Effects of chronic amphetamine on central serotonergic and dopaminergic systems in rats of various ages,

The central neurochemical effects of chronic low doses (0.5 or 5 mg/kg s.c., every 12 h for 2 wk) if d-amphetamine (AMPH) were studied in rats of varying ages. Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activities in neostriata of male offspring of dams exposed to 5 mg/kg AMPH during gestation showed significant depressions at 3 weeks of age, while corresponding neurotransmitters were at control levels. In contrast, the striatal enzyme activities in the dams were normal, but significant depressions were observed in the levels of striatal 5-hydroxytryptamin (5-HT), 5-hydroxyindoleacetic acid 5-HIAA, dopamine (DA) 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA). All parameters returned to normal by 7 weeks. Neurochemical changes were minimal in juvenile and adult males subjected to the same AMPH dosing regimen. The hypothalamic concentrations of 5-HT, 5-HIAA, and DA in adult males were reduced at 16 h, but these parameters were unaffected in the juveniles. Recovery in the adults was complete by 3 wks post dosing. Not effects were seen with the lower dose of AMPH (0.5 mg/kg) in any age group. The chronic doses of AMPH used in this study are not as toxic to the central serotonergic or dopaminergic systems of the rat as higher doses that have previously been studied in acute and subacute dosing regimens. Under these condition, the age of the animal at the time of drug exposure did not significantly alter the severity of the toxicity that was observed. Further, our data suggest that that Neurotoxicity of AMPH is highly depend on dose and duration of exposure.

EFFECTS OF CHRONIC AMPHETAMINE ON CENTRAL SEROTONERGIC AND DOPAMINERGIC SYSTEMS IN RATS OF VARIOUS AGES by Elaine Wyne Snowhill A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biochemical Pha~~cology and Toxicology The University of Utah June 1983 © 1983 Elaine W. Snowhill All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by Elaine W. Snowhill This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. March 4, 1983 Ph.D. March 4, 1983 March 4, 1983 Ralph Karler, Ph.D. March 4, 1983 Glen R. Hanson, D.D.S., Ph.D. March 4, 1983 THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of The University of Utah: I have read the dissertation of Snowhill its final form and have found that (1) its format, citations, and bibliographic style are consistent and acceptable; (2) its illustrative materials ihcluding figures, tahles, and charts are in place; and (3) the final manuscript is satisfactory to the Supervis­ory Committee and is ready for submission to the Graduate School. ~D(--e-~--~D~--- ~~~1~~·-------- ~perViSOry Committee Approved for the Major Department Chairman I Dean Approved for the Graduate Council ABSTRACT The central neurochemical effects of chronic low doses (0.5 or 5 mg/kg, s.c., every 12 h for 2 wk) of d-amphetamine (AMPH) were studied in rats of varying ages. Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activities in neostriata of male offspring of dams exposed to 5 mg/kg AMPH during gestaton showed significant depressions at 3 weeks of age, while corresponding neurotransmitters were at con­trol levels. In contrast, the striatal enzyme activities in the dams were normal, but significant depressions were observed in the levels of striatal 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), dopamine (DA) , 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA). All parameters returned to normal by 7 weeks. Neurochemical change s were minimal in juvenile and adul t males subjected to the same AMPH do sing regimen. The hypothalamic concentrations of 5-HT, 5-HIAA, and DA in adult males were reduced at 16 h, but these parameters were unaffected in the juveniles. Recovery in the adults was complete by 3 wks post dosing. No effects were seen with the lower dose of AMPH (0.5 mg/kg) in any age group. The chronic doses of AMPH used in this study are not as toxic to the central sero­tonergic or dopaminergic systems of the rat as higher doses that have previously been studied in acute and subacute dosing regimens. Under these conditions, the age of the animal at the time of drug exposure did not significantly alter the severity of the toxicity that was ob- served. Further, our data suggest that the neurotoxicity of AMPH is highly dependent on dose and duration of exposure. v ABSTRACT LIST OF TABLES ACKNOWLEDGEMENTS INTRODUCTION . MATERIALS AND METHODS . RESULTS DISCUSSION REFERENCES CONTENTS iv vi .. viii 1 3 8 21 28 LIST OF TABLES Table I. Levels of 5-HT, DA, and metabolites (~g/mg tissue) in adult male control rats. . • •.