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Home , Tryptophan

Advances in Biochemical , Vol. 10 Raven Press, New York © 1974

Brain After Tolerance

_6-557. okod DosageofLSD rties of m istry, reening Daniel X. Freedman and WilliamO. Boggan Neuro- d high University of Chicago, Chicago, Illinois 6"0637 d cord.

Ltbrain :646m I. INTRODUCTION of t, _rnalian Some 15 years ago we studied tolerance to LSD (Freedman,

•hal_zymicof Aghajanian, Ornitz, and Rosner, 1958) because of intrinsic interest and as a "lever" to aid in defining the role of neurohumors and mechanisms Le_ay. of LSD action. Operationally, tolerance is a decrement of effect _phrine contingent upon prior dosage schedule; the mechanisms axe obscure. ;6-467. With appropriate behavioral measures in rat, we observe a diminished H and response intensity or duration, or both, after daily doses of LSD; alciumemical increased dosage restores the initial response. A less distinct "short- term" tolerance occurs with LSD at hourly intervals (Freedman and f brain Aghajanian, 1959).

'sRavenycho- Tolerance is not "absolute"; there is an interaction of dosage, behavioral task, and drug effect (Freedman, Appel, Haxtman, and fitters. Molliver, 1964a). In the rat, sympathomimetic effects (mydriasis, 5strate piloerection, pyrexia), EEG alerting, and hind-limb ataxia (lasting 45 to don in 60 min after moderate doses) show tolermnce; centrally mediated parasympathomimetic effects (salivation and bradycardia--lasting 90 ?",NewJ- J--- min or more) do not (Freedman et al., 1958). Tolerance is not evident with noxious reinforcement such as escape behavior (Appel, 1968; '. aasay Hamilton, 1960). Thera- In man, subjective and sympathomimetic effects of 200 pg or less of dne in LSD are absent by the thh'd or fourth daily dose and return after 3 or 4 them- days of abstinence. In the rat, a behavioral effect gradually diminishes mid is clear-cut after foul" to seven daily doses; full sensitivity returns ctricalLmino- after 3 or 4 drug-free days. The "acute" effect is a tendency to cease responding for 30 to 45 min immediately after 130 pg/kg of LSD (longer periods with higher dosage m_d shorter periods with lower :.yand dosage). Positive reinforcement (food) seems not as '"mterestmo"_,'",the animal can, but is not disposed to exert organized behavior under these

