Collateral sprouting and reduced activity of the rat mesocortical dopaminergic neurons after selective destruction of the ascending noradrenergic bundles

Neuroscience Vol. 4. pp. 1569 lo 1582 Pergamon Press Ltd 1979. Printed in Great 0 IBRO

0306-4522/79/l

IOI- I569102.00/0

Britain

COLLATERAL SPROUTING AND REDUCED ACTIVITY OF THE RAT MESOCORTICAL DOPAMINERGIC NEURONS AFTER SELECTIVE DESTRUCTION OF THE ASCENDING NORADRENERGIC BUNDLES J. P. TASSIN, S. LAVIIUE, D. HERW?,G. BLANC,A. M. THIERRY,

C. ALVAREZ,~B. BERGIZR’and J. GLOWINSKI Groupe NB, INSERM U.114, College de France, 11, place Marcelin Berthelot, 75231 Paris cedex 05, France Abstract-The properties of the mesocortical dopaminergic neurons projecting to the pregenual and anterior supragenual cortices were examined 3-6 months after the degeneration of ascending noradrenergic pathways caused by bilateral multiple or single microinjections of Chydroxydopamine made laterally to the pedunculus cerebellaris superior. In all rats and in all cortical areas examined, noradrenaline levels were reduced by more than 75%. A similar decrease in noradrenaline levels was obtained in the ventral tegmental area. As indicated by the increases in cortical levels of dopamine and in C3H]dopamine specific uptake sites as well as by his&hem&al analysis, these lesions induced a collateral sprouting of the mesocortical dopaminergic neurons. The intensity of the elIect varied from one animal to another and even from one anteromedial hemicortex to another. When present, the increase in dopamine levels was observed in all the cortical areas investigated. As suggested by the decreased ratio of the amount of dihydroxyphenylacetic acid to dopamine in the cortex, the activity of the mesocortical dopaminergic neurons was reduced in the rats with lesions. This effect was even seen in rats in which the cortical levels of dopamine were only slightly increased. Both the collateral sprouting and the reduced activity of the mesocortical dopaminergic neurons were related to the degeneration of the noradrenergic neurons and not to a non-specific effect of bhydroxydopamine, since both phenomena did not occur in rats pretreated with desipramine, a treatment which prevented the decline in noradrenaline levels. Thus, a lesion of the ascending noradrenergic pathways can lead to sprouting of dopaminergic neurons in the cortex and a reduced activity of these dopaminergic neurons. The respective role of the disappearance of the noradrenergic innervation in the cerebral cortex and in the ventral @mental area in the collateral sprouting and in the reduced activity of the mesocortical dopaminergic neurons is discussed.

THE PRESNCEof doparninergic

nerve terminals in the also been found in the anteromedial cerebral cortex of cerebral cortex was first demonstrated in biochemical the rat after large unilateral 6-OHDA lesions of the studies using rats with bilateral 6-hydroxydopamine ascending noradrenergic bundles (EMSON& KOOB, (6-OHDA) lesions made laterally to the pedunculus 1978). These effects could be attributed to a reduced cerebellaris superior in order selectively to destroy the activity of the mesocortical dopaminergic neurons ascending noradrenergic pathways (TWRRY, BLANC, and/or to modifications of the cortical dopaminergic HOTEL,STNIS & GL~WINSKI, 1973). This approach innervation. The present study was undertaken to was also selected to facilitate the visualization of the determine more precisely the changes which could dopaminergic arborizations with the his&hem&al occur in the properties of the mesocortical dopafluorescent technique (BERGW, TASSIN, BLANC, minergic neurons projecting to the pregenual and MOYNE& THIERRY, 1974). Surprisingly, in some of anterior supragenual cot&es after the long term dethese experiments, we observed that the number of generation of the ascending noradrenergic neurons in[‘H]dopamine specific uptake sites (TA.w~~,THIERFCY, duced by bilateral 6-OHDA lesions. A preliminary BLANC& GLWINSKI, 1974) and the levels of dopareport of some of the results of this study was premine in the cerebral cortex were higher in rats with sented at the 4th International Catecholamine Symlesions than in control rats (BERGERet al., 1974). posium (TASSIN, LAVIELLE, BLANC, THIEWY & More recently, an increase in dopamine levels haa BEnoER, 1979). ‘INSERM U.134, Laboratoire de Neuropathologie Charles Foix, Hospital Salpfttribe, 75634 Paris cedex 13, France. Abbreufutions: DOPAC, dihydroxyphenylacttic acid; 6-OHDA, dhydroxydopamine. Nsc. 4/l l-c

