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Jr n ooom _ LIT sanoq 3 Ned siunoo J282V A 88 17 activity at mid-day was the result of disturbances associated with the changing of food and water. For all experiments, counts were only recorded between 7:00 p.m. and 7:00 a.m. and expressed as counts per hour. Two days of control readings were taken before drugs were added to the diet. Control activity counts varied between groups (1000-1600 counts per hour), but within any one group the difference was usually less than 200 counts per hour. When it became necessary to isolate the mice, only two animals were housed in the cage. They were separated by a clear plexiglass panel with holes large enough to allow only nasal contact. c. Shuttle box conditioning in rats and mice Discriminated conditioned avoidance was in— vestigated as described by Moore and Rech (1967). For rats, a light stimulus was presented automatically by a timer and an observer recorded the responses of each rat. Each trial was initiated by activating a small light on the Side of the cage occupied by the animal. After 5 sec the grid floor on that side was electrified for an additional 5 sec after which both the light and shock were terminated together. If the rat moved to the unlighted side during the initial 5 sec, the response was termed an avoidance; if the animal shuttled during the latter 5 sec of the trial, when the grid floor was electrified, the reSponse was an escape. The trials 18 were repeated every 30 sec and 20 such trials constituted a test session. Only the rats which consistently averaged more than 15 avoidances per session were used. A similar training procedure was employed in mice using a two compartment shuttle box designed for this Species (Lehigh Valley). Either a light or a tone served as the conditioning stimulus. The entire procedure was automated and an observer was not required to record the responses. 3. Chemical assays a. Analysis of norepinephrine and dopamine The concentrations of dopamine and norepinephrine in whole mouse brain were determined in several ways using modifications of previously described methods (Moore and Rech, 1967). Two or 4 pooled brains were homogenized in 6 m1 of cold 0.4N perchloric acid and kept in ice. After 30 min the homogenate was centrifuged at 5,000 x g for 5 min. The supernatant was collected and the pellet rehomo— genized in 6 ml of 0.4N perchloric acid and centrifuged as before. The supernatants were combined and the pH adjusted to 4 with 10N KOH and the potassium perchlorate precipitate was removed by centrifugation. The tissue extracts were transferred to 50 m1 glass stoppered centrifuge tubes containing approximately 400 mg of alumina oxide (Woelm) and 0.5 m1 of 0.2M disodium ethylenediaminetetraacetate (EDTA). The pH was 19 adjusted to 8.6-8.7 with 5M and 0.2M K CO and the 2 3 tubes shaken for 5 min. The supernatant was removed by aSpiration and the alumina was Shaken for 5 min with 10 ml of distilled H20. The water was aspirated off and the catecholamines eluted from the alumina with 8 m1 of 0.2N acetic acid or 4 m1 of 0.05N perchloric acid if 2 brains were pooled. For this last step, shaking time was 10 min and the supernatant was removed after centrifugation. A standard tube containing 0.8 ug norepinephrine and 2.0 pg dopamine was carried through the procedure. 1. Norepinephrine assay in acetic acid (4 m1) a. adjust pH to 6.5—6.8 with 5M K CO 2 3 b. add 0.8 m1 of 0.1M phosphate buffer (KH2P04) pH 6.5 c. take 2.4 ml aliquot for sample and the remainder for the blank Sample d. add 0.05 ml of 0.25% potassium ferricyanide (K3FeCN6) e. after 2 min add 0.25 ml of freshly prepared alkaline sulfite (20 mg ascorbic acid + 1.0 ml H20 + 9.0 m1 5N NaOH) Read fluorescence 10 min later with activation- fluorescence wave lengths at 391-510 mu respectively. 20 9.1.2311): Eliminate step (d). Recovery of 0.8 pg of norepinephrine standard was approximately 80%. ii. Norepinephrine assay in perchloric acid (2 m1) a. adjust pH to 6.5 with 0.2M K CO 2 b. add 0.4 ml of phOSphate buffer 3 c. take 1.2 ml for sample and remainder for the blank Steps d, e, f, same as above Recovery of 0.4 pg of norepinephrine was approximately 75%. iii. DOpamine assay in acetic acid (4 ml) a. add 2.0 m1 of 0.5M phosphate buffer (KZHPO4) pH 8.0 b. take 3.0 ml for sample and remainder for the blank Sample c. add 0.2 m1 of 0.5% sodium periodate (freshly prepared) d. after 1 min add 1.0 m1 of alkaline sulfite (2.65 g of Na SO + 10 ml 2 3 H20 + 9 ml of 5N NaOH) e. in rapid succession add 2.8 m1 H20; 1.0 ml of 0.5M citrate buffer pH 4.0 and 1.7 ml of 3M phOSphoric acid mixing after each addition iv. 21 Read fluorescence 10 min later with activation-fluorescence wave lengths at 335—395 mp respectively. The recovery of 2.0 pg dopamine standard was approximately 85%. RARE: Step (c) substitute 0.2 m1 H20 and repeat steps d, e. DOpamine assay in perchloric acid (2 ml) a. add 1 m1 0.5M phosphate buffer (KH2P04) pH 7.0 b. take a 1.5 ml aliquot for the sample and the remainder for the blank Sample c. add 0.2 ml of 0.5% sodium periodate d, e, f. Same as above except the volumes of reagents are only half of those described 1.3122: Same as above The recovery of 1.0 pg standard of dOpamine was approximately 55%. b. Analysis of catecholamines and a-methyldopa In the studies with d-methyldOpa, 4 pooled mouse brains or individual rat brains were homogenized in cold 0.4N perchloric acid as described above. Ascorbic acid 22 (0.1 ml of 20 mg/ml solution) was added to the super— natant and the pH was adjusted to 4 with 10N KOH and to 6.0 Wlth 5M K2CO3. was poured on a column of Dowex ion-exchange resin After centrifugation the supernatant (5W X-8 200—400 mesh, Na+ form, 6 mm x 34 mm). The resin was treated as described by Uretsky and Seiden (1969). It was cycled through washes with 2N NaOH, distilled H 0 until the pH was 7; 2N HCL and distilled 2 H20 until the pH was 7. When the columns were prepared, the Na+ form was achieved by passing through 0.1M NaH2P04 was 6.5 (about 15 ml). After 10 ml of H20 was added to the column and discarded, the tissue supernatant was buffer pH 6.5 until the pH of the effluent poured on the column. The effluent and subsequent 10 m1 H20 wash were combined and contained a-methyldopa. Five m1 of 1N HCL were added to the column and discarded. Norepinephrine and a-methylnorepinephrine were then eluted with 8 m1 of 1N HCL. An additional 2 m1 of 1N HCL were collected and discarded. DOpamine and d- methyldOpamine were then eluted in 12.5 ml of 2N HCL. i. a—Methyldopa assay Ten m1 of the combined column effluent and H20 wash was adjusted to pH 8.6—8.7 and shaken with alumina (see pg 18). a- Methyldopa was eluted from the alumina with 8 m1 of 0.2N acetic acid and an apprOpriate 23 aliquot was assayed for a-methyldopa exactly as described for norepinephrine (see pg 19). Fluorescence was maximal 45 min after oxidation. The recovery for 2.0 pg of d-methyldOpa carried through the procedure was approximately 40%. ii. Norepinephrine assay in 1N HCL Dowex eluate a. adjust pH of a 2 ml aliquot to 6.5 with saturated, l and 0.5M K2CO3 b. Take a 1.15 ml aliquote for the sample and the remainder for the blank Sample c. add 0.2 m1 of 0.1M phOSphate buffer (KH2P04) pH 6.5 d. add 0.05 ml of potassium ferricyanide e. after 2 min add 0.12 ml alkaline ascorbate (10 mg ascorbic acid, 0.5 ml H20 and 4.5 ml 5N NaOH) Recovery for 0.4 pg norepinephrine standard was approximately 75%. Blank e. add 0.14 ml of 5N NaOH f. after 15 min add 0.2 ml of ascorbic buffer (6 mg ascorbic acid in 5 m1 of phOSphate buffer) 24 Table 1. Fluorescence of d-methylnorepinephrine (dMNE) in the presence and absence of norepinephrine (NE) Fluorescence Units 1* 2+ 3+ 4 Erythro— dMNE aMNE aMNE Column 2 (i) dMNE + + minus pg 0.1 pg NE 0.1 pg NE Column 1 0.05 22 28 6 6 0.1 23 35 13 12 0.15 23 43 20 20 0.2 24 49 26 25 Activating and fluorescence peaks: 390 and 510 mp; meter multiplier = 0.03; sensitivity = 0. ‘1: not heated +heated 50 min in boiling water bath 25 iii. a—Methylnorepinephrine assay in 1N HCL Dowex iv. eluate a. heat a 2 m1 aliquot in a boiling water bath for 50 min b. repeat the entire procedure described above for norepinephrine The amount of a-methylnorepinephrine in the heated samples was calculated by subtracting from the total fluorescent units in these samples, the amount of fluorescence measured in unheated samples (compare columns 3 and 4, Table 1). The activation—fluorescence wave lengths are those for norepinephrine. The recovery for 0.8 pg of racemic a-methyl— norepinephrine was approximately 55%. Alumina procedure -dopamine and d—methyl— dopamine a. transfer 12 ml of the 2N HCL Dowex eluate to 50 ml glass stoppered test tubes containing approximately 400 mg of alumina and 0.5 ml of EDTA b. add 2.6 ml of 5M K CO3 and adjust pH 2 to 8.6—8.7 with 5M K2C03 c. alumina procedure and elution with 4 m1 of 0.05N perchloric acid as described on pg 18 26 v. a-Methyldopamine assay a. take a 2 m1 aliquot of the perchloric acid eluate and assay using the tri- hydroxyindole procedure for norepinephrine (pg 20) Fluorescence is maximal 15 min after oxidation. Activation-fluorescence wave lengths are those for norepinephrine. Recovery for 1.2 pg of a-methyldopamine carried through the entire procedure was approximately 40%. Vi. Dopamine assay a. Dopamine was assayed in the remaining 2 m1 aliquot exactly as described on pg 21. Recovery for a 2.0 pg standard was approxi- mately 45%. c. Radioactive catecholamine analysis Cl4-Tyrosine U.L. (337 mc/mmole, New England Nuclear Corp., Boston, Mass.) stored in 1N HCL, was evaporated to dryness in a rotary evaporator (Buchler Instruments). The dry residue was dissolved in saline and administered (10 pC in 0.1 m1 saline) to mice by injection into the tail vein. One half hour later, the mice were decapitated and individual brains homogenized in 10 ml of 0.4N perchloric acid. One pg of unlabeled norepinephrine, dopamine and normetanephrine was added 27 as a carrier and the catecholamines were adsorbed on alumina as described on pg 18. The amines were eluted initially with 3 ml of 0.2N acetic acid and a second time with 1 ml. The alumina effluent, which contained the O-methylated catechols was passed through a Dowex column (6 mm x 34 mm) in the Na+ form. After a 10 ml water wash, 3 ml of 2N HCL were added to the column and discarded; normetanephrine was then collected in 12 ml of 2N HCL. Labeled norepinephrine and dopamine in the alumina eluate were separated on a Dowex ion exchange resin (50 W X-8 200—400 mesh Na+ form, 6 mm x 38 mm). After a 10 m1 H O wash,5 m1 of 1N HCL were added to the 2 column and discarded. Norepinephrine was then collected in 14 ml of 1N HCL. One ml of 2N HCL was discarded and dOpamine was eluted with 14 m1 of 2N HCL. The radio— activity in the combined column effluent and H20 wash was considered to be that of the deaminated catechols. In all cases, the compounds were collected in scintillation vials and dried in a laboratory hood under a stream of air. The dry residue was dissolved in 1 ml of H20. Ten ml of modified Bray's solution (6 g of 2,5—diphenyloxazole and 100 g of naphthalene/liter of dioxane) was added and the samples were counted in a Beckman DPM—lOO liquid scintillation counter. Counting efficiency corrected for by means of an external standard was 88-90%. Recoveries for labeled standards carried through the 28 entire procedure were approximately 73% for norepinephrine; 60% for dopamine; 60% for normetanephrine. d. o-Methyltyrosine analysis d—Methyltyrosine was assayed fluorometrically by a modification (Carr and Moore, 1968) of the method described by Porter et al. (1966). i. Plasma a—methyltyrosine a. combine 0.1 ml of plasma with 1.0 ml of 6% trichloroacetic