151 L_

152 5-HT METABOLISM AFTER LSD

contingencies (Appel, Whitehead, and Freedman, 1968). A simple at 9:30 a.m. on food-rew_ded F-R schedule demonstrates the temporal characteristics differences in of this dose-contingent tolerance and cross tolerance of LSD, was , and (Freedman and Aghajanian, 1959; Appel and Lewander, 197( Freedman, 1968). Conceptually, "periods" of neurochemJcal change after drug injection may be correlated with the occurrence, intensity, and duration of different physiological and behavioral responses. A significant period for such correlation is the first 60 min after a The results s: single dose of LSD. There is an increase of 12 to 20% of 5-hydroxy- X 7 occurs dis -- (5-HT) localized in the particulate fraction of whole brain This shift of th (Freedman, 1961a), and further to a vesicle-rich fraction (Halaris, doses of LSDq Lovetl, and Freedman, 1972; Halaris and Lovell, 1973); a slight were acting lik( decrease in (Freedman, 1961b; Freedman, Barchas, and 5-HIAA (30 mi Schoenbrun, 1962; Freedman, 1963); a decrease in 5-hydroxy- 60 minis inter: indoleacetic acid (5-HIAA) and slowed turnover (Lovell, Rosecrans, and in time, this Freedman, 1967; Rosecrans, Lovell, and Freedman, 1967; Diaz, Ngai, tryptophan in I and Costa, 1968; And6n, Corrodi, Fuxe, and Hokfelt, 1968; And_n, to L X 1 (Bo! Corrodi, and Fuxe, 1971); a small increase of brain tryptophan (Tonge diminished rest and Leonard, 1970; Boggan and Freedman, 1973); brain levels of response of tr3 drug--following the plasma--arrive at half-life values by 45 min daily doses of: (Freedman, Aghajanian, and Coquet, 1964b; Freedman and Coquet, metabolism wit 1965; Freedman, 1966); raphe firing is inhibited with roughly similar onset and duration as the amine changes (Aghajanian, Foote, and Sheard, 1970). Synthesis is decreased (Lin, Ngai, and Costa, 1969; Schubert, Nyback, and Sedvall, 1970; Shields and Eccleston, 1973) as The increas, early as 21 min after drug (Shields and Eccleston, 1973), and recently, decreased syn we find, at 15 min after drug. The increased 5-HT, decreased 5-HIAA, Huttunen (197 and clearance of drug peak at the termination of the "acute" behavioral multiple oral ( effects. The 5-HT and 5-HIAA effect occurs with psychotomimetic when there are but not with phenylethylamines or nonpsychotomimetic reduced turnov congeners(Freedman, Gottlieb, and Lovell,1970). It now seel We now report initial attempts to determine whether tolerance dosa_ metabolism changes with tolerance dosage schedules during tim same the earlier pe_ period that physiological and behavioral effects show tolerance. We find tryptophan at significant alterations in brain 5-HT, 5-HIAA, and tryptophan concen- events seem t tration after daily administration of LSD, as preliminarily reported by dissimilar to BogganandFreedman(1973). (Freedmanet effects and pea II.METHOD Aftera singl (1) cessation Male Sprague-Dawley rats (175 to 250 g) were randomly divided into mentalization , three equal groups: (1) S X 7--daily saline for 7 days; (2) L X 1--saline whatever the for 6 days and LSD (520 pg/kg) on the seventh day; and (3) L X (3) undefined 7--LSD (520 pg/kg) daily for 7 days. Injections were at 5:00 p.m. and inhibition (Fr_ !

5-IIT METABOLISM AFTER LSD 153

ple at 9:30 a.m. on the day of sacrifice. The groups showed no significant tics differences in weight. Analysis of whole-brain 5-HT, 5-HIAA, and _D, tryptophan was then done (And_n and Magnusson, 1967; Jonsson and md Lewander, 1970; Denckta and Dewey, 1967). r!_je ity, III. RESULTS

_r a The results show the following: (1) The peak elevation of 5-HT in L xy- × 7 occurs distinctly earlier than for L × 1 (30 vs. 45 min) (Fig. 1). ain This shift of the 5-HT peak "to the left" also occurs with single lower ris, - doses of LSD (Rosecrans et al., 1967) (as expected if multiple doses _ht were acting like a single smaller dose). (2) The time of peak decrease in md 5-HIAA (30 min) is the same, but the concentration in L X 7 at 45 and xy- 60 rain is intermediate--between controls and L X 1; ra_her than a shift md in time, this represents a diminished response. (3)By 60 min, _ai, tryptophan in L X 7 returns to control concentrations--in sharp contrast, en, to L X 1 (Boggan and Freedman, 1973). As with 5-HIAA, this is a _ge diminished response to multiple doses. We now find similarly reduced of response of tryptophan after three daily doses of 520 pg/kg or seven nin daily doses of 260 pg/kg. We, as Winter (1971), find no change in drug ,et, metabolism with tolerance dosage. [lar md IV. COMMENT 39; as The increase in brain tryptophan after LSD, during a time of ly, decreased synthesis, will require future examination. Diaz and ,A, Huttunen (1971) showed an increase in turnover 13 hr after the last of ral multiple oral doses (20 /_g/kg); the general question is whether and tic when there are compensatory changes after one or several episodes of tic - reduced turnover. It now seems evident that there are neurochemieal responses to _le tolerance dosage schedules of LSD, manifest both in shifts in time (e.g., .he the earlier peak of 5-HT) and in magnitude (e.g., the differences in nd tryptophan at 60 min and the 5-HIAA at 45 and 60 min). In general, m- events seem to occur more ra_idly and to terminate sooner--not by dissimilar to what is obselwed in behavioral tests of tolerance (Freedman et al., 1964a). Termination of certain acute behavioral effects and peak changes in 5-HT seems roughly correlated. After a single dose of LSD, the mechanisms affecting indoles include _1) cessation of raphe neuronal firing; (2) "binding" or compart- to mentalization of 5-HT (an increase of no more than 100 ng/gm brain, ne whatever the dosage) almost specifically in nerxe-ending vesicles; X _3) undefined inaccessibility to MAO without evidence of direct nd inhibition (Freedman, 1961a; Freedman et al., 1970; Collins, Lovell, 154 5-HT METABOLISM AFTER LSD

