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Developmental characteristics of central monoamine neurons and their reciprocal relations
EXPERIMENTAL
NEUROLOGY
Developmental Neurons
53, 801-814 (1976)
Characteristics of Central Monoamine and Their Reciprocal Relations G. JONSSONl
Department
of Histology,
Karolinska Received
Institutet, July
S-104
01 Stockholm,
Sweden
7,1976
The effects of neonatal administration of the monoamine neurotoxins 6hydroxydopamine and 5,7-dihydroxytryptamine on the postnatal development of monoamine nerve terminals in various regions of rat brain have been investigated. 6-Hydroxydopamine is known to induce a selective degeneration of catecholamine neurons, and 5,7-dihydroxytryptamine has a preferential action on 5-hydroxytryptamine neurons. Monoamine nerve density was monitored by measuring the in vitro uptake of [‘Hlmonoamines in brain tissue homogenates, combined with pharmacological analysis and fluorescence histochemistry. It was consistently observed that the relative reductions of [‘HImonoamine uptake in the cerebral cortex and neostriatum were quantitatively the same 6 to 7 days after the neurotoxin treatment compared with the situation when the uptake analysis was performed on tissues from adult rats. These results indicate that the neurotoxin in these regions produces damage and permanent denervation of a certain number of monoamine nerve terminals, whereas spared nerves undergo an apparent normal development. It was confirmed that the marked and permanent reduction in [aH]noradrenaline uptake in the cerebral cortex induced by systemic 6-hydroxydopamine treatment at birth was associated with a considerable increase in [“Hlnoradrenaline uptake in the pons-medulla. This treatment had no effects on uptake in 5-hydroxytryptamine and dopamine nerve terminals. Analogous changes with respect to 5-[‘Hlhydroxytryptamine uptake was observed after neonatal 5,7-dihydroxytryptamine treatment. These plastic changes are most likely due to a “pruning effect” induced by the neurotoxins. The activity of phenylethanolamine N-methyltransferase, a marker of adrenaline neurons, developed normally, independent of the changes in noradrenaline neurons induced by 6-hydroxydopamine. The present results favor the view that the postnatal development of monoamine nerve terminals is strictly ordered. Furthermore, the neurons exhibit an apparently high degree of intrinsic growth regulation, especially with respect to quantity of nerve terminal arborization. There was no evidence for any interaction between growing monoamine neurons. 1 The present study has been supported by the Swedish Medical (04X-2295), Nathorsts, Groschinskys and Bergvalls Stiftelse. 801
Copyright 1976 by Academic Press, Inc. rights o9 reproduction in my formreserved.
All
Research Council
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JONSSON
INTRODUCTION Central monoamine neurons have been shown to appear relatively early in gestation in several species including humans (13, 14, 21, 22, 28, 30, 31, 47, 48). These neuronal structures have thus been reported to appear first on day 12 to 14 during gestation in the mouse, rat, and rabbit. Biochemical studies have also provided evidence for the presence of monoamine neurons early in the development (1, 3-6, 27). The monoamine cell body groups are fully developed at birth (43) whereas the arborization of the nerve terminal projections occurs to a large extent after birth (24). Although there is evidence suggesting a functional balance between certain monoamine neuron systems in the adult stage (34, 40) and Svendgaard et al. (45) have reported data pointing to a considerable interaction between regrowing central monoamine fiber systems into iris transplants, very little is known at present about any interaction between growing monoamine fibers during development. The present study was therefore undertaken to elucidate the interaction possibility by the use of the monoamine neurotoxins 6-hydroxydopamine (6-OH-DA) and 5,7-dihydroxytryptamine (5,7-HT), which can be utilized to induce selective degenerations of monoamine neurons in newborn rats (19, 35, 36, 39). MATERIAL
AND
METHODS
Sprague-Dawley rats were used and were housed in an air-conditioned room with controlled temperature and a standarized dark-light schedule (14/10 hr, light on 6: 00 AM, and off 8: 00 PM). Each litter contained not more than eight pups which were separated at 21 days of age. Three types of experiments were carried out : (i) newborn rats treated with 6-hydro.rydopam& (2 X 100 mg/kg, 24-hr interval) administered subcutaneously in a volume of 0.05 ml 0.9% NaCI, the first dose being given within 4 hr after birth, (ii) 5-day old rats treated with 100 pg 6-hydroxydopamine administered intracisternally in a volume of 10 ~1 0.9% NaCl (44) ; and (iii) newborn rats treated with 5,7-dihydroxytryptanaine (100 mg/kg, subcutaneous) administered in a volume of 0.05 ml 0.9% NaCl. The rats in the control groups received an equal volume of the vehicle. In groups (ii) and (iii), animals pretreated with the uptake blocker desipramine (20 mg/kg, subcutaneous) 30 min before the administration of the manoamine neurotoxin were included in the experiment. No separation between male and female rats were made, and both sexes were used randomly in the same experiment. The animals were killed 6 to 7 days or 6 to 7 weeks after the treatment by cervical dislocation under light chloroform anesthesia. The brains were rapidly removed and placed in beakers with cold Krebs-Ringer buffer. The various brain regions were dissectedout for further analysis. [SH]Monoamine Uptake Measurements. The brain tissue was homoge-
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nized in 10 volumes (w/v) cold 0.3 M sucrose and the homogenates were centrifuged at 1OOOg for 10 min in a refrigerated centrifuge (Sorval RCZB) . The supernatant solutions (containing monoamine synaptosomes) were taken for subsequent in vitro uptake studies. Aliquots (100 ~1) of the supernatant solutions were added to 1.9 ml Krebs-Ringer bicarbonate buffer (pH 7.4, containing 1.l mM ascorbic acid and 0.01 rnM pargyline) in centrifuge tubes and preincubated at +37”C for 5 min. Thereafter 10 pl [3H]monoamine (0.05 PM, final concentration) was added and the incubation continued for another 5 min. Incubation was terminated by adding 8 ml ice-cold Krebs-Ringer buffer, and the tubes were centrifuged at 10,OOOg for 10 min. Radioactivity was extracted from the synaptosome pellets with 1 ml absolute ethanol and determined by liquid scintillation spectrometry [for details, see ( 18) 1. Quenching was monitored by an internal standard procedure using E3H]toluene. Each homogenate was analyzed in triplicate, both at +37”C and O”C, and means were calculated. In experiments determining [ 3H] dopamine and 5- [ 3H] hydroxytryptamine uptake, the uptake values at 0°C were subtracted from those obtained at +37”C to correct for “extraneuronal” uptake (a “active uptake”). In experiments analyzing [ 3H] noradrenaline uptake, homogenates were in addition analyzed in triplicate at +37’C with 50 PM desipramine