•• 9 II. TPH and TH activities (nMoles substrate oxidized/ g tissue/hour) in adult male control rats • . . • • • 10 III. Levels of 5-HT, DA, and metabolites (~g/mg tissue) of control rats ••••• • . . . • . • 11 IV. TPH and TH activities (nMoles substrate oxidized/ g tissue/hour) of control rats. . • • • . • 12 V. 5-HT, DA, and metabolite levels in neostriata of treatment grouos at 16 hand 3 wk post-dosing (5.0 mg/kg AMPH, twice daily for two weeks). • .. .• 14 VI. TPH and TH activities in neostriata of treatment groups at 16 hand 3 wk post-dosing (5.0 mg/kg VII. VIII. IX. X. AMPH, twice daily for two weeks).. •••..•. 15 5-HT and 5-HIAA levels and TPH activity in cerebral cortex of treatment groups at 16 hand 3 wk post­dosing (5 mg/kg AMPH, twice daily for two weeks) •.. 16 5-HT, 5-HlAA, and DA levels in hypothalamus of treatment groups at 16 hand 3 wk post-dosing (5 mg/kg AMPH, twice daily for two weeks) • • . · • . 18 5-HT, 5-HIAA, and DA levels and TPH activities in cerebral cortex and hypothalamus of juvenile- and adult-treated males at 16 hand 3 wk post-dosing (10 mg/kgAMPH, twice daily for two weeks) ••.••. 19 5-HT, DA, and metabolite levels and TPH and TH activities in neostriata of juvenile- and adult­treated males at 16 hand 3 wk post-dosing (10 mg/kg AMPH, twice daily for two weeks). • . • • • • . 20 ACKNOWLEDGEMENTS I would like to express my gratitude to the members of my commit­tee, Drs. James W. Gibb, Harold H. Wolf, Ralph Karler, Glen Hanson, and James K. Wamsley, for their guidance in developing this project. Special thanks are extended to my family and close friends for their continuous support and confidence in me, and to Dr. L. T. Lais, whose dynamic approach to science and teaching made a lasting impact on my training. This work was supported, in part, by a grant from The Thrasher Research Fund and fellowship support from the American Foundation for Pharmaceutical Education and the Robert Wood Johnson Foundation. INTRODUCTION The neurochemical changes induced by high-dose, short-term exposure ~o amphetamines have been examined in great detail 1 ' 9- 12,14,15,17,18,33,38 Multiple injections of methamphetamine (METH) (10-15 mg/kg/ do se) over 18-36 hours cause severe depres sions of the serotonergic and dopaminergic systems in several discrete brain regl. ons of t h e rat1 0,14,15,33 . This is not a generalized neurotoxic effect, as cholinergic and GABA-ergic systems in the same areas are not similarly affectedl5 . Furthermore, striatal tyrosine hyroxylase (TH) 1,9 and tryptophan hydroxylase (TPH) 1 activities and 5-hydroxy-tryptamine (5-HT) levels1 remain depressed as long as 110 days after dosing. Similarly, a single dose of amphetamine (AMPH) to iprindole-treated rats causes a decrease in striatal dopamine (DA) that persists at least 7 daysl1,12,38. Amphetamine-induced neurotoxicity has also been approached from a developmental standpoint. Wagner et- al. 40 found that JO-day old rats exposed to very high doses of AMPH (25 mg/kg) twice daily for 30 days had depressed caudate DA levels at 2 weeks post-dosing. How-ever, the magnitude of the change was significantly less than that previously reported in adul t animals39 • Lal and Feldmuller20 calcu-lated the whole brain half-life of AMPH in 12-day old rats to be 150 minutes, compared to 66 minutes in adul ts. In spite of this slower clearance, peak AMPH levels after a single injection of the drug were 2 found to be lower in the immature than in the adult anima1s20 • High-dose acute or subacute studies are important in de1ineat-ing the mechanisms of drug-induced toxicity and for providing animal models for the study of drug abuse. We were interested, however, in the neurochemical consequence~ of more clinically relevant AMPH exposure. AMPH is used therapeutically in low doses for the treatment of such diverse disorders as obesity, narcolepsy, and attention deficit disorder (ADD; i. e. minimal brain dysfunction, hyperkine- . )41 S1S • The latter condition, ADD, is most frequently recognized and trea ted in young children. The current research was designed to examine the neurochemical effects of chronic, low doses of AMPH and to determine whether any of the drug-induced changes are dependent on the age of the animal at the time of exposure. MATERIALS AND METHODS Adult male (180-200g), juvenile male (I-week old), and timed­pregnant