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Effects of prenatal methylazoxymethanol treatment on the development of central monoamine neurons
Developmental Brain Research, 2 (1982) 513-530
513
Elsevier/North-HollandBiomedicalPress
EFFECTS OF PRENATAL METHYLAZOXYMETHANOL TREATMENT ON THE DEVELOPMENT OF CENTRAL MONOAMINE NEURONS
GOSTA JONSSON and H/t~KANHALLMAN Department of Histology, Karolinska lnstitutet, S-10401 Stockholm (Sweden)
(Accepted May 1lth, 1981) Key words: noradrenaline-- dopamine-- serotonin-- development-- receptors
SUMMARY The effects of prenatal treatment with the antimitotic agent methylazoxymethanol (MAM; 25 mg/kg i.v., gestation day 15) on the development of monoamine neurons and their receptors have been investigated by neurochemical techniques. The MAM treatment led to a forebrain microencephaly with an approximately 50~ weight reduction of the cerebral cortex and hippocampus, 30 ~o of the striatum while the other CNS regions were reduced by about 5-15 ~. Endogenous noradrenaline (NA) dopamine (DA) and 5-hydroxytryptamine (5-HT) concentrations in the cerebral cortex and hippocampus were increased by about 100 ~o and to a similar extent for all amines, whereas the total amine content in each brain region analyzed was more or less unchanged after MAM treatment. The DA concentration in the striatum was increased by 40 ~o without any change in the total DA content. The subcellular distribution of NA and DA in the cerebral cortex and striatum was similar in MAMtreated and control rats. The effect of the NA denervation agent DSP4 was identical in MAM-treated and controls, showing very pronounced NA reductions in the cerebral cortex, hippocampus, cerebellum and the spinal cord, while the DA levels in various brain regions were not or only to a minor degree affected. Analysis of [3H]NA and [3H]5-HT uptake in slices from the cerebral cortex in vitro, demonstrated an approximate doubling of the uptake (calculated per weight) for both amines after MAM, while total uptake was not notably changed. MAM treatment also led to a significant reduction of the in vitro binding of various radioligands for monoamine (a, fl, 5-HT) receptors. The most pronounced reduction was observed for fl-receptors. Administration of a tyrosine-hydroxylase (a-methyl-p-tyrosine) or a tryptophan hydroxylase (a-propyldopacetamide) inhibitor led to marked NA/DA and 5-HT reductions after MAM in all regions analyzed, indicating that the monoamine neurons are active in synthesizing and releasing their neurotransmitter. The results suggest that 0165-3806/82/00004)000/$02.75 © Elsevier/North-HollandBiomedicalPress
514 MAM treatment leads to a monoamine hyperinnervation in the atrophic regions without markedly changing the total number of monoamine nerve terminals. All the monoamine nerves appear to develop independent of the formation of the postsynaptic receptors. The results imply that monoamine neurons in the CNS are strictly programmed to produce a certain quantity of nerve terminal arborizations in regions they innervate during the development relatively independent of the effector cells, pointing to a high degree of intrinsic growth regulation. INTRODUCTION Administration of pregnant rats with the antimitotic agent methylazoxymethanol (MAM) has been shown by several investigators to cause forebrain microencephaly in the offspringS,6,~0,29,32. MAM is a potent alkylating agent that kills dividing cells4,2z. This compound will therefore cause a marked and permanent atrophy in a CNS region where the cells undergo rapid proliferation at the time of administration. Injection of MAM at gestation day 15 will produce an atrophy mainly in the forebrain, in particular of the cerebral cortex, with preservation of the brain stem, where the neurons have an earlier birth date than those in the forebrain ~s,a0. It has recently been shown that prenatal MAM treatment leads to an elevated NA concentration and a hyperinnervation of noradrenaline (NA) nerve terminals in the atrophic neocortex810,zl, suggesting that the locus coeruleus NA neurons, which are formed on day 13 during gestation19, 23, are programmed to produce a certain quantity of nerve terminal arborizations during the development. The view that locus coeruleus NA neurons as well as central 5-hydroxytryptamine (5-HT) neurons possess a high degree of intrinsic growth regulation with the formation of a determined number of nerve terminals that is relatively independent of the regions they innervate have also emerged from histoand neurochemical studies on the effect of monoamine neurotoxins on the postnatal development of central monoamine neurons14,15,25,27. These studies thus demonstrated that the monoamine neurotoxins produced permanent denervations of distant N A and 5-HT nerve terminal projections and concomitantly a hyperinnervation in cell body near regions without any significant alteration of the total number of nerve terminals. It was therefore considered of interest to further study the relationship between the development of monoamine nerve terminals and their effector regions in microencephalic rat brain induced by MAM treatment in the foetal stage. A detailed regional analysis of the effects of prenatal MAM administration on the development of monoamine neurons and associated postsynaptic receptors using neurochemical techniques is reported here. Parts of the present results have been communicated in a preliminary form 11. MATERIALS AND METHODS Pregnant rats (Sprague-Dawley) were injected with MAM acetate (25 mg/kg i.v., Schwartz/Mann) under light ether anaesthesia on gestation day 15. MAM was diluted in 0.9 ~ NaCI and injected in a concentration of 10 mg/ml of the solvent.
515 Controls received an equal i.v. injection of the solvent alone. The litters were born on gestation day 22 and each litter was raised to contain not more than 8 pups which were separated from their mother at the age of about 3 weeks. The animals were housed in an air-conditioned room with controlled temperature and kept on a standardized light-dark schedule (14/10 h; light on 06.00 h and off 20.00 h). The animals were sacrificed by decapitation using a Harvard animal decapitator. The brains and spinal cords were rapidly dissected out and placed in cold saline for about 3-5 min. The regional dissection was made mainly as described by Jonsson and Sachs 15. Fresh tissue was taken for uptake studies and subcellular distribution studies whereas tissue taken for monoamine and receptor binding assays were frozen on dry-ice, packed in aluminium foil and stored at --80 °C pending neurochemical analyses. In some experiments the tissue samples taken for catecholamine assay were immediately placed in tubes containing standard amounts of 0.1 M perchloric acid and a-methyldopamine (a-methyl-DA; vide infra). The tubes were stored at --20 °C. Statistical evaluation of the results was performed using Student's t-test. Catecholamine assay The tissue pieces were homogenized by sonication in 310/~1 0.1 M perchloric acid containing 10--50 pmol a-methyl-DA (internal standard) using a Branson B30 sonifier. After a clean-up step of the extracts using an A12-Os-adsorption-desorption procedure, NA and dopamine (DA) were assayed by liquid chromatography with electrochemical detection (LCEC) according to Keller et al. 17. The values were expressed as ng/g wet weight of the tissue, based on measurements using a-methyl-DA as internal standard. 5-HT assay The frozen tissue pieces were homogenized by sonication in 5-10 vol. (w/v) of acidified n-butanol. After centrifugation, 80 #1 of the supernatant was added to 200 #1 heptane and 25/zl of 0.1 M perchloric acid in a tube and the mixture shaken for transfer of 5-HT to the aqueous phase. Aliquots (5-10/A) of this latter phase was taken for 5-HT assay using LCEC according to the procedure of Ponzio and Jonsson 26. The values were corrected for recovery, determined by adding authentic 5HT to homogenates, and expressed as ng/g wet weight of the tissue. [3H]monoamine uptake measurements in vitro Thin slices (thickness 250/~m) were prepared from fresh tissue (neocortex) using a Mcllwain tissue chopper. The slices were placed in beakers containing 5 ml Ringer bicarbonate buffer (pH 7.4) and preincubated for 5 min at +37 °C. Thereafter, 25 #1 [ZH]NA or [3H]5-HT was added to give a final concentration of 0.05/~M and the incubation continued for another 5 min. The incubation was terminated by placing the beakers in ice-water; the slices were rapidly washed in cold Krebs-Ringer buffer, blotted on filter paper, weighed and dissolved in vials by adding 0.5-1 ml tissue solubilizer (Protosol, NEN). After addition of scintillation fluorophor, radioactivity taken up in the tissue was determined by liquid scintillation spectrometry. The values
516 were corrected for 'extraneuronal uptake' by subtracting blank values, obtained by performing the incubations at + 4 °C or including 10/~M desipramine (for [3H]NA uptake) or 10 #M chlorimipramine (for [3H]5-HT uptake) in the incubation medium. Subcellular distribution studies Fresh tissue from the neocortex and striatum were homogenized (8-10 strokes) in 20 vol. (w/v) of cold 0.3 M sucrose using a glass homogenizer with a teflon pestle (clearance 0.05 mm) at a speed of about 1000 rpm. Four fractions were obtained by differential centrifugation; P1 ~ 1000 × g (20 min) pellet; P2 = 12,000 g (20 min pellet; Pz = 100,000 g (90 min) pellet and Sup. ~ high speed supernatant. The pellets and an aliquot of Sup. were taken for catecholamine assay as described above. In vitro receptor binding assays The procedure of Bylund and Snyder a was employed for fl-receptor binding studies using [3H]dihydroalprenolol ([aH]DHA) as radioligand. The homogenates were incubated in vitro in 1 nM [3H]DHA. The amount of non-specific binding was determined by including 1 #M (--)-alprenolol in the incubation mixture. Specific [aH]DHA binding was defined as the difference between total and non-specific binding; a-receptor assay was carried out according to U'Prichard et al. 31 by incubating brain homogenates in 0.2 nM [3H]WB4101. Non-specific binding was determined by including 100/~M 1-NA in the incubation mixture and specific 5-HTreceptor assay was performed by using [3H]5-HT or [3H]spiroperidol ([sH]Spi) as radioligands according to Bennett and Snyder 2 and Peroutka and Snyder 24. The homogenates were incubated in 6 nM [3H]5-HT or 0.3 nM [3H]Spi and non-specific binding determined by adding 1 #M unlabeled 5-HT or 100 #M 5-HT respectively. Drugs and substances Drugs and substances used were: MAM acetate (Schwarz/Mann); N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, (DSP4, obtained from Dr. R. Dahlbom, University of Uppsala, Uppsala); pargyline-HC1 (Sigma); [ZH]5-hydroxytryptamine creatinine sulphate (10 Ci/mmol, Radiochemical Center); [ZH]noradrenaline-HCl (12 Ci/mmol, Radiochemical Center); [3H]spiroperidol (79 Ci/mmol, NEN); [3H]dihydroalprenolol (45 Ci/mmol, NEN); [aH]WB-4101 (25 Ci/mmol, NEN); a-methyl-ptyrosine methylester (H44/68, AB H/issle); a-propyldopacetamide (H22/54, AB Hassle); desipramine-HC1 (Pertofran, Ciba-Geigy); chlorimipramine-HC! (Anafranil, Ciba-Geigy).