in the incubation medium. These latter values were subtracted from those obtained at +37”C to a measure [3H]noradrenaline uptake in noradrenaline nerve terminals. The desipramine values after subtraction of the 0°C uptake values were considered to reflect [3H]noradrenaline uptake in dopamine nerve terminals. Desipramine is known to block amine uptake in noradrenaline but not in dopamine nerve terminals, whereas the transport mechanism of both neuron types is completely inhibited at 0°C (15). The [3H] amine uptake was expressed as nanocuries per milligram of protein, which was determined on aliquots of the 1OOOg supernatant solution according to Lowry et al. (25) as modified by Miller (26). Cathecholamine Assay. The brain tissue was homogenized in 20 to 200 volumes (w/v) cold 0.1 M perchloric acid. After centrifugation, 300 ~1 of the supernatant solution was taken for noradrenaline and dopamine assay according to the radioenzymatic technique of Coyle and Henry (6) and Palkovitz et al. (33). Endogenous noradrenaline and dopamine were expressed as nanograms per gram wet weight of tissue, calculated on the basis of internal standard measurements. Phenylethanolamine N-Methyltransferase (PNMT) Assay. The transferase activity was assayed using a modification of the procedure of Deguchi and Barchas (7) as described previously (20). The tissue was homogenized in 5 to 10 volumes (w/v) distilled water and the homogenates centrifuged at 100,OOOg for 60 min. One hundred microliters of the supernatant solution was then taken for assay using phenylethanolamine (2.4 mM) as substrate
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and S- [nzetlzylJ*C] adenosyl-1-methionine (2 nmol) as cofactor. Enzyme activity was expressed as nanomoles of product formed per gram per hour. Flourescence HistoclaeuListry. Brain slices (thickness, about 0.5 mm) were incubated in eifro in Krebs-Ringer bicarbonate buffer (pH 7.4) containing 1 or 5 pM noradrenaline or G-hydroxytryptamine for 20 min at +37”C. After a brief rinse in cold buffer, the slices were processed according to the Falck-Hillarp technique for the histochemical demonstration of monoamines (2, 10, 12, 32 ) . Drugs and Substances lised. [ 3H] noradrenaline. HCl (6-12 Ci/mmol) ; [ 3H] dopamine . HCI (2.4 C/1 mmol) ; 5- [ 3H] hydroxytryptamine creatinine sulfate (16 Ci/mmol) ; 3H- and [‘“Cl methyl-labeled S-adenosyl-l-methionine (7.2 and 0.055 Ci/mmol ; Radiochemical Centre, Amersham) ; Ghydroxydopamine. HCl (888/32, AB Hassle, Giiteborg, noradrenaline 9HCl (Sigma) ; 6-hydroxytryptamine creatinine sulfate (AB HBssle, Giiteborg) ; desipramineeHC1 (Pertofran, Geigy) ; chlorimipramine. HCl (Geigy). RESULTS In agreement with previous studies (l&20, 35-38)) neonatal 6-hydroxydopamine treatment (2 x 100 mg/kg, subcutaneous) produced a veq pronounced reduction in [ 3H] noradrenaline uptake in noradrenaline nerve terminals of the cerebral cortex (Fig. 1). The relative decrease (about 30% of littermate control) was the same for the 6-day-old and the adult rats, although the latter animals had a total cortical tissue mass which was three to four times larger than that of the younger animals. The desipramine-resistant uptake of [3H] noradrenaline, most likely representing uptake in dopamine nerve terminals (16, 49) was unchanged in the cerebral cortex in both groups of animals. 5- [ 3H] hydroxtryptamine uptake was similarly unchanged after the 6-hydroxydopamine treatment (Fig. 1). Fluorescence histochemical analysis (according to Falck and Hillarp) of sectioned slices from the cerebral cortex showed that the neonatal 6hydroxydopamine treatment produced an almost complete disappearance of the catecholamine nerve terminals normally present (11). Some single catecholamine terminals could be detected. The standard Falck-Hillarp procedure used can only demonstrate noradrenaline nerve terminals in the cerebral cortex (23). In vitro incubation of cortical slices in 1 to 5 PM noradrenaline for 20 min at +37”C did not restore the network of catecholamine nerve terminals, although some additional terminals could be observed. However, incubation of slices from 6-hydroxydopamine-treated animals in 1 or 5 PM 6-hydroxytryptamine resulted in the apearance of a dense network of fine yellow-green fluorescing nerve terminals due to uptake and accumulation of 6-hydroxytryptamine, (Fig. 2), in all probability representing 5-hydroxytryptamine nerve terminals (17). This net-
CENTRAL
MONOAMINE
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CORTEX
PONS
MEDULLA
NEOSTRIATUM
%+DA
3H5HT
‘H-NA
3H 5 HT
FIG. 1. Effects of neonatal 6-hydroxydopamine treatment (2 X 100 mg/kg, subcutaneous, 24-hr interval) on [‘HIamine uptake (0.05 PM, 5 min) in homogenates from the cerebral cortex, neostriatum, and pons-medulla. The animals were killed 6 days and 6 weeks (adult) after treatment. Each column represents the mean f the standard error of four or five determinations, expressed as a percentage of littermate control. ‘DA’-desipramine-resistant uptake of [“HI noradrenaline, most likely reflecting uptake in dopamine nerve terminals (see Material and Methods).
work was considerably denser than that of catecholamine nerve terminals seen in slices from untreated control animals with or without incubation in noradrenaline [ c.f. (8) 1. The uptake-accumulation of 6-hydroxytryptamine could be almost completely blocked by including a 50 PM concentration of the 5-hydroxytryptamine uptake blocker chlorimipramine in the incubation medium. The systemic 6-hydroxydopamine treatment at birth had no effect on the [3H] amine uptake in vitro in the neostriatum (nucleus caudatus putamen) (Fig. 1). The samewas the case for 5- [ 3H] hydroxytryptamine in the pons and medulla oblongata, although the [ 3H]noradrenaline uptake in this region was markedly increased in the adult stage (Fig. 1) . The biochemical analysis of tissue from adult animals was in complete accordance with the [ 3H]amine uptake data, showing about an 80% (18 + 4% of control ; N = 4) reduction of the endogenous noradrenaline in the cerebral cortex after 6-hydroxydopamine without affecting endogenous dopamine (95 * 4% of control ; N = 4). Control values for cerebral cortex were noradrenaline ; 250 + 12 rig/g wet weight of tissue (N = 4) and dopamine, 48 f 5 rig/g (N = 3). Endogenous dopamine was unchanged in the nucleus
FIG. 2. Fluorescence histochemical demonstration of uptake and accumulation of 6-hydroxytryptamine in nerve terminals of the cerebral cortex after 6-hydroxydopamine treatment (2 X 100 mg/kg, subcutaneous, as in Fig. 1) at birth. The animals were killed 8 weeks after birth. A-Dense network of nerve terminals seen after in vitro incubation in 1 pM 6-hydroxytryptamine for 20 min. in all probability representing S-hydroxytryptamine nerve terminals. B-Same conditions as in A except that 50 PM chlorimipramine was present in the incubation medium. X160.
CENTRAL
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CEREBRAL 100. %
807
NEURON DEVELOPMENT
CORTEX
- -- - - - -
SO-
o-
6OH-DA
DMI* SO&DA
@OH-DA
3H-NA
DMI* 6.0~DA
DA
Dl$ 6,Oli-DA
,
3H.5-HT
‘DA’
1 NEOSTRIATUM
6.0H-
,
50.