female (day 2 of gestation) Sprague-Dawley rats were obtained from Simonsen 'Labs. Animals were housed in groups except for the pregnant females, which were housed separately. All rats were main­tained in temperature-controlled rooms (24 0 C) and were allowed accesS to food and water ad libitum. Experimental animals received d-amphetamine (0.5 or 5 mg/kg, s.c.) every 12 hours for two weeks; controls received equivalent vol­umes of· the saline vehicle. Twice daily do sing with· 0.5 mg/kg AMPH was selected for study as an approximation of the regimen used clini­cally in children for the treatment of ADD. The higher dose,S mg/kg, was included for purposes of direct comparison with the effects of the lower do sea Without assuming direct correIa tions between human use and our animal model, 5 mg/kg AMPH twice daily represents a dose level somewhat higher than is routinely used clinically in adults (e.g. nar­colepsy, obesity). Higher doses could not be employed in this study due to an exceptionally high mortality rate in the pregnant f em.ale s. Under the conditions described here mortality was less than 5 percent. Figure 1 is a schematic representation of the dosing regimen and sacrifice schedule used in this study. Juvenile males were dosed beginning on postnatal day 14. Females were dosed from day 7 of ges-tation through day 20; the last dose was given 12-24 hours before par- [EXPElITr1ENTAC DESIGN} JUVENILE MALES ---Y~t-....&Jj_ _________. L.--_________. ...I " 1\ 1\ ADULT MALES r---r~:.4 " 1\ 1\ PREGNAtIT FEMALES I ""8Z~ - - ] (DAMS) DAyl DAy7 TERM " 1\ r1,\!...E OFFSPRING - - - I Figure 1. BIRTH .". " • I .--------------- -- I -3 -2 0 3 7 TIME (WEEKS) IZA DURATION OF DOSING A SACRIFICE '& OFFSPRING \\EANED Experimental Design. Amphetamine was administered subcutaneously every 12 hours for 2 weeks (shown by l2j.t :41l). Offspring of the treated darns were weaned on postnatal day 21 ('6.). Individual animals from each group were sacri­ficed at time points designated by )\. Sacrifice at Time 0 in the juvenile and adult males represents a time of 16 hours post-dosing. .+:-- 5 turition. Mal e of f spring of these dams, which were included in the neurochemical analyses, were exposed to AMPH in utero only. Indi-vidual animals from each group were sacrificed at the times shown in Figure 1. To allow ample differentiation of the brain for dissection, the earlie st time of sacrifice of the offspring was three weeks. Females were required for nursing of the young until weaning at 21 days. Rats were sacrificed between 09.00 and 12.00 hours to minimize the effects of diurnal and/or circadian rhythms on neurochemical ac-tivity. Rats were decapitated and the whole brain placed immediately on ice. Neostriatum, cerebral cortex, and hypothalamus were dissected out and frozen separately (-70 0 C) in paraf ilm until analyses were performed. These brain regions were selected for study since the cortex and striatum have been reported to be particularly sensi­tive1 ,14,23,33 and the hypothalamus more resistant l ,33 to the toxicity of the amphetamines. In addi tion, the developmental state of the hypothalamic hormonal influence was of interest for the comparison of the responses observed in the immature versus adult animals. In neostriatum and cortex, enzyme activities were measured as f o 11o ws: TPH act~.v.~t y by a mo d ~. f·~ e d 14CO 2-trapp~. ng met h 0 d 16 ,37 as described previously by Hotch~iss et al. 15 , TH activity by the method of Naga tsu et a 1. 25 In hypothalamus, as well as neostriatum and cortex, selected monoamines and metabolites were also measured. High performance liquid chromatography with electrochemical detection (HPLC-EC) was utilized to quantitate simultaneously 5-HT, 5-HIAA, DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA), 6 27 using a modification of the method described by Neilsen and Moore . The HPLC conditions were as follows: Waters microbondapak C-18 reverse-phase analytical column (10 micron) with a mobile phase con-sisting of 0.1 M citric acid (anhydrous), 0.1 M potassium phosphate dibasic, 12.5% methanol, 0.004% sodium heptane sulfonic acid, and 1 mM EDTA, adjusted to a final pH of 3.5. Use of the present counter-ion in this procedure resulted in a different separation (Figure 2) than was previously described27 using sodium octane sulfonic acid. Elec-trochemical detection (Bioanalytical Systems) was at a potential of +0.73V. Brain regions were homogenized in the mobile phase buffer and centrifuged at 41,300 x g for 20 