EXPERIMENTAL PROCEDURES Animalsand surgery Male Spragu*Dawley rats weighing 250-2808 were used. They were kept in a controlled environment (24°C 60% humidity, alternate cycles of 12 h light, 7 a.m.-7 p.m.

1569

1570

J. P.

TASSIN et d.

and darkness) and received food and water ud lihirum. Lesions of the noradrenergic ascending pathways were performed by bilateral microinjections of 6-OHDA made laterally to the pedunculus superior. Rats anaesthetized with sodium pentobarbital were placed in a Stoelting stereotaxic apparatus (Krieg model 51200; incisor bars. 3.4 mm above the interaural line) and single (4 pg in 2 ~1) or multiple (4 x 1~1, 2 p&l) 6-OHDA microinjections were done at the following coordinates: 6 mm posterior to the bregma: l.4mm on either side of the midline and 7.7 mm (single) or 5.3, 6.8. 8.3 and 9.8 mm (multiple) deep under the skull surface. 6-OHDA dissolved in an isotonic saline solution containing ascorbic acid (2 mgiml) adjusted to pH 4 was delivered at a rate of I pl/5 min. Finally, some rats received an intraperitoneal injection of desipramine (20 mg/kg) 30 min before 6-OHDA in order to prevent the effects of 6-OHDA on the noradrenergic ascending pathways (MALMFORS& SACHS, 1968).

Rats were killed by decapitation between I1 a.m. and 12 a.m. 12-16 weeks after the 6-OHDA lesions. Each brain was rapidly removed, split into two parts in a frontal plane which were either frozen in dry ice (for dihydroxyphenylacetic acid (DOPAC), dopamine and noradrenaline determinations) or immediately frozen on sodium chloride (0.9”~) on a Leitz Wetzler microtome stage refrigerated at - 7’C using a Leitz Kryomat (uptake studies) (TASSIN.

ide according to the method of LOWKY, Roswwrwf. FARR & RANDALL(1951). Catechol derivatives were first isolated by adsorption on alumina microcolumns. The eluates were then evaporated to dryness and catechol derivatives were simultaneously transformed into their radioactive methylated derivatives by catechol-O-methyl transferase, using C3H]S-adenosylmethionine (10.2 Ci/mmol, Amersham) as the methyl dorm (GAWHY, TAS~IN,GLOWINSKI& CHERAMY, 1976). Radioactive 3-methoxy-4-hydroxyphenylethylamine. 3-methoxy-4hydroxyphenylethanolamine and homovanillic acid wcrc separated by organic extraction and isolated by chromatography either on paper (methoxyaminest or on silica gci (homovanillic acid), according to a procedure which will be published in detail elsewhere (J. P. TASSIN.S ~..AVIELI.I~. c;. BLANC,D. HERW&A. M. THERRY & J. GI.OWINSKI, unpublished observations). In the conditions used (3 PM [‘HJSadenosyhnethionine) the enzymatic reactton was linear up to at least 4.5 ng of total catechol derivattves and therefore changes in tissue noradrenaline levels did not influence the estimation of dopamine and DOPAC. The initial separation of catechol derivatives by adsorption on alumma avoids the use of internal recov?ery standards. The limit of sensitivity of the technique (twice the blank values) was 40 pg for dopamine. 100 pg for noradrenaline and 60 pg for DOPAC. Uprake