Od
6-OH-DA
-XL----
DMI 6.OiLDA
O-OH-DA
DMI s.o&DA
JH-5-HT
FIG. 3. Effects of intracranial administration of 6hydroxydopamine (100 pg in 10 ~1) to S-day-old rats with or without desipramine pretreatment (20 mg/kg, subcutaneous) on rH]amine uptake in homogenates from the cerebral cortex and neostriatum. The animals were killed 7 days or 6 weeks (adult) after the injection. Each column represents the mean f the standard error four or five determinations, expressed as a percentage of untreated littermate control.
caudatus putamen (104 -C 5% of control, N = 4 ; control value 7.1 f 0.6 pg/g), whereas noradrenaline was markedly increased in the pons-medulla (168 + 157o of control; 720 * 59 rig/g). The phenylethanolamine Nmethyltransferase activity, marker of adrenaline neurons, in this latter region was also unaltered after neonatal 6-hydroxydopamine treatment in both groups of animals : 98 Ifr 8% (N = 4) of littermate control at 6 days and 103 * 9% (N = 4) of control in adults. In another set of experiments, 5-day-old rats were treated with 100 pg 6-hydroxydopamine administered intracranially (44) and [3H] amine uptake analyzed in the cerebral cortex and neostriatum 5 days and 6 weeks afterward (Fig. 3). The results show that this treatment led to a very pronounced decrease in [3H]noradrenaline uptake (about 10% of control) in the cerebral cortex, .and the relative decrease was quantitatively similar for both age groups studied. The desipramine-resistant (“dopamine”) and 5- [ 3H] hydroxytryptamne uptakes were unchanged. The reduction in [ 3H] noradrenaline uptake could be completely abolished by pretreatment of the
808
G.
CEREBRAL
JOXSSOS
CORTEX
50-
o-
5.7.HT
5.7.HT
DMI + 57.HT
DMI 5.7.+HT
5.7.HT
DMI $7.+H T
‘DA’
5.7 HT L
DMI &H T
----
3ti-
DA
5.7. HT L-
~~ ~~~
DMI 5.7fHT -
3H.5-HT
FIG. 4. Effects of systemic treatment of newborn rats with 5,7-dihydroxytryptaminc (100 mg/kg, subcutaneous) on [“HIamine uptake in homogenates from the cerebral cortex and neostriatum. One group of animals was pretreated with desipramine (20 mg/kg, intraperitoneal) 30 min before the injection of 5,7-dihydroxytryptamine. The rats were killed 7 days or 7 weeks afterward. Each column represents the mean -C the standard error of four to six determinations, expressed as a percentage of untreated littermate control.
rats with the uptake blocker, desipramine. This treatment had practically no effect on the desipramine-resistant uptake of [ 3H] noradrenaline and on the 5- [3H] hydroxytryptamine uptake as well. The intracranial 6hydroxydopamine treatment also led to a marked decrease in [3H]dopamine uptake in the neostriatum which was quantitatively similar for both experimental groups. Consistent with the view of clesipramine not affecting uptake in dopamine neurons (15)) desipramine pretreatment did not modify the 6-hydroxydopamine-induced changes in [ 3H] dopamine uptake. No effects on 5- [3H] hydorxytryptamine uptake were seen in this region. Systemic treatment of newborn rats with .5,7-dihydroxytryptamine (100 mg/kg subcutaneous) known to act preferentially on S-hydroxytryptamine neurons (39) led to an approximately 50% reduction of 5- [3H]hydroxytryptamine uptake in the cerebral cortex and the changes were similar in 7day-old and adult rats (Fig. 4). In agreement with previous studies (39), this treatment was also found to increase 5- [ 3H] hydroxytryptamine uptake
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NEURON
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by about 50% in the pons-medulla (data not shown). The effects of 5,7dihydroxytryptamine on 5-hydroxytryptamine neurons are thus analogous to those of 6-hydroxydopamine on [SH]noradrenaline uptake. The 5,7dihydroxtryptamine treatment was furthermore observed to produce a small reduction of [3H]noradrenaline uptake in the cerebral cortex which, however, could be completely counteracted by desipramine pretreatment. Desipramine had no effect on the 5- [3H]llydroxytryptamine uptake. The action of 5,7-dihydroxytryptamine is thus selective for 5-hydroxytryptamine neurons when the animals were pretreated with desipramine [cf. (39) ] which is known to have a preferential action on the uptake mechanism in noradrenaline neurons ( 17). The neonatal 5,7-dihydroxytryptamine had no or minute changes in [SH]amine uptake in the neostriatum (Fig. 4). DISCUSSION Measurement of the initial rate of [3H]monoamine uptake was used in the present study to quantitate 6-hydroxydopamineor 5,7-dihydroxytryptamine-induced changes in the relative number of monoamine nerve terminals or nerve density. This technique is based on the assumption that the number of uptake sites is constant per unit area of the axonal membrane. In view of the fact that the high-affinity mechanism of growing and developing monoamine neurons has the same kinetic and pharmacological properties as that of adult mature nerves (3, 18, 29, 37), it can be considered that observed changes in [ 3H] monoamine uptake quantitatively reflect alterations in the density of monoamine nerve terminals. The general experience from this laboratory is also that the technique is a reliable method for quantitation of the relative number of monoamine nerve terminals. The present results show two consistent features after the treatments with monoamine neurotoxins. First, in regions where the treatments led to permanent denervations (cerebral cortex and neostriatum), it was observed that the relative reductions in [3H]monoamine uptake were quantitatively the same 6 or 7 days after the neurotoxin administration compared to the results obtained from animals which had reached the adult stage. Previous studies have shown that the neurotoxic actions of 6-hydroxydopamine and 5,7-dihydroxytryptamine are completed within 2 to 6 hr after their administration to newborn rats (37, 39). Second, a change in the [aH]amine uptake in a particular type of monoamine neuron after neurotoxin treatment was never accompanied with a systematic alteration of [ SH]amine uptake in another type of monoamine neuron. To exemplify these general observations it may be of relevance to discuss the results obtained from the cerebral cortex. It was thus observed in this region that the relative reductions in [SH] noradrenaline uptake
810
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JONSSON
FIG. 5. Schematic representation of the changes which noradrenaline neurons of the locus coerzclens may undergo during the postnatal development following a systemic injection of 6-hydroxydopamine at birth. The upper drawing (1.) shows the normal situation. The perikarya are localized in the nucleus locz~ coerzrle~s in the pons. aDistant nerve terminal projections (e.g., in the cerebral cortex) ; b-projections closer to the cell bodies (e.g., in the pons-medulla). The neonatal 6-hydroxydopamine treatment in the lower drawing (2.) produces permanent degeneration of a substantial number of nerve terminals in a (represented by neurons 1 and 2) leading to a compensatory sprouting and outgrowth of nerve terminals in b. The unaffected neurons (represented by neuron 3) has an unaltered postnatal development.