RESULTS The MAM-treated animals did not show any marked changes in gross behaviour. In agreement with Johnston and Coyles it was observed that the prenatal MAM treatment only marginally affected the body weight gain (Table I) with a reduction in body weight of 10-20~ in the adult stage. No sex differences were noticed. There were, however, very marked differences in the weight development of the various brain
517 TABLE I
Mean body weightof 6- and 9-week-oMrats treatedprenatally with MAM (25 mg/kg i.v.) (n = 8-10)
6 weeks old Male Female 9 weeks old Male Female
Control
MAM
(g)
(g)
% change
159 143
142 121
--11
297 220
251 172
--15 --22
--15
regions after MAM. While the weight of the whole CNS was reduced by about 25 ~o, the most pronounced weight reductions were found in the forebrain regions, cerebral cortex and hippocampus, being about half of the control weight (Table II). The olfactory bulb and striatum were reduced in weight by 20-30 ~o, while hypothalamus and mesencephalon were affected only to a minor degree (reduction by approximately 10~o) after MAM. Cerebellum, pons-medulla oblongata and the spinal cord were affected hardly at all. Regional effects o f M A M on endogenous N A and DA The p r e n a t a l M A M t r e a t m e n t resulted in m a r k e d regional alterations o f the e n d o g e n o u s N A a n d D A concentrations, f r o m p r o n o u n c e d increases to n o o r only m a r g i n a l effects (Table I I I a n d Fig. 1). I n regions with altered c a t e c h o l a m i n e concentrations, it was n o t e d t h a t the changes were quantitatively similar for N A a n d D A . The m o s t m a r k e d increases in N A a n d D A c o n c e n t r a t i o n s after M A M were f o u n d in those regions displaying the m o s t p r o n o u n c e d weight reductions, n a m e l y the cerebral cortex, h i p p o c a m p u s a n d the striatum. The N A a n d D A c o n c e n t r a t i o n s were TABLE I!
Wet weight (means) of various CNS regions of 9-week-oMrats treatedprenatally with MAM (25 mg/kg i.v.) (n=16-20) Region Olfactory bulb Cerebral cortex Hippocampus Striatum Hypothalamus Mesencephalon Pons-medulla oblongata Cerebellum Rest of brain Spinal cord Whole CNS
Control (mg)
M.,IM (me)
~ change
42 660 104 51 60 141 183 267 220 537
33 310 54 35 52 124 172 254 166 520
--21 --53 --48 --31 --13 --12 -- 6 -- 5 --24 -- 3
2265
1720
--24
TABLE III
Effects of prenatal M A M treatment on the regional NA and D,4 concentrations in the brain of rats raised to adulthood Pregnant rats were treated with MAM (25 mg/kg i.v.) on gestation day 15. The pups were raised to adulthood (9 weeks of age) when sacrificed and samples taken for catecholamine assay using LCEC. n -- 8-10. Each value (mean i S.E.M.) includes determinations of samples from an equal number of male and female.
Region Olfactory bulb Cerebral cortex Hippocampus Striatum Hypothalamus Mesencephalon Pons-medulla oblongata Cerebellum Spinal cord Rest of brain * 0.05
>
P
>
NA DA NA DA NA DA NA DA NA DA NA DA NA DA NA DA NA DA NA DA
Control (ng/g)
MAM
234 12.3 345 13.5 422 6.8 69 9716 2146 288 507 136 716 38 156 5.2 335 18.3 608 1517
273 17.1 713 25.0 701 13.4 132 13.840 2357 260 607 156 757 41 168 5.8 336 19.2 806 1711
± 27 ± 1.1 i 38 ~- 2.4 i 55 ± 1.3 ± lI ± 388 ± 107 ± 20 ± 14 i 6 i 30 ~ 1.2 ± 5.0 ± 0.2 ± 6 :i 0.6 ± 53 £ 74
% change ± 16 ~_ 2.5 ± 46*** ± 3.5* ± 48** ± 2.5" ± 14"* ~ 846** ± 187 i 15 ± 15"** ~ 8 ~ 18 ± 1.3 ± 8.2 ± 0.5 ~ 14 zb 1.0 ± 91 _+ 118
+17 +39 +107 +85 +66 ±97 +91 ÷42 -+ 10 ~ 11 ±20 ~ 15 ~6 +8 +8 F12 -4 5 ÷33 +13
0.01.
** 0.01 > P > 0.001. *** P < 0.001.
20O %
10¢
NA
iN OB
CI
Hipp
OConc. l~Jlotal
Str
HypQ. Mes.
P-re.
Cb.
S~c.
200"
%
1 OB
-
Cx,
Hipp.
Str.
H y p o . Mel.
P-re.
Cb.
Sp.c.
R
Fig. 1. Effects of prenatal MAM treatment (25 mg/kg i.v.) on the regional NA and DA levels in the brain of rats raised to adulthood (9 weeks of age). Each bar represents the mean 5z S.E.M. of 6-8 determinations, expressed either as a percentage of the concentration (cone., ng/g) or as a percentage of the total amount (Total, ng per region) of respective control value for each region. OB, olfactory bulb; Cx., cerebral cortex; Hipp., hippocampus; Str., striatum; Hypo., hypothalamus; Mes., mesencephalon; P-m., pons-medulla oblongata; Cb., cerebellum; sp.c., spinal cord; R, rest of brain.
519 increased by 4 0 - 1 0 0 ~ in these regions. Intermediate elevations were n o t e d in the olfactory b u l b a n d mesencephalon, whereas n o or m i n o r effects were seen in the h y p o t h a l a m u s , p o n s - m e d u l l a oblongata, cerebellum a n d the spinal cord. The latter regions also showed n o or m i n u t e changes in wet weight after M A M . However, i n d e p e n d e n t o f the degree of elevation of N A a n d D A concentration, n o significant difference between M A M - t r e a t e d a n d control was f o u n d when calculating the values as total a m o u n t of N A or D A per b r a i n region (Fig. 1). N o sex differences of the effects of M A M o n the development of e n d o g e n o u s catecholamines were observed. The alteration in cortical N A a n d D A concentrations described above for adult rats were n o t e d already one week (8 days) after birth, whereas only m i n o r changes were seen in other regions (Table IV). Similar results were f o u n d in 30-day-old rats.
Effects o f D S P 4 on endogenous catecholamines in M A M - t r e a t e d and controls rats The effect of the N A n e u r o t o x i n DSP4 o n the regional N A a n d D A levels in the TABLE IV
Effects of prenatal M A M treatment on the regional NA and DA levels in the CNS of 8- and 30-day-old rats (n = 4-6) MAM treatment and NA/DA analyses as in Table III. Changes expressed either as per cent of NA or DA concentration (Conc.) or as per cent of the total amount (Total) of respective control value for each region.
Region
Control
MAM (ng/g)
8 days old: Cerebral cortex Mesencephalon Pons-medulla oblongata Cerebellum Spinal cord 30 days old: Cerebral cortex Striatum Mesencephalon Cerebellum Spinal cord * 0 . 0 5 > P > 0.01. ** 0.01 > P > 0.001. *** P < 0.001.
% changes Conc. Total
NA DA NA DA
93 + 12 4.2 4- 0.2 173 4- 9 113 4- 34
197 4- 6*** 8.4 4- 1.2" 209 4- 10" 114 4- 34
+112 +100 +21 --
+31 +27 +17 --
NA DA NA DA NA DA
222 4- 4 16 4- 1.5 93 4- 2 0.7 4- 0.2 204 4- 11 8.7 ± 0.7
283 4- 14" 16 4- 2.0 110 + 9 0.8 4- 0.1 253 4- 14' 9.8 4- 0.8
+27 -+18 +14 +24 +13
+27 -+19 +15 +21 +10
NA DA NA DA NA DA NA DA NA DA
148 4- 4 12 4- 1.0 73 4- 12 5881 4- 377 477 4- 29 316 4- 4 105 4- 3 2 4- 0.5 347 4- 36 23 4- 3
328 4- 18"** 28 4- 6* 114 4- 9* 8120 4- 142"* 559 4- 19" 354 4- 26 119 4- 6 2.5 4- 0.5 350 4- 41 24 4- 2
+121 +133 +56 +38 +17 +12 +13 +25 +1 +4
+8 +14 --6 --17 --7 -~21 --13 --16 --13
520 100 %
N_A. [ ] NaCI*DSP4 [ ] MAM+DSP4
80
I
60" 4020" O"
Cx.
H~.
Str.
Hypo.
IVies
P-m
Cb
Sp.¢
R
120-
I
100%
_L
i~-
-
8060" ?
40 20
0
OB
Cx
Hi~
Str
Hypo.
Mes
P-m
Cb
SOc.
Fig. 2. Effect of DSP4 (50 mg/kg i.p., 3 days) on the endogenous NA and DA concentrations (ng/g) in various regions of the brain from adult rats (9 weeks of age) treated with MAM or NaCI prenatally (25 mg/kg i.v., on gestation day 15). Each bar represents the mean ! S.E.M. of 3-4 determinations, expressed as a percentage of respective control values for each group and region. CNS of MAM-treated and control rats was investigated. It was found that the relative (comparison between DSP4-treated and respective group controls) N A reductions induced by DSP4 (50 mg/kg i.p., 3 days) were generally very similar in MAM-treated and control rats (Fig. 2), although the absolute N A levels were higher in regions from MAM-treated rats which after M A M alone displayed marked increases in the NA concentration, e.g. the cerebral cortex and the hippocampus. No or minor effects of DSP4 were noted on the DA levels both in MAM-treated and control rats.
Effects of prenatal M A M treatment on the subeellular distribution of NA and DA in the cerebral cortex and the striatum The relative distribution of N A and D A in both regions analyzed was very similar for MAM-treated and controls (Fig. 3) indicating that the amines are stored in identical structures in the two groups of animals. Most of the N A (about 50 ~ ) of the cerebral cortex was recovered in the Pz-fraction (containing synaptosomes), while most of the D A (40-50 ~ ) originating both from the striatum and the cerebral cortex were found in the high speed supernatant. Relatively less D A from these regions were found in the P3 fraction compared to cortical NA. The D A distribution in tissue from the neocortex was on the whole more similar to that of the striatum, indicating that the main part of the D A present in the cerebral cortex is present in D A nerve terminals.
521
%
[] ~1
[] ~M
L 5!
;!i
i ili
o
NA
DA
DA
NA
DA
c'x.
DA
DA
NA
st~
Cx.
i
DA
c;
St~
~.
Fig. 3. Subcellular distribution of endogenous N A and D A in cerebral cortex (Cx.) and striatum (Str.) of M A M and saline (NaCl)-treated rats. M A M (25 mg/kg i.v.) was administered on gestation day 15 and the rats were sacrificed at the age of 3 weeks. The tissue was fractionated by differential centrifugation to obtain a P1 (1000 g pellet), P2 (12,000 g pellet), P8 (100,000 g pellet) and Sup. (highspeed supernatant). Each bar represents the mean 4- S.E.M. of 3 determinations, and the values expressed as a percentage of the total amount of N A or D A recovered from each tissue sample analyzed.