acid (TCA) (mix) b. transfer supernatant to 15 ml glass centrifuge tubes containing 0.1 ml pyridine and 0.5 ml 1% ninhydrin c. heat in boiling H20 bath for 10 min and cool immediately d. add 0.1 m1 of concentrated HCL (Shake) e. add 2.0 m1 of ethylacetate (stOpper and shake) f. aspirate ethylacetate layer and transfer 1.0 ml of the remaining solution to 15 ml glass centrifuge tubes g. add 0.5 ml of nitric acid reagent (1 ml 2.5% NaNO + 49 ml HNO 2 3 of conc HNO3) and 0.5 m1 nitroso-napthal (1:5 dilution reagent (100 mg 1-nitroso—2—naptha1 in 100 ml 95% ethanol) h. heat for 30 min at 55°C 29 after cooling add 2.5 ml of ethylene dichloride and shake for 5 min centrifuge and read the fluorescence of the supernatant Activation—fluorescence wave lengths were 456—560 mp respectively. The concentration in the plasma was determined from a standard curve . ii. Brain o-methyltyrosine a. homogenize 4 pooled brains in 6 ml 6% TCA after centrifugation transfer 3 ml of the supernatant to 15 m1 glass centri— fuge tubes containing 1.5 ml ninhydrin and 0.3 m1 pyridine and Shake heat in boiling water bath for 10 min and then cool add 0.35 ml of concentrated HCL and Shake add 5.0 ml of ethylacetate; shake aspirate the supernatant and transfer 2 ml of the remaining solution to 15 ml glass stOppered centrifuge tubes add 1 ml of nitric acid reagent and 1 m1 of nitroso—napthal reagent and shake 30 The remaining steps are the same as those described for the assay in plasma. For all of the fluorometric assays the meter multiplier and sensitivity of the Aminco- Bowman Spectrophotofluorometer were set at 0.03 and 0 respectively. e. Statistical analysis The significance of all data was determined with Student's t test. P values less than 0.01 were considered significant. CHAPTER III ACUTE EFFECTS OF u-METHYLDOPA Aromatic L—amino acid decarboxylase was the first of the three enzymes involved in the biosynthesis of catecholamines to be studied. It was also the first for which inhibitors were developed (Clark, 1959). The addition of a methyl group to the a carbon atom of dOpa produced a compound (d—methyldopa) that inhibited the Ho . (.2943 no.— cufc — m2 q-thvwom I .COOH decarboxylase enzyme both in vitra (Sourkes, 1954) and in viva (Stone and Porter, 1967). a—Methyldopa is thought to prevent decarboxylation of aromatic amino acid substrates by competitive inhibition (Stone and Porter, 1967). Clinically methyldopa has antihypertensive as well as sedative properties (Oates at al., 1960; Gillespie at aZ., 1962). Although marked reductions in blood pressure were not seen in animal studies, sedation and behavioral depression are well documented (see Sourkes, 1965; Kadzielawa, 1967; Stone and Porter, 1967). Attempts to correlate these effects with biochemical 31 32 changes have not produced a clear understanding of the antihypertensive or sedative effects of a-methyldopa. Acute administration of a—methyldopa to animals resulted in a reduction of the dopamine and 5-HT levels in the brain and a much longer lasting depletion of norepinephrine from the brain and peripheral tissues (Hess at aZ., 1961; Porter at aZ., 1961). These investigators concluded that inhibition of the decarboxy— lase enzyme was primarily responsible for the effects on dOpamine and 5-HT, but the depletion of norepinephrine resulted from some other mechanism. Carlsson and Lindqvist (1962) found that d-methyldOpa was converted to d—methyldopamine and a-methylnorepinephrine in the brain and suggested that these compounds could displace endogenous norepinephrine. In addition to displacement, Day and Rand (1963, 1964) suggested that the @- methylamines, particularly d-methylnorepinephrine, could actually be released from peripheral adrenergic nerve terminals and act as less effective or "false" transmitters. o-Methylnorepinephrine was subsequently identified by a number of investigators in animal tissues and in the urine of man (see Stone and Porter, 1967) and was Shown to be released by adrenergic nerve stimulation (Muscholl and Maitre, 1963). The "false" transmitter hypothesis has been exten— sively investigated in peripheral adrenergic systems in 33 an attempt to elucidate the mechanism of the anti- hypertensive action of d—methyldopa (Stone and Porter, 1966). The antihypertensive effect is not due to inhibition of the decarboxylase enzyme because other inhibitors of this enzyme do not lower blood pressure (Levine and Sjoerdsma, 1964). The reduction in blood pressure is not the result of a peripheral action of the decarboxylated products of a—methyldOpa, because the antihypertensive effect is not altered when the decarboxylation of this compound is selectively blocked in extracerebral tissues (Sjoerdsma at aZ., 1963; Davis at aZ., 1963; Henning, 1969). Furthermore, administration of d-methyldopamine does not lower blood pressure in man (Buhs at aZ., 1964). Comparing dose response curves, Holtz and Palm (1967) reported that @- methylnorepinephrine had a slightly less pressor effect than norepinephrine, but following phenoxybenzamine was much more potent in producing vasodilation. They suggested that this vasodilating effect of a-methylnor- epinephrine could eXplain the blood pressure reduction following d-methyldOpa. Brunner et a1. (1967), however, were unable to lower blood pressure of renal hypertensive rats with the administration of a-methylnor— epinephrine. Recent evidence has emphasized a central site of action. The antihypertensive effect of a—methyldOpa is 34 greatly attenuated if its decarboxylation is prevented in the brain (Henning, 1969). Therefore, it would appear that the reduction of blood pressure is due to an effect of either a-methyldopamine or d—methylnorepinephrine in the central nervous system. d—Methyldopa also has prominent central depressant properties. In man, this is manifested as sedation but it has not been a problem clinically, because tolerance to this effect develops rapidly. In animals, acute admini- stration of a—methyldOpa causes a reserpine—like ptosis and sedation, and disrupts performance in a number of behavioral tests (Sourkes, 1965; Stone and Porter, 1967; Kadzielawa, 1967). No chronic behavioral studies and therefore no tolerance in animals has been reported. The mechanism of the central depressant action of a—methyldopa is not well understood; more confusing is the report that this compound can reverse the behavioral depression produced by reserpine (Uretsky and Seiden, 1969). The general impression is that the central depressant effects are not temporally related to the depletion of norepinephrine since this amine is still depleted when behavior returns to normal (Stark et aZ., 1964). There have been no studies relating the time course of the behavioral effects to the depletion of brain amines or to the formation of d—methylamines in the brain. The reason for this is that both a-methyldopa and 35 Figure 4. 20 >< 2 A/s... //o “I : '5 :x: S 5 I I I I l A A II I l o 2 4 T" 12 24 i 0.3- , D 1 j . d ; F,4r’/+ 1 .m- ; i (“s/9)!!!" NE ‘ f ' ‘ i E 02(- _ 4 l 2 [ |-——o/. S 0 I 1 l I I a 1 JL 1 a . g o 2 4 a ll 12 24 U fi‘ . < U z 0.4 " 7t g . n 9 (ea/Jab . i 2 4 b 12 24 ‘ HOURS AFTER a M DOPA (zoo rug/kg) ’ s———747—744'~7 ,1, ,1 ~ Time course of the effects of a—methyldopa(a-MDOPA) on rat shuttle box performance and brain catecholamines. Avoidances per session (A/S), escapes lost per session (EL/S), and the brain concentrations of norepinephrine (NE), dopamine (D), a-methyldopamine (a-MD) and a-methylnorepinephrine (a-MNE) were determined at various times after the i.p. administra- tion of QFMDOPA 200 mg/kg. Each point represents the mean and the vertical lines indicate f 1 S.E.M. The solid points are significantly different from control at the 1% level. Shuttle box, n=7-9; brain catecholamines, n=6. 36 d-methyldOpamine have fluorescent characteristics similar to norepinephrine and must be separated from each other and from norepinephrine in order to be measured. .Further- more, d-methylnorepinephrine is only weakly fluoreSCent and its measurement has been primarily with bioassay techniques. Waldeck (1968) showed that the fluorescence of this compound was much improved if its stereoisomeric configuration was changed by boiling in HCL. This principle was further examined and a method was developed whereby dopamine and norepinephrine as well as d—methyl- dope and its metabolites, d-methyldopamine and @- methylnorepinephrine, could be analyzed in a single tissue extract (see Methods). This method was utilized to investigate the relationship between a-methyldopa-induced behavioral depression and the concentration of these amines in the brain. A. Conditioned avoidance responding_in rats The effect of 200 mg/kg of d—methyldopa on shuttle box behavior and brain amine content is summarized in Figure 4. Following d—methyldOpa, the number of avoidances per session (A/S) was reduced when the animals were tested 1—6 hours later, but was not different from control at 12 hours. At no time was there a significant loss of escapes (EL/S) indicating that although sedated, the animals were still capable of reacting to the LOCOMOTOR ACTIVITY coums/w MIN (Hg/9) BRAIN CATECHOLAMINES 12 HOURS AFTER om DOPA (200 mg/kg) _.._— _ ._._ Figure 5. Time course of the effects of a-methyldopa (a-MDOPA) on loco- motor activity and brain catecholamines in mice. Locomotor activity (n=l2) and brain catecholamines (n=6-9) were deter- mined at various times after the i.p. administration of as MDOPA 200 mg/kg. See legend to Figure 4 for additional details. 38 electrical shock with an escape response. The time course in the reduction of the dopamine content, and the accumulation of a-methyldOpamine in the brain appeared to coincide with the depression of learned behavior. In contrast, the depletion of brain norepinephrine was Slow in onset, reaching a maximum at 6 hours, and per— sisting for at least 24 hours. The change in the norepinephrine content was accompanied by an accumulation of a-methylnorepinephrine in the brain; both events, because of the slow onset and long duration, were temporally unrelated to the behavioral depression. B. Exploratory locomotor activity in mice A Single intraperitoneal injection of 200 mg/kg d- methyldopa depressed locomotor activity in mice when tested 1—6 hours later, but had no effect 12 hours after administration (Figure 5). The decrease in brain dOpamine and the concentration of a—methyldOpamine were temporally more related to the depression of activity than was the replacement of norepinephrine by d—methyl— norepinephrine. By comparing the data in Figures 5 and 6 it would appear that the reduced locomotor activity in mice also coincides with the concentration of o-methyldOpa in the brain. Therefore it was necessary to determine if @- methyldopa or its decarboxylated product, produced the 40— 7 A U5 as\ 30- . L g 20- - O z X z 101- - < II CD 0_l l #L . % d O 2 4 6 12 HOURS AFTER ocMDOPA Figufgw6. Theflconcentration of atmethyldopa (a-MDOPA) in mouse brain at various times after the i.p. injection of 200 mg/kg of the drug. Each point represents the mean and the vertical lines indicate i l S.E.M. for 5 determinations. I I..- . {y'all-Ml; I . .-|I..lll'.l. 31"}, .‘lr II.) . . Ill. :5 Ila .I‘ (1 ‘_§.v- l 1“; U I . 40 Figure 7. 800? 