Neurochemical changes following the administration of depleters of biogenic monoamines

Life Sciences, Vol . 19, pp .1663-1668, 1976 . Printed in the U .S .A .

Pergamon Press

I~UROCE~QCAL CIiAN(~5 PDLIAWING TI-D; ALhIINISTRATIDri OF nFpr .~s OF BIOGENIC 1~VOAI~üNES J. D . Lane, J . E. Smith, P . A. Shea and W. J. McBride Section of Basic Neural Sciences, The Institute of Psychiatric Research and Departments of Biochemistry std Psychiatry Indiana University School of Medicine, Indianapolis, Indiana 46202 (Received in final form October 11, 1976) MARY

A variety of drags which were thought to selectively effect the synthesis, release or reuptake of the biogenic motxmnnines, and thereby deplete their stores in brain, were administered to rats . The animals were given intraperitoneal injections of reserpine, d-methyl-mats-tyrosine, d-methyl-pare-tyrosine, parachlorophenylalanine and tetrabenazine, and were killed at intervals known to cause maximum depletion of serotonin, dopamine and norepinephrine . When Ga~ared to saline controls, the drugs depleted the respective aionoamines, as expected, but changes in the levels of acetylcholine, alanine, glycine, Î-aminobutyric acid, aspartate std glutamate were also observed . Depleters of the biogenic tronomnines have traditionally been studied in the context of drugs which effect the synthesis, reuptake and release of serotonin, dopamine and norepinephrine . Data fran this laboratory have shown that more than one transmitter system can be affected by manipulating another (1), and several other investigators have begun to re-evaluate the actions of same of these drugs and have reported that reserpine, among others, effects the amino acid putative neurot ~n~~ tters (2,3) . To expand this idea, preliminary studies were carried out using whole brain samples to determine if reserpine, o(-methyl-mete-tyrosine, of methyl-pats-tyrosine, parachloraphertylalanine and tetrabenazine when administered to rats in acute doses had any effects on the content of acetylcholine, alanine, glycine, ~-aminobutyric acid, aspartate and glutamate, in addition to their reported effects on serotanin, dopamine and norepinephrine . METFDDS Wistar male rats (200-250 g) were used in studies . They were housed in groups of six and given food and water ad libitum, and were maintained on a 12 hour light (6 A.M . to 6 P .M .) - 12 hour dark schedule . To minimize artifacts due to the sacrifice procedure, the animals were also handled and accoamodated to a dipping cage of the near-freezing apparatus (4) . The animals received an intraperitaneal injection of a saline solution or suspension of the respective drug (injection wlumes of 0 .5 to 1 .0 ml) and were returned to the dipping cage . At the appropriate time, (usually between 11 A.M . and 12 A.M .) each animal was killed by the near-freezing technique (5) . The whole brain was removed in a refrigerated Goldbox at -4°C (6) and stored in liquid nitrogen until analyzed . The choices for doses and kill times were determined from previous publi1663