induced by 6-hydroxydopamine (administered systemically or intracranially) were very marked (70 to 90% decrease) and similar after the two time intervals investigated, in spite of the fact that the total cortical tissue masshad increased three to four times at the later time interval. Quite analogous changes were seen on 5- [3H] hydroxytryptamine uptake after desipramine + 5,7-dihydroxytryptamine, a treatment selectively affecting 5hydroxytryptamine neurons (39). These results would then imply that the 6-hydroxydopamine and 5,7-dihydroxytryptamine treatments cause a permanent denervation of a certain number of noradrenaline and S-hydroxytryptamine nerve terminals, respectively, and that the nerves which are spared undergo an apparently normal development with respect to the quantity of nerve terminal arborization (Fig. 5). The spared nerves thus seemto develop normally in spite of the fact that they should theoretically be exposed to increased numbers of vacated synaptic sites. Therefore it seemslikely that the types of denervations used in the present study are
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not associated with any substantial “collateral sprouting” of the nerves not damaged by the neurotoxin, although the present data cannot exclude that this occurs locally to a certain extent. The results, rather, point to a strict regulation of the developmental program that the monoamine neurons are designated to perform. This might be related to the early development in gestation of mechanisms involved in the stability of the brain’s internal environment (41). In line with the view of a strict regulation of the developmental program for the monoamine neurons were the findings that the 6-hydroxydopamineinduced changes of [ 3H ] noradrenaline uptake in noradrenaline nerve terminals in the cerebral cortex and pons-medulla were in no case associated with a change in 5- [ 3H] hydroxytryptamine uptake or desipramine-resistant uptake of [ 3H]noradrenaline (reflecting uptake in dopamine nerve terminals) . The .5-hydroxytryptamine and dopamine nerve terminal networks thus seem to undergo a normal development in situations where the noradrenaline network is markedly changed (cf. 46). These results as well as those obtained after 5,7-dihydroxytryptamine indicate that there is little or no interaction between growing monoamine nerves during the postnatal development. The results from phenylethanolamine N-methyltransferase determinations in the pons-medulla are also consistent with this view. It should be kept in mind, however, that the present data can only be interpreted from a structural point of view and therefore do not preclude the existence of a functional balance or interaction between the growing monoamine neurons. In the pons-medulla, the monoamine cell body-containing region, the situation is somewhat more complex, because the systemic treatment with neurotoxin led to selective increases in [ 3H] noradrenaline and 5- [3H]hydroxytryptamine uptake, depending on the type of neurotoxin administered. These changes have previously been analyzed (18-20, 36-39) and have been interpreted to be due mainly to a “pruning effect” (9, 42) (Fig. 5). The permanent denervation in the forebrain leads to a compensatory sprouting and increased outgrowth of nerve terminals in the pons-medulla in projections close to the cell bodies, which survive the neurotoxin treatment (Fig. 5). It has been shown that systemic 6-hydroxydopamine treatment in the neonate stage has a specific action on the locus coerzrleus noradrenaline system which is highly collateralized, having nerve terminal projections to several brain regions. Although this treatment leads to very marked regional changes in noradrenaline nerve density, it has been found that the total number of noradrenaline nerve terminals in the central nervous system is very little affected, again pointing to a strict regulation of the developmental program, especially with respect to the quantity of nerve terminal aborization.
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REFERENCES P. C., and W. B. QUAY. 1969. 5-Hydroxytryptamine metabolism in early embryogenesis, and the development of brain and retinal tissues. A review. Bra& Res. 12: 273-295. CORRODI, H., and G. JONSSON. 1967. The formaldehyde fluorescence method for the histochemical demonstration of biogenic monoamines-A review of the methodology. J. Histochem.. Cyfochem 15 : 65-78. COYLE, J. T., and J. AXELROD. 1971. Development of the uptake and storage of L-3H-norepinephrine in the rat brain. J. Nrurochenr. 18: 2061-2075. COYLE, J., and J. AXELROD. 1972. Tyrosine hydroxylase in rat brain: Developmental characteristics. 1. Nellrorhef?l. 19 : 1117-1123. COYLE, J., and J. AXELROD. 1972. Dopamine-j3-hydroxylase in the rat brain: Developmental characteristics. J. Nez~~ochern. 19 : 449-459. COYLE, J. T., and D. HENRY. 1973. Catecholamines in fetal and newborn rat brain. J. Neurochem. 21: 61-67. DEGUCHI, T., and J. BARCHAS. 1971. Inhibition of transmethylations of biogenic amines by S-adenosylhomocysteine. J. Biol. Chew. 246 : 3175-3181. 1975. Selective deafferentaDESCARRIES, L., A. BEAUDET, and J. DECHAMPLAIN. tion of rat neocortex by destruction of catecholamine neurons with intraventricular 6-hydroxydopamine, pp. 101-106. Itt “Chemical Tools in Catecholamine Research,” Vol. I. G. Jonsson, T. Malmfors, and Ch. Sachs [Eds.]. North-Holland, Amsterdam. DEVOR, M., and G. E. SCHNEIDER. 1975. Neuroanatomical plasticity: The principle of conservation of total axonal arborization, pp. 191-200. In “Aspects of Neural F. Vital-Durand and M. Jennerod [Eds.]. Editions INSERM, Plasticity.” Paris. FALCK, B., K.-A. HILLARP, G. THIEME, and A. TOKP. 1962. Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. HistoCkCWl. Cytoch~l. 10 : 348-354. FUXE, K., B. HAMBERGER, and T. HORFELT. 1968. Distribution of noradrenaline nerve terminals in cortical areas of the rat. Brair~ Res. 8: 125-131. FUXE, K., T. HGKFELT, G. JONSSON, and U. UNGEKSTEDT. 1970. Fluorescence microscopy in neuroanatomy, pp. 275-314. Ilt “Contemporary Research in Neuroanatomy.” W. J. H. Nauta and S. 0. E. Ebbesson [Eds.]. Springer-Verlag, Berlin/Heidelberg/New York. GOLDEN, G. S. 1972. Embryological demonstration of a nigro-striatal projection in the mouse. Brais Res. 44: 278-282. GOLDEN, G. S. 1973. Prenatal development of biogenic amine systems of the mouse brain. Dev. Biol. 33: 300-311. HAMBERGER, B. 1967. Reserpine-resistant uptake of catecholamines in isolated tissues of the rat. Acta Physiol. Scad. [Suppl. 2951 : l-64. HOKFELT, T., K. FUXE, 0. JOIIANSSON, and I%. LJUXWAHI.. 1974. Pharmacohistochemical evidence of the existence of dopamine nerve terminals in the limbic cortex. Eur. J. Phamacol. 25 : 108-112. JONSSON, G., K. FUXE, B. HAMBERGER, and T. H~KFELT. 1969. 6-Hydroxytryptamine-A new ,tool in monoamine fluorescence histochemistry. Bvairz Rcs. 13 : 190-195. JONSSON, G., CH. PYCOCK, K. FUXE, and CH. SACHS. 1974. Changes in the development of central noradrenaline neurons following neonatal administration of 6-hydroxydopamine. J. Nr~trockcn~. 22 : 419-426. JONSSON, G.; CH. PYCOCX, and CH. SACHS. 1973. Plastic changes of central
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noradrenaline neurons after 6-hydroxydopamine, pp. 459-461. In “Frontiers in Catecholamine Research.” E. Usdin and S. Snyder [Eds.]. Pergamon Press, New York. JONSSON, G., and CH. SACHS. 1976. Regional changes in *H-noradrenaline uptake, catecholamines and catecholamine synthetic and catabolic enzymes in rat brain following neonatal 6-hydroxydopamine treatment. Med. Biol. 54 : 286-297. LAUDER, J. M., and F. E. BLOOM. 1974. Ontogeny of monoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat. I. Cell differentiation. I. Comb. Neural. 155: 469-481. LAUDER, J. M., and F. E. BLOOM. 1975. Ontogeny of monoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat. II. Synaptogenesis. J. Camp. Neural. 163: 251-264. LIDBRINK, P., and G. JONSSON. 1974. Noradrenaline nerve terminals in the cerebral cortex-Effects on noradrenaline uptake and storage following axonal lesion with 6-hydroxydopamine. J. Newochem. 22 : 617-626. LOIZOU, L. A. 1972. The postnatal ontogeny of monoamine-containing neurones in the central nervous system of the albino rat. Bruin Res. 40: 395-418. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL. 19.51. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 26.5-275. MILLER, G. L. 1959. Protein determination for large numbers of samples. Anal. Chem. 31: 964. MIRKIN, B. L. 1972. Ontogenesis of the adrenergic nervous system: Functional and pharmacologic implications. Fed. Proc. 31 : 65-73. NOBIN, A., and A. BJ~RKLUND. 1973. Topography of monoamine neuron systems in the human brain as revealed in fetuses. Acta PhysioE. Stand. [Suppl. 3881 : l-40. NOMURA, Y., F. NAITOH, and T. SEGAWA. 1976. Regional changes in monoamine content and uptake of the rat brain during postnatal development. Brain Res. 101: 305-31s. OLSON, L., L. 0. BOREUS, and A. SEIGER. 1973. Histochemical demonstration and mapping of S-hydroxytryptamine and catecholamine-containing neuron systems in the human fetal brain. 2. Anat. Entzoicklzlngsgesch. 139: 259-282. OLSON, L., and hi. SEIGER. 1972. Early prenatal ontogeny of central monoamine neurons in the rat : Fluorescence histochemical observations. Z. Anat. Entwicicklungsgesch. 137 : 301-316. OLSON, L., and U. UNGERSTEDT. 1970. A simple high capacity freeze-drier for histochemical use. Histochemie 22 : 8-19. PALKOVITZ, M., M. BROWNSTEIN, J. M. SAAVEDRA, and J. AXELROD. 1974. Norepinepbrine and dopamine content of hypothalamic nuclei of the rat. Brain Res. 77 : 137-149. PUJOL, J. F., J. P. KAN, M. BUDA, F. PETITJEAN, J. MOUSET, and M. JOUVET. 1975. Is 6-hydroxydopamine a specific tool for the study of functional roles of catecholaminergic neurons in the sleep-waking cycle? pp. 259-271. 1~ “Chemical Tools in Catecholamine Research,” Vol. I. G. Jonsson, T. Malmfors, and Ch. Sachs, [Eds.]. North-Holland, Amsterdam. SACHS, CH., and G. JONSSON. 1972. Degeneration of central noradrenaline neurons after 6-hydroxydopamine in newborn animals. Res. Commun. Chem. Pathol. Pharmacol.
