation in the ability of isolated brain stem nuclei to synthesize RNA in the in vitro assay (Fig. 4). The increase was 54.3% ± 1.2 after 30 min of nuclear incubation expressed over parallel controls which received saline injections (average of four trials). Brain stem is the total brain minus cerebral hemispheres and cerebellum. Cerebral hemisphere nuclei also showed an enhancement in their ability to synthesize RNA (Fig. 5). The stimulation was 13.2% i 0.2 (average of four trials). In these experiments LSD was injected intravenously into the rabbits 2.5 hr prior to removal of the brain for nuclear isolation. No
x4 2
x/ -6
2
4
10
20
30
Time (min)
2
0
1
2
3
4
MnCl(mM)
FIG. 2. Effect of MnCl2 concentration on RNA synthesis in brain nuclei. Cerebral hemisphere nuclei were incubated under standard assay conditions with the exception of variation in concentration of MnCl2.
FIG. 4. Time course of RNA synthesis in isolated brain stem nuclei after LSD administration in vivo. LSD (100 ,Ag/kg) was injected intravenously into young rabbits, which were killed 2.5 hr later. Nuclei purified from the brain stem (total brain minus cerebral hemispheres and cerebellum) were incubated under standard assay conditions, as described in Methods. *, LSD treatment; 0, saline control.
Proc. Nat. Acad. Sci. UASA 72
RNA Synthesis in Isolated Brain Nuclei after LSD
(1975)
TABLE 1. a-Amanitin-resistant RNA synthesis in isolated brain nuclei
839
165 >1 co
% a-Amanitin-resistant RNA
synthesis Brain region
Cerebral hemisphere nuclei Brain stem nuclei
Saline control LSD treatment 27.3 4 1.0 28. 1 4+ 1. 3 26.9 i 1.2 27.9 i 1.3
Brain nuclei were incubated under standard assay conditions with or without a-amanitin at 0.5 .g per assay. Each value is the mean of five experiments i SEM.
stimulation was observed in either brain region when nuclei were isolated 30 min after drug administration. To determine whether the LSD effect was due solely to increased ribosomal RNA synthesis, I analyzed the percentage of a-amanitin-resistant synthesis. As shown in Table 1, percentage a-amanitin-resistant RNA synthesis was the same in nuclei from control and LSD-treated animals. This result suggested that the drug-induced stimulation was due to proportionate increases in both nucleoplasmic and nucleolar RNA synthesis. DISCUSSION Isolated nuclei systems have been useful for the study of transcription in neural tissue: maturation-dependent changes in template ability of brain nuclei have been demonstrated (9), the presence of poly(A)-containing RNA suggested (10), and a report made of multiple RNA polymerases (11). We have utilized an isolated nuclei system to test whether LSD affects transcription in regions of thewrabbit brain. Under the conditions of our in vitro assay, the major synthetic activity is due to nucleoplasmic RNA polymerase. The drug was found to stimulate the ability of nuclei from two brain regions to synthesize RNA. The effect was pronounced in nuclei from the brain stem while less increase was observed in cerebral hemisphere nuclei. Both nucleoplasmic and nucleolar RNA synthesis were stimulated. The location of the maximal effect correlates with our previous findings on LSD stimulation of brain histone acetylation in which the midbrain demonstrated the greatest increase (6). The change in RNA synthesis at 2.5 hr after intravenous LSD administration is subsequent to the histone acetylation alteration, which occurs at 30 min. Modification of chromosomal proteins has been associated with regulation of transcription (7). At the
4A 4A
m
122
IM6
x
E3
E
0.
0
a
I
I 11
11%
20
30
10
if%
Time (min)
FIG. 5. Time course of RNA synthesis in isolated cerebral hemisphere nuclei after LSD administration in vivo. Procedure was as given in legend of Fig. 4. 0, ISD treatment; 0, saline control.
physiological level, LSD has been reported to specifically affect neuron firing and serotonin metabolism in the raphe nucleus region (12). This area is included in our brain stem fraction. Transcription in the brain is known to be complex. More transcripts of nonrepeated DNA are present in neural tissue than in other organs (1-5). LSD may provide a convenient method for inducing a change in RNA synthesis which can be used to advantage in studies that attempt to analyze basic mechanisms of neural transcription. The excellent technical assistance of Miss S. W. Lee is gratefully acknowledged. This project was supported under the program of Research on Drug Abuse administered by the NonMedical Use of Drugs Directorate, Health and Welfare, Canada. 1. Brown, I. R. & Church, R. B. (1971) Biochem. Biophys. Res. Commun. 42, 850-856. 2. Brown, I. R. & Church, R. B. (1972) Develop. Biol. 29, 7384. 3. Hahn, W. E. & Laird, C. D. (1971) Science 173, 158-161. 4. Grouse, L., Chilton, M. D. & McCarthy, B. J. (1972) Biochemistry 11, 798-805. 5. Grouse, L., Omenn, G. A. & McCarthy, B. J. (1973) J. Neurochem. 20, 1063-1073. 6. Brown, I. R. & Liew, C. C. (1975) Science, in press. 7. Allfrey, V. G. (1971) in Histones and Nucleohistones, ed. Phillip, D. P. (Plenum Press, New York), pp. 241-294. 8. Reeder, R. H. & Roeder, R. G. (1972) J. Mol. Biol. 67, 433441. 9. Banks, S. P. & Johnson, T. C. (1972) Brain Res. 41, 155169. 10. Banks, S. P. & Johnson, T. C. (1973) Science 181, 10641065. 11. Thompson, R. J. (1973) J. Neurochem. 21, 19-40. 12. Sourkes, T. L. (1972) in Basic Neurochemistry, eds. Albers, R. W. et al. (Little, Brown & Co., Boston), pp. 581-606.