Regional effects of tyrosine hydroxylase inhibition (Till) on the NA and DA reduction in brain of MAM-treated and control rats The effects of administration of the tyrosine hydroxylase inhibitor H44/68 (amethyl-p-tyrosine methylester) on the catecholamine content of various brain regions in MAM-treated and control rats was investigated (Figs. 4 and 5). From the cerebral 8
Front.
Cing=
Occ
Hip_p~
xlO2 NA~ ng~.
2
0. 10
C 'MAM
C
MAM
DA
C
MAM
C
MAM
e
M~"~t
[ ] NaCI [ ] H44/68
0 J
C
' MAM
C
' MAM
C
MAM
Fig. 4. Effects of H44/68 (250 mg/kg i.p., 2 h) on the N A and D A concentrations in frontal (Front), cingulate (Cing.), occipital (Occ.) cortex and hippocampus (Hipp.) of MAM-treated and control (C) rats. M A M (25 mg/kg i.v.) was administered on gestation day 15 and the rats sacrificed at the age of 6 weeks. Each bar represents the mean 4- S.E.M. of 6 determinations, expressed as ng/g wet weight of the tissue. Each group of animals consisted of 3 males and 3 females. The figures above the hatched bars denote the relative N A or D A values after H44/68 as a percentage of respective control (NaCI) value (open bars).
522
st,i..,%~
Ports-reed
Cefebefl
Sp cord
NA
[ ] NaCI
,1022 ng/g
~ 3 H44/68 92 2
./
~4 C
~103
ng/g
MAM
:)A
C ' MAM
C
MAM
C
C
MAM
' MAM
5~
,10 -~ J
10
,10 ~
,1012
4t
I
] ,t
,,
3~ -+-
tlt
t C
MAM
C
MAM
C ' MAM
' MAM
C
MAM
MAM
Fig. 5. Effects of H44/68 on the NA and DA concentrations in striatum, hypothalamus (Hypo.) mesencephalon (Mes.), pons-medulla, cerebellum and spinal cord of MAM-treated and control (C) rats. Experimental conditions as in Fig. 4. Each bar represents the mean :k S.E.M. of 6 determinations, expressed as ng/g wet weight of the tissue. The figures above the hatched bars denote the relative NA or DA values after H44/68 as a percentage of respective control (NaCI) value (open bars). cortex 3 regions were sampled: frontal, cingulate and occipital. The M A M treatment led to an increase in N A concentration in all these regions to about the same absolute level, although the highest relative increase was seen in the occipital cortex ( + 2 8 0 ~ ) , and the lowest in the cingulate cortex ( + 6 0 ~ ; see Fig. 4). All regions showed an approximately 3-fold increase in D A concentration. Calculating these values as total amount per region it was again found that this value was approximately similar for MAM-treated and controls for all regions. The relative H44/68-induced reduction of N A was less pronounced in all cortical regions and hippocampus after M A M treatment compared to controls, indicating a reduced N A 'turn-over' in each N A nerve terminal after M A M . This effect was significant in the frontal (P < 0.01) and cingulate (P < 0.05) cortex, while only tendencies were noted in the occipital cortex and hippocampus (P < 0.1). The absolute amounts of N A that disappeared after H44/68 was clearly greater after M A M in all regions, indicating that the absolute amounts of N A 'turned over' in these regions is increased after M A M . The relative reduction in D A concentration caused by H44/68 was similar in all cortical regions and hippocampus for MAM-treated and controls except in the occipital cortex (Fig. 4), although as in the case of N A the absolute reduction of D A were greater, suggesting that these regions are exposed to a larger amount o f D A in absolute terms. In most of the other CNS regions analyzed, no clear-cut differences in relative H44/68-induced reductions of both N A and D A between M A M and control rats were observed, except in the mesencephalon and pons-medulla for N A and pons-medulla
523 TABLE V
Effects of prenatal M A M treatment on the regional 5-HT concentrations in the brain of rats raised to adulthood Pregnant rats were treated with MAM (25 mg/kg i.v.) on gestation day 15. The pups were raised to adulthood (9 weeks of age), sacrificed and samples were taken for 5-HT assay using LCEC. n = 8-10. Each value includes determinations of samples from an equal number of male and female rats.
Region
Control
Olfactory bulb Cerebral cortex Hippocampus Striatum Hypothalamus Mesencephalon Pons-medulla oblongata Cerebellum Spinal cord Rest of brain
284 260 337 180 680 772 608 48 305 472
MAM (ng/g)
-4- 21 ± 15 -4- 35 -4- 33 dz 52 ± 29 -4- 35 ± 9 ± 18 ± 27
444 533 690 345 860 872 744 61 326 504
% change
~: 54* ± 46*** ± 50*** ± 36** -4- 48* ± 56 -4- 44* 4- 8 ± 36 ± 48
+56 +128 +105 +92 +27 +13 +22 +27 +7 +7
* 0.05 > P > 0.01. ** 0.01 > P > 0.001. *** P < 0.001. an d spinal c o r d f o r D A (Fig. 5). T h e results indicated an increased N A t u r n - o v e r in m e s e n c e p h a l o n , b o t h in relative a n d absolute terms, while the reverse seem to be the case f o r pons-medulla.
Regional effects o f M A M treatment on endogenous 5 - H T Th e p r e n a t a l M A M t r e a t m e n t caused very m a r k e d regional alterations o f the 5H T c o n c e n t r a t i o n , similar to that seen for e n d o g e n o u s N A and D A (Table V an d Fig. 6). Th e m o s t p r o n o u n c e d increases with an a p p r o x i m a t e d o u b l i n g o f the 5 - H T -HT.
..~
[ ] Conc. ~ Total
20O
%
I •
OB
Cx,
1
Hipp.
Str.
v
A.
r.,i
Hypo. Mes. P-m.
Cb.
SpC,
R
Fig. 6. Effects of prenatal MAM treatment (25 mg/kg i.v.) on the regional 5-HT levels in the brain of rat raised to adulthood (9 weeks of age). Each bar represents the mean -4- S.E.M. of 6-8 determinations, expresssed either as a percentage of the concentration (Conc., ng/g) or as a percentage of the total amount (Total; ng per region) of respective control value for each region, OB, olfactory bulb; Cx., cerebral cortex; Hipp., hippocampus; str., striatum; Hypo., hypothalamus; Mes., mesencephalon; P-m., ports-medulla oblongata; Cb., cerebellum; Sp.C., spinal cord; R, rest of brain.
524 concentration was found in the cerebral cortex, hippocampus and striatum, while moderate increases (20-50 ~) were observed in the olfactory bulb, hypothalamus and pons-medulla. Calculating these data as total amount per region, it was found that the 5-HT content in MAM and control rats did not differ significantly for any region. Similar results were noticed when analyzing the rats 8 days and 30 days after birth (Table VI). In one set of experiments the effect of the tryptophan hydroxylase inhibitor H22/54 (a-propyldopacetamide) on the regional 5-HT levels in brain from MAMtreated and control rats was investigated (Fig. 7). It was observed that the 5-HT concentration was markedly increased in all cortical regions analyzed and as in the case of NA, the highest relative 5-HT increase was noted in the occipital cortex and the lowest in the cingulate cortex. Administration of H22/54 led to marked reductions of 5-HT both in MAM-treated and control rats in all regions analyzed. There did not appear to be any consistent alterations of the relative H22/54 reduction when comparing the various regions of MAM-treated and control rats. It is clear, however, that the absolute reduction of 5-HT after H22/54 is increased in most atrophic regions of MAM-treated rats compared to control, indicating an increase in 5-HT turn-over in absolute terms.
Effects of prenatal M A M treatment on Jail]amine uptake in the cerebral cortex Analysis of the in vitro uptake of [aH]NA or [SH]5-HT in slices from the ceiebral cortex of MAM-treated and control rats showed that there was an about doubling of the uptake of both [SH]amines when calculating the uptake per weight unit (Fig. 8). However, when expressing the uptake as total uptake, there were no differences between MAM-treated and controls for both [aH]NA and [aH]5-HT.
Effects of prenatal M A M treatment on the in vitro binding of various receptor ligands to homogenates from the cerebral cortex The effect of prenatal MAM treatment on the specific in vitro binding of various labeled receptor ligands, (a, fl, and 5-HT receptors) to homogenates from the cerebral cortex was investigated (Fig. 9). It was found that the only ligand that showed a significantly reduced binding per weight unit of the tissue was the fl-adrenoceptorligand, [SH]DHA. Expressing the results as total binding, all of them showed a significantly reduced binding, which was most pronounced for [SH]DHA. In displacement experiments it was found that the IC50 values for various receptor compounds (concentration needed to inhibit specific [SH]ligand binding by 50 ~) was similar for homogenates from MAM-treated and control rats for all radioligands. This indicates that the affinity of the various receptors towards the receptor-ligand was not markedly changed after MAM. The changes in binding are therefore most likely mainly related to an alteration in the number of receptor sites.
525 TABLE VI
Effects of prenatal M . 4 M treatment on the regional 5-HT levels in the C N S of 8- and 30-day-old rats (n = 4-6) M A M treatment and 5-HT analysis as in Table V. Changes expressed either as per cent of 5-HT concentration (Conc.) or as per cent of the total amount (Total) of respective control value for each region.
Region
Control
8 days old: Cerebral cortex Mesencephalon Pons-medulla oblongata Spinal cord Rest of brain 30 days old: Cerebral cortex Striatum Pons-medulla oblongata Spinal cord Rest of brain
MAM (he~g)
% changes Conc. Total
109 394 199 326 69
4± ± ± ±
2.4 60 15 23 4.6
176 381 164 314 82
± ± ± ± ±
8.8** 20 7.2 17 5.0
+62 --2 --18 ---4 + 19
+6 --2 --5 +4 + 19
184 250 560 369 286
± ± ± ± ±
27 18 41 19 14
340 419 452 302 288
± ± ± ± ±
24* 29* 39 23 15
±85 +68 --19 --18 +1
--11 +26 --11 --9 ÷5
* 0.05 > P > 0.01. > P > 0.001.
** 0.01
14. t~ c>
OB.
Front.
Cing~
Pariet.
Occ.
Hip_~
12.
0 C 16-
MAM
Striatum
C
MAM
HylJO~
C
MAM
I~
C
NAM
Ports,reed.
C
MAM
Cerebell.