4 : 203-220.
36. SACHS, CH., CH. PYCOCK, and G. JONSSON. 1974. Altered development of central noradrenaline neurons during ontogeny by 6-hydroxydopamine. Med. Biol. 52 : 55-65.
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37, SACHS, CH., and G. JONSSON. 197.5. Effects of 6-hydroxydopamine on central noradrenaline neurons during ontogeny. Brain Res. 99: 277-291. 38. SACHS, CH., and G. JONSSON. 1975. Changes in the development of central noradrenaline neurons following neonatal 6-OH-DA administration, pp. 163171. In “Chemical Tools in Catecholamine Research,” Vol. I. G. Jonsson, T. Malmfors, and Ch. Sachs [Eds.]. North-Holland, Amsterdam. 39. SACHS, CH., and G. JONSSON. 1975. 5,7-Dihydroxytryptamine induced changes in the postnatal development of central 5-hydroxytryptamine neurons. Med. Biol. 53 : 156164. 40. SAMANIN, R., and S. GARATTINI. 1976. The serotonergic system in the brain and its possible connections with other aminergic systems. Life Sci. 17: 1201-1210. 41. SAUNDERS, N. R., and M. W. B. BRADBURY. 1973. The development of the internal environmem of the brain, pp. 93-109. Zrt “Fetal Pharmacology.” L. Boreus [Ed.]. Raven Press, New York. 42. SCHNEIDER, G. E., and S. R. JHAVERI. 1974. Neuroanatomical correlates of spared or altered function after brain lesions in the newborn hamster, pp. 65 109. In “Plasticity and Recovery of Function in the Central Nervous System.” D. G. Stein, J. J. Rosen, and N. Butters [Eds.]. Academic Press, New York. 43. SEIGER, A., and L. OLSON. 1973. Late prenatal ontogeny of central monoamine neurons in the rat: Fluorescence histochemical observations. 2. Anat. En&t&kZnngsgesch. 140 : 281-318. 44. SMITH, R. D., B. R. COOPER, and G. R. BREESE. 1973. Growth and behavioural changes in developing rats treated intracisternally with 6-hydroxydopamine : Evidence for involvement of brain dopamine. J. Plzar~rzacoZ. 185 : 609-619. 45. SVENDGAARD, N. A., A. BJ~RKLUND, and U. STENEVI. 1975. Regenerative properties of central monoamine neurons. A&r. Anat. E~rrOryol. Cell Biol. 51 (Fast. 4) : l-77. 46. TASSIN, J. P., L. VELLEY, L. STIKUS, G. BLANC, J. GLOWINSKI, and A. M. THIERRY. 197.5. Development of cortical and nigro-neostriatal dopaminergic systems after destruction of central noradrenergic neurons in foetal or neonatal rats. Brain Res. &3: 93-106. 47. TENNYSON, V. M., R. E. BARRETT, G. COHEN, L. COTE, R. HEIKKILA, and C. MYTILINEOU. 1972. The developing neostriatum of the rabbit: Correlation of fluorescence histochemistry, electron microscopy, endogenous dopamine levels and ‘H-dopamine uptake. Brain Res. 46: 251-285. 48. TENNYSON, V. M., C. MYTILINEOU, and R. E. BARRETT. 1973. Fluorescence and electron microscopic studies of the early development of the substantia nigra and area ventralis tegmenti in the fetal rabbit. J. Co@. Nenrol. 149 : 233-258. 49. THIERRY, A. M., L. STINUS, G. BLANC, and J. GLOWINSKI. 1973. Some evidence for the existence of dopaminergic neurons in the rat cortex. Braiu Rcs. 50: 230-234.
NEUROLOGY
Developmental Neurons
53, 801-814 (1976)
Characteristics of Central Monoamine and Their Reciprocal Relations G. JONSSONl
Department
of Histology,
Karolinska Received
Institutet, July
S-104
01 Stockholm,
Sweden
7,1976
The effects of neonatal administration of the monoamine neurotoxins 6hydroxydopamine and 5,7-dihydroxytryptamine on the postnatal development of monoamine nerve terminals in various regions of rat brain have been investigated. 6-Hydroxydopamine is known to induce a selective degeneration of catecholamine neurons, and 5,7-dihydroxytryptamine has a preferential action on 5-hydroxytryptamine neurons. Monoamine nerve density was monitored by measuring the in vitro uptake of [‘Hlmonoamines in brain tissue homogenates, combined with pharmacological analysis and fluorescence histochemistry. It was consistently observed that the relative reductions of [‘HImonoamine uptake in the cerebral cortex and neostriatum were quantitatively the same 6 to 7 days after the neurotoxin treatment compared with the situation when the uptake analysis was performed on tissues from adult rats. These results indicate that the neurotoxin in these regions produces damage and permanent denervation of a certain number of monoamine nerve terminals, whereas spared nerves undergo an apparent normal development. It was confirmed that the marked and permanent reduction in [aH]noradrenaline uptake in the cerebral cortex induced by systemic 6-hydroxydopamine treatment at birth was associated with a considerable increase in [“Hlnoradrenaline uptake in the pons-medulla. This treatment had no effects on uptake in 5-hydroxytryptamine and dopamine nerve terminals. Analogous changes with respect to 5-[‘Hlhydroxytryptamine uptake was observed after neonatal 5,7-dihydroxytryptamine treatment. These plastic changes are most likely due to a “pruning effect” induced by the neurotoxins. The activity of phenylethanolamine N-methyltransferase, a marker of adrenaline neurons, developed normally, independent of the changes in noradrenaline neurons induced by 6-hydroxydopamine. The present results favor the view that the postnatal development of monoamine nerve terminals is strictly ordered. Furthermore, the neurons exhibit an apparently high degree of intrinsic growth regulation, especially with respect to quantity of nerve terminal arborization. There was no evidence for any interaction between growing monoamine neurons. 1 The present study has been supported by the Swedish Medical (04X-2295), Nathorsts, Groschinskys and Bergvalls Stiftelse. 801
Copyright 1976 by Academic Press, Inc. rights o9 reproduction in my formreserved.