minutes. The supernatant was fil-tered across densely packed glass wool and a 50 1-11 aliquot inj ected directly onto the chromatography column. Quantitation of samples was achieved by comparison of peak heights to those of standard solutions injected daily. The Student's t-test6 was used to compare the appropriate con-trol and treated groups, and in all cases a p value of < 0.05 was used to assign statistical significance. Each of the paradigms were performed in a minimum of two sepa-rate experiments; the data were pooled, providing values of 12-18 ani-mals per group unless otherwise stated. w z :E a:: 0 0 ~ 0 a. 0 l- X I LO o l- X I LO (.) ~ o o 7 HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION OF STANDARDS (C-t8 ANALYTICAL COLUMN, WATERS ASSOC.) ELECTROCHEMICAL DETECTION (810ANAL YT1CAL SYSTEMS) AT A POTENTIAL OF + 0.73 V. c::( c::( X I LO 10 20 TIME (min) Figure 2. HPLC-EC separation of stand'ards. RESULTS Most of the parameter.:s measured exhibited a wide range of control values between age groups. Tables I and II give the mean absolute values ( ± S.E.M.) observed in adult male control animals. These figures are similar to those previously reported in the litera-t 1,29 ure • Except for a slightly enhanced striatal DA concentration (p <.05), values obtained in the control dams were not signif icantly d iff erent from tho se in the adul t males (see Table s III and IV). Due to considerable postnatal development of monoaminergic systems in the t 7 ,8,24,28 ra , control values in the offspring and juvenile groups changed as a function of their age at the time of sacrifice (see Tables III and IV). Most of the dopaminergic parameters reached adul t levels by 3 weeks of age, i.e., striatal TH, DOPAC, and HVA and hypo-thal ami c DA. Striatal DA, however, did increase slightly between 3 and 7 weeks postnatally, indicating a lag period between maturation of the rate-limiting synthetic enzyme and its product. Most of the serotonergic parameters increased towards adult values during this time period (i.e., striatal and cortical TPH; striatal, cortical, and hypothalamic s-HT). In contrast, s-HIAA did not change wi th time in any of the areas studied. All parameters reached adult levels by 7 weeks of age, which is consistent with data reported else-h 24,28,31 were • Because of these normal fluctua tions in the absolute values of the parameters in control tissues, experimental data Table I. Levels of 5-HT, DA, and metabolites (~g/mg tissue) in adult male control rats. t 5-HT 5-HIAA Neostriatum 0.537 + .010 0.522 + .030 -Co-r-te-x 0.323 + .019 0.178 + .005 Hyp~c:t~~.J-amus 0.712 + .015 0.698 + .033 tValues are expressed as means + S.E.M.; (N ~ 12) tt Not detected DA 00 PAC HVA 8.07 + 0.25 1.10 + 0.07 0.781 + .063 tt tt tt 0.593 + .034 tt tt ~ Table II. Neostriatum Cortex TPH and TH activities (~wles substrate oxidized/g tissue/hour) in adult male control rats.t TPH TIl 32.5 + 1.3 3173 + 270 29.3 + 1.4 tt t Values are expressed as means + S.E.M.; N 12 ttNot determined ...... o Table III. Levels of S-HT, DA, and metabolites (~g/mg tissue) of control rats. t _._5--lI-T --- 5-HIAA DA OOPAC HVA Dams !!~~trlatum O. 620 :t .0 3Q 0.545 :t .037 10.2:t 0 •4 1.1O:t.07 0.856 + .040 Cortex 0.242 + .011 0.201.+' .012 !I y~o.~l~~ ~a.m~ 0.822 :t .041 0.632 + .073 0.488 :t.051 Juvenile males --16h---- Neostriatum 0.416 + .013 0.723 + .064 5.72 :t .20 1.00 + .046 0.761 + .030 Cortex 0.183 + .010 0.200 :t .011 Hypothalamus 0.720 + .030 0.750 + .022 0.601 :t .021 3 wk Neostriatum 0.606 + .039 0.663 :t .060 8.70 :t .60 1.14 :t .09 0.747 ± .087 Cortex 0.222 + .005 0.171 + .011 Hypothalamus 0.830 :t .037 0.618 + .028 0.625:t .040 Malt:.~f_~ring 3 wk Neostriatum 0.421 + .024 O. 781 + .033 6.10 + .15 0.950 :t .048 0.950 :t .060 Cortex 0.112 + .012 0.177 :t .017 Hypothalamus O. 616 :t .0 72 0.696 + .036 0.629 :t .036 7 wk Neostriatum 0.550 + .020 0.760 :t .052 8.98 + .31 0.916 + .080 0.850:t .081 Cortex 0.201 + .010 0.175 :t .014 Hypothalamus 0.840 :t .024 0.640 + .020 0.621 + .031 t Values are expressed as means :t S.E.M.; N = 12 - 18 - Not detected t--' t--' Table IV. TPH and TH activities (nMole oxidized/g tissue/hour) of control rats.+ Dams -Neos t ria tum Cortex Juvenile males -[6-h--- Neostriatum Cortex 3wk Neostriatum Cortex Male offspring 3 wk NeOStriatum Cortex 7 wk Neostriatum Cortex TPH 28.3 + 1.2 28.2 + 1. 5 22.8 + 1. 9 19.5+1.4 34.6 + 1. 8 26. 7 + 1. 4 21.2!1.4 19.2 + 1.0 30.6 + 1. 8 24.3 ! 0.3 tVa1ues are expressed as means! S.E.M.; ~ = 12 - 18 ttNot determined TH 3140 + 249 tt 2933! 