C
MAM
Sp.cord
i ~ i ~r~~'I'-~H~22/ NaCI ~54
12 ¸
6~
75
C
MAM
C
MAM
C
MAM
C
MAM
C
MAM
C
MAM
Fig. 7. Effects of H22/54 (500 mg/kg i.p. 2 h) on the 5-HT concentrations in various brain regions of MAM-treated and control (C) rats. M A M (25 mg/kg i.v.) was given on gestation day 15 and the rats were sacrificed at the age of 5 weeks. Abbreviations for the various CNS regions as in Figs. 4 and 5. Each bar represents the mean 4- S.E.M. of 3-4 determinations. The figures above the hatched bars denote the relative 5-HT value after H22/54 as a percentage of respective control (NaCI) value (open bars).
526 300
3H-NA
3H.5-HT
[]
20C
NaCI
%
w
T
w
T
Fig. 8. Effect of prenatal M A M treatment on the in vitro uptake of [SH]NA (0.05/~M, 5 min) or [sI-I]5H T (0.05/zM, 5 rain) in slices from the cerebral cortex. M A M treatment as in Fig. 4. Each bar represents the mean ± S.E.M. of 4-6 determinations, expressed as a percentage of the neuronal Jail]amine uptake per mg of tissue (W) or total uptake (T) in control.
-.L F'-] NaCI
_L.
5
F~MAM
_L_, r-,
!2 1 oJ
3H-OHA P,
3H~WB-4101 (z
3H-5HT
4-
~
3H-SPI
5~HT
3.d.
J
O-
3H-DHA fl
,,,
3H-WB-4101 o
3H-5HT
3H-SPI 5-HT
Fig. 9. Effects of prenatal M A M treatment on the specific binding of [SH]DHA, [sH]WB-4101, [SH]5H T or [sH]Spi to homogenates from the cerebral cortex. Each bar represents the mean J: S.E.M. of 4-6 determinations, and the values expressed as pmol of [sI-I]ligand bound/g of tissue (upper part) and total amount bound to the tissue (lower part). ** 0.01 > P > 0.001 ; *** P < 0.001.
527 DISCUSSION In agreement with previous studies it was found that prenatal administration of the antimitotic agent MAM led above all to a weight reduction of the forebrain regions, the cerebral cortex and the hippocampus s,2a. Marked weight reductions were also noted in the olfactory bulb and the striatum. The most pronounced changes in the concentration of endogenous monoamines were found in the atrophic regions. The concentration changes of neurotransmitters were closely associated with the weight reductions, so when calculating the results as monoamine content per region it was found that there were no significant differences between MAM-treated and controls for any monoamine. These results indicate that the monoamine nerve terminal fields develop relatively independent of the region they innervate and that in spite of a marked reduction of the size of the region, the nerve terminal net forms a normal extent of arborizations. This will then lead to marked hyperinnervations in the regions where the most pronounced weight reductions are at hand. This interpretation is strongly supported by the uptake experiments showing an increase in [3H]amine uptake (measure of the axonal membrane area and thus an index of the number of nerve terminals) per weight unit in the cerebral cortex quantitatively similar to the increase in endogenous NA and 5-HT concentration, while total uptake was not notably affected. The present results are in agreement with previous reports on the effect of MAM on monoamine neuronsT-1O,lS,21. It can be argued that the observed increase in DA concentration in the cerebral cortex is not associated with DA nerves, but rather due to an increase in NA nerves and hence an increase in content of DA precursor. The results from the experiments with DSP4, a selective NA denervating agent 12,13, do not support this view. It was thus observed that the reduction of DA after DSP4 was similar and only marginal (about 30 ~) in cerebral cortex of both MAM-treated and control rats. It should be noted that the DSP4 treatment led to an approximately 90 ~ NA reduction in the cerebral cortex of both groups. These data thus indicate that the quantitative relation between NA and DA nerve terminals is unaltered after MAM treatment and hence also DA nerve terminals display a hyperinnervation pattern in all regions where they are present and where MAM treatment leads to an atrophy. Based on the results from the DSP4 experiments it could be calculated that about 10 ~ of the endogenous DA found in the cerebral cortex is associated with NA nerve terminals, serving'as"precursor in NA biosynthesis. The subcellular distribution results are also in agreement with the view that both NA and DA nerves form a denser nerve net and hyperinnervation in the cerebral cortex after MAM. It was thus found that the DA distribution was more similar to that of DA in the striatum (containing predominantly DA nerves) than the subcellular distribution of NA in the cerebral cortex for both MAM-treated and control rats. From a developmental viewpoint it appears as if the hyperinnervation pattern is present very early in ontogeny, since the altered monoamine concentrations observed in atrophic cerebral cortex in the adult stage were seen already one week after birth, which is in agreement with data recently reported by Johnston and Coyle9.
528 Concerning the analysis of various monoamine receptors in the cerebral cortex after MAM treatment, it was found that the density of receptors was approximately the same for 5-HT and a-receptors after MAM, while the fl-receptors were the only type with markedly reduced density. However, calculated on a total basis all receptors analyzed were markedly reduced after MAM. These results thus suggest that the cerebral cortex is devoid of a significant number of effector cells after prenatal MAM treatment and that the monoamine neurons develop independent of the postsynaptic receptors (cf. ref. 16). The studies of the effect of tyrosine or tryptophan hydroxylase inhibition on the endogenous monoamines showed that inhibition of these enzymes caused a significant reduction in the catecholamines and 5-HT respectively in MAM-treated rats and in many regions to the same extent as in controls. This indicates that the monoamine neurons, also in the atrophic regions, are functionally active in synthesizing and releasing their neurotransmitter by nerve impulses (see And6n et al.1). Although the present results do not allow any precise analysis as to any quantitative differences between release and turn-over of monoamine neurotransmitters after MAM, there appears to be a consistent pattern with respect to NA in the cerebral cortex. It was thus observed that the relative NA reductions induced by H44/68 were less pronounced in all the cortical regions and also in the hippocampus compared to controls. This could indicate that the amount of NA released per nerve terminal is reduced in these hyperinnervated regions. The reason for this could be that the inhibitory presynaptic a-receptor-mediated control of NA release is more pronounced in these hyperinnervated regions. Consistent with this view it was observed that the absolute amounts of NA turned-over in these regions was increased, possibly providing a situation of increased concentrations of NA in the extracellular space acting on the presynaptic a-receptors. Concerning the DA and 5-HT systems, no such pattern could be observed, possibly indicating that presynaptic DA and 5-HT receptors are not as sensitive as presynaptic a-receptors in such an autoregulation. However, in view of the rather extensive changes occurring in several neurotransmitter systems after prenatal MAM treatmentT,9,1°, it is clear that much more work is needed to be able to adequately interpret alterations in transmitter release and turn-over in atrophic regions induced by MAM. In conclusion, the present study has shown that an atrophic development of certain forebrain regions induced by prenatal MAM treatment leads to a hyperinnervation of monoamine nerve terminals which normally innervate these structures. This hyperinnervation is closely related to the atrophy with formation of a normal network of nerves from a quantitative viewpoint. These results strongly suggest that the monoamine neurons are strictly programmed to produce a certain quantity of nerve terminal arborizations in regions they innervate during the development, which to a large extent appears to be independent of the effector cells and their postsynaptic receptors. The monoamine neurons thus seem to possess a remarkably high degree of intrinsic growth regulation.
529 ACKNOWLEDGEMENTS
The present study has been supported by grants from the Swedish MRC (04X2295), Expressen's Prenatalforskningsn~imnd, Karolinska Institutet, M. Bergvall's and Jeansson's Foundations. The skillful technical assistance of E. Lindqvist, B. Drevinger and B. K/iller is gratefully acknowledged. The authors wish to thank B. Frideen for secretarial help. REFERENCES 1 And6n, N.-E., Corrodi, H. and Fuxe, K., Turnover studies using synthesis inhibition. In G. Hooper (Ed.), Metabolism of Amines in the Brain, MacMillan, London, 1969, pp. 38-47. 2 Bennett, J. P. and Snyder, S. H., Serotonin and lysergic acid diethylamide binding in rat brain membranes: relationship to postsynaptic serotonin receptors, Molec. PharmacoL, 12 (1976) 373-389. 3 Bylund, D. B. and Snyder, S. H., Beta adrenergic receptor binding in membrane preparations from mammalian brain. Molec. Pharmacol., 12 (1976) 568-580. 4 Druckrey, H. and Lange, A., Carcinogenicity of azoxymethane dependent on age in BD rats, Fed. Proc., 31 (1972) 1482-1485. 5 Fischer, M. H., Welker, C. and Waisman, H. A., Generalized growth retardation in rats induced by prenatal exposure to methylazoxymethanol acetate, Teratology, 5 (1972) 223-232. 6 Haddad, R. K., Rabe, A., Laquer, G. L., Spatz, M. and Valsamis, M. P., Intellectual deficit associated with transplacentally induced microcephaly in the rat, Science, 163 (1969) 88-90. 7 Johnston, M. V., Carman, A. B. and Coyle, J. T., Effects of fetal treatment with methylazoxymethanol acetate at various gestational dates on the neurochemistry of the adult neocortex of the rat, J. Neurochem., 36 (1981) 124-128. 8 Johnston, M. V. and Coyle, J. T., Histological and neurochemical effects of fetal treatment with methylazoxymethanol on rat neocortex in adulthood, Brain Res., 170 (1979) 135-155. 9 Johnston, M. V. and Coyle, J. T., Ontogeny of neurochemical markers for noradrenergic, GABAergic and cholinergic neurons in neocortex lesioned with methylazoxymethanol acetate, J. Neurochem., 34 (1980) 1429-1441. 10 Johnston, M. V., Grzanna, R. and Coyle, J. T., Methylazoxymethanol treatment of fetal rats results in abnormally dense noradrenergic innervation of neocortex, Science, 203 (1979) 369-371. 11 Jonsson, G. and Hallman, H., Effect of prenatal methylazoxymethanol (MAM) treatment on the development of central monoamine neurons, Neurosci. Lett., Suppl. 5 (1980) $305. 12 Jonsson, G., Hallman, H., Ponzio, F. and Ross, S., DSP4-(N-2-chloroethyl-N-ethyl-2-bromobenzylamine) - - a useful denervation tool for central and peripheral noradrenaline neurons, Europ. J. PharmacoL, 72 (1981) 173-188. 13 Jonsson, G., Pycock, Ch., Fuxe, K. and Sachs, Ch., Changes in the development of central noradrenaline neurons following neonatal administration of 6-hydroxydopamine, J. Neurochem., 22 (1974) 621-626., 14 Jonsson, G., Ponzio, F. and Ross, S., A new neurotoxic compound as denervation tool for noradrenaline neurons. Abstract (2810). In 7th lntern. Congress PharmacoL, Paris, 1978. 15 Jonsson, G. and Sachs, Ch., Regional changes in aH-noradrenaline uptake, catecholamines and catecholamine synthetic and catabolic enzymes in rat brain following neonatal 6-hydroxydopamine treatment, Med. BioL, 54 (1976) 286-297. 16 Jonsson, G., Wiesel, F.-A. and Hallman, H., Developmental plasticity of central noradrenaline neurons after neonatal damage - - changes in transmitter functions, J. Neurobiol., 10 (1979) 337-353. 17 Keller, R., Oke, A., Mefford, I. and Adams, R. N., Liquid chromatographic analysis of catecholamines - - routine assay for regional brain mapping, Life Sci., 19 (1976) 995-1004. 18 Lai, H., Quock, R. M., Makous, W., Hovita, A. and Jen, L. S., Methylazoxymethanol acetate: effect of postnatal injection on brain amines and behaviour, PharmacoL Biochem. Behav., 8 (1978) 251-257. 19 Lauder, J. M. and Bloom, F. E., Ontogeny of monoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat. I. Cell differentiation, J. comp. NeuroL, 155 (1979) 469-481.