All
Research Council
802
G.
JONSSON
INTRODUCTION Central monoamine neurons have been shown to appear relatively early in gestation in several species including humans (13, 14, 21, 22, 28, 30, 31, 47, 48). These neuronal structures have thus been reported to appear first on day 12 to 14 during gestation in the mouse, rat, and rabbit. Biochemical studies have also provided evidence for the presence of monoamine neurons early in the development (1, 3-6, 27). The monoamine cell body groups are fully developed at birth (43) whereas the arborization of the nerve terminal projections occurs to a large extent after birth (24). Although there is evidence suggesting a functional balance between certain monoamine neuron systems in the adult stage (34, 40) and Svendgaard et al. (45) have reported data pointing to a considerable interaction between regrowing central monoamine fiber systems into iris transplants, very little is known at present about any interaction between growing monoamine fibers during development. The present study was therefore undertaken to elucidate the interaction possibility by the use of the monoamine neurotoxins 6-hydroxydopamine (6-OH-DA) and 5,7-dihydroxytryptamine (5,7-HT), which can be utilized to induce selective degenerations of monoamine neurons in newborn rats (19, 35, 36, 39). MATERIAL
AND
METHODS
Sprague-Dawley rats were used and were housed in an air-conditioned room with controlled temperature and a standarized dark-light schedule (14/10 hr, light on 6: 00 AM, and off 8: 00 PM). Each litter contained not more than eight pups which were separated at 21 days of age. Three types of experiments were carried out : (i) newborn rats treated with 6-hydro.rydopam& (2 X 100 mg/kg, 24-hr interval) administered subcutaneously in a volume of 0.05 ml 0.9% NaCI, the first dose being given within 4 hr after birth, (ii) 5-day old rats treated with 100 pg 6-hydroxydopamine administered intracisternally in a volume of 10 ~1 0.9% NaCl (44) ; and (iii) newborn rats treated with 5,7-dihydroxytryptanaine (100 mg/kg, subcutaneous) administered in a volume of 0.05 ml 0.9% NaCl. The rats in the control groups received an equal volume of the vehicle. In groups (ii) and (iii), animals pretreated with the uptake blocker desipramine (20 mg/kg, subcutaneous) 30 min before the administration of the manoamine neurotoxin were included in the experiment. No separation between male and female rats were made, and both sexes were used randomly in the same experiment. The animals were killed 6 to 7 days or 6 to 7 weeks after the treatment by cervical dislocation under light chloroform anesthesia. The brains were rapidly removed and placed in beakers with cold Krebs-Ringer buffer. The various brain regions were dissectedout for further analysis. [SH]Monoamine Uptake Measurements. The brain tissue was homoge-
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nized in 10 volumes (w/v) cold 0.3 M sucrose and the homogenates were centrifuged at 1OOOg for 10 min in a refrigerated centrifuge (Sorval RCZB) . The supernatant solutions (containing monoamine synaptosomes) were taken for subsequent in vitro uptake studies. Aliquots (100 ~1) of the supernatant solutions were added to 1.9 ml Krebs-Ringer bicarbonate buffer (pH 7.4, containing 1.l mM ascorbic acid and 0.01 rnM pargyline) in centrifuge tubes and preincubated at +37”C for 5 min. Thereafter 10 pl [3H]monoamine (0.05 PM, final concentration) was added and the incubation continued for another 5 min. Incubation was terminated by adding 8 ml ice-cold Krebs-Ringer buffer, and the tubes were centrifuged at 10,OOOg for 10 min. Radioactivity was extracted from the synaptosome pellets with 1 ml absolute ethanol and determined by liquid scintillation spectrometry [for details, see ( 18) 1. Quenching was monitored by an internal standard procedure using E3H]toluene. Each homogenate was analyzed in triplicate, both at +37”C and O”C, and means were calculated. In experiments determining [ 3H] dopamine and 5- [ 3H] hydroxytryptamine uptake, the uptake values at 0°C were subtracted from those obtained at +37”C to correct for “extraneuronal” uptake (a “active uptake”). In experiments analyzing [ 3H] noradrenaline uptake, homogenates were in addition analyzed in triplicate at +37’C with 50 PM desipramine in the incubation medium. These latter values were subtracted from those obtained at +37”C to a measure [3H]noradrenaline uptake in noradrenaline nerve terminals. The desipramine values after subtraction of the 0°C uptake values were considered to reflect [3H]noradrenaline uptake in dopamine nerve terminals. Desipramine is known to block amine uptake in noradrenaline but not in dopamine nerve terminals, whereas the transport mechanism of both neuron types is completely inhibited at 0°C (15). The [3H] amine uptake was expressed as nanocuries per milligram of protein, which was determined on aliquots of the 1OOOg supernatant solution according to Lowry et al. (25) as modified by Miller (26). Cathecholamine Assay. The brain tissue was homogenized in 20 to 200 volumes (w/v) cold 0.1 M perchloric acid. After centrifugation, 300 ~1 of the supernatant solution was taken for noradrenaline and dopamine assay according to the radioenzymatic technique of Coyle and Henry (6) and Palkovitz et al. (33). Endogenous noradrenaline and dopamine were expressed as nanograms per gram wet weight of tissue, calculated on the basis of internal standard measurements. Phenylethanolamine N-Methyltransferase (PNMT) Assay. The transferase activity was assayed using a modification of the procedure of Deguchi and Barchas (7) as described previously (20). The tissue was homogenized in 5 to 10 volumes (w/v) distilled water and the homogenates centrifuged at 100,OOOg for 60 min. One hundred microliters of the supernatant solution was then taken for assay using phenylethanolamine (2.4 mM) as substrate
804
G.