167 tt 2820 + 187 tt 3041 :t 107 tt 3111 ! 167 tt 12 13 throughout this discussion are expressed as percents of their corre­sponding controls. The lowest dose of AMPH (0.5 mg/kg) caused no significant neurochemical changes at any time point tested (data not shown). The effects of 5.0 mg/kg AMPH in the neostriatum are shown in Tables V and VL The dams were the only group to show uniform depressions in all monoamine and metabolite levels. At 3 weeks, the decreases were to approximately 80% of the control concentrations of 5-HT, 5-HIAA, DA, DO PAC , and HVA. The male offspring sacrificed at the same time showed only a slight depletion of DA (to 86%). Whereas striatal TPH and TH activities were normal in the dams, the enzymes were signifi­cantly depressed (to 73 and 80%, respectively) in their offspring. Although adult and juvenile males exhibited no changes 16 hours post dosing, TPH activity in the adults was increased to 134% of control at 3 weeks and recovered by 7 weeks. There were few changes observed in the cerebral cortex of any of the groups (see Table VII). Adult males showed a significant de­pression of 5-HIAA (to 75% of control) at 16 hours which had recovered by 3 weeks. In addi tion, the only changes seen in this study at 7 weeks post-dosing (data not shown) were in the cortical 5-HT (80 ± 4%) and 5-HIAA (86 ± 3%) levels in juvenile-treated males (data not shown) • The hypothalamus was also relatively insensitive to the drug, with juvenile males showing more resistance than the adult-treated groups. At 16 hours post dosing, adult 5-HT, 5-HIAA, and DA were de­pressed to 79%, 68%, and 82% of controls, respectivly, while juvenile- Table V. 5-HT, DA, and metabolite levels in neostriata of treatment groups at 16 hand 3 wk post~osing (5.0 mg/kg AMPH, twice daily for two weeks).t 5-HT 5-HlAA DA 00 PAC HVA 16 h Juvenile males 91 +- 7 98 + 5 96 +- 4 95 + 4 90 + 4 Adult males 92 +- 4 97 + '7 92 +- 4 101 + 5 96 +- 6 3 wk Juvenile males 101 +- 6 98 +- 6 92 +- 4 92 + 5 95 + 5 Adult males 95 +- 3 96 +- 4 103 + 5 105 + 4 95 +- 7 Dams 83 + 3 * 83 -+ 4 * 81 +- 2* 80 +- 5 * 79 + 5 * Male offspring 96 + 8 86 + 11 86 +- 3 * 95 + 5 94 + 6 tValues are expressed as mean percent of corresponding controls ± SEM; N =12-18 per group. * Significantly different from control, p < 0.05 t-' ~ Table VI. TPH and TH activities in neostriata of treatment groups at 16 hand 3 wk post-dosing (5.0 mg/kg AMPH, twice daily for two weeks).t TPH TH 16 h Juvenile males 96 +- 6 116 -+ 6 Adult males 106 + 7 109 + 7 3 wk Juvenile males 101 + 6 106 + 5 Adult males 134 + 8 * 106 +- 5 Dams 106 + 4 95 + 6 Male offspring 73 + 6 * 80 + 3* t Values are expressed as mean percent of corresponding controls ± SEMj N == 12-18 per group. * Significantly different from control, p < 0.05 I-' In Table VII. 5-HT and 5-HlAA levels and TPH activity in cerebral cortex of treatment groups at 16 hand 3 wk post­dosing (5 mg/kg AMPH, twice daily for two weeks).t 5-HT 5-HlAA TPH 16 h Juvenile males 90 ~ 5 92 + 6 98 ~ 3 Adult males 90 ~ 5 75 ~ 7* 77 + 10 ~ Juvenile males 92 + 6 89 + 7 99 + 4 Adult males III + 6 110 + 6 105 + 5 Dams 88 + 7 94 + 8 84 ~ 5 Male offspring 98 :t 8 104 + 8 98 :t 6 tValues are expressed as mean percent of corresponding controls ± SEM; N = 12-18 per group * Significantly different from control, p < 0.05 16 17 treated rats did not differ from their controls (see Table VIII). To determine whe ther a larger do se of AMPH in this do sing regimen would induce more severe or longer-lasting neurochemical changes, one additional dosing experiment was performed (N = 6-8 per . group). Juvenile and adult males were dosed with 10 mg/kg AMPH twice daily for 2 weeks (as described above). Pregnant females could not be used due to an exceptionally high mortality rate at this dose level. From these preliminary data (see Tables IX and X), a significant dose­effect component of AMPH-induced toxicity in this dosing paradigm was apparent. Quantitatively, more significant changes were seen in both groups at this higher dose. In the striata of juvenile males 5-HT and 5-HIAA were greatly depleted, being 64% and 68% of respective controls at 16 hours. These had completely recovered by 3 weeks, whereas smaller decreases in DA and RiA (each to 84% of controls) at 16 hours had not recovered by 3 weeks. Striatal TPH was elevated in the adult males at both time points and TH was significantly increased at 3 weeks, without accompanying changes in monoamine levels. Juvenile males exhibited striatal TH activity of 137% of control at 16 hours, with recovery to control by 3 weeks. Cortical 5-HIAA was decreased in both groups at 16 hours; this was the only change observed in this brain region at the high (10 mg/kg) dose. Hypothalamic levels of 5-HT and DA that were depleted at 16 hour s in adul ts and j uvenil es, respectively, had returned to con­trol by 3 weeks. Table VIII. 5-HT, 5-HIAA, and DA levels in hypothalamus of treatment groups at 16 hand 3 wk post-dosing (5 mg/kg AMPH, twice daily for two weeks). 