1664

Neurochemistry Following Depleters

Vol . 19, No . 11

cations : a) A saline vehicle control was injected intraperitoneally (0 .5 ml whims) and the animals were killed at 4 hours ; b) Reserpine (Ciba Pharmaceutical Co .) was dissolved according to Pletscher et al . (7) and was administered in a dose of 5 mg/kg and the animals were-ki3led at 4 hours . d-methyl-mats-tyrosine monohydrate (Regis Chemical Co.) was administered in a dose of 400 mg/kg and the animals were killed at 4 hours as described by Hess et al . (8) ; d) d-methyl-pare-tyrosine methyl ester HC1 (Sigma Chemical Co.) wâs administered in a dose of 100 mg/kg and the animals were killed at 4 hours as described by Spector et al . (9) ; e) Para-chlorophenylalanine methyl ester FiCl (Regis Chemical Co .) was a~ninistered in a dose of 300 mg/kg~and the animals were killed at 72 hours as described by Koe and Weissman (10) ; and f) Tetrabenazine (Fioffman LaRoche, Inc .) was administered in a dose of 2 mg/kg and the animals were killed at 30 minutes as described by Pletscher (11) and Aprison and Hingtgen (12) . The criteria for each dose and time was a canbination whidi yielded a maxims depletion of the respective biogenic monoamines . The whole brain samples were analyzed according to the procedure described by 9mi.th et a1 . (13) for content of serotonin, dopamine, norepinep~hrine, acetylcTiol~ne, alanine, glycine, Î-aminobutyric acid, aspartate and glutamate . RF.S[A.TS Compared to the saline vehicle controls, the drugs caused statistically significant depletians (p ~ 0 .001) of the biogenic mmoamines, to varying degrees (Table 1) . The administration of an acute dose of reserpine caused an 88 percent decrease in level of serotonin, a 98 percent decrease in the level of dopamine and an 87 percent decrease in the level of norepinephrine . The administration of an acute dose ofo(rmethyl-mete-tyrosine caused a 70 percent decrease in the level of serotonin, a 81 percent decrease in the level of dopamine and a 78 percent decrease in the level of norepinephrine . The administratia~n of an acute dose ofd-methyl-pare-tyrosine caused a 74 percent decrease in the level of dopamine and a 45 percent decrease in the level of mrepinephrine, while the level of serotonin did not change . The administration of an acute dose of par~-chlorophenylalanine caused a 90 percent decrease in the level of serotonin, and also caused a 32 percent decrease in the level of dopamine and a 16 percent decrease in the level of norepinephrine . The administration of an acute dose of tetrabenazine caused a 44 percent decrease in the level of serotonin, a 40 percent decrease in the level of dopamine and a 41percent decrease in the level of norepinephrine . Data similar to these have bean reported by other investigators (4-12) . Sane statistically significant changes (p ~ 0 .05) in the levels of acetylcholine and the aanino acids were also observed in the same samples (Table 1) . The administration of an acute dose of reserpine caused a 9 percent decrease in the level of acetylcholine, a 12 percent decrease in the level of glycine, and a 9 percent decrease in the level of ~-aminobutyric acid and a 7 percent decrease in the level of glutamate . The administration of an acute dose of par~-chlorophenylalanine caused an 8 percent decrease in the level of acetylcholine, a 20 percent decrease in the level of alanine and a 12 percent decrease in the level of ~-amir~obutyric acid . The administration of an acute dose of tetrabenazine caused a 7 percent decrease in the level of acetylcholirue, a 16 percent decrease in the level of aspartate and an 11 percent decrease in the level of glutamate . Three animals were injected with saline and killed after 30 minutes to see if the time interval effected the tetrabenazine data. Values for content of the canpounds for these saline controls were essentially identical to the 4-hour animals . No changes in the levels of

Neurochemistry Following Depleters

Vol . 19, No . 11

1665

TABLE 1 EFFECTS OF INJECTIONS OF RFSHRPINE, d-METHYL-META-TYROSII~, d-1~1NYL-PARA-TYR06INE, PARANE AND TETRABIIZ4ZINE ON THE ODrTIENT OF BIOGFNIC AMINES AND M~QPD ACIDS IN WtDLE BRAIN OF RATS

Content

Saline

Reserpine

d-DMI'