JONSSON
and S- [nzetlzylJ*C] adenosyl-1-methionine (2 nmol) as cofactor. Enzyme activity was expressed as nanomoles of product formed per gram per hour. Flourescence HistoclaeuListry. Brain slices (thickness, about 0.5 mm) were incubated in eifro in Krebs-Ringer bicarbonate buffer (pH 7.4) containing 1 or 5 pM noradrenaline or G-hydroxytryptamine for 20 min at +37”C. After a brief rinse in cold buffer, the slices were processed according to the Falck-Hillarp technique for the histochemical demonstration of monoamines (2, 10, 12, 32 ) . Drugs and Substances lised. [ 3H] noradrenaline. HCl (6-12 Ci/mmol) ; [ 3H] dopamine . HCI (2.4 C/1 mmol) ; 5- [ 3H] hydroxytryptamine creatinine sulfate (16 Ci/mmol) ; 3H- and [‘“Cl methyl-labeled S-adenosyl-l-methionine (7.2 and 0.055 Ci/mmol ; Radiochemical Centre, Amersham) ; Ghydroxydopamine. HCl (888/32, AB Hassle, Giiteborg, noradrenaline 9HCl (Sigma) ; 6-hydroxytryptamine creatinine sulfate (AB HBssle, Giiteborg) ; desipramineeHC1 (Pertofran, Geigy) ; chlorimipramine. HCl (Geigy). RESULTS In agreement with previous studies (l&20, 35-38)) neonatal 6-hydroxydopamine treatment (2 x 100 mg/kg, subcutaneous) produced a veq pronounced reduction in [ 3H] noradrenaline uptake in noradrenaline nerve terminals of the cerebral cortex (Fig. 1). The relative decrease (about 30% of littermate control) was the same for the 6-day-old and the adult rats, although the latter animals had a total cortical tissue mass which was three to four times larger than that of the younger animals. The desipramine-resistant uptake of [3H] noradrenaline, most likely representing uptake in dopamine nerve terminals (16, 49) was unchanged in the cerebral cortex in both groups of animals. 5- [ 3H] hydroxtryptamine uptake was similarly unchanged after the 6-hydroxydopamine treatment (Fig. 1). Fluorescence histochemical analysis (according to Falck and Hillarp) of sectioned slices from the cerebral cortex showed that the neonatal 6hydroxydopamine treatment produced an almost complete disappearance of the catecholamine nerve terminals normally present (11). Some single catecholamine terminals could be detected. The standard Falck-Hillarp procedure used can only demonstrate noradrenaline nerve terminals in the cerebral cortex (23). In vitro incubation of cortical slices in 1 to 5 PM noradrenaline for 20 min at +37”C did not restore the network of catecholamine nerve terminals, although some additional terminals could be observed. However, incubation of slices from 6-hydroxydopamine-treated animals in 1 or 5 PM 6-hydroxytryptamine resulted in the apearance of a dense network of fine yellow-green fluorescing nerve terminals due to uptake and accumulation of 6-hydroxytryptamine, (Fig. 2), in all probability representing 5-hydroxytryptamine nerve terminals (17). This net-
CENTRAL
MONOAMINE
CEREBRAL
NEURON
DEVELOPMENT
805
CORTEX
PONS
MEDULLA
NEOSTRIATUM
%+DA
3H5HT
‘H-NA
3H 5 HT
FIG. 1. Effects of neonatal 6-hydroxydopamine treatment (2 X 100 mg/kg, subcutaneous, 24-hr interval) on [‘HIamine uptake (0.05 PM, 5 min) in homogenates from the cerebral cortex, neostriatum, and pons-medulla. The animals were killed 6 days and 6 weeks (adult) after treatment. Each column represents the mean f the standard error of four or five determinations, expressed as a percentage of littermate control. ‘DA’-desipramine-resistant uptake of [“HI noradrenaline, most likely reflecting uptake in dopamine nerve terminals (see Material and Methods).
work was considerably denser than that of catecholamine nerve terminals seen in slices from untreated control animals with or without incubation in noradrenaline [ c.f. (8) 1. The uptake-accumulation of 6-hydroxytryptamine could be almost completely blocked by including a 50 PM concentration of the 5-hydroxytryptamine uptake blocker chlorimipramine in the incubation medium. The systemic 6-hydroxydopamine treatment at birth had no effect on the [3H] amine uptake in vitro in the neostriatum (nucleus caudatus putamen) (Fig. 1). The samewas the case for 5- [ 3H] hydroxytryptamine in the pons and medulla oblongata, although the [ 3H]noradrenaline uptake in this region was markedly increased in the adult stage (Fig. 1) . The biochemical analysis of tissue from adult animals was in complete accordance with the [ 3H]amine uptake data, showing about an 80% (18 + 4% of control ; N = 4) reduction of the endogenous noradrenaline in the cerebral cortex after 6-hydroxydopamine without affecting endogenous dopamine (95 * 4% of control ; N = 4). Control values for cerebral cortex were noradrenaline ; 250 + 12 rig/g wet weight of tissue (N = 4) and dopamine, 48 f 5 rig/g (N = 3). Endogenous dopamine was unchanged in the nucleus
FIG. 2. Fluorescence histochemical demonstration of uptake and accumulation of 6-hydroxytryptamine in nerve terminals of the cerebral cortex after 6-hydroxydopamine treatment (2 X 100 mg/kg, subcutaneous, as in Fig. 1) at birth. The animals were killed 8 weeks after birth. A-Dense network of nerve terminals seen after in vitro incubation in 1 pM 6-hydroxytryptamine for 20 min. in all probability representing S-hydroxytryptamine nerve terminals. B-Same conditions as in A except that 50 PM chlorimipramine was present in the incubation medium. X160.
CENTRAL
MONOAMINE
CEREBRAL 100. %
807
NEURON DEVELOPMENT
CORTEX
- -- - - - -
SO-
o-
6OH-DA
DMI* SO&DA
@OH-DA
3H-NA
DMI* 6.0~DA
DA
Dl$ 6,Oli-DA
,
3H.5-HT
‘DA’
1 NEOSTRIATUM
6.0H-
,
50.
Od
6-OH-DA
-XL----
DMI 6.OiLDA
O-OH-DA
DMI s.o&DA
JH-5-HT
FIG. 3. Effects of intracranial administration of 6hydroxydopamine (100 pg in 10 ~1) to S-day-old rats with or without desipramine pretreatment (20 mg/kg, subcutaneous) on rH]amine uptake in homogenates from the cerebral cortex and neostriatum. The animals were killed 7 days or 6 weeks (adult) after the injection. Each column represents the mean f the standard error four or five determinations, expressed as a percentage of untreated littermate control.
caudatus putamen (104 -C 5% of control, N = 4 ; control value 7.1 f 0.6 pg/g), whereas noradrenaline was markedly increased in the pons-medulla (168 + 157o of control; 720 * 59 rig/g). The phenylethanolamine Nmethyltransferase activity, marker of adrenaline neurons, in this latter region was also unaltered after neonatal 6-hydroxydopamine treatment in both groups of animals : 98 Ifr 8% (N = 4) of littermate control at 6 days and 103 * 9% (N = 4) of control in adults. In another set of experiments, 5-day-old rats were treated with 100 pg 6-hydroxydopamine administered intracranially (44) and [3H] amine uptake analyzed in the cerebral cortex and neostriatum 5 days and 6 weeks afterward (Fig. 3). The results show that this treatment led to a very pronounced decrease in [3H]noradrenaline uptake (about 10% of control) in the cerebral cortex, .and the relative decrease was quantitatively similar for both age groups studied. The desipramine-resistant (“dopamine”) and 5- [ 3H] hydroxytryptamne uptakes were unchanged. The reduction in [ 3H] noradrenaline uptake could be completely abolished by pretreatment of the
808
G.