125- _ L x7 Thed, O I.SD ma 120- -. -- [] [] TP, Lxl r,x',.ptor ",/" _\ _ TP, Lx7 ,I_...nces. 115- _...... o \\ * p < .05 _..!_:vior: Ct' . _ a:l_)ther , %, 5 - HT h

0 105- after re_ or" wtraben: I-- Z }"rt.edllla: _j _ the fact O 100 . ltF/0b) },

O n it't.;.l}_O[_,: LI_ to attvtr_l O 95 m'tion, :..rotonin 90-

85- A_i'taF_ntw • 5-HIAA -- (_lo_y d

dJ,'Lh)l_ L,f," .'_'<" At:d.rL N 75 1; 5'0 4'5 6'0 7'5 9'0 105 120 ,,,,.,;,, [Aru,t,-n, N TIME (MIN) OF SACRIFICE AFTER LAST INJECTION :.,.,.,..,., J.*t_r_:.N FIG. 1. Effect of multiple and single injections of LSD (520 #g/kg} on rat brain 5-HT, 5-HIAA, An,t," ) .% and tryptophan. Control valuesfor 5-HT and 5-HIAA are as follows (mean and standarderror in nanograms per gram): 5-HT, 502.t ± 5.1; 5-HIAA, 321.5 -+3.9. The control value for d,.t,._m_ tryptophan (mean _ standard error in micrograms/gram) is 4.96 + 0.06. The number of animals _:_ ;(J _ - ,%p;,,.! J used wasat least 20 per group per time point. The asterisks represent significance (p < 0,05) as t,-,c_,,; compared to the other LSD-treated group. E':,,_, I A;_;,,q. J

Boggan, and Freedman, 1970" Ziegler, Lovell, and Freedman, 1973); (4) some effects on uptake or reuptake of 5-HT (Ziegler et al., 1973) ,_.;,.,-: of and inhibition release (Chase, Breese, and Kopin, 1967); (5) a _:, _...... -:*. slowdown of synthesis of tryptophan to serotonin; and (6) increase of ,'_ :,.: j brain tryptophan. Precise effects of multiple dosage on these must yet '_ :-_. be defined. ::" '": _ 5-HT METABOLISM AFTER LSD 155

: 7 The decrement in magnitude and duration, not only of physiological : 1 and behavioral but also biochemical response, to several daffy doses of LSD may all be related to a single process--an as yet tmdiscerned , Lxl receptor or macromolecular mechanism which, in its "molar" conse- , L x7 quences, we call tolerance. But the fact that neurochemical as well as :.05 behavioral response shows "tolerance" indicates--at the least---yet another con'elation of LSD with mechanisms governing 5-HT metabo- lism and behavior. Other such linkages are apparent in the exquisite sensitivity of the raphe cell body to LSD (Aghajanian, Haigler, and Bloom, 1972) and in the fact that decreases in 5-HT enhance the effect of LSD [whether ""o after reserpine (Freedman, 1961b; Appel and Freedman, 1964), tetrabenazine (Appel and Freedman, 1964), PCPA (Appel, Lovell, and Freedman, 1970a) or raphe lesions (Appel, Sheard, and Freedman, 1970b)]. While the mechanism of the effect of multiple doses on amine metabolism is obscure, such dosage schedules provide yet another tool to attempt to delineate behavioral and biochemical mechanisms of LSD action, and their link to intrinsic regulatory sequences in brain serotonin metabolism.