530 20 Matsumoto, H., Spatz, M. and Laquer, G. L., Quantitative changes with age in the DNA content of methylazoxymethanol-induced microencephalic rat brain, J. Neurochem., 19 (1972) 297-306. 21 Matsutani, T., Nagayoshi, M. and Tsukada, T., Elevated monoamine levels in the cerebral hemispheres of microencephalic rats treated prenatally with methylazoxymethanol or cytosine arabinoside, J. Neurochem., 34 (1980) 950-956. 22 Nagata, Y. and Matsumoto, H., Studies on methylazoxymethanol; methylation of nucleic acids in fetal rat brain, Proc. Soc. exp. Biol. (N. Y.), 132 (1971) 383-385. 23 Olson, L. and Seiger, A, Early prenatal ontogeny of central monoamine neurons in the rat: fluorescence histochemical observations, Z. Anat. Entwickl.-Gesch., 137 (1972) 301-316. 24 Peroutka, S. J. and Snyder, S. H., Multiple serotonin receptors" differential binding of aH-5hydroxytryptamine, aH-lysergic acid diethylamide and aH-spiroperidol, Molec. Pharmacol., 16 (1979) 687-699. 25 Ponzio, F. and Jonsson, G., Effects of neonatal 5,7-dihydroxytryptamine treatment on the development of serotonin neurons and their transmitter metabolism, Develop. Neurosci., 1 (1978) 80-89. 26 Ponzio, F. and Jonsson, G., A rapid and simple method for the determination of picogram levels of serotonin in brain tissue using liquid chromatography with electrochemical detection, J. Neurochem., 32 (1979) 129-132. 27 Sachs, Ch. and Jonsson, G., 5,7-dihydroxytryptamine induced changes in the postnatal development of central 5-hydroxytryptamine neurons, Med. BioL, 53 (1975) 156-164. 28 Schultz, B., Nowak, B. and Mauser, W., Cyclic times of the neural epithelial cells of various types of neurons in the rat. An autoradiographic study, J. comp. Neurol., 158 (1974) 207-218. 29 Spatz, M. and Laquer, G. L., Transplacental chemical induction of microencephaly in two strains of rats, Proc. Soc. exp. Biol. ( N. Y.), 129 (1968) 705-710. 30 Taber, E., Histogenesis of brain stem neurons studied autoradiographically with aH-thymidine in the mouse, Anat. Rec., 145 (1963) 291. 31 U'Prichard, D. C., Greenberg, D. A. and Snyder, S. H., Binding characteristics of a radiolabelled agonist and antagonist at central nervous system alpha noradrenergic receptors, Molec Pharmacol., 13 (1977) 454-473. 32 Welker, C., Fischer, M. H. and Waisman, H. A., Postnatal development of the brain in micro encephalic rats, Anat. Rec., 169 (1971) 452.
513
Elsevier/North-HollandBiomedicalPress
EFFECTS OF PRENATAL METHYLAZOXYMETHANOL TREATMENT ON THE DEVELOPMENT OF CENTRAL MONOAMINE NEURONS
GOSTA JONSSON and H/t~KANHALLMAN Department of Histology, Karolinska lnstitutet, S-10401 Stockholm (Sweden)
(Accepted May 1lth, 1981) Key words: noradrenaline-- dopamine-- serotonin-- development-- receptors
SUMMARY The effects of prenatal treatment with the antimitotic agent methylazoxymethanol (MAM; 25 mg/kg i.v., gestation day 15) on the development of monoamine neurons and their receptors have been investigated by neurochemical techniques. The MAM treatment led to a forebrain microencephaly with an approximately 50~ weight reduction of the cerebral cortex and hippocampus, 30 ~o of the striatum while the other CNS regions were reduced by about 5-15 ~. Endogenous noradrenaline (NA) dopamine (DA) and 5-hydroxytryptamine (5-HT) concentrations in the cerebral cortex and hippocampus were increased by about 100 ~o and to a similar extent for all amines, whereas the total amine content in each brain region analyzed was more or less unchanged after MAM treatment. The DA concentration in the striatum was increased by 40 ~o without any change in the total DA content. The subcellular distribution of NA and DA in the cerebral cortex and striatum was similar in MAMtreated and control rats. The effect of the NA denervation agent DSP4 was identical in MAM-treated and controls, showing very pronounced NA reductions in the cerebral cortex, hippocampus, cerebellum and the spinal cord, while the DA levels in various brain regions were not or only to a minor degree affected. Analysis of [3H]NA and [3H]5-HT uptake in slices from the cerebral cortex in vitro, demonstrated an approximate doubling of the uptake (calculated per weight) for both amines after MAM, while total uptake was not notably changed. MAM treatment also led to a significant reduction of the in vitro binding of various radioligands for monoamine (a, fl, 5-HT) receptors. The most pronounced reduction was observed for fl-receptors. Administration of a tyrosine-hydroxylase (a-methyl-p-tyrosine) or a tryptophan hydroxylase (a-propyldopacetamide) inhibitor led to marked NA/DA and 5-HT reductions after MAM in all regions analyzed, indicating that the monoamine neurons are active in synthesizing and releasing their neurotransmitter. The results suggest that 0165-3806/82/00004)000/$02.75 © Elsevier/North-HollandBiomedicalPress
514 MAM treatment leads to a monoamine hyperinnervation in the atrophic regions without markedly changing the total number of monoamine nerve terminals. All the monoamine nerves appear to develop independent of the formation of the postsynaptic receptors. The results imply that monoamine neurons in the CNS are strictly programmed to produce a certain quantity of nerve terminal arborizations in regions they innervate during the development relatively independent of the effector cells, pointing to a high degree of intrinsic growth regulation. INTRODUCTION Administration of pregnant rats with the antimitotic agent methylazoxymethanol (MAM) has been shown by several investigators to cause forebrain microencephaly in the offspringS,6,~0,29,32. MAM is a potent alkylating agent that kills dividing cells4,2z. This compound will therefore cause a marked and permanent atrophy in a CNS region where the cells undergo rapid proliferation at the time of administration. Injection of MAM at gestation day 15 will produce an atrophy mainly in the forebrain, in particular of the cerebral cortex, with preservation of the brain stem, where the neurons have an earlier birth date than those in the forebrain ~s,a0. It has recently been shown that prenatal MAM treatment leads to an elevated NA concentration and a hyperinnervation of noradrenaline (NA) nerve terminals in the atrophic neocortex810,zl, suggesting that the locus coeruleus NA neurons, which are formed on day 13 during gestation19, 23, are programmed to produce a certain quantity of nerve terminal arborizations during the development. The view that locus coeruleus NA neurons as well as central 5-hydroxytryptamine (5-HT) neurons possess a high degree of intrinsic growth regulation with the formation of a determined number of nerve terminals that is relatively independent of the regions they innervate have also emerged from histoand neurochemical studies on the effect of monoamine neurotoxins on the postnatal development of central monoamine neurons14,15,25,27. These studies thus demonstrated that the monoamine neurotoxins produced permanent denervations of distant N A and 5-HT nerve terminal projections and concomitantly a hyperinnervation in cell body near regions without any significant alteration of the total number of nerve terminals. It was therefore considered of interest to further study the relationship between the development of monoamine nerve terminals and their effector regions in microencephalic rat brain induced by MAM treatment in the foetal stage. A detailed regional analysis of the effects of prenatal MAM administration on the development of monoamine neurons and associated postsynaptic receptors using neurochemical techniques is reported here. Parts of the present results have been communicated in a preliminary form 11. MATERIALS AND METHODS Pregnant rats (Sprague-Dawley) were injected with MAM acetate (25 mg/kg i.v., Schwartz/Mann) under light ether anaesthesia on gestation day 15. MAM was diluted in 0.9 ~ NaCI and injected in a concentration of 10 mg/ml of the solvent.