d-MPT

PCPA

TBZt

~oles/g 5-HT

3.31 +0 .44

0 .39** +0 .15

0 .99** +0 .46

3 .06 +0 .39

0 .33** +0 .24

1 .84** +0 .38

NE

2.53 +0 .12

0 .33** +0 .07

0 .56** +0 .07

1.39** +0 .10

2 .12** +0 .15

1.49** +0 .29

Il4

5 .76 +0 .77

0.12** +0 .07

1 .08** +0 .41

1 .49** +0 .59

3 .91** +0 .58

3 .47** +0 .79

ACh

19 .9 +1 .9

18 .1* +2 .1

18 .8 +1 .1

19 .0 +1 .0

18 .4* +1 .0

18 .5* +0 .6

Ala

0 .414 +0 .054

0 .441 +0 .069

0 .457 +0 .090

0.424 +0 .038

0.332* +0 .065

0.438 +0 .033

Gly

1 .52 +0 .16

1 .34* +0 .22

1.66 +0 .57

1.43 +0 .16

1.50 +0 .34

1.58 +0 .31

GABA

1 .61 +0 .18

1 .47* +0 .14

1 .58 +0 .11

1.51 +0 .16

1.41* +0 .21

1.50 +0 .08

Asp

2 .46 +0 .16

2 .56 +0 .22

2 .57 +0 .24

2.55 +0 .21

2.36 +0 .24

2.06* +0 .18

Glu

12 .6 +0 .8

11 .7* +0 .8

13 .1 +1 .3

13 .1 +0 .9

12 .2 +1 .0

11 .2* +1 .4

~rooles/8

Data represent the means + standard deviations, N ~ 12, tN ~ 6. Statistically significant differences with respect to saline controls are: **pC 0 .001 ; *p~ 0 .05 . acetylcholine nor the amino acids were observed following the administration of d-methyl-mete-tyrosine or ofmethyl-pare-tyrosine . DISCUSSION Drugs which were thought to selectively deplete biogenic monoamines were observed to deplete acetylcholine a1d the amino acid putative neurotransmitters. These observations support and supplement the work of other investi gators . Reserpine has been shown to cause a decrease in the levels of giu-

1666

Ne ;srochesnistry Following Depleters

Vol . 19, No .ll

tamate, glutamine, aspartate and alanine/glycine (measured as one spot by thin layer chromatography) in sympathetic ganglia and spinal cord of the cat (2) . Berl and Frigyesi also showed that resezpine caused a three-fold in crease in turnover of amino acids in brain areas of the cat (3) . McGeer et al . demonstrated that reserpine caused a 33 percent decrease in the levelsof acetylcholine in rat striatum, when given in three 1 ~kg doses at 24 hour intervals (14) and Malhotra and Rmdlik observed changes in acetylcholine in brain areas of dogs following adtnini .stration of reserpine (15) . Wurtman et al . observed a decrease in the levels of tyrosine and dihydroxyphenylalanne brain of rats following the administration of pare-chloropherylalanine, and suggested that this might explain the observed decreases in levels of dopamine and norepinephrine (16) . Doteuchi et al, s~gested that 0(methylpara-tyrosine was converted to pare-hydraxyamrmhétamine which causes the release of dopamine (17) . o(-Methyl-mete-tyrosine is apparently decarbaxylated to its metabolite, 0(-methyl-mete-tyramine, which acts as a false tn+~~ tter to cause release of serotonin and other compounds (18,19) . The results fran injections of o(-methyl-pare-tyrosine suggest that its actions are fairly specific for the adrenergic system, while the other drugs seem to have multiple effects .