5-Hf 5-HlAA DA 16 h Juvenile males 94 + 2 96 + 3 90 + 3 Adult males 79 + 2* 68 :t 3 * 82 + 5 * 3 wk Juven He ma les 96 + 3 95 :t 3 96 + 3 Adult males 119 + 8 128 + 11 92 + 10 Dams 88 + 4 96 + 3 96 + 5 Male offspring 106 + 7 101 + 4 95 + 5 t Values are expressed as mean percent of corresponding controls ! SEMj N 12-18 per group. * Significantly different from control, p < 0.05 ....... 00 Table IX. 5-HT, 5-HIAA, and DA levels and TPH activities in cerebral cortex and hypothalamus of juvenile- and adult-treated males at 16 hand 3 wk post-dosing (10 mglkg AMPH, twice daily for two weeks).t 5-HT Cortex 16 h Juvenile males 92 + 4 Adult males 89 + 9 3 wk Juvenile males 98 + 5 Adult males 105 + 6 Hypothalamus 16 h Juvenile males 90 + 3 Adult males 79 ± 2 3 wk Juvenile males 98 + 1 Adult males 96 + 6 * 5-HIAA *- 75 + 3 81 + 5 * 114 + 4 115 + 11 81 + 3 '* 71 + 4 '* 98 + 2 104 + 4 DA 87 + 4 '* 99 + 9 100 ± 7 105 + 4 t Values are expressed as mean percent of corresponding controls ± Significantly different from control, p < 0.05 - Not detected tt Not determined TPH 98 + 4 74 + 12 97 + 3 112+9 tt tt tt tt SEMi N - 6 -8 per group ...... \0 Table X. 5-HT, DA, and metabolite levels and TPH and TH activities in neostriata of juvenile- and adult-treated males at 16 hand 3 wk post-dosing (10 mg/kg AMPH, twice daily for two weeks),t 5-IIT 5-HlAA DA DO PAC HVA TPH -T-H - 16 h .bvenile males 64 + 10 #I 68 + 8 * 84 + 3 * 88 + 7 841"5* 104 + 3 137 + 8 * Adult males 87 + 3 98 + 8 89 + 5 106 + 6 125 + 9 * 129 + 9 * 110 + 7 3 wk Juvenile males 94 + 2 96 + 4 84 + 3 * 90 + 3 85 :! 4 * 104 + 6 100 + 4 Adult males 128 + 25 95 + 16 102 + 8 123 :! 13 126 + 13 124 + 9 * 119 + 6 * -------- tV.. alues are expressed as mean percent of corresponding controls :!: SEM; N ... 6 - 8 per &roup Si~nificantly different from control. p < 0.05 N o DISCUSSION The control data document the developmental changes that occur in the dopaminergic and serotonergic systems in three discrete brain regions of the rat (Tables I, II, III, and IV). As both systems were monitored simultaneously in the same animals, several generalizations can be made. First, the neuronal systems studied here are not mature at birth in the rat, which is widely accepted. Secondly, different areas of the brain do not mature at the same rate. For example, hypothalamic DA was at adult levels by 3 weeks postnatally, whereas striatal DA reached matute levels only after 7 weeks. Finally, the different neurotransmitter systems do not mature at the same time; in the regions examined the dopaminergic system, generally, matured earlier than the serotonergic system. This has obvious significance for the development of any physiological functions, such as behavior, that depend upon the relative balance between such neurotransmitter systems for their expression. The most striking observation from the experimental data is tha t AMPH, in this do sing regimen, caused only minimal, rever si ble neurochemical changes. This is in sharp contrast to the severe, long-lasting depressions of both the serotonergic and dopaminergic systems that have been observed after high-dose subacute drug exposure1 "9 14 , 33,39 Short-term exposure to low doses of METH does not cause toxicity in these neurotransmitter systems 10 ' 14 ; a s~. ngle dose of up 22 to 1 mg/kg AMPH actually increases the synthesis of DA in the striatum19 ,2l. The low doses used in this study (0.5 or 5 mg/kg AMPH) may be below the threshold required to produce neuronal depletion. This is an important finding, clinically, as AMPS is prescribed within this approximate dose range for chronic use in humans4l • Administration of AMPH (5 mg/kg) to pregnant rats produced some intriguing resul ts (Tabl es V and VI). At 3 weeks of age, male offspring of these dams exhibited normal striatal monoamine levels while the corresponding synthetic enzymes were significantly de-pressed. Results from similar studies have been variable5 ,13,26,32, possibly due to wide discrepancies in doses and dosing regimens