515 Controls received an equal i.v. injection of the solvent alone. The litters were born on gestation day 22 and each litter was raised to contain not more than 8 pups which were separated from their mother at the age of about 3 weeks. The animals were housed in an air-conditioned room with controlled temperature and kept on a standardized light-dark schedule (14/10 h; light on 06.00 h and off 20.00 h). The animals were sacrificed by decapitation using a Harvard animal decapitator. The brains and spinal cords were rapidly dissected out and placed in cold saline for about 3-5 min. The regional dissection was made mainly as described by Jonsson and Sachs 15. Fresh tissue was taken for uptake studies and subcellular distribution studies whereas tissue taken for monoamine and receptor binding assays were frozen on dry-ice, packed in aluminium foil and stored at --80 °C pending neurochemical analyses. In some experiments the tissue samples taken for catecholamine assay were immediately placed in tubes containing standard amounts of 0.1 M perchloric acid and a-methyldopamine (a-methyl-DA; vide infra). The tubes were stored at --20 °C. Statistical evaluation of the results was performed using Student's t-test. Catecholamine assay The tissue pieces were homogenized by sonication in 310/~1 0.1 M perchloric acid containing 10--50 pmol a-methyl-DA (internal standard) using a Branson B30 sonifier. After a clean-up step of the extracts using an A12-Os-adsorption-desorption procedure, NA and dopamine (DA) were assayed by liquid chromatography with electrochemical detection (LCEC) according to Keller et al. 17. The values were expressed as ng/g wet weight of the tissue, based on measurements using a-methyl-DA as internal standard. 5-HT assay The frozen tissue pieces were homogenized by sonication in 5-10 vol. (w/v) of acidified n-butanol. After centrifugation, 80 #1 of the supernatant was added to 200 #1 heptane and 25/zl of 0.1 M perchloric acid in a tube and the mixture shaken for transfer of 5-HT to the aqueous phase. Aliquots (5-10/A) of this latter phase was taken for 5-HT assay using LCEC according to the procedure of Ponzio and Jonsson 26. The values were corrected for recovery, determined by adding authentic 5HT to homogenates, and expressed as ng/g wet weight of the tissue. [3H]monoamine uptake measurements in vitro Thin slices (thickness 250/~m) were prepared from fresh tissue (neocortex) using a Mcllwain tissue chopper. The slices were placed in beakers containing 5 ml Ringer bicarbonate buffer (pH 7.4) and preincubated for 5 min at +37 °C. Thereafter, 25 #1 [ZH]NA or [3H]5-HT was added to give a final concentration of 0.05/~M and the incubation continued for another 5 min. The incubation was terminated by placing the beakers in ice-water; the slices were rapidly washed in cold Krebs-Ringer buffer, blotted on filter paper, weighed and dissolved in vials by adding 0.5-1 ml tissue solubilizer (Protosol, NEN). After addition of scintillation fluorophor, radioactivity taken up in the tissue was determined by liquid scintillation spectrometry. The values
516 were corrected for 'extraneuronal uptake' by subtracting blank values, obtained by performing the incubations at + 4 °C or including 10/~M desipramine (for [3H]NA uptake) or 10 #M chlorimipramine (for [3H]5-HT uptake) in the incubation medium. Subcellular distribution studies Fresh tissue from the neocortex and striatum were homogenized (8-10 strokes) in 20 vol. (w/v) of cold 0.3 M sucrose using a glass homogenizer with a teflon pestle (clearance 0.05 mm) at a speed of about 1000 rpm. Four fractions were obtained by differential centrifugation; P1 ~ 1000 × g (20 min) pellet; P2 = 12,000 g (20 min pellet; Pz = 100,000 g (90 min) pellet and Sup. ~ high speed supernatant. The pellets and an aliquot of Sup. were taken for catecholamine assay as described above. In vitro receptor binding assays The procedure of Bylund and Snyder a was employed for fl-receptor binding studies using [3H]dihydroalprenolol ([aH]DHA) as radioligand. The homogenates were incubated in vitro in 1 nM [3H]DHA. The amount of non-specific binding was determined by including 1 #M (--)-alprenolol in the incubation mixture. Specific [aH]DHA binding was defined as the difference between total and non-specific binding; a-receptor assay was carried out according to U'Prichard et al. 31 by incubating brain homogenates in 0.2 nM [3H]WB4101. Non-specific binding was determined by including 100/~M 1-NA in the incubation mixture and specific 5-HTreceptor assay was performed by using [3H]5-HT or [3H]spiroperidol ([sH]Spi) as radioligands according to Bennett and Snyder 2 and Peroutka and Snyder 24. The homogenates were incubated in 6 nM [3H]5-HT or 0.3 nM [3H]Spi and non-specific binding determined by adding 1 #M unlabeled 5-HT or 100 #M 5-HT respectively. Drugs and substances Drugs and substances used were: MAM acetate (Schwarz/Mann); N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, (DSP4, obtained from Dr. R. Dahlbom, University of Uppsala, Uppsala); pargyline-HC1 (Sigma); [ZH]5-hydroxytryptamine creatinine sulphate (10 Ci/mmol, Radiochemical Center); [ZH]noradrenaline-HCl (12 Ci/mmol, Radiochemical Center); [3H]spiroperidol (79 Ci/mmol, NEN); [3H]dihydroalprenolol (45 Ci/mmol, NEN); [aH]WB-4101 (25 Ci/mmol, NEN); a-methyl-ptyrosine methylester (H44/68, AB H/issle); a-propyldopacetamide (H22/54, AB Hassle); desipramine-HC1 (Pertofran, Ciba-Geigy); chlorimipramine-HC! (Anafranil, Ciba-Geigy).
RESULTS The MAM-treated animals did not show any marked changes in gross behaviour. In agreement with Johnston and Coyles it was observed that the prenatal MAM treatment only marginally affected the body weight gain (Table I) with a reduction in body weight of 10-20~ in the adult stage. No sex differences were noticed. There were, however, very marked differences in the weight development of the various brain
517 TABLE I
Mean body weightof 6- and 9-week-oMrats treatedprenatally with MAM (25 mg/kg i.v.) (n = 8-10)
6 weeks old Male Female 9 weeks old Male Female
Control
MAM
(g)
(g)
% change
159 143
142 121
--11
297 220
251 172
--15 --22
--15
regions after MAM. While the weight of the whole CNS was reduced by about 25 ~o, the most pronounced weight reductions were found in the forebrain regions, cerebral cortex and hippocampus, being about half of the control weight (Table II). The olfactory bulb and striatum were reduced in weight by 20-30 ~o, while hypothalamus and mesencephalon were affected only to a minor degree (reduction by approximately 10~o) after MAM. Cerebellum, pons-medulla oblongata and the spinal cord were affected hardly at all. Regional effects o f M A M on endogenous N A and DA The p r e n a t a l M A M t r e a t m e n t resulted in m a r k e d regional alterations o f the e n d o g e n o u s N A a n d D A concentrations, f r o m p r o n o u n c e d increases to n o o r only m a r g i n a l effects (Table I I I a n d Fig. 1). I n regions with altered c a t e c h o l a m i n e concentrations, it was n o t e d t h a t the changes were quantitatively similar for N A a n d D A . The m o s t m a r k e d increases in N A a n d D A c o n c e n t r a t i o n s after M A M were f o u n d in those regions displaying the m o s t p r o n o u n c e d weight reductions, n a m e l y the cerebral cortex, h i p p o c a m p u s a n d the striatum. The N A a n d D A c o n c e n t r a t i o n s were TABLE I!
Wet weight (means) of various CNS regions of 9-week-oMrats treatedprenatally with MAM (25 mg/kg i.v.) (n=16-20) Region Olfactory bulb Cerebral cortex Hippocampus Striatum Hypothalamus Mesencephalon Pons-medulla oblongata Cerebellum Rest of brain Spinal cord Whole CNS
Control (mg)
M.,IM (me)
~ change
42 660 104 51 60 141 183 267 220 537
33 310 54 35 52 124 172 254 166 520
--21 --53 --48 --31 --13 --12 -- 6 -- 5 --24 -- 3
2265
1720
--24
TABLE III
Effects of prenatal M A M treatment on the regional NA and D,4 concentrations in the brain of rats raised to adulthood Pregnant rats were treated with MAM (25 mg/kg i.v.) on gestation day 15. The pups were raised to adulthood (9 weeks of age) when sacrificed and samples taken for catecholamine assay using LCEC. n -- 8-10. Each value (mean i S.E.M.) includes determinations of samples from an equal number of male and female.
Region Olfactory bulb Cerebral cortex Hippocampus Striatum Hypothalamus Mesencephalon Pons-medulla oblongata Cerebellum Spinal cord Rest of brain * 0.05
>
P
>
NA DA NA DA NA DA NA DA NA DA NA DA NA DA NA DA NA DA NA DA
Control (ng/g)
MAM
234 12.3 345 13.5 422 6.8 69 9716 2146 288 507 136 716 38 156 5.2 335 18.3 608 1517
273 17.1 713 25.0 701 13.4 132 13.840 2357 260 607 156 757 41 168 5.8 336 19.2 806 1711
± 27 ± 1.1 i 38 ~- 2.4 i 55 ± 1.3 ± lI ± 388 ± 107 ± 20 ± 14 i 6 i 30 ~ 1.2 ± 5.0 ± 0.2 ± 6 :i 0.6 ± 53 £ 74
% change ± 16 ~_ 2.5 ± 46*** ± 3.5* ± 48** ± 2.5" ± 14"* ~ 846** ± 187 i 15 ± 15"** ~ 8 ~ 18 ± 1.3 ± 8.2 ± 0.5 ~ 14 zb 1.0 ± 91 _+ 118
+17 +39 +107 +85 +66 ±97 +91 ÷42 -+ 10 ~ 11 ±20 ~ 15 ~6 +8 +8 F12 -4 5 ÷33 +13
0.01.
** 0.01 > P > 0.001. *** P < 0.001.
20O %
10¢
NA
iN OB
CI
Hipp
OConc. l~Jlotal
Str
HypQ. Mes.
P-re.
Cb.
S~c.
200"
%
1 OB
-
Cx,
Hipp.
Str.
H y p o . Mel.
P-re.
Cb.
Sp.c.
R
Fig. 1. Effects of prenatal MAM treatment (25 mg/kg i.v.) on the regional NA and DA levels in the brain of rats raised to adulthood (9 weeks of age). Each bar represents the mean 5z S.E.M. of 6-8 determinations, expressed either as a percentage of the concentration (cone., ng/g) or as a percentage of the total amount (Total, ng per region) of respective control value for each region. OB, olfactory bulb; Cx., cerebral cortex; Hipp., hippocampus; Str., striatum; Hypo., hypothalamus; Mes., mesencephalon; P-m., pons-medulla oblongata; Cb., cerebellum; sp.c., spinal cord; R, rest of brain.
519 increased by 4 0 - 1 0 0 ~ in these regions. Intermediate elevations were n o t e d in the olfactory b u l b a n d mesencephalon, whereas n o or m i n o r effects were seen in the h y p o t h a l a m u s , p o n s - m e d u l l a oblongata, cerebellum a n d the spinal cord. The latter regions also showed n o or m i n u t e changes in wet weight after M A M . However, i n d e p e n d e n t o f the degree of elevation of N A a n d D A concentration, n o significant difference between M A M - t r e a t e d a n d control was f o u n d when calculating the values as total a m o u n t of N A or D A per b r a i n region (Fig. 1). N o sex differences of the effects of M A M o n the development of e n d o g e n o u s catecholamines were observed. The alteration in cortical N A a n d D A concentrations described above for adult rats were n o t e d already one week (8 days) after birth, whereas only m i n o r changes were seen in other regions (Table IV). Similar results were f o u n d in 30-day-old rats.
Effects o f D S P 4 on endogenous catecholamines in M A M - t r e a t e d and controls rats The effect of the N A n e u r o t o x i n DSP4 o n the regional N A a n d D A levels in the TABLE IV
Effects of prenatal M A M treatment on the regional NA and DA levels in the CNS of 8- and 30-day-old rats (n = 4-6) MAM treatment and NA/DA analyses as in Table III. Changes expressed either as per cent of NA or DA concentration (Conc.) or as per cent of the total amount (Total) of respective control value for each region.
Region
Control
MAM (ng/g)
8 days old: Cerebral cortex Mesencephalon Pons-medulla oblongata Cerebellum Spinal cord 30 days old: Cerebral cortex Striatum Mesencephalon Cerebellum Spinal cord * 0 . 0 5 > P > 0.01. ** 0.01 > P > 0.001. *** P < 0.001.