n

Since the drugs studied here cause gross depletion of the biogenic monoamines, one might question whether a 7 to 16 percent depletion of acetylcholine and/or amino acids could bring about behavioral or physiological changes . 9nith et al . (20) observed a significant 14 percent decrease in ~-aminobutyric aciéin the telencephalon and a significant 16 percent decrease i.n glycine in the midbrain of rats subjected to one day of shock avoidance training (compared to their yoked-shocked controls) . Hingtgen et al . (21) observed a significant 8 percent increase in acetylcholine in thé te~encephalon and a significant 9 percent increase in acetylcholine in the midbrain of rats subjected to conditioned suppression (conditioned emotional response) training . Admittedly neuxnchemical-behavioral data of this type are sparse, but suggest that small but significant changes in airy of the putative neurotransmitters could bring about a change in behavior. The changes in the levels of acetylcholine and the amino acids observed after the administration of drugs which are thogght to act primarily on the biogenic mo~wamines suggest that the dnigs lack specificity of action and/or there are extensive and complex interactions between neurons which release different transmitter substances . In these preliminary experiments, the effects of the various drugs were studied on whole brain samples at only one time point . Therefore, it is possible that changes may be occurring which are not demonstrated by the present experiments . Consequently, more detailed time worse studies with small regions of the brain are required before re liable statements can be made regarding ineffectiveness of a drug. It is not currently known whether these changes represent primary responses, or metabolic and compensatory mechanism at work, but this study suggests that future reference to the specificity of action of certain drugs might be evaluated in a necessarily cautious manner . ACIQ~lQYL~~Nf This work was supported in part by Research Grant MH-03225-16,17 fro® N.I .M.F1 ., a Research Grant fron the Association for the Advancement of Mental health Research and Fducation, Inc ., Indianapolis, Indiana and Postdoctoral T~sining Grant MH 10695 .

Vol. 19, No . 11

Neurochemistry Following Depletere

1667

REFERII~ICES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 .

M.H . APRISON and J.N . HINGIC~N, Life Sci. 5 1971-1978 (1966) . M. SANfINI and S . BERL, Brain Re~TGI-TI6 (1972) . S . BERL and T.L . FRIGYESÎSra~es. 14 683-695 (1969) . R. TAKAEU44II and M.H . APRISUN,.T.~etu~e~ . 11 887-898 (1964) . P.A . SIDA and M.H . APRISON, Anal. Sioc~-5665-177 (1973) . M.H . APRISOTi, P .A . SHEA and 7~ Ri(iilEl~ incholine and Ace lcholine : Handbook of Cheaical Assa Methods (I . Hanin, e . , pp . - , ven ress , I~éwYork 1 T A. PLE1~~R, P.A . SH'JRE and B .B . BImDIE, J. Pharmacol . Exptl . Ther . 11 6 84-89 (1956) . S.M . HESS, R.H . CONVAMACE~t, M . OZAKI and S . UDE[~RIEND, J_. Pharmacol . tl . Ther . 134 129-138 (1961) . A~JOERA4~IA and S . UDENFRIEND, J_. fharmacol . Exptl . Ther . 147 86-95 (1964) . gK. ImE and A. WEIS4r1AN, J. Pharmacol . Exptl . Ther . _154 499-516 (1966) . A. PLE15(~OïtR, Science 126 ~5b7~.M.H . APRISOtd a~.f~ AI~IGEN, Recent Advances in Biolo ical P chut Vol . VIII, pp . 87-100, Plenen Press, ew or J.E . SMITH, J .D . LANE, P .A . SFIPA, W.J . McBRIDE and M.H . APRISOri, Anal . Biochem. 64 149-169 (1975) . ~FrcGEEIF, D.S . GRII~fVAAL and E.G . McGEER, Brain Res . 80 211-217 (1974) . C.L . MAI~DIRA and P .G . P[l1mLIK, Brit . J . Pha~~4 ~C-55 (1959) . R.J . W[IRIMAN, F. LARIN, S. bUSTAFA~JR . . , Science _185 183-184 (1974) . M. DOTHJCHI, C. WANG and E . COSTA, Biochem. Pharmacol . 10 225-236 (1974) W.J . McBRIDE, M.H . APRI90N and J .N . col . 12 769 ; 6 (1973) . G.L . GPSSA, E . COSTA, R. KUNfZMAN and B .B . BRODIE, Life Sci. 1 353-360 (1962) . J.E . SMITH, J .D . LANE, P .A . SHEA, J .N . HINGTGEN, W .J . McBRIDE and M.H . APRI90N, Fed. Pros . 34 292 (1975) . J.N . HING3~EtQ,7~. 5PiI1H, P.A . SHEA, M.H . APRISOtd and T .M . GAFF, Science 193 332-334 (1976) .