600L ‘ grmml L counts/1o MIN. D & O O O O ' I I LOCOMOYCR ACIIVITY 0.6 L 3 E 0.4 ' I . I v-\ < u . * . ;}~ 5 5 a; .. . “I i x I '6 o. _____ PRETREATMENT H20 HMD Re +4602 TREATMENT NONE “ M DOPA “M DOPA “M DOPA iEffect of aromatic L-amino acid decarboxylase inhibitors on the depression of locomotor activity and formation of brain a-methyl amines following a-methyldopa (OSMDOPA). One half hour after an i.p. injection of either H20, hydrazino @- MDOPA (HMD) or R0 4-4602 (500 mg/kg), mice were injected with 200 mg/kg a-MDOPA. Locomotor activity (n=12) and brain con- centrations of a—methyldopamine (open bars, n=6) and a—methyl- norepinephrine (solid bars, n=6) were determined 4 hrs later. The height of the bars represents the mean and the vertical lines indicate i l S.E.M. Asterisks for locomotor activity represent those values that are significantly different from no treatment (ngLOl); for a-methyl amine concentrations, the asterisks represent those values that are significantly different from H20 —a-MDOPA treatment (p- » I '2 22' ’ a h- . 2; 400'” * «I. ' 3E . . . . .— o 0% go 200- « u § . . Oh -|.—_1 .—_ .—_i J r l . q d MEIHYL AMINES (us/9 ) p N I L. 4* . o. h 1 PRETREATMENT U-14, 624 MC U-14,624 TREATMENI H20 o( M DOPA a: M DOPA Effect of a dopamine B-hydroxylase inhibitor on the depression of locomotor activity and formation of brain atmethyl amines following atmethyldopa (ahMDOPA). One half hour after the oral administration of either 1% methylcellulose (MC) or Url4,624 (75mg/kg), mice were injected i.p. with 200 mg/kg GPMDOPA. Locomotor activity (n=12) and brain concentrations of armethyl dopamine (open bars, n=6) and aimethylnorepinephrine (solid bars, n=6) were determined 4 hrs later. The height of the bars represents the mean and the vertical lines indicates i 1 S.E.M. The asterisk for motor activity indicates that value which is significantly different from U‘14,624 - H20 treatment (p<:0.01); for armethylnorepinephrine, the asterisk indicates that value which is significantly different from MC - QPMDOPA treatment (p< 0.01) . 43 AS indicated in Figure 8, U—14,624 alone did not depress locomotor activity. It did, however, greatly reduce the formation of d-methylnorepinephrine and it completely antagonized the behavioral depression produced by d— methyldopa 4 hours after injection. In addition, when a—methyldopa was administered to mice on a 24 hour diet containing 0.3% U—l4,624, no depression of locomotor activity was observed 2 hours after the administration of the drug. In 6 experiments, the mean activity counts i l S.E.M. per 10 min were: d-methyldOpa, 131 i 10; U-14,624 + d—methyldopa, 523 i 64. Thus, formation of d~methylnorepinephrine appears to be primarily reSponsible for the sedation produced in mice by a-methyldOpa. Since a high concentration of d—methylnorepinephrine is maintained in the brain for some time after the behavioral effects have subsided, it is apparently only a newly synthesized source of_d-methylnorepinephrine that is producing a functional deficit in adrenergic transmission. If this is true,then the formation of norepinephrine from tyrosine should be reduced at early times after d-methyldopa administration but not at later times when the precursors of d—methylnorepinephrine are no longer available. In order to investigate this hypothesis, the effect of d-methyldopa on the formation of dopamine and norepinephrine from Cl4-tyrosine was determined (Table 2). The formation of both dopamine 44 Table 2. Effect of d—methyldopa (d-MDOPA) on the conver— sion of Cl4-tyrosine to Cl4—dopamine (D), C14— norepinephrine (NE), Cl4—normetanephrine (NM) and deaminated catecholamines (DEAM) in mouse brain :2::: Agiiiity Cl4—D Cl4-NE Cl4-NM Cl4—DEAM OL-MDOPA X 10 0 1358i47 1254i 73 550i25 494$ 26 683i70 1.5 1413:57 614tl66* 213i30* 249i 54* 615I73 12.5 1306i70 1090i 55 391f29* 438i145 570i11 * p<0.01 when compared to control. 10 pc of 12 hours 1/2 hour the mean tyrosine—Cl4 were administered by tail vein 1 and after dMDOPA 200 mg/kg i.p. Animals were killed later. The numbers are in DPM/g and represent i 1 S.E.M. of 4 determinations. 45 and norepinephrine was markedly reduced one hour after d—methyldopa. At 12 hours, when sedation was no longer evident, the formation of dopamine from tyrosine was normal but the conversion to norepinephrine, although much greater than at 1 hour, was still lower than control. The brain content of normetanephrine was significantly reduced at 1 hour but not different from control at 12 hours (Table 2). There was no significant change in the amount of deaminated metabolites (DEAM; Table 2). The mechanisms by which d—methyldopa may lower catecholamine concentrations in the brain require further discussion. The accumulation of Cl4 catechol— amines in the brain following administration of C14- tyrosine is not an exclusive measurement of synthesis, but is the product of a dynamic process involving synthesis, release, uptake, retention and metabolism. Presynaptically released norepinephrine is thought to be partially inactivated by O—methylation, forming normetanephrine, at or near the receptor site (KOpin, 1966). Thus the formation of this metabolite may reflect the amount of neuronally released norepinephrine. According to Table 2, the decreased amount of normetanephrine accompanying the reduced formation of dopamine and norepinephrine 1 hour after d—methyldopa, indicates that their synthesis is inhibited. Andén et a2. (1969) 46 and KOpin at al. (1969) also feel that a-methyldopa inhibits catecholamine synthesis shortly after administration. This is probably due to competition between the endogenous and false substrates for the decarboxylating and B-hydroxylating enzymes. Kopin at al. (1969) have prOposed that the inhibition of synthesis may be due to a release of norepinephrine into the cytOplasm by the d-methylamines resulting in a feedback inhibition on tyrosine hydroxylase. A direct inhibition of this enzyme by d—methyldopa or its metabolites was also suggested. The presence of a methyl group on the a carbon atom of the catecholamines retards deamination by intra- neuronal monoamine oxidase. Thus d—methyldopamine and d-methylnorepinephrine accumulate in the neuron and by occupying storage sites prevent the intraneuronal retention of the endogenous amines, particularly nor- epinephrine (KOpin, 1968). The fact that d-methyl- dOpamine disappears from the brain much more rapidly than d-methylnorepinephrine suggests that most of it is located in norepinephrine containing areas where it is converted to a—methylnorepinephrine. Thus,retention of Cl4-norepinephrine may be impaired 12 hours after d—methyldOpa (Table 2), but actual synthesis and release, as indicated both by the formation of normetanephrine and the lack of sedation, may be normal. An increase in 47 the deaminated metabolites was not detected (Table 2), but would be expected if binding were impaired (Glowinski at aZ., 1966). However, the a—methylamines may inhibit monoamine oxidase (Kopin, 1968). Further- more, only total deaminated catecholamines were measured and specific changes in norepinephrine metabolism may have been masked. Although inhibition of norepinephrine synthesis accompanies the formation of d-methylnorepinephrine, the latter appears to be responsible for the sedative effects of d—methyldopa. Microiontophoretic studies have suggested a possible mechanism. In the cat brain stem, d—methylnorepinephrine produced less excitation than norepinephrine, but it could also block the excitatory effects of norepinephrine (Boakes at aZ., 1968). Thus, the marked sedative action of d—methyldOpa may result from the combination of a reduced synthesis and release of norepinephrine and a blockade of nor— epinephrine effects by d—methylnorepinephrine. Persistent blockade, however, requires continuing synthesis of d—methylnorepinephrine. The brain stem reticular formation is a likely site of action. C. Summary Acute administration of d—methyldopa decreases con- ditioned avoidance responding in rats and exploratory 48 locomotor activity of mice, and lowers the brain concentrations of dopamine and norepinephrine. The Central depressant effect, at least in mice, appears to be due to the formation of d-methylnorepinephrine but is evident only during the period of its actual synthesis when the formation of norepinephrine is inhibited. When the supply of precursor is exhausted, d—methylnorepinephrine remains in the nerve terminals where it impairs the storage of norepinephrine but no longer inhibits its synthesis. CHAPTER IV ACUTE STUDIES WITH a-METHYLTYROSINE Following the report that the hydroxylation of tyrosine to dopa was the rate limiting step in the biosynthesis of catecholamines (Levitt at aZ., 1965) and the characterization of the enzyme tyrosine hydroxy— lase (Nagatsu at aZ., 1964), a number of compounds were shown to inhibit this enzyme in vitra (Moore and Dominic, 1970). One of these compounds, the d—methyl analogue of tyrosine (d—methyltyrosine, d—MT) was subsequently C": l PDQ «(1.2.2- ”‘2 d-MEYHYLYYIOSUNE 0°". _ i found to be an effective inhibitor of tyrosine hydroxylase in viva (Spector at aZ., 1965; Figure 2). Administration of d-methyltyrosine produced a selective decrease in the concentration of dopamine and norepinephrine in the brain (5-HT was not affected) and a depletion of nor— epinephrine in several adrenergically innervated tissues. It was evident from this study that a pharmacological tool was now available which could effectively inhibit catecholamine synthesis in viva. Once synthesis was inhibited and sufficient drug remained to maintain 49 50 inhibition, catecholamine depletion would proceed at a rate determined by utilization. Numerous studies have since attempted to assess the consequences of brain catecholamine depletion by a—methyltyrosine on the behavioral performance of animals. Acute administration of d-methyltyrosine decreases conditioned responding in cats (Hanson, 1965); suppresses hypothalamic self— stimulation in rats (Poschel and Ninteman, 1966); reduces motor activity, rotarod and Shuttle box performance in rats (Rech at aZ., 1966); disrupts conditioned behavior in guinea pigs (Moore, 1966) and inhibits operant responding in rats (Schoenfield and Seiden, 1967, 1969). a-Methyltyrosine also decreases REM (rapid eyeball movement) sleep time in monkeys (Weitzman at aZ., 1967) and increases the voltage of the rat electrocorticogram (Pirch and Rech, 1968). A. Effects on locomotor activity and brain catecholamines in mice The relatively small amount of d-methyltyrosine initially available and the high cost of the drug once it became commercially available, necessitated that studies with this drug, especially the chronic effects, be carried out in mice. When d—methyltyrosine is added to the diet of mice, depletion of brain catecholamines and behavioral depression result (Johnson at aZ., 1967; Moore, 1968). Acute studies 51 Figure 9. (D o. 0 JS 0 O MOTOR ACTIVITY no 0 9 . b -I H H (Ti 25 2'5 5'0 IOO 200 O P .8- D - BRAIN AMINES' (pg/g) 2 I'll O‘oh'lTés 55 5D IOO zoo DOSE OF OIMT (mg/kg) “DSEE:EéspdfiSé effects Of armethyltyrosine (QLMT) on lOcomOtor activity and brain catecholamine levels. Points represent values obtained 4 hrs after the i.p. injection of various doses of a-MT. Each point represents the mean and the vertical line indicates i 1 S.E.M. Solid symbols are those values which are significantly different from control at the 1% level. Locomotor activity is expressed as counts per 10 min and each point represents the mean of 12 determinations; brain cate- cholamine levels (D, dopamine; NE, norepinephrine) are the means of 6-9 determinations. 