employed. In addition, many of the biochemical data reported to date for in- utero AMPH exposure have been data obtained from whole brain homogena tes5 '· 26 which could easily mask the localized neurochemical changes that are detected in the present study. Another important facet of these data is that they allow direct comparisons of effects on the offspring wi th effects on the treated dams. At a time that only enzymatic changes were present in the offspring (3 weeks), enzyme activities were normal in the dams but 5-HT, 5-HIAA, DA, DOPAC, and HVA levels were depressed. Since the data reflect only two time points after dosing, a complete explanation of the maternal-fetal interaction in response to chronic AMPS chal-lenge during gestation is not possible. Enzyme activities in the dams may have been decreased during dosing and/or shortly thereafter but recovered by 3 weeks after dosing. A lag period in restoration of neurotransmitter levels after TIl or TPH inhibition and recovery has 23 b een reporte d 1. n centra1 do pam.1 nerg1. c 2 and serotonerg1. c3 0 systems, respectively. This could explain the depression in monoamines and metabolites observed here at 3 weeks. The offspring were exposed to -AMPH in utero during the critical periods for development of the cerebral cortex (days 13-17 of gestation) and striatum (days 15-18 of gestat1. 0n )3 • Clearly, however, the neurochemical changes seen in these areas postnatally were minimal and temporary. The fetus may have a decreased sensitivity to this drug because of the immaturity of, or an increased plasticity in, the neuronal systems on which AMPH is known to act. Offspring and dams fully recovered from the drug insult, as all parameters were normal in both groups by 7 weeks post dosing. With the low doses of AMPH used in this chronic dosing regimen, the drug does not exhibit potent biochemical teratogenicity. A comparison of the juvenile- and adult-treated males in the present study (5 mg/kg) shows that the neurotoxic effects in the hypothalamus appeared to be dependent upon the age of the animal at the time of exposure. At 16 hours post dosing, DA, 5-HT, and 5-HlAA were signif icant ly depressed only in the adul ts. Preliminary data using 10 mg/kg per dose suggest that immature animals are not totally protected from the toxicity. Changes similar to those seen in the adults were produced in the juveniles at this higher dose. The depen-dence upon age for low-dose production of toxicity in the hypothalamus was not apparent in the other brain regions studied. Time- and dose-response studies are required to evaluate the importance of this finding. The present data underline, however, the importance of dose-response in the production of AMPH-induced neurotoxicity. Under the 24 conditions of this study, the severity of AMPH-induced toxicity did not increase dramatically in magnitude nor duration as a function of the age of the animal at the time of exposure. Sex-related differences in behavioral sensitivity to AMPH have been studied in detail. Female rats show greater increases in activi­ty levels and stereotypy than do males given equivalent doses4 ,35 • AMPH is metabolized more rapidly in males, but when the administered dose is adjusted to give equivalent concentrations of AMPH in the brains of both sexes, females still show the greater behavioral response4• The present study extends these findings to a biochemical level. The females in this study {Table V} were more sensitive to chronic AMPH exposure than either of the treated male groups {juvenile or adult}. The striatal depressions observed in the dams at 3 weeks post dosing (5 mg/kg) were, however, reversed by 7 weeks. Intra-regional differences in sensitivity to AMPH were appar­ent, the hypothalamus showing a relative resistance, as reported else­wherel ,33. One possible explanation for the small magnitude and short duration of neurochemical changes in this area involves the availability of hydroxylase cofactor. Levine et 81. 