% changes Conc. Total
NA DA NA DA
93 + 12 4.2 4- 0.2 173 4- 9 113 4- 34
197 4- 6*** 8.4 4- 1.2" 209 4- 10" 114 4- 34
+112 +100 +21 --
+31 +27 +17 --
NA DA NA DA NA DA
222 4- 4 16 4- 1.5 93 4- 2 0.7 4- 0.2 204 4- 11 8.7 ± 0.7
283 4- 14" 16 4- 2.0 110 + 9 0.8 4- 0.1 253 4- 14' 9.8 4- 0.8
+27 -+18 +14 +24 +13
+27 -+19 +15 +21 +10
NA DA NA DA NA DA NA DA NA DA
148 4- 4 12 4- 1.0 73 4- 12 5881 4- 377 477 4- 29 316 4- 4 105 4- 3 2 4- 0.5 347 4- 36 23 4- 3
328 4- 18"** 28 4- 6* 114 4- 9* 8120 4- 142"* 559 4- 19" 354 4- 26 119 4- 6 2.5 4- 0.5 350 4- 41 24 4- 2
+121 +133 +56 +38 +17 +12 +13 +25 +1 +4
+8 +14 --6 --17 --7 -~21 --13 --16 --13
520 100 %
N_A. [ ] NaCI*DSP4 [ ] MAM+DSP4
80
I
60" 4020" O"
Cx.
H~.
Str.
Hypo.
IVies
P-m
Cb
Sp.¢
R
120-
I
100%
_L
i~-
-
8060" ?
40 20
0
OB
Cx
Hi~
Str
Hypo.
Mes
P-m
Cb
SOc.
Fig. 2. Effect of DSP4 (50 mg/kg i.p., 3 days) on the endogenous NA and DA concentrations (ng/g) in various regions of the brain from adult rats (9 weeks of age) treated with MAM or NaCI prenatally (25 mg/kg i.v., on gestation day 15). Each bar represents the mean ! S.E.M. of 3-4 determinations, expressed as a percentage of respective control values for each group and region. CNS of MAM-treated and control rats was investigated. It was found that the relative (comparison between DSP4-treated and respective group controls) N A reductions induced by DSP4 (50 mg/kg i.p., 3 days) were generally very similar in MAM-treated and control rats (Fig. 2), although the absolute N A levels were higher in regions from MAM-treated rats which after M A M alone displayed marked increases in the NA concentration, e.g. the cerebral cortex and the hippocampus. No or minor effects of DSP4 were noted on the DA levels both in MAM-treated and control rats.
Effects of prenatal M A M treatment on the subeellular distribution of NA and DA in the cerebral cortex and the striatum The relative distribution of N A and D A in both regions analyzed was very similar for MAM-treated and controls (Fig. 3) indicating that the amines are stored in identical structures in the two groups of animals. Most of the N A (about 50 ~ ) of the cerebral cortex was recovered in the Pz-fraction (containing synaptosomes), while most of the D A (40-50 ~ ) originating both from the striatum and the cerebral cortex were found in the high speed supernatant. Relatively less D A from these regions were found in the P3 fraction compared to cortical NA. The D A distribution in tissue from the neocortex was on the whole more similar to that of the striatum, indicating that the main part of the D A present in the cerebral cortex is present in D A nerve terminals.
521
%
[] ~1
[] ~M
L 5!
;!i
i ili
o
NA
DA
DA
NA
DA
c'x.
DA
DA
NA
st~
Cx.
i
DA
c;
St~
~.
Fig. 3. Subcellular distribution of endogenous N A and D A in cerebral cortex (Cx.) and striatum (Str.) of M A M and saline (NaCl)-treated rats. M A M (25 mg/kg i.v.) was administered on gestation day 15 and the rats were sacrificed at the age of 3 weeks. The tissue was fractionated by differential centrifugation to obtain a P1 (1000 g pellet), P2 (12,000 g pellet), P8 (100,000 g pellet) and Sup. (highspeed supernatant). Each bar represents the mean 4- S.E.M. of 3 determinations, and the values expressed as a percentage of the total amount of N A or D A recovered from each tissue sample analyzed.
Regional effects of tyrosine hydroxylase inhibition (Till) on the NA and DA reduction in brain of MAM-treated and control rats The effects of administration of the tyrosine hydroxylase inhibitor H44/68 (amethyl-p-tyrosine methylester) on the catecholamine content of various brain regions in MAM-treated and control rats was investigated (Figs. 4 and 5). From the cerebral 8
Front.
Cing=
Occ
Hip_p~
xlO2 NA~ ng~.
2
0. 10
C 'MAM
C
MAM
DA
C
MAM
C
MAM
e
M~"~t
[ ] NaCI [ ] H44/68
0 J
C
' MAM
C
' MAM
C
MAM
Fig. 4. Effects of H44/68 (250 mg/kg i.p., 2 h) on the N A and D A concentrations in frontal (Front), cingulate (Cing.), occipital (Occ.) cortex and hippocampus (Hipp.) of MAM-treated and control (C) rats. M A M (25 mg/kg i.v.) was administered on gestation day 15 and the rats sacrificed at the age of 6 weeks. Each bar represents the mean 4- S.E.M. of 6 determinations, expressed as ng/g wet weight of the tissue. Each group of animals consisted of 3 males and 3 females. The figures above the hatched bars denote the relative N A or D A values after H44/68 as a percentage of respective control (NaCI) value (open bars).
522
st,i..,%~
Ports-reed
Cefebefl
Sp cord
NA
[ ] NaCI
,1022 ng/g
~ 3 H44/68 92 2
./
~4 C
~103
ng/g
MAM
:)A
C ' MAM
C
MAM
C
C
MAM
' MAM
5~
,10 -~ J
10
,10 ~
,1012
4t
I
] ,t
,,
3~ -+-
tlt
t C
MAM
C
MAM
C ' MAM
' MAM
C
MAM
MAM
Fig. 5. Effects of H44/68 on the NA and DA concentrations in striatum, hypothalamus (Hypo.) mesencephalon (Mes.), pons-medulla, cerebellum and spinal cord of MAM-treated and control (C) rats. Experimental conditions as in Fig. 4. Each bar represents the mean :k S.E.M. of 6 determinations, expressed as ng/g wet weight of the tissue. The figures above the hatched bars denote the relative NA or DA values after H44/68 as a percentage of respective control (NaCI) value (open bars). cortex 3 regions were sampled: frontal, cingulate and occipital. The M A M treatment led to an increase in N A concentration in all these regions to about the same absolute level, although the highest relative increase was seen in the occipital cortex ( + 2 8 0 ~ ) , and the lowest in the cingulate cortex ( + 6 0 ~ ; see Fig. 4). All regions showed an approximately 3-fold increase in D A concentration. Calculating these values as total amount per region it was again found that this value was approximately similar for MAM-treated and controls for all regions. The relative H44/68-induced reduction of N A was less pronounced in all cortical regions and hippocampus after M A M treatment compared to controls, indicating a reduced N A 'turn-over' in each N A nerve terminal after M A M . This effect was significant in the frontal (P < 0.01) and cingulate (P < 0.05) cortex, while only tendencies were noted in the occipital cortex and hippocampus (P < 0.1). The absolute amounts of N A that disappeared after H44/68 was clearly greater after M A M in all regions, indicating that the absolute amounts of N A 'turned over' in these regions is increased after M A M . The relative reduction in D A concentration caused by H44/68 was similar in all cortical regions and hippocampus for MAM-treated and controls except in the occipital cortex (Fig. 4), although as in the case of N A the absolute reduction of D A were greater, suggesting that these regions are exposed to a larger amount o f D A in absolute terms. In most of the other CNS regions analyzed, no clear-cut differences in relative H44/68-induced reductions of both N A and D A between M A M and control rats were observed, except in the mesencephalon and pons-medulla for N A and pons-medulla
523 TABLE V
Effects of prenatal M A M treatment on the regional 5-HT concentrations in the brain of rats raised to adulthood Pregnant rats were treated with MAM (25 mg/kg i.v.) on gestation day 15. The pups were raised to adulthood (9 weeks of age), sacrificed and samples were taken for 5-HT assay using LCEC. n = 8-10. Each value includes determinations of samples from an equal number of male and female rats.
Region
Control
Olfactory bulb Cerebral cortex Hippocampus Striatum Hypothalamus Mesencephalon Pons-medulla oblongata Cerebellum Spinal cord Rest of brain
284 260 337 180 680 772 608 48 305 472
MAM (ng/g)
-4- 21 ± 15 -4- 35 -4- 33 dz 52 ± 29 -4- 35 ± 9 ± 18 ± 27
444 533 690 345 860 872 744 61 326 504
% change
~: 54* ± 46*** ± 50*** ± 36** -4- 48* ± 56 -4- 44* 4- 8 ± 36 ± 48
+56 +128 +105 +92 +27 +13 +22 +27 +7 +7
* 0.05 > P > 0.01. ** 0.01 > P > 0.001. *** P < 0.001. an d spinal c o r d f o r D A (Fig. 5). T h e results indicated an increased N A t u r n - o v e r in m e s e n c e p h a l o n , b o t h in relative a n d absolute terms, while the reverse seem to be the case f o r pons-medulla.
Regional effects o f M A M treatment on endogenous 5 - H T Th e p r e n a t a l M A M t r e a t m e n t caused very m a r k e d regional alterations o f the 5H T c o n c e n t r a t i o n , similar to that seen for e n d o g e n o u s N A and D A (Table V an d Fig. 6). Th e m o s t p r o n o u n c e d increases with an a p p r o x i m a t e d o u b l i n g o f the 5 - H T -HT.
..~
[ ] Conc. ~ Total
20O
%
I •
OB
Cx,
1
Hipp.
Str.
v
A.
r.,i
Hypo. Mes. P-m.
Cb.
SpC,
R
Fig. 6. Effects of prenatal MAM treatment (25 mg/kg i.v.) on the regional 5-HT levels in the brain of rat raised to adulthood (9 weeks of age). Each bar represents the mean -4- S.E.M. of 6-8 determinations, expresssed either as a percentage of the concentration (Conc., ng/g) or as a percentage of the total amount (Total; ng per region) of respective control value for each region, OB, olfactory bulb; Cx., cerebral cortex; Hipp., hippocampus; str., striatum; Hypo., hypothalamus; Mes., mesencephalon; P-m., ports-medulla oblongata; Cb., cerebellum; Sp.C., spinal cord; R, rest of brain.