52 were therefore initiated in order to establish that these two events are causally related. Dose responSe curves for d-methyltyrosine are depicted in Figure 9. Four hours after drug administration, exploratory (spontaneous) locomotor activity counts and the concen- tration of dopamine in the brain fell progressively as the dose of a—methyltyrosine was increased. At a dose of 25 mg/kg, inhibition of norepinephrine synthesis appeared to be maximal and larger doses produced no further depletion of this brain amine. The marked depression of activity following doses of 100 and 200 mg/kg may be the consequence, in part, of the toxicity of a-methyltyrosine (Moore at aZ., 1967). Following a single intraperitoneal injection of d-methyltyrosine (Figure 10a), the brain concentrations of dopamine and norepinephrine fell progressively with time. The more rapid decline of dOpamine is to be eXpected considering its higher rate of turnover (Brodie et al., 1966). By 12 hours, the concentration of both amines had returned to the control values. Depression of spontaneous eXploratory locomotor activity roughly paralleled brain catecholamine depletion. A portion of the depression at the one and two hour points could be due in part to some irritating effect of the insoluble amino acid because a similar pattern was not seen when the soluble ethyl ester of u-methyltyrosine 53 a. q—MT b. q-MTethyl ester 600- 600» r//5D 400- Y//S> 400- < E —l V) 200- 200- 0‘o 2 4 o H n 0‘0 2 4 o H n .1” / iii /‘ OI 3 < 4' U 3 I/0 g .2. 2- a: I I 1—‘¥I I I llg o.I #1 1 I L I I o o 2 A o H n o 4 o d n 2 HOURS AFTER INJECTION Figure 10. Time course of the depression of spontaneous locomotor act- ivity (SLMA) and the depletion of brain dopamine (D) and norepinephrine (NE) after a) 50 mg/kg a-methyltyrosine (a-MT) and b) 100 mg/kg QPMT ethyl ester HCL. See legend to Figure 9 for further details. 54 was administered in a parallel study (Figure 10b). With the soluble form of the drug, a good temporal relation— ship, with regard to duration and extent, was observed between behavioral depression and brain catecholamine depletion. Because of a limited supply of the soluble ester, subsequent studies were carried out with the insoluble amino acid. B. Adrenergic blocking properties It has been suggested, with no documentation, that some of the pharmacological effects of d—methyltyrosine may be due to adrenergic blockade (Muscholl, 1966; Dewhurst, 1969). If this were the case,the behavioral depression would be eXpected to parallel the concentration of the drug in the brain. As indicated in Figure 11, the amount of d-methyltyrosine in the brain following a single dose of 50 mg/kg is unrelated to the pattern of locomotor depression following a Similar dose (Figure 10a). That is, marked depression is observed when only a small amount of a—methyltyrosine remains in the brain (compare 6 hour value, Figures 10a and 11). Furthermbre, no effect was observed when a-methyltyrosine was tested for adrenergic blocking prOperties on an in vitra preparation of the mouse spleen (Figure 12). At a bath concentration of 10_3M d-methyltyrosine did not significantly alter the contractile response to norepinephrine. This concentration is nearly ten times 5 5 DO 0 __"_. ES 0 . ¢\+ _ \‘|\ I—R 2 BRAIN OIMT (AG/g) O O 2 4 6 HOURS AFTER OIMT Figure 11. Brain concentration of a-methyltyrosine (OPMT) following a single i.p. injection of 50 mg/kg of this drug. Each point and vertical line represents the mean i 1 S.E.M. of 6 separate determinations. Figure 12. 100r- . I . A 80- . III In 72 O a. 3 a: 60L _‘ . ‘< E X if K 40- _ O .— 12 I“ C) 5 IL 20b 0-—O CONTROL '3 H 10 M aI-MT D—D 158M CPZ 0 ()~ 3————---" I . I J -9 -8 -7 -6 _5 . LOG MOLARITY OF NE (8") Effect of flamethyltyrosine (a-MT) and chlorpromazine (CPZ) on the contractile response of the isolated mouse spleen to cumulative additions of norepinephrine (NE). The values are expressed as the percent of the maximal response ob- tainable with NE before the addition of the antagonists to the bath. The points and vertical lines represent the mean + 1 S.E.M. of 5 experiments. The molar concentrations are those in the bath. 57 that found in the brain one hour after a single dose of 50 mg/kg. In contrast, the potent tranquilizer» chlorpromazine produced marked competitive inhibition at a concentration of 10_8M; phentolamine (not shown) in the same concentration had a similar effect. Possible 8 adrenergic blockade was also examined with the mouse spleen preparation. At a concentration of 10_3M, d— methyltyrosine did not significantly alter the reduction in tension produced by 10'-7 M iSOproterenol measured as a downward deflection of the recorder pen. ISOproterenol alone produced a 62.3 I 9.7 mm deflection; in the presence of d—methyltyrosine the deflection was 51 i 5.2 mm. These values are the mean i l S.E.M. for 4 experiments. Propranalol (10_7M) completely blocked the response to isoproterenol. C. Pretreatment with a monoamine oxidase inhibitor The intraneuronal accumulation of catecholamines following inhibition of monoamine oxidase probably exerts a feedback inhibition of tyrosine hydroxylase resulting in a marked reduction of synthesis (Neff and Costa, 1968). Under these conditions it would be expected that inhibition of synthesis would produce less functional impairment. Therefore, pretreatment with a monoamine oxidase inhibitor should slow the rate of catecholamine depletion and antagonize the behavioral depression that follows the administration of d—methyltyrosine. The monoamine oxidase inhibitor pheniprazine (10 mg/kg), 58 .8 f; S 15-6” : \ L) c» 4 a. V4 III E Z !- E2- 0 a: 2 m . - . . _L I ll J .L l 1 J i 0O I 2+6 00 I 27"76 HOURS AFTER OIMT Figure 13. Effect of pretreatment with a monoamine oxidaSe inhibitor on Gamethyltyrosine (a-MT) - induced behavioral depression and brain catecholamine depletion. Non-pretreated animals [5, and those treated 18 hrs previously with 10 mg/kg of pheniprazine C): were injected with 50 mg/kg of OPMT. One, 2 and 6 hours later animals were tested in the motor activity cages and then sacrificed for brain catecholamine analysis. Each point was compared statistically with zero time values from non-pretreated mice. See legend to Figure 9 for addit— ional details. 59 administered 18 hours prior to a-methyltyrosine, had just such an effect (Figure 13). Pheniprazine alone (0 time) had no effect on motor activity or on the concentration of brain dopamine but significantly ele— vated the level of norepinephrine. It did, however, retard the rate of depletion of both dopamine and norepinephrine and antagonized the reduction of motor activity produced by d-methyltyrosine. Pargyline (100 mg/kg) had similar effects. The effects of monoamine oxidase inhibition in mice extend and support previous studies in rats (Moore and Rech, 1967) which suggested a causal relationship between the behavioral depressant and brain catecholamine depleting actions of d-methyltyrosine. Evidence from earlier studies lends further support to this proposal. Behavioral depression following acute administration of d-methyltyrosine is not related to its concentration in the brain (Rech et aZ., 1966) but can be reversed by the catecholamine precursor L—dOpa (Seiden and Hanson, 1964; Moore and Rech, 1967). Prior depletion of catecholamine stores with reserpine or tetrabenazine enhance the behavioral effects of @— methyltyrosine (Rech at aZ., 1968). Other factors have been considered in interpreting the cause of behavioral depression. Certainly with the higher doses (200 mg/kg or more), a—methyltyrosine 60 toxicity is encountered in the form of renal damage and it can be lethal (Moore at aZ., 1967). Peripheral effects of the drug appear to be minimal. d- or B— adrenergic blocking effects were not observed. Pressor responses to tyramine and carotid occlusion are reduced but hypotensive effects are lacking (Bhaghat and Shein, 1965; Spector at aZ., 1965). The pituitary—adrenal reSponse to stress is not reduced (Carr and Moore, 1968; Hirsch and Moore, 1969). Although future studies may discover some as yet unknown action of d—methyltyrosine, such as a possible effect on amino acid tranSport, the evidence to date strongly suggests that brain catecholamine depletion and behavioral depression following the administration of this drug are causally related. D. Antagonism of amphetamine stimulation The concept that newly synthesized norepinephrine is more readily released by nerve stimulation (Kopin at aZ., 1968) and indirect acting sympathomimetic amines (Alousi and Weiner, 1966) is widely supported. The interactions between d—methyltyrosine and amphetamine—like drugs has further supported this hypothesis. As first demonstrated by Weissman at al. (1966), d-methyltyrosine blocked the stereotyped excitatory symptoms and locomotor stimulating actions of amphetamine, methamphetamine, and phenmetrazine, but not the excitatory actions of a number of other CNS stimulants (e.g., metrazol). It has since been a. 50 mg / kg b. 6.25 mg / kg . - I Azooo- - 2000. g . ~ . § I600- - I600- I ' I I >- - I- I a I 3 IZOO- - I200. ; :9: 800- ~ 800» 4 I. - . .I z (kc—0.4 (7'; 400»\\/{/. 400’ M‘ O _ I I I fin: I Ii 0 . gr i #'L J _. 0 I 2 6 I2 0 I 2 6 I2 I- " 1' «J BRAIN AMINES (pg/g) , ' , 'e . e JfZ,K//::> '7 J i O _ g 4 l I I J O I 2 6 l2 0 l 2 6 l2 HOURS AFTER OIMT - -m..—~.._ —-—.- .. Figure 14. Amphetamine stimulation, Spontaneous activity and brain catecholamines after akmethyltyrosine (aFMT). Amphetamine- stimulated motor activity (AMPH-MA), spontaneous locomotor activity (SLMA) and brain catecholamine levels were deter- mined at various times after intraperitoneal injections of a) 50 and b) 6.25 mg/kg of QFMT. See Methods and legend to Figure 9 for additional details. w—‘r-u 62 demonstrated that a—methyltyrosine effectively reduces or blocks a variety of central effects of amphetamine or amphetamine-like drugs: the restoration of avoidance responding in reserpine-treated cats (Hanson, 1965); the lowering of threshhold for convulsive agents (Wolf at aZ., 1969; Spencer and Turner, 1969); hyperthermia (Morpurgo and Theobald, 1968) and self-administration reinforcement (Pickens at aZ., 1968). In man, a— methyltyrosine blocks both the central euphoriant effects and the blood pressure elevation following the intra- venous administration of amphetamine (Jonsson at aZ., 1969). In an effort to further characterize the nature of the a-methyltyrosine—amphetamine interaction, two doses of the amino acid (Figure 14) were compared for their effects on amphetamine stimulated activity, spontaneous eXploratory locomotor activity and brain catecholamine depletion. The dependency of amphetamine stimulation on uninterrupted catecholamine synthesis, rather than the stored amine pool, is evident from the fact that both doses of d-methyltyrosine produce similar reduc— tions of stimulation at l and 2 hours, but only the higher dose caused a decrease in spontaneous activity and brain catecholamine concentrations. A partial dependency of amphetamine stimulation on catecholamine stores, however, is suggested Since maximal antagonism 63 n “A *I‘ 3200, . N 4:. O O l 1 0_- - -4:—' -I O l 2 6 l2 Q HOURS AFTER OIMT , Figure 15. Effect of reserpine-pretreatment on aFmethyltyrosine antag- onism of amphetamine stimulation. Amphetamine-stimulated motor activity (AMPHFMA) was determined in non-pretreated mice A , and in mice pretreated with reserpine O (2 mg/kg 4 days prior to the experiment)at Various times after an i.p. injection of aFMT (50 mg/kg) and 20 min after a s.c. injection of d-amphetamine sulfate (2mg/kg). See Methods and legend to Figure 9 for additional details. 64 by d—methyltyrosine is seen only after a latent period when brain dopamine and norepinephrine reach their lowest values (6 hours, Figure 14a). E. Effect of reserpine on antiamphetamine action of a—methyltyrosine Depletion of amine stores by reserpine enhances catecholamine synthesis (Neff and Costa, 1968). This suggests a greater functional dependency of a catechol- amine—depleted neuron on the process of synthesis. The antiamphetamine effect of d—methyltyrosine was tested in reserpine-treated animals. Four days after a single injection of 2 mg/kg reserpine, mouse brain concentrations of dopamine and norepinephrine were less than 50% of control (0.29 i .02 pg/g and 0.16 i .02 pg/g respectively; mean i 1 S.E.M. for 6 determinations), but locomotor activity approached control values. At this time amphetamine-stimulated motor activity was enhanced in reserpine-pretreated animals (0 time, Figure 15). The enhancement of amphetamine stimulation following reserpine may be a reflection of a higher rate of catechol— amine synthesis (Stolk and Rech, 1968). d—Methyltyrosine reduced the amphetamine—stimulated activity in these mice to the same level as found in non—pretreated mice; reser— pine treatment, however, did hasten the onset of the d—methyltyrosine blockade (compare one hour values, Figure 15). 