22 showed a strong correlation between total hydroxylase activity {m + TPH} and the concentration of the natural cofactor, tetrahydrobiopterin (BH4 ) , in ten discrete regions of the rat brain. Most of the areas studied, including the striatum and cerebral cortex, exhibit hydroxylase ac­tiv ity linearly proportional to the amount of cofactor present. In the hypothalamus, however, there is a great excess of pterin for the amount of hydroxylase activity present. Our laboratory is currently 25 investigating the possibility that the neurotoxicity of the amphet-amines involves depletion or inactiviation of this cofactor. If this theory is correct, areas such as the hypothalamus, containing a rela-tive abundance of BH4 would not be expected to show extensive depres­sions in hydroxylase activity (nor the corresponding depletions in neurotran8mitters) in response to amphetamine challenge. The two brain areas selected for their reported sensi tivi ty to AMPH, the cortex and striatum, also showed very few significant changes. The changes that did occur were minor and short-lived. This cannot be attributed solely' to a dose-response effect, as the highest dose used (10 mg/kg) has been shown to cause severe depletions when administered subacutely38. The present data support the idea that AMPH neurotoxicity can be greatly affected not only by dose level but also by duration of dosing. The importance of the relative contributions of dose and dura-tion of exposure on the neurotoxicity of amphetamines was illustrated in an early report by Koda and Gibb17 • At least 10 or 15 mg/kg per dose of METH was necessary to decrease striatal TH when the drug was given every 6 hours for 8 doses (sacrifice 6 hours after the last dose). A similar paradigm has been used by many researchers to induce severe neuroc h em.l ca 1 d ep 1 etl.o ns 1,10,14,33 • Ho wever, W.l t h contl.n ue d dosing of METH (every 6 hours for 66 hours), TH activity in the striatum returned to normal levels by 60 hours, and appeared augmented by 72 hours, after the first dose l7 • These results were confirmed and extended by Kogan et al. 18 Data were also obtained from the substan-tia nigra, a site of dopaminergic cell bodies which project axons to 26 the striatum. With continued dosing of METH, TH activity was depressed ear 1l· er, and recovere d f aster, l. n t he n·l gra t han ·ln t h e strl.a tum1 8 • Additional research is needed to elucidate the mechanism of dopaminergic system recovery with continued amphetamine exposure. This study lends preliminary support to the possibility that a similar recovery phenomenon occurs in the serotonergic system. Adult-treated males showed an apparent rebound increase in striatal TPH activity 3 weeks after the moderate dose (5 mg/kg, Table VI) and at 16 hours and 3 weeks after the highest dose (10 mg/kg, Table X). Some of the effects observed in this study could have been caused by drug-induced changes in receptor populations. Several dif-ferences in receptor-mediated events occur as functions of dose level and duration of dosing of AMPH. High-dose subacute exposure decreases t h e num b er 0 f d opam.l nerg.l c agon.l st and antagon.l st bI· nd l· ng sl. tes 36 and the density of 3(H)-DA uptake sites39 • Conversely, lower dose, chronic AMPH exposure increases striatal DA binding34 • Certainly, the neuronal system can respond differently at the molecular level to distinct types of drug challenge. Receptor changes must be included as offering plausible, if partial, explanations for the neurochemical effects reported here. A single dose of 9.2-36.8 mglkg AMPH administered with iprindole, which greatly slows elimination of the former drug, causes pers.l stent d ep 1et·lo ns l. n t h e strl. ata 1 d opam.lne.rglc system1 1,12,38 • This has been used as a model for the neurotoxic effects of continuous central AMPH exposure. Implantation of AMPH-releasing pellets for 7 days depletes striatal TH activity for as long as 110 days. 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Wagner, G.C., Ricaurte, G.A., Johnson, C.E., Schuster, C.R., and Seiden, L.S., Amphetamine induces depletion of dopamine and loss of dopamine uptake sites in caudate, Neurology, 30 (1980) 547-5'50. 40. Wagner, G.C., Schuster, C.R., and Seiden, L.S., Neurochemical consequences following administration of CNS stimulants to the neonatal rat, Pharm. Bioch. Beh., l! (1981) 117-119. 41. Weiner, N., Norepinephrine, epinephrine and the sympathomimetic amines. In A.G. Gilman, L.S. Goodman, and A. Gilman, (Eds), The Pharmacological Basis of Therapeutics, 6th edition, Macmillan, New York, 1980, pp. 138-175. EFFECTS OF CHRONIC AMPHETAMINE ON CENTRAL SEROTONERGIC AND DOPAMINERGIC SYSTEMS IN RATS OF VARIOUS AGES by Elaine Wyne Snowhill An abstract of a dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy James W. Gibb Chairman, Supervisory Committee Professor of Biochemical Pharmacology and Toxicology Department of Biochemical Pharmacology and Toxicology The University of Utah June 1983 © 1983 Elaine W. Snowhill All Rights Reserved