524 concentration was found in the cerebral cortex, hippocampus and striatum, while moderate increases (20-50 ~) were observed in the olfactory bulb, hypothalamus and pons-medulla. Calculating these data as total amount per region, it was found that the 5-HT content in MAM and control rats did not differ significantly for any region. Similar results were noticed when analyzing the rats 8 days and 30 days after birth (Table VI). In one set of experiments the effect of the tryptophan hydroxylase inhibitor H22/54 (a-propyldopacetamide) on the regional 5-HT levels in brain from MAMtreated and control rats was investigated (Fig. 7). It was observed that the 5-HT concentration was markedly increased in all cortical regions analyzed and as in the case of NA, the highest relative 5-HT increase was noted in the occipital cortex and the lowest in the cingulate cortex. Administration of H22/54 led to marked reductions of 5-HT both in MAM-treated and control rats in all regions analyzed. There did not appear to be any consistent alterations of the relative H22/54 reduction when comparing the various regions of MAM-treated and control rats. It is clear, however, that the absolute reduction of 5-HT after H22/54 is increased in most atrophic regions of MAM-treated rats compared to control, indicating an increase in 5-HT turn-over in absolute terms.
Effects of prenatal M A M treatment on Jail]amine uptake in the cerebral cortex Analysis of the in vitro uptake of [aH]NA or [SH]5-HT in slices from the ceiebral cortex of MAM-treated and control rats showed that there was an about doubling of the uptake of both [SH]amines when calculating the uptake per weight unit (Fig. 8). However, when expressing the uptake as total uptake, there were no differences between MAM-treated and controls for both [aH]NA and [aH]5-HT.
Effects of prenatal M A M treatment on the in vitro binding of various receptor ligands to homogenates from the cerebral cortex The effect of prenatal MAM treatment on the specific in vitro binding of various labeled receptor ligands, (a, fl, and 5-HT receptors) to homogenates from the cerebral cortex was investigated (Fig. 9). It was found that the only ligand that showed a significantly reduced binding per weight unit of the tissue was the fl-adrenoceptorligand, [SH]DHA. Expressing the results as total binding, all of them showed a significantly reduced binding, which was most pronounced for [SH]DHA. In displacement experiments it was found that the IC50 values for various receptor compounds (concentration needed to inhibit specific [SH]ligand binding by 50 ~) was similar for homogenates from MAM-treated and control rats for all radioligands. This indicates that the affinity of the various receptors towards the receptor-ligand was not markedly changed after MAM. The changes in binding are therefore most likely mainly related to an alteration in the number of receptor sites.
525 TABLE VI
Effects of prenatal M . 4 M treatment on the regional 5-HT levels in the C N S of 8- and 30-day-old rats (n = 4-6) M A M treatment and 5-HT analysis as in Table V. Changes expressed either as per cent of 5-HT concentration (Conc.) or as per cent of the total amount (Total) of respective control value for each region.
Region
Control
8 days old: Cerebral cortex Mesencephalon Pons-medulla oblongata Spinal cord Rest of brain 30 days old: Cerebral cortex Striatum Pons-medulla oblongata Spinal cord Rest of brain
MAM (he~g)
% changes Conc. Total
109 394 199 326 69
4± ± ± ±
2.4 60 15 23 4.6
176 381 164 314 82
± ± ± ± ±
8.8** 20 7.2 17 5.0
+62 --2 --18 ---4 + 19
+6 --2 --5 +4 + 19
184 250 560 369 286
± ± ± ± ±
27 18 41 19 14
340 419 452 302 288
± ± ± ± ±
24* 29* 39 23 15
±85 +68 --19 --18 +1
--11 +26 --11 --9 ÷5
* 0.05 > P > 0.01. > P > 0.001.
** 0.01
14. t~ c>
OB.
Front.
Cing~
Pariet.
Occ.
Hip_~
12.
0 C 16-
MAM
Striatum
C
MAM
HylJO~
C
MAM
I~
C
NAM
Ports,reed.
C
MAM
Cerebell.
C
MAM
Sp.cord
i ~ i ~r~~'I'-~H~22/ NaCI ~54
12 ¸
6~
75
C
MAM
C
MAM
C
MAM
C
MAM
C
MAM
C
MAM
Fig. 7. Effects of H22/54 (500 mg/kg i.p. 2 h) on the 5-HT concentrations in various brain regions of MAM-treated and control (C) rats. M A M (25 mg/kg i.v.) was given on gestation day 15 and the rats were sacrificed at the age of 5 weeks. Abbreviations for the various CNS regions as in Figs. 4 and 5. Each bar represents the mean 4- S.E.M. of 3-4 determinations. The figures above the hatched bars denote the relative 5-HT value after H22/54 as a percentage of respective control (NaCI) value (open bars).
526 300
3H-NA
3H.5-HT
[]
20C
NaCI
%
w
T
w
T
Fig. 8. Effect of prenatal M A M treatment on the in vitro uptake of [SH]NA (0.05/~M, 5 min) or [sI-I]5H T (0.05/zM, 5 rain) in slices from the cerebral cortex. M A M treatment as in Fig. 4. Each bar represents the mean ± S.E.M. of 4-6 determinations, expressed as a percentage of the neuronal Jail]amine uptake per mg of tissue (W) or total uptake (T) in control.
-.L F'-] NaCI
_L.
5
F~MAM
_L_, r-,
!2 1 oJ
3H-OHA P,
3H~WB-4101 (z
3H-5HT
4-
~
3H-SPI
5~HT
3.d.
J
O-
3H-DHA fl
,,,
3H-WB-4101 o
3H-5HT
3H-SPI 5-HT
Fig. 9. Effects of prenatal M A M treatment on the specific binding of [SH]DHA, [sH]WB-4101, [SH]5H T or [sH]Spi to homogenates from the cerebral cortex. Each bar represents the mean J: S.E.M. of 4-6 determinations, and the values expressed as pmol of [sI-I]ligand bound/g of tissue (upper part) and total amount bound to the tissue (lower part). ** 0.01 > P > 0.001 ; *** P < 0.001.
527 DISCUSSION In agreement with previous studies it was found that prenatal administration of the antimitotic agent MAM led above all to a weight reduction of the forebrain regions, the cerebral cortex and the hippocampus s,2a. Marked weight reductions were also noted in the olfactory bulb and the striatum. The most pronounced changes in the concentration of endogenous monoamines were found in the atrophic regions. The concentration changes of neurotransmitters were closely associated with the weight reductions, so when calculating the results as monoamine content per region it was found that there were no significant differences between MAM-treated and controls for any monoamine. These results indicate that the monoamine nerve terminal fields develop relatively independent of the region they innervate and that in spite of a marked reduction of the size of the region, the nerve terminal net forms a normal extent of arborizations. This will then lead to marked hyperinnervations in the regions where the most pronounced weight reductions are at hand. This interpretation is strongly supported by the uptake experiments showing an increase in [3H]amine uptake (measure of the axonal membrane area and thus an index of the number of nerve terminals) per weight unit in the cerebral cortex quantitatively similar to the increase in endogenous NA and 5-HT concentration, while total uptake was not notably affected. The present results are in agreement with previous reports on the effect of MAM on monoamine neuronsT-1O,lS,21. It can be argued that the observed increase in DA concentration in the cerebral cortex is not associated with DA nerves, but rather due to an increase in NA nerves and hence an increase in content of DA precursor. The results from the experiments with DSP4, a selective NA denervating agent 12,13, do not support this view. It was thus observed that the reduction of DA after DSP4 was similar and only marginal (about 30 ~) in cerebral cortex of both MAM-treated and control rats. It should be noted that the DSP4 treatment led to an approximately 90 ~ NA reduction in the cerebral cortex of both groups. These data thus indicate that the quantitative relation between NA and DA nerve terminals is unaltered after MAM treatment and hence also DA nerve terminals display a hyperinnervation pattern in all regions where they are present and where MAM treatment leads to an atrophy. Based on the results from the DSP4 experiments it could be calculated that about 10 ~ of the endogenous DA found in the cerebral cortex is associated with NA nerve terminals, serving'as"precursor in NA biosynthesis. The subcellular distribution results are also in agreement with the view that both NA and DA nerves form a denser nerve net and hyperinnervation in the cerebral cortex after MAM. It was thus found that the DA distribution was more similar to that of DA in the striatum (containing predominantly DA nerves) than the subcellular distribution of NA in the cerebral cortex for both MAM-treated and control rats. From a developmental viewpoint it appears as if the hyperinnervation pattern is present very early in ontogeny, since the altered monoamine concentrations observed in atrophic cerebral cortex in the adult stage were seen already one week after birth, which is in agreement with data recently reported by Johnston and Coyle9.
528 Concerning the analysis of various monoamine receptors in the cerebral cortex after MAM treatment, it was found that the density of receptors was approximately the same for 5-HT and a-receptors after MAM, while the fl-receptors were the only type with markedly reduced density. However, calculated on a total basis all receptors analyzed were markedly reduced after MAM. These results thus suggest that the cerebral cortex is devoid of a significant number of effector cells after prenatal MAM treatment and that the monoamine neurons develop independent of the postsynaptic receptors (cf. ref. 16). The studies of the effect of tyrosine or tryptophan hydroxylase inhibition on the endogenous monoamines showed that inhibition of these enzymes caused a significant reduction in the catecholamines and 5-HT respectively in MAM-treated rats and in many regions to the same extent as in controls. This indicates that the monoamine neurons, also in the atrophic regions, are functionally active in synthesizing and releasing their neurotransmitter by nerve impulses (see And6n et al.1). Although the present results do not allow any precise analysis as to any quantitative differences between release and turn-over of monoamine neurotransmitters after MAM, there appears to be a consistent pattern with respect to NA in the cerebral cortex. It was thus observed that the relative NA reductions induced by H44/68 were less pronounced in all the cortical regions and also in the hippocampus compared to controls. This could indicate that the amount of NA released per nerve terminal is reduced in these hyperinnervated regions. The reason for this could be that the inhibitory presynaptic a-receptor-mediated control of NA release is more pronounced in these hyperinnervated regions. Consistent with this view it was observed that the absolute amounts of NA turned-over in these regions was increased, possibly providing a situation of increased concentrations of NA in the extracellular space acting on the presynaptic a-receptors. Concerning the DA and 5-HT systems, no such pattern could be observed, possibly indicating that presynaptic DA and 5-HT receptors are not as sensitive as presynaptic a-receptors in such an autoregulation. However, in view of the rather extensive changes occurring in several neurotransmitter systems after prenatal MAM treatmentT,9,1°, it is clear that much more work is needed to be able to adequately interpret alterations in transmitter release and turn-over in atrophic regions induced by MAM. In conclusion, the present study has shown that an atrophic development of certain forebrain regions induced by prenatal MAM treatment leads to a hyperinnervation of monoamine nerve terminals which normally innervate these structures. This hyperinnervation is closely related to the atrophy with formation of a normal network of nerves from a quantitative viewpoint. These results strongly suggest that the monoamine neurons are strictly programmed to produce a certain quantity of nerve terminal arborizations in regions they innervate during the development, which to a large extent appears to be independent of the effector cells and their postsynaptic receptors. The monoamine neurons thus seem to possess a remarkably high degree of intrinsic growth regulation.
529 ACKNOWLEDGEMENTS
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