65 400 F 200 r PERCENT OF CONTROL 100 ' " " ‘— 0- — U-14 U-14 + d-A L. d—A 0(- MT x—MT + d—A d-A Figure 16. Amphetamine stimulation following inhibition of tyrosine hy- droxylase or dopamine B-hydroxylase. calculated as counts per hour and plotted as percent of con— trol. All drugs were added to a ground diet in the following percentages: d-amphetamine $04 (d—A, 0.05%); DL-Okmethyl— tyrosine (QFMT, 0.4%) and U-l4,624, 0.3%. The height of the bars and vertical lines indicate the mean i experiments. Control activity was: U-14,624, a—MT, 1229 i 146 counts per hour. * p< 0.01 when compared to control 1 S.E.M. for 3-4 1292 i 135; Continuous activity was 66 These data support the prOposal that amphetamine exerts its locomotor stimulant action by preferential release of a newly synthesized pool of catecholamines (Weissman at al., 1966; Dingell at aZ., 1967; Sulser at aZ., 1967). The antiamphetamine action of d—methyl— tyrosine is not related to the concentration of the drug in the brain (compare Figure 11 and Figure 14), and adrenergic blockade is not a property of d-methyl- tyrosine (Figure 12). In addition, u-methyltyrosine does not alter the level of amphetamine in the brain (Dingell at aZ., 1967). F. U-14,624 - amphetamine interaction a—Methyltyrosine inhibits the synthesis of both dOpamine and norepinephrine in the brain. It is difficult, therefore, to determine which of these amines may be more important for the central motor stimulant effects of amphetamine. The dopamine B-hydroxylase inhibitors, disulfiram and diethyldithiocarbamate, have been used in attempts to answer this question. The results of these studies led some investigators to believe that dOpamine was more important for the stereotyped behavior while norepinephrine is involved with the stimulation of locomotor activity produced by amphetamine (Randrup and Scheel—Kruger, 1966, 1967; Maj and Przegalinski, 1967; Maj at aZ., 1968). On the 67 Aoema .muooz can Hoosmaumflo>co> Eonmv+ HOHucoo OD COMMQEOO £033 ao.ovmR wm.o mama +Hnm.ma .mAe.Nm +Hpmm +INA em www.4AI: wv.o mnwma an mm «m.mnom mHHS «NHv.mH «moozIa «Hva RH.onm.mm «Snow RmHmm RAH em we.o BZIB cHE om\mpssoo CHE oa\mpasoo H£\mpcdoo GOHDBHSEHDm MDH>HDO¢ MDH>HDO¢ m\m: m\m1 poaw SH mnflfimumsmfid SHODMHOmem msoscflusoo Q mz w msuo aoupcou mo ucmouom COHROQ M503 em o How OOHE mo DOHU 05¢ OD cocoa cons wmm.vHID can A£DmEIa MO muoowmm mpsom may mo humaadm .m OHQBB 68 other hand, Van Rossum and Hurkmans (1964) feel that dopamine is more involved with locomotor stimulation. d—Methyltyrosine and the dopamine B-hydroxylase inhibitor U—14,624 were compared for their ability to block the central stimulant action of amphetamine on continuous locomotor activity. As indicated in Figure 16, both U—l4,624 and d—methyltyrosine, when added to the diet of mice, significantly reduced the level of continuous activity recorded between the hours of 7:00 p.m. and 7:00 a.m. Both drugs produced an equivalent reduction in the brain concentration of norepinephrine, but only d—methyltyrosine lowered the dopamine level (see Table 3). When amphetamine was combined with these drugs in the diet, only d—methyltyrosine anta- gonized its stimulant effect. These results suggest that dopamine rather than norepinephrine may be more important for the locomotor stimulant effects of amphetamine. The same conclusions can be drawn from an analysis of the data in Table 3. Summarized here are the acute effects of d-methyltyrosine, d—methyldopa, and U—14,624 when added to the diet of mice for a 24-hour period. All three agents depleted the brain of norepinephrine. The marked effect of d—methyldopa can be explained by the accumulation of d—methylnorepinephrine which inter— feres with the intraneuronal retention of norepinephrine. 69 The reduction of continuous activity by all three of these drugs appears to be related to their ability to deplete the brain of norepinephrine. Only a-methyl- tyrosine, which significantly inhibits the synthesis of dopamine, lowers exploratory locomotor activity and blocks the stimulant effect of amphetamine. There may be another explanation for the specific antiamphetamine effects of d-methyltyrosine. Enhance- ment of norepinephrine synthesis accompanying peripheral nerve stimulation occurs at the tyrosine hydroxylase step; that is, formation of norepinephrine from tyrosine, but not from the precursors dOpa or dOpamine, is enhanced (Sedvall, 1969). Amphetamine increases the synthesis of both dopamine and norepinephrine in the brain (Besson at al., 1969). If this is also the result of increased tyrosine hydroxylase activity, then amphetamine stimulation would only be blocked by inhibitors of this enzyme (e.g., d—methyltyrosine). In support of this hypothesis is the fact that cold stress enhances brain catecholamine depletion following d—methyltyrosine (Gordon at aZ., 1966) but not disulfiram (Moore, 1969). In other words, stresscm amphetamine can stimulate nor- epinephrine synthesis in Spite of inhibition of dopamine B-hydroxylase. In order to test this theory, the conversion of Cl4—tyrosine to dopamine and norepinephrine in the brain was measured twenty min after 4 mg/kg 70 .mOHmEMm map mo HHm cH HDHHEHm mmz >HH>HHOBOHUDH HBHOH one .mpcOEHuomxm a mo .2.m.m H H acme map pcmmmhmmu can panoz CHMHQ mo m\2mo sH mum mHOQESq one .mHmmHmsm OGHEMHOQOOHBO MOM om>oEmu mums mcHwHQ OQH HOHDH H50: HHS: Odo .Amx\ma av mIo MO SOHpoomcH .m.H cm Howwm QHE om GHO> HHBH 03p Och COHooth mcz AU: OHV osHmOHSHIvHO filo + SETS OH COHBQEOO c033 Ho.ova« Houpsoo OH cmmmmaoo coca Ho.ova memmHH HNHHovv meHSmmH *mNHmmv mmHHNHNH mHHHHmm QIwHU mmHmSv «w HSmH «ASH Hmmm RHmHSwN Rom HHmH SH Room szvHO 4Io HHOD OQH cH coomHm mHoumHooEEH OHOB OOHE OQH can .Q.H cogomnsH Duos macho auom .mQOHHmGHEHOHOU w How .E.m.m H H mHGSOO smog map pcmmmumon mosHm> one Houusoo Op oonmmaoo cons Ho.ovmR CHE om\m#fiDoo I I I I omHAmomm NmHAmmHm OOHASHON mx\me m HOHUmHmHm .mmaeemm Iaemfloeem HONHNmSN SmHHmwom HHS om\mpasoo mx\ms N mcHEmuosmam SHE OH\mHGDOO mmflmmm mm Ewes S4 Hmmm em amee SDH>HD0< msomcmpsomm H + Spa a man a SEQ H HOHHQOU HOMO flmomzla mHH>Huom HOHOE ompmHDEHumImsuo can msoocwucomm no lamoozlav BaoeHSBDaBIS no HOHBBADHAHAHHB DAaonao no pommpm .e magma 81 Table 7. Tolerance to d—methyldopa (d-MDOPA)—induced depression of continuous activity of individual mice Percent of control activity Drug Drug included in diet Day 0 Day 1 Day 2 Day 3 Day 4 d—MT 100 98.5 78.6 85.5 96.0 d—MDOPA 100 31.8 54.7 127.7 90.3 d-MT + d—MDOPA 100 26.7 45.0 94.6 116.8 Values represent percent of control activity counts per hour recorded from 7 p.m. to 7 a.m. Control values were d—methyltyrosine (d—MT; n = l), 687; d—MDOPA (n = l), 674; d—MT + d—MDOPA (n = 2), 696, 701 counts/hr. Percentage of drug in diet a—MT — 0.2%; a—MDOPA - 0.4%. 82 exploratory locomotor activity either on day 4 of the diet or 1 day after the 4 day diet was terminated (4 + 1) (Table 6). However, the level of amphetamine stimu— lation in these same mice was enhanced at these times; the stimulant effects of pipradrol were not. The data from the Cl4—tyrosine experiments in Table 5, and the enhancement of amphetamine, but not pipradrol, stimulation suggests that an increased synthesis rate of nor— epinephrine accompanies the development of tolerance to the central depressant effect of u—methyldopa. Enhancement of norepinephrine synthesis in peripheral adrenergic systems generally occurs at the tyrosine hydroxylase step (Sedvall, 1969). If this were the situation in the brain after chronic administration of u—methyldopa, then addition of d-methyltyrosine to the diet might retard the development of tolerance. d— Methyltyrosine, in an amount which when given alone was insufficient to reduce the level of activity, failed to alter the pattern of tolerance development when added to an u—methyldOpa diet (Table 7). The aggressive behavior also was not attenuated by combining d—methyl— tyrosine with d—methyldopa in the diet. B. Discussion This study demonstrated that tolerance develops in mice to the depression of continuous activity resulting from the addition of d—methyldopa to the diet. The 83 development of tolerance after 3—4 days could not be explained by a reduction in the intake of the drug nor by a change in the brain concentrations of the catechol- amines or their d—methyl analogues when compared to the 1 day values. Compared to day 1, however, there was a significant increase in the formation of norepinephrine from Cl4—tyrosine. This could result from a reduction in the formation of d—methylnorepinephrine or an increased hydroxylation of tyrosine. The unavailability of a radioisotope of a—methyldopa prevented testing of the first theory. The latter possibility could result from an increase in the activity of tyrosine hydroxylase. The activity of this enzyme is thought to be regulated by the concentration of norepinephrine through a process of feedback inhibition (Spector at aZ., 1967). Sustained low levels of norepinephrine in the brains of mice on the a—methyldopa diet would likely remove this inhibition. In addition, postulated receptor blockade by u—methyl— norepinephrine could result in a reflex increase of adrenergic nerve activity and an accompanying enhancement of norepinephrine synthesis as occurs with phenoxy— benzamine (Bigelow at aZ., 1969). If tolerance is associated with an increase in tyrosine hydroxylase activity, it should be blocked by d—methyltyrosine. This did not occur. However, since d—methyltyrosine alone can reduce the activity of mice, only a small 84 amount was added to the diet which may not have been sufficient to block synthesis. The aggressive behavior normally apparent by the third day of the diet was not attenuated when d-methyl- tyrosine was included in the diet. One eXplanation is that the dose of the drug was insufficient. Another possibility is that norepinephrine may have an inhibitory function in certain cortical and subcortical areas of the brain (Lynch at aZ., 1969). For example, activity in the amygdala is thought to facilitate aggressive behavior in the rat (Valzelli, 1967). Stein (1967) has proposed that norepinephrine has an inhibitory action in the amygdala. If a deficiency of norepinephrine at this or other brain areas results in a removal of inhibition and unopposed aggressive behavior predominates, then administration of a—methyltyrosine would not be expected to block it. Interestingly, agents that antagonize the muricidal response in rats are presumed to increase the availability of norepinephrine at central receptor Sites (Sofia, 1969). This explanation would assume that the increased synthesis of norepinephrine on day 4 of the diet does not occur at these inhibitory sites and is restricted to brain areas where norepinephrine may have an excitatory action (e.g., brain stem). Brain 5-hydroxytryptamine (5-HT) is also decreased by u-methyldopa (Hess at aZ., 1961) and its turnover 85 rate is somewhat decreased in isolated aggressive mice (Giacalone at aZ., 1968). Thus it is conceivable that d-methyldopa-induced aggressive behavior may be related to alterations of brain 5—HT metabolism. The development of tolerance to