CEREBRAL
JOXSSOS
CORTEX
50-
o-
5.7.HT
5.7.HT
DMI + 57.HT
DMI 5.7.+HT
5.7.HT
DMI $7.+H T
‘DA’
5.7 HT L
DMI &H T
----
3ti-
DA
5.7. HT L-
~~ ~~~
DMI 5.7fHT -
3H.5-HT
FIG. 4. Effects of systemic treatment of newborn rats with 5,7-dihydroxytryptaminc (100 mg/kg, subcutaneous) on [“HIamine uptake in homogenates from the cerebral cortex and neostriatum. One group of animals was pretreated with desipramine (20 mg/kg, intraperitoneal) 30 min before the injection of 5,7-dihydroxytryptamine. The rats were killed 7 days or 7 weeks afterward. Each column represents the mean -C the standard error of four to six determinations, expressed as a percentage of untreated littermate control.
rats with the uptake blocker, desipramine. This treatment had practically no effect on the desipramine-resistant uptake of [ 3H] noradrenaline and on the 5- [3H] hydroxytryptamine uptake as well. The intracranial 6hydroxydopamine treatment also led to a marked decrease in [3H]dopamine uptake in the neostriatum which was quantitatively similar for both experimental groups. Consistent with the view of clesipramine not affecting uptake in dopamine neurons (15)) desipramine pretreatment did not modify the 6-hydroxydopamine-induced changes in [ 3H] dopamine uptake. No effects on 5- [3H] hydorxytryptamine uptake were seen in this region. Systemic treatment of newborn rats with .5,7-dihydroxytryptamine (100 mg/kg subcutaneous) known to act preferentially on S-hydroxytryptamine neurons (39) led to an approximately 50% reduction of 5- [3H]hydroxytryptamine uptake in the cerebral cortex and the changes were similar in 7day-old and adult rats (Fig. 4). In agreement with previous studies (39), this treatment was also found to increase 5- [ 3H] hydroxytryptamine uptake
CENTRAL
MONOAMINE
NEURON
DEVELOPMENT
809
by about 50% in the pons-medulla (data not shown). The effects of 5,7dihydroxytryptamine on 5-hydroxytryptamine neurons are thus analogous to those of 6-hydroxydopamine on [SH]noradrenaline uptake. The 5,7dihydroxtryptamine treatment was furthermore observed to produce a small reduction of [3H]noradrenaline uptake in the cerebral cortex which, however, could be completely counteracted by desipramine pretreatment. Desipramine had no effect on the 5- [3H]llydroxytryptamine uptake. The action of 5,7-dihydroxytryptamine is thus selective for 5-hydroxytryptamine neurons when the animals were pretreated with desipramine [cf. (39) ] which is known to have a preferential action on the uptake mechanism in noradrenaline neurons ( 17). The neonatal 5,7-dihydroxytryptamine had no or minute changes in [SH]amine uptake in the neostriatum (Fig. 4). DISCUSSION Measurement of the initial rate of [3H]monoamine uptake was used in the present study to quantitate 6-hydroxydopamineor 5,7-dihydroxytryptamine-induced changes in the relative number of monoamine nerve terminals or nerve density. This technique is based on the assumption that the number of uptake sites is constant per unit area of the axonal membrane. In view of the fact that the high-affinity mechanism of growing and developing monoamine neurons has the same kinetic and pharmacological properties as that of adult mature nerves (3, 18, 29, 37), it can be considered that observed changes in [ 3H] monoamine uptake quantitatively reflect alterations in the density of monoamine nerve terminals. The general experience from this laboratory is also that the technique is a reliable method for quantitation of the relative number of monoamine nerve terminals. The present results show two consistent features after the treatments with monoamine neurotoxins. First, in regions where the treatments led to permanent denervations (cerebral cortex and neostriatum), it was observed that the relative reductions in [3H]monoamine uptake were quantitatively the same 6 or 7 days after the neurotoxin administration compared to the results obtained from animals which had reached the adult stage. Previous studies have shown that the neurotoxic actions of 6-hydroxydopamine and 5,7-dihydroxytryptamine are completed within 2 to 6 hr after their administration to newborn rats (37, 39). Second, a change in the [aH]amine uptake in a particular type of monoamine neuron after neurotoxin treatment was never accompanied with a systematic alteration of [ SH]amine uptake in another type of monoamine neuron. To exemplify these general observations it may be of relevance to discuss the results obtained from the cerebral cortex. It was thus observed in this region that the relative reductions in [SH] noradrenaline uptake
810
G.
JONSSON
FIG. 5. Schematic representation of the changes which noradrenaline neurons of the locus coerzclens may undergo during the postnatal development following a systemic injection of 6-hydroxydopamine at birth. The upper drawing (1.) shows the normal situation. The perikarya are localized in the nucleus locz~ coerzrle~s in the pons. aDistant nerve terminal projections (e.g., in the cerebral cortex) ; b-projections closer to the cell bodies (e.g., in the pons-medulla). The neonatal 6-hydroxydopamine treatment in the lower drawing (2.) produces permanent degeneration of a substantial number of nerve terminals in a (represented by neurons 1 and 2) leading to a compensatory sprouting and outgrowth of nerve terminals in b. The unaffected neurons (represented by neuron 3) has an unaltered postnatal development.
induced by 6-hydroxydopamine (administered systemically or intracranially) were very marked (70 to 90% decrease) and similar after the two time intervals investigated, in spite of the fact that the total cortical tissue masshad increased three to four times at the later time interval. Quite analogous changes were seen on 5- [3H] hydroxytryptamine uptake after desipramine + 5,7-dihydroxytryptamine, a treatment selectively affecting 5hydroxytryptamine neurons (39). These results would then imply that the 6-hydroxydopamine and 5,7-dihydroxytryptamine treatments cause a permanent denervation of a certain number of noradrenaline and S-hydroxytryptamine nerve terminals, respectively, and that the nerves which are spared undergo an apparently normal development with respect to the quantity of nerve terminal arborization (Fig. 5). The spared nerves thus seemto develop normally in spite of the fact that they should theoretically be exposed to increased numbers of vacated synaptic sites. Therefore it seemslikely that the types of denervations used in the present study are
CENTRAL
MONOAMINE
NEURON
DEVELOPMENT
811
not associated with any substantial “collateral sprouting” of the nerves not damaged by the neurotoxin, although the present data cannot exclude that this occurs locally to a certain extent. The results, rather, point to a strict regulation of the developmental program that the monoamine neurons are designated to perform. This might be related to the early development in gestation of mechanisms involved in the stability of the brain’s internal environment (41). In line with the view of a strict regulation of the developmental program for the monoamine neurons were the findings that the 6-hydroxydopamineinduced changes of [ 3H ] noradrenaline uptake in noradrenaline nerve terminals in the cerebral cortex and pons-medulla were in no case associated with a change in 5- [ 3H] hydroxytryptamine uptake or desipramine-resistant uptake of [ 3H]noradrenaline (reflecting uptake in dopamine nerve terminals) . The .5-hydroxytryptamine and dopamine nerve terminal networks thus seem to undergo a normal development in situations where the noradrenaline network is markedly changed (cf. 46). These results as well as those obtained after 5,7-dihydroxytryptamine indicate that there is little or no interaction between growing monoamine nerves during the postnatal development. The results from phenylethanolamine N-methyltransferase determinations in the pons-medulla are also consistent with this view. It should be kept in mind, however, that the present data can only be interpreted from a structural point of view and therefore do not preclude the existence of a functional balance or interaction between the growing monoamine neurons. In the pons-medulla, the monoamine cell body-containing region, the situation is somewhat more complex, because the systemic treatment with neurotoxin led to selective increases in [ 3H] noradrenaline and 5- [3H]hydroxytryptamine uptake, depending on the type of neurotoxin administered. These changes have previously been analyzed (18-20, 36-39) and have been interpreted to be due mainly to a “pruning effect” (9, 42) (Fig. 5). The permanent denervation in the forebrain leads to a compensatory sprouting and increased outgrowth of nerve terminals in the pons-medulla in projections close to the cell bodies, which survive the neurotoxin treatment (Fig. 5). It has been shown that systemic 6-hydroxydopamine treatment in the neonate stage has a specific action on the locus coerzrleus noradrenaline system which is highly collateralized, having nerve terminal projections to several brain regions. Although this treatment leads to very marked regional changes in noradrenaline nerve density, it has been found that the total number of noradrenaline nerve terminals in the central nervous system is very little affected, again pointing to a strict regulation of the developmental program, especially with respect to the quantity of nerve terminal aborization.
812
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