REFERENCES

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Appel, J. B., Whitehead, W. E., and Freedman, D. X. (1968): Motivation and the behavioral effects of LSD. Psychonomic Science, 12(No. 7):305-306. co.- Boggan, W.O., and Freedman, D. X. (1973): LSD tolcrance and brain serotonin metabolism. Federation Proceedings, 32:694 (abstract). l.,,'.mi Chase, T. N., Breese, G. R., and Kopin, I. J. (1967): Serotonin release from brain ,br._ slices by electrical stimulation: Regional differences and effects of LSD. Science, i_, ,,_ 157:1461-1463. _"" tc_i Collins, B. J., Lovell, R. A., Boggan, W. O., and Freedman, D. X. (1970): Effect_s of :', h_, hallucinogens on rat brain monoamine oxid_.se activity. The Pharmacologist, _,f 12(No.2):256. I';: : Denckla, W. D., and Dewey, H. K. (1967): The determination of tryptophan in plasma, and urine. Journal of Laboratory and Clinical Medicine, :;:m-!,'.: 69:160-I69. f,t_: _ Diaz, J. L., and Huttunen, M. O. (1971): Persistent increase in brain serotonin ,_:rr: turnover after chronic administration of LSD in the rat. Science, 174:62-64. 'l',mg,., Diaz, P. M., Ngai, S. H., and Costa, E. (1968): Factors modulating brain serotonin 9_ I turnover. Advances in Pharmacology, 6B :75-92. \_m,,-r Freedman, D. X. (1961a): Effects of LSD-25 on brain serotonin. Journal of :m,.! Pharmacology and Experimental Therapeutics, 134:160-166. ,l,,u, Freedman, D. X. (1961b): Studies of LSD-25 and serotonin in the brain. Z:,._,h.r Proceedings of 3rd World Congress of Psychiatry, 1:653-658. tt,_ t_ Freedman, D. X. (1963): Psychotomimetic drugs and brain biogenic anaines, amt American Journal of Psychiatry, 119:843-850. Freedman, D. X. (1966): Aspects of the biochemical pharmacology of psychotropic drugs. In: Psychiatric Drugs, edited by P. Solomon, pp. 32-57. Grune and Stratton, New York. Freedman, D. X., and Aghajanian, G. K. (1959): Time parameters in acute tolerance, cross tolerance, and antagonism to psychotogens. Federation Pro- ceedings, 18:390 (abstract). Freedman, D. X., Aghajanian, G. K., and Coquet, C. A. (1964b): Effect of reserpine on plasma binding and brain uptake of LSD-25, Federation Proceedings, 23:147. Freedman, D. X., Aghajanian, G. K., Ornitz, E. M., and Rosner, B. S. (1958): Patterns of tolerance to lysergic acid diethylamide and mescaline in rats. Science, 127:1173-1174. Freedman, D. X., Appel, J. B., Hartman, F. R., and Molliver, M.D. (196,ta): Tolerance to the behavioral effects of LSD-25 in rat. Journal of Pharmacology and Experimental Therapeutics, 143:309-313. Freedman, D. X., Barchas, J. B., and Schocnbrun, R. L. (1962): Response of brain to exhaustion-stress or LSD-25. Federation Proceedings, 21:337 (ab- stract). Freedman, D. X., and Coquet, C. A. (i965): Regional subcellular distribution of LSD and effects on 5-HT levels. The Pharmacologist, 7:183. Freedman, D. X., Gottlieb, R., and Lovell, R. A. (1970): Psychotomimetic drugs and brain 5-hydroxytryptamine metabolism. Biochemical Pharmacology, 19(3):1181-1188. - Halaris, A. E., and Lovell, R. A. (1973): On the nature of the serotonin binding after LSD. Federation Proceedings, 32:694. Halaris, A. E., LoveIl, R. A., and Freedman, D. X. (1972): Subcellular studies on the effect of LSD on rat brain serotonin. Program and Abstracts, Society for Neuroscience, Second Annual Meeting, p. 114. Hamilton, C. L. (1960): Effects of LSD-25 and on a running response in the rat. Archives of General Psychiatry, 2:104-109. Jonsson, J., and Lewander, T. (1970): A method for the simultaneous determina- tion of 5-hydroxy-3-indoleacetie acid (5-HIAA) and 5-hydroxytryptamine (5-ttT) in brain tissue and eerebrospinal fluid. Acta PhysioIogica _._candinavica. 78:43-51. Lin, R. C., Ngai, S. H., and Costa, E. (1969): hysergic acid diethylamide: Role i_1 L

5-tIT METABOLISM AFTER LSD 157

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