d-methyldopa was accompanied by a supersensitivity to the locomotor stimulant effects of amphetamine but not pipradrol which is not known to be a catecholamine releasing agent (Dominic and Moore, 1969). The central stimulant effects of amphetamine, but not pipradrol, are enhanced following acute administration of u-methyldOpa (Smith, 1963; Quinton and Halliwell, 1963). The enhanced response to amphetamine remained 1 day after the termination of a 4 day diet of d-methyldopa. An increased synthesis rate of norepinephrine could explain the amphetamine effect as well as the development of tolerance. A post synaptic change such as increased receptor sensitivity is also a possibility. However, the rapid occurrence of toler- ance with d—methyldOpa is not characteristic of enhanced drug responses attributed to ‘ organ sensitivity; the latter generally requires 1—2 weeks to develOp (Trendelenburg, 1963). In addition, the lack of "rebound" or withdrawal hyperactivity in either the continuous or eXploratory locomotor measurements upon cessation of the u-methyldopa diet is not consistent 86 with receptor theories of tolerance (Sharpless, 1969; see also Chapter VI). C. Summary d—MethyldOpa, when administered in the diet for 24 hours, reduced continuous activity and lowered the con- centration of norepinephrine in the brain; dopamine, exploratory activity and amphetamine stimulation were not altered. Tolerance to the depression of activity was seen after 3-4 days of drug administration. It was accompanied by the appearance of aggressive behavior and an enhancement of amphetamine stimulation. Neither exploratory activity nor pipradrol stimulation was increased. The development of tolerance could be best explained by an increased synthesis of nor— epinephrine. CHAPTER VI CHRONIC EFFECTS OF a-METHYLTYROSINE A. Development of tolerance In a limited number of clinical trials, the only significant effect of d—methyltyrosine was a reduction in blood pressure and general improvement of patients with diagnosed pheochromocytoma (Sjoerdsma at aZ., 1965; Engelman at aZ., 1968b; Jones at aZ., 1968). In essential hypertension no consistent beneficial effect was observed. In patients suffering from a variety of mental disorders, primarily schizophrenia, a—methyltyrosine had none of the effects anticipated from a reduction of brain catecholamines; it did not produce depression nor alleviate psychoses (Gershon at aZ., 1967; Charalampous and Brown, 1967). Mild sedation was a consistent observation in all clinical trials with d-methyltyrosine. This was most apparent during the first few days of treatment but tended to disappear with continued administration of the drug (Engelman at aZ., 1968b). In an attempt to explain the lack of a central depressant effect in man with con— tinued administration of d-methyltyrosine, Moore (1968) examined the effects of chronic administration of the 87 8'8 ( I I; V1 2900+ - SE <9 I‘ - \ a: 03200 I '— 08 23 - . 0- a - - . - -HI—- 0 2 4 I0 I4 I.0- Io“J Z a 8 8 .8- 8>-- 3 E< .6. .sm go 21: u 35 I LU" \ . F: g I So ~4' DOPAMINE '41. a 5 EV ‘+ < < _J 30: .2- -2°- l—o NOREPINEPHRINE 0,: I I I I I I I I I I II ._ ‘ o 2 4 6 B IO “ I40 ‘ DAYS Figure 18WOTE% Ot-methyltyrosine on spon— taneous locomotor activity, plasma levels of the drug and concentrations of catecholamines in the brain. Plasma at methyltyrosine A and brain catecholamine 0 represent the mean of 8 determinations and motor activity 0 represents the mean of 16 determinations; the vertical lines project- ed upon each point represents 1 standard error of that mean. Solid points represent those values that are significantly different from control (zero time) at P<:0.01. (Moore, 1968). 89 drug to mice (Figure 18). When L—a—methyltyrosine was added to the diet of mice for 1 day, exploratory loco- motor aCtivity as well as the brain concentrations of dopamine and norepinephrine were significantly reduCed. Mice maintained on the a—methyltyrosine diet gradually showed tolerance to the depression of activity so that by 10—14 days of continuous consumption of the drug, their level of activity was normal (Figure 18). At this time, however, brain concentrations of dopamine and norepinephrine were at the same low level as seen on day l of the diet. Thus tolerance to the behavioral depressant but not the brain catecholamine depleting action of d-methyltyrosine was demonstrated in mice. Food intake and the plasma level of a-methyl— tyrosine remained relatively constant throughout the experiment. A similar study was repeated with DL-a- methyltyrosine and the same result were obtained. As indicated in Table 8, brain catecholamine depletion was maintained during 13 days of an d-methyltyrosine diet and there was no significant alteration in the amount of the drug in either the brain or plasma during the course of the diet. Therefore, the develOpment of tolerance cannot be explained by an increased rate of metabolism or excretion of the drug. 90 Table 8. Brain and plasma concentrations of a-methyl— tyrosine (d-MT) and the brain concentrations of dOpamine (D) and norepinephrine (NE) during a chronic diet of DL-d—MT Days of Brain d—MT Plasma d-MT Brain NE Brain D a-MT diet ug/g ug/ml ug/g ug/g 0 0 0 0.33:.01 0.74:.06 l 6.2i0.3 5.0:0.8 0.22:.01* 0.49:.05* 13 5.1i1.2 5.2i0.4 0.16:.Ol* 0.38:.04* *p<0.01 when compared to day 0 The values represent the mean i l S.E.M. for 6-8 determinations. 91 Figure 19. Effects of chronic administration of QFmethyltyrosine (a—MT) on spontaneous (SLMA) and amphetamine-stimulated locomotor activity (d-A MA). Motor activity was determined before (0 time), during (days 1-13) and l and 3 days after (days 14 and 16) the addition of 0.3% DLea-MT to the diet. Each point and vertical line represents the mean i 1 S.E.M. of 12 determinations. Solid points are those values which are significantly different from control at the 1% level. 92 B. Tolerance to the antiamphetamine effect of d— methyltyrosine If the ability of d—methyltyrosine to produce behavioral depression and block the central stimulant _effects of amphetamine is the result of inhibition of catecholamine synthesis in the brain, then with chronic administration tolerance should develop to the anti— amphetamine effect as well as to the locomotor depression. As depicted in Figure 19, both the loco- motor depressant (lower half) and antiamphetamine (upper half) actions of d—methyltyrosine are evident after 1 day of the diet. After 13 days of the d— methyltyrosine diet, however, neither parameter was different from control. In addition, 1 day after the drug was witheld from the diet (day 14), both meaSures of activity were significantly higher than control. This activated state was only transient and by day 16 the mice responded in a normal manner. A similar occurrence has been reported in man. Cessation of d- methyltyrosine administration caused patients to experience anxiety, restlessness, and insomnia for a period of 2—3 days (Engelman at aZ., 1968b). Several possible mechanisms that could explain the development of tolerance were considered and subjected to further investigation. Specifically, it was felt that tolerance could occur as a result of 93 Table 9. Effects of a chronic diet of DL—u-methyltyrosine (d—MT) on brain catecholamines Days of 439/9 d-MT diet ' NE d-MNE D 0 .33i.01 0 .74i.06 l .22i.01* 0 .49i.06* 1 (+1) .31i.01 0 .83i.10 13 .16i.01* .08i.03 .38i.04* 13 (+1) .25i.01* .04i.02 .76i.04 *p<0.01 when compared to day 0 The concentrations of norepinephrine (NE), d-methylnorepinephrine (d-MNE) and dopamine (D) were determined in 4 pooled mouse brains. The values represent the mean i l S.E.M. for 6 determinations. (+1) indicates 1 day following the termination of a 0.3% d-MT diet. 94 1) an accumulation of "false" adrenergic transmitters in the brain, 2) an alteration in catecholamine synthesis rates or 3) an increase in catecholamine receptor sensitivity. C. Accumulation of "false" adrenergic transmitters In man and the rat most of the administered a- methyltyrosine is excreted unchanged in the urine (Engelman at aZ., 1968a; Moore at aZ., 1967); it is filtered and passively reabsorbed in the proximal tubule (Hook and Moore, 1969). Nevertheless, small amounts of d—methyldopamine and d—methylnorepinephrine have been detected in the brains of rats after acute administration of a-methyltyrosine (Maitre, 1965). Following an injection of H3—u-methy1tyrosine, H3—d—methy1norepinephrine has also been identified in the heart and brain of guinea pigs (Udenfriend et aZ., 1966) and in human urine (Engelman at aZ., 1968a). With chronic administration of d—methyltyrosine, these compounds may accumulate and restore function by acting as substitute adrenergic transmitters. To investigate this possibility, the brains of mice on a chronic diet of d—methyltyrosine were analyzed for u—methyldopamine and d—methyl— norepinephrine (Table 9). One day of the a-methyltyrosine diet reduced the brain concentrations of norepinephrine and dopamine but no d—methylnorepinephrine was detected. In similarly treated animals which were allowed an 95 additional day of normal diet (1+1), dopamine and norepinephrine had returned to control concentrations. After 13 days of the d—methyltyrosine diet, there was a small accumulation of d—methylnorepinephrine. No d—methyldopamine was detected in the brain. Since a—methylnorepinephrine appears to have central depressant properties in the mouse (see Figure 8), it is probably not a significant factor in the development of tolerance. It may, however, occupy binding sites and retard the restoration of norepinephrine, but not dopamine, to control levels 1 day after the termination of the 13 day diet (13+1). This becomes evident when the brain concentrations of norepinephrine and dopamine are compared on days 1+1 and 13+1. D. Alteration of catecholamine synthesis rate As previously indicated, the brain concentrations of dopamine and norepinephrine remain low throughout the period of chronic d-methyltyrosine administration. However a Change in the actual rate of synthesis could have occurred but not be detected by measuring the steady state brain concentration of endogenous catecholamines. To test this possibility the formation of catecholamines from tracer doses of Cl4—tyrosine was used as an indi— cation of the rate of synthesis (Sedvall at aZ., 1968). The effects of a chronic diet of u—methyltyrosine on l I 800- - ' I l $ MOTOR ACTIVITY (COUNTS/10min) fiF-I (“R/I) 600 I- ' .. 400 T C - CATECHOLAMINES 14 200 " 0L NE D NE D NED NED DAYS ON ct-MT DIET O 1 13 13 61) Figure 20. Effects of a chronic diet of OFmethyltyrosine (a-MT) on exploratory locomotor activity and on the conversion of Cl ~tyrosine to dOpamine (D) and norepinephrine (NE). Cl4-tyrosine (10 pC) was injected into the tail vein. One hour later, the mice were decapitated and individual brains analyzed for labeled catecholamines (n=6). Motor activity (n512) was determined in a separate group of mice. (13'+1) represents 1 day after cessation of a 13 day DL-a-MT diet (0.3%). 97 l4-tyrosine to Cl4-dopamine and C14— the conversion of C norepinephrine are summarized in Figure 20 (the lower portion). The spontaneous locomotor activity counts of Similarly treated mice are depicted in the upper portion. After one day of the diet, the synthesis of norepinephrine and dOpamine was reduced but not com— pletely blocked. On day 13, when tolerance to the behavioral depression had developed (upper half, Figure 20), catecholamine synthesis from Cl4-tyrosine was inhibited to the same extent as on day l. The hyperactivity seen 1 day after the drug was witheld from the diet (13+1), was not accompanied by greater than normal catecholamine synthesis. Therefore, the tolerance and withdrawal hyperactivity could not be explained by an increase in tyrosine hydroxylating activity as measured by catecholamine formation from Cl4—tyrosine in viva. E. Alterations in receptor sensitivity Chronic interruption of the normal transmission process in peripheral nervous systems results in "supersensitivity" to the transmitter. In skeletal muscle this may be due to an enlargement of the chemo— sensitive zone (see Sharpless, 1969). A similar change may occur in the central nervous system (see Collier, 1969). Specifically, persistent blockade of catecholamine 98 7: OF CONTROL Figure I80 I60 I40 I20 I00 80 60 40 20 21. i + - I I4 I I4 I I4 I l4 .1...) EPHEDRINE d-AMPHETAMINE PIPRADROL METHYLPHENIDATE Stimulation of motor activity by ephedrine, amphetamine, pip- radrol and methylphenidate after 1 day of an ahmethyltyrosine (a-MT) diet and 1 day following the cessation of a 13 day diet of 0.3% DL-a-MT. The values for each drug treatment are plotted as the percentage of stimulation for that drug in animals on a control diet. The actual 20 min motor activity counts in control animals were: no drug, 557 i 107; ephedrine, 1005 i 79; amphetamine, 1491 i 162; pipradrol, 2031 f 177; methylphenidate, 2410 i 157 (mean i 1 S.E.M. for 12 determinations} Each bar and vertical line represents the mean i 1 S.E.M. Asterisks indicate those values that are significantly different from control at the 1% level. 99 synthesis may induce a receptor conformational change or an increase in the number of receptors resulting in an enhanced response to a given amount of catechol— amine. To test this possibility, the effects of central stimulant drugs, which presumably act by releasing cate— cholamines, were examined. The data in Figure 21 indicate that the actions of such drugs are enhanced following the cessation of a 13 day diet of a-methyl— tyrosine. After 1 day of the diet, the stimulant effects of ephedrine and amphetamine, which presumably act by releasing newly synthesized norepinephrine and/ or dopamine, were markedly reduced. The responses to pipradrol and methylphenidate were unaltered. One day following the termination of a 13 day diet of d- methyltyrosine (day 14), the responses to ephedrine and amphetamine were greatly enhanced while those of pipradrol and methylphenidate were not different from control. This suggested that postulated receptor changes were restricted to those Sites sensitive to catecholamines. To further test the theory of receptor sensitivity, attempts were made to produce direct stimulation of central catecholamine receptors by the administration of L—dOpa. To increase the amount of L—dopa in the brain, the decarboxylation of this amino acid in peripheral organs was blocked by the administration of either HMD or R0 4-4602, which in the doses used, 100 Figure 22. 100i go A W U! z 0 O. V! W a 60 P .1 < E x < H- O .— z I.“ U g 20 . O——O CONTROL . A-——-A 13 DAY III-MT + 1 I I OL L_ L 1 I __I J -9 -a -7 -6 +5 LOG MOLARITY OF NE (5) 0 Effect of a chronic diet of a-methyltyrosine (a-MT) on the contractile response of the isolated mouse spleen to cum- ulative additions of norepinephrine (NE). Spleens were re- moved 1 day after a 13 day diet of 0.3% DLja-MT had been terminated. The values are expressed as the percent of the maximal response obtainable with NE. The points and vert- ical lines represent the mean i 1 S.E.M. of 9 experiments. The concentrations of NE are those in the bath. 101 selectively block extracerebral aromatic L-amino acid decarboxylase (see Figure 3; Bartholini at aZ., 1969; LOtti, 1969). Forty-five min after i.p. or s.c. ad- ministration of 25-50 mg/kg of either drug, L—dOpa (25-100 mg/kg i.v. or 50-200 mg/kg i.p. or s.c.) produced such variable stimulation in regard to degree, peak reSponse time and duration that no meaningful data were obtained. In some animals, depression rather than stimulation was observed. Similar difficulties in attempts to produce central stimulation with L—dOpa in mice have been encountered (A. Rudzik, personal communication). Lotti (1969) has reported that the central stimulant effects of L—dOpa were enhanced when administered to mice in combination with the peripheral decarboxylase inhibitor HMD. Similar results have been reported in rats (Bartholini at aZ., 1969; Butcher and Engel, 1969). Species and strain differences could eXplain this inconsistency. A peripheral adrenergic neuroeffector organ, the mouse spleen, was examined in vitra for sensitivity changes in the contractile reSponse to norepinephrine following inhibition of catecholamine synthesis by a—methyltyrosine. The dose response curve for norepinephrine in the Spleens taken from mice 1 day following the termination of a 13 day d—methyltyrosine diet was identifical to control (Figure 22). Furthermore, 102 no supersensitivity could be demonstrated in the spleens from 24 hour reserpine-treated mice or when cocaine or desmethylimipramine was added to the in vitra bath (not shown). F. Tolerance in other behavioral tests Depression of shuttle box conditioned avoidance behavior in mice occurred when d—methyltyrosine was added to the diet. Of the eight mice that reached a satisfactory level of performance, the development of tolerance to the depressant effect of a-methyltyrosine was observed in two animals. The record of one of these is illustrated in Figure 23. After 1 day of the diet the number of avoidances per session was markedly reduced. With continued administration, tolerance developed rapidly to the behavioral depression and was complete within 3—4 days. During a week of an d— methyltyrosine-free diet the animal continued to perform at a satisfactory level. The drug was then added to the diet again and the pattern was duplicated. The performance of the 6 remaining trained animals, when fed the d-methyltyrosine diet, was markedly disrupted; Figure 24 is the record of one of these mice. Toler- ance did not develop; but the pre—drug level of performance was quickly restored when the diet was terminated. 20 P ' 15- ' A/5 10 - - DAYS 1‘ 0 5 10 15 20 4- MT o<~MT Figure 23. Development of tolerance to the depression of shuttle box behavior during a chronic diet of 0.4% DL—a—methyltyrosine (a—MT). Depicted is the performance of a single mouse. Daily test sessions consisted of 20 trials and the number of avoidances per session (A/S) was recorded. Solid bars indicate the days when OFMT was added to the diet. 104 20- - l 15 ' Io- - A/S 5b - 0.. llllllLllllllllJlJLllJ .- 0 5 10 15 20 DAYS — OQ'MT Figure 24. Lack of tolerance to the depression of shuttle box behav- ior during a chronic diet of 0.4% DL-aFmethyltyrosine (OFMT). See legend to Figure 23 for details. 105 With the continuous activity measurement, the results were also variable (Figure 25). In 3 groups tested, a 24 hour diet of 0.4% a-methyltyrosine significantly reduced the level of continuous activity. In 2 groups there was no indication of tolerance during a 2 week diet. In addition, withdrawal hyperactivity was not observed when the diet was terminated. In the remaining group tolerance develOped by the ninth day. This group also showed withdrawal hyperactivity. G. Discussion In attempting to eXplain the develOpment of toler— ance to the behavioral depressant actions of 0— methyltyrosine several possibilities were eliminated. Measurement of plasma and brain levels of a—methyl- tyrosine,as well as drug consumption,indicated that there was no alteration in the intake, metabolism or excretion of this drug. The small accumulation of d-methylnorepinephrine in the brain may act to impair the intraneuronal retention of norepinephrine. It is unlikely, however, that u-methylnorepinephrine plays any role in the restoration of normal behavior since this compound appears to have sedative properties (see Chapter III). Despite the fact that the brain catecholamines remained low while motor activity returned to control, an actual increase in tyrosine hydroxylase activity and 106 150 ,. .1 PERCENT OF CONTROL ACTIVITY 50 r A A I ‘ ‘ A - A 0 g I l J I 1 I I I J l l l l J.‘ DAYS 0 2 LI 6 8 10 12 — OGMETHYLTYROSINE DIET Figure 25. Continuous activity of mice fed a chronic diet of 0.4% DL— armethyltyrosine (OFMT). Each curve represents the data from a single group of 4 mice. The values were calculated as counts per hour from 7 p.m. to 7 a.m. and plotted as the percent of control. Control activity:[]-C]l,203; ()——{) 1,655; Ak-i§ 2,128 counts per hour. 107 catecholamine synthesis could have occurred. It has been reported that this does take place as a result of a compensatory increase in nerve activity following administration of reserpine or 6-hydroxyd0pamine (Mueller et aZ., 1969a and b) and can be prevented by cycloheximide or actinomysin D (Mueller et al., 1969c). The lack of an increase in catecholamine synthesis from C14-tyrosine after a 13 day diet of a—methyltyrosine suggests that either an increase in tyrosine hydroxylase activity did not occur, or if it did, and was a result of de novo synthesis, there was sufficient drug available to inhibit it. Pharmacological blockade of excitatory impulses to peripheral effector organs results in a slowly developing type of drug enhancement or supersensitivity (Trendelenburg, 1963, 1966) to which Sharpless (1964) has applied the term "disuse" supersensitivity. It can be produced by ganglion blocking drugs, catecholamine depleting agents, or compounds that prevent the liberation of norepinephrine or acetylcholine (Trendelenburg, 1963, 1966). In autonomic effector organs, disuse supersensitivity has certain salient features which have been recently summarized (Sharpless, 1969): 1) it is caused by inactivity of the effector organ resulting from blockade of nerve impulse flow; 2) it develOps slowly (2-3 weeks); 3) it is relatively 108 nonspecific; the effector organ becomes sensitized to any excitatory agent including K+ and Ca++; 4) it is produced by blockade of excitatory but not inhibitory influences (denervation supersensitivity is not this selective); 5) it is reversible and declines when excitatory input is restored; 6) if the action of the blocking agent can be terminated rapidly "rebound" or withdrawal hyperactivity occurs. The first demon— stration of this withdrawal phenomenon was in the CNS; the hypothermic response to pilocarpine was exaggerated after termination of chronic sc0polamine administration (Friedman et aZ., 1969). The characteristics of the tolerance to d—methyltyrosine meet all of these criteria except the reference to Specificity. The enhanced responses following the termination of chronic a— methyltyrosine administration appear to be Specific for the catecholamine releasing agents amphetamine and ephedrine. Non-specificity, however, cannot be eliminated since the particular receptor sites involved may also be supersensitive to other excitatory drugs or ions if they were applied directly. Systemically administered pipradrol or methylphenidate may not be acting at the same sites in the brain as amphetamine or ephedrine. Alternatively, the characteristics defined for receptors on smooth muscle cells may not fully apply to neurons of the CNS. 109 The develOpment of tolerance to a—methyltyrosine in behavioral tests other than eXploratory locomotor activity was inconsistent. This could partially be explained by the relatively small number of animals tested which would accentuate individual variations. Certainly, the ability of animals to learn a test varies widely, and the predrug level of performance, eSpecially in regard to the latent period, could influence the reSponseS to the drug. With chronic reserpine treatment, tolerance develops to locomotor depression but not to the disruption of Shuttle box and rotarod performance of rats (Pirch and Rech, 1968). Tolerance to the depressant effects of d—methyltyrosine on lever pressing for food has been reported in rats (Beaton and Crow, 1969). In this study of two animals, uneXplained and unusual apparent tolerance developed when single doses of a-methyltyrosine were administered once every 14 days. Another consideration is the theory that postulated receptor changes may occur in certain neuronal pathways which may be important for Specific behavioral reSponses. As suggested earlier, certain data indicated that dOpamine may be more important for exploratory locomotor activity while norepinephrine may be a chemical mediator in CNS pathways maintaining the level of con- tinuous unstimulated activity. Chronic studies with dOpamine B-hydroxylase inhibitors which deplete the llO brain of norepinephrine, but not dopamine, would be interesting in regard to possible tolerance develop— ment, withdrawal hyperactivity, and amphetamine supersensitivity. The inability to demonstrate supersensitivity of the isolated mouse Spleen to norepinephrine following a chronic diet of u—methyltyrosine or other procedures (reserpine pretreatment, addition of cocaine or DMI in vitro) is consistent with studies of the cat spleen. Chronic reserpine administration did not produce supersensitivity to norepinephrine in isolated strips of Spleen. Green and Fleming (1968) concluded that a postsynaptic type of supersensitivity could not be produced in this preparation even with chronic surgical denervation. It is possible that, in the spleen where the norepinephrine turnover rate is low, reduced transmitter availability may have little functional consequences and adaptive receptor changes may not occur. Another possibility is that supersensitivity actually is present in viva but cannot be demonstrated in vitro. Such a Situation has been reported in studies of the cat nictitating membrane (Tsia et aZ., 1968). Other mechanisms have been prOposed to explain tolerance in the CNS. The "redundancy theory" suggests that depression of synaptic activity by prolonged lll transmitter deficiency results in a hypertrOphy of a neuronal shunt around the depressed synapse (Martin, 1970). This hypothesis could certainly explain the results obtained with d—methyltyrosine but, like the receptor theory, it is not presently amenable to direct validation. H. Summary Chronic administration of d-methyltyrosine produced tolerance to the depression of exploratory activity and amphetamine antagonism but not to the depletion of brain catecholamines. Transient rebound hyperactivity was observed when the drug was discontinued. Less consistent tolerance developed to the depression of continuous activity and shuttle box behavior. Evidence suggested that presynaptic mechanisms such as enhanced catecholamine synthesis rate and accumulation of "false" adrenergic transmitters were not involved; a post synaptic change resulting in increased receptor sensitivity was a more likely occurrence. CHAPTER VII DISCUSSION: THEORIES OF CEREBRAL CATECHOLAMINE FUNCTION Despite the accumulation of knowledge during the past 15 years concerning the distribution and metabolism of catecholamines in the brain, there is no general agreement regarding the functions of these amines. The results of this study support the conclusions of earlier investigations that inhibition of catechol- amine synthesis is associated with behavioral depression in animals. Aspects of this study can be discussed in relation to current theories of cerebral amine function. Histochemical and electrophysiological evidence suggests that norepinephrine in the brain stem is involved in the activity of the reticular formation. Availability of norepinephrine at certain synapses in this system may maintain the level of tonic activity necessary for normal unstimulated daily activity patterns of animals. Thus agents which block the synthesis of norepinephrine or antagonize its action at receptor sites would be eXpected to decrease the level of activity. Conversely, an excess of norepinephrine at these same receptor sites resulting from the administration of amphetamine 112 113 or ephedrine could explain the increased activity in animals and enhanced alertness in man. The reduction in continuous activity in mice accompanying a deficiency of norepinephrine can probably best be related to signs of sedation and tranquilization in man. The marked effects of d—methyldopa in mice certainly parallel those seen initially, at least, in man (Gillespie et aZ., 1962) while the rather mild de- pression of continuous activity by d—methyltyrosine is consistent with its weak sedative effect in man (Engelman et al., 1968b). Possible explanations for the differential effects of these two drugs have been discussed (see Chapter IV). The exploratory locomotor activity of animals exposed for a short period to a novel environment may also be an indication of activity in the reticular formation and resulting state of cortical excitability. The transient Withdrawal hyperactivity upon termination of a chronic d—methyltyrosine diet in mice seems analagous to the signs of suggested reticular hyper— activity (insomnia, anxiety, restlessness) observed in man after cessation of d-methyltyrosine administration (Engelman et aZ., 1968b). This study suggested that an increase in receptor sensitivity could explain this phenomenon but future investigations are necessary in order to ascribe these events to Specific changes in norepinephrine receptors. 114 A deficiency of norepinephrine at certain receptor Sites in the brain is thought to be the underlying cause of some, if not all, depressive states in man (Schildkraut, 1965). Sites in the brain where a possible deficiency of norepinephrine may exist are not generally discussed. The greatest concentration of norepinephrine is in the hypothalamus (Fuxe et al., 1969). The emotional Signs of depression suggest that this structure, which is an integral component of the limbic system, could be the locus of a norepinephrine deficiency. The apathy, decreased alertness and responsiveness of depressed patients, also suggests a reduction of activity in the reticular center (Kraines, 1963). The evidence for Schildkraut's theory is primarily pharmacological. Reserpine—induced depressions are regarded as indistinguishable from the naturally occurring disorders (Schildkraut and Kety, 1967). Although there is no animal model for depression, studies with antidepressant drugs in a number of Species have provided the basis for their prOposed mechanism of action. Monoamine oxidase inhibitors, which allow the presynaptic accumulation of norepinephrine and the tricyclic antidepressants which, among other actions, block the reuptake of presynaptically released nor— epinephrine are effective therapeutic agents in the 115 treatment of depression (Schildkraut, 1969). These drugs have other actions which may contribute to their antidepressant effect. Monoamine oxidase inhibitors also elevate the brain level of 5-HT and their therapeutic effect is reported enhanced when combined with tryptOphan (Coppen, 1968). The tricyclic compounds block the uptake of 5—HT (Carlsson et aZ., 1968, 1969) as well as norepinephrine and also have cholinergic and adrenergic blocking effects. Adaptative changes occurring as a result of receptor blockade could explain the effectiveness of these agents in depressed states Since clinical improvement occurs only after 1 to 4 weeks of treatment (Schildkraut, 1969). The relative ineffectiveness of amphetamine, which can release norepinephrine as well as block its uptake (Glowinski at aZ., 1966), in major depressive disorders seems inconsistent with the catecholamine theory of depression. One explanation is that amphetamine is partially metabolized to p-hydroxynorephedrine which may accumulate in presynaptic adrenergic terminals and serve as a "false" transmitter (Gropetti and Costa, 1969). Another possibility is that depression is not associated with a deficiency of brain norepinephrine. An extension of the catecholamine theory of affective disorders is the idea that an excess of 116 norepinephrine at Specific receptor Sites in the brain is associated with elation or possibly mania (Schildkraut, 1965). A somewhat related theory is that schiZOphrenia can be produced by a continuous state of central excitation or overarousal probably emanating in the brainstem reticular formation (Kornetsky and Mirsky, 1966). Studies with inhibitors of catecholamine synthesis have not supported these prOposals. Although clinical trials were limited, d—methyltyrosine had no beneficial effect in schizophrenic patients (Gershon et aZ., 1967; Charalampous and Brown, 1967). d— Methyldopa was equally ineffective and in some cases aggravated the condition (Mosher et aZ., 1966; Herkert and Keup, 1969). Proponents of the above theories could argue that catecholamine synthesis cannot be completely inhibited by these agents. Another possibility is that the development of tolerance by any of the mechanisms explored in this study obscures any initial therapeutic effect; this problem will probably be encountered with any attempt to chronically inhibit catecholamine synthesis. The View that norepinephrine has primarily depressant properties in the central nervous system continues to be supported by a few researchers (Mandell et aZ., 1968, 1969; Dewhurst, 1968, 1969). Accordingly, depletion of brain norepinephrine Should lead to central excitation. 117 The data presented in this study refute these ideas. Nevertheless, norepinephrine may have an inhibitory function in the brain since it cannot be established at this time if the proposed stimulant effects of this amine are actually due to inhibition of tonic excitatory systems. There is ample evidence to suggest that norepinephrine is an excitatory mediator of a median forebrain bundle reward system in the brain of the rat (Stein, 1967, Wise and Stein, 1969). Stein (1967), however, has also suggested that activation of this reward system could be the result of widespread nor- adrenergic inhibition of forebrain suppressor cell groups. An excess of norepinephrine at these sites could be one explanation for the taming effect of amphetamine and methamphetamine in aggressive rats (Sofia, 1969) and its calming action in hyperkinetic children (Weiss et aZ., 1968). The high concentration of dOpamine in the caudate nucleus (Carlsson, 1959) suggests that it may play a role in the function of this nucleus. There are indications that the caudate nucleus is involved in the cerebral control of movement (Liles and Davis, 1969a, b). Microiontophoretic studies suggest that dopamine acts as an inhibitory synaptic transmitter within the caudate (McLennan and York, 1967). Electrical stimulation of different areas of the caudate nucleus can either 118 facilitate or inhibit cortically—induced movement (Liles and Davis, 1969a). The symptoms of Parkinson's disease (akinesia, rigidity and tremor) have been attributed to a deficiency of striatal dOpamine (Hornykiewicz, 1966). Akinesia, best defined as the "difficulty in initiating voluntary movements,‘ could result from a loss of facilitation; whereas rigidity and tremor might indicate a release from tonic inhibition. Administration of L—dopa to Parkinsonian patients has been reported to alleviate most effectively the akinesia while the tremor and rigidity were less improved (Cotzias et al., 1968). This would suggest that dOpamine has primarily a facilitory function in locomotor activity. Inhibition of catecholamine synthesis by d-methyltyrosine produced tremor in monkeys which was alleviated by L—dopa (Bédard et aZ., 1970). In a few cases, d-methyltyrosine produced slight tremor in man (Engelman et aZ., 1968b). These data support an inhibitory role for dopamine. 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