srudies

Uptake studies were carried out as described previously CHERAMY,BLANC, THIERRY& GLOWINSKI, 1976). (TASSIN,BOCKAERT,BLANC, STINUS,THI~RRY, LAV1fd.II.. In the first experiment, the anteromedial cortices of PREMONT& GLOWINSKI,1978). Briefly, five microdiscs were control rats and those with lesions were dissected by hand punched out from each anteromedial hemicortex ot from frontal slices (Fig. I) obtained with a microtome lesioned or control rats (Fig. 5). Each microdisc was blown refrigerated at - fO”C. Each slice (5OOpm thick) was into a 1.5ml Eppendorf plastic tube containing I5 ~1 0.25 M denominated by its rostra1 plane; the beginning of the sucrose. It was homogenized for 10 s with a piston made of striatum at 9800 pm was used as a landmark according to dental cement (Stellon from Detrey) using a I.5 ml Eppenthe atlas of K~NW & KLIPPEL.(1963). dorf tube as a matrix. Then 400 ~1 of a physiological In the second experiment, the anteromedial cortices were medium (in mM: glucose, 5: NaCl, 136: KCI, 5.6; MgCl,, punched out with a cooled stainless tube with an equi1.2: CaCI,, 2.2; and NaHCO,, 16.2) containing in all cases lateral triangular shape (3.7mm side) at its extremity on I.1 mM ascorbic acid, 1.25 x low4 v pargyline and the 10,000 and 9500 Itm slices as described previously 5 x lo-’ M desipramine were added III each homogenate. (LAVIELLE.TASSIN,THIERRY,BLANC, HER&, BARTHELEMY Particular attention was paid to rinsing the piston. & GLOWINSKI,1979). Furthermore, the caudal part of the After a 5 min preincubation at 37°C 10 111of [G-‘H]3,4. ventral tegmental area was dissected by hand from the dihydroxyphenylethylamine, 9.3 Ci/mmol, NEN Chemicals slices corresponding to the planes 2100 and 1600 pm of the (final concentration 6 x lo-so), were added to each atlas of KOENIG & KLIPPEL(1963). The substantia nigra was sample and the incubation was continued for another carefully removed to avoid any contamination. 5 min. The incubation was stopped by cooling the tubes in When uptake studies were performed, tissues were not a water bath at 0°C. Tubes were then centrifuged for frozen below - 7°C (TASSINet al.. 1976). The samples were 30min at 20,OOOg;the sediments were washed twice with punched out from six successive frontal slices as illustrated 400$ of sodium chloride (0.9”/,) and finally suspended in in Fig. 5 either with a cylindric tube (1.4 mm diameter) for 50 pl of Triton X-100 (1%). Radioactivity was measured by the estimation of C3H]dopamine uptake activity or with a transferring this suspension to vials containing 10ml of tube exhibiting an equilateral triangular shape at its exscintillation medium. All blanks were obtained both by tremity (3.7 mm side) for the estimation of catecholamines. adding 10m6M benztropine to the preincubation medium Tissues used for the estimations of dopamine, norand by cooling and centrifuging the samples immediately adrenaline and DOPAC levels were in all cases immersed after the addition of [sH]dopamine (TASSIN et ul.. 1978). in 110 ~1 of a 0.1 N perchloric acid, 0.01 N thioglycolic acid solution and kept frozen at - 20°C. Fluorescence histochemistq Estimations

qf rhr

dih~drox:phenplac,eti~

lervls

qf

dopamine,

noradrenaline

and

wit/

Samples were sonicated with an Annemasse sonifier equipped with a 2.5 mm sonotrode. After centrifugation (2O,C00~, 20min) supernatants were used for the estimation of noradrenaline, dopamine and DOPAC and proteins were determined in pellets dissolved in 1 N sodium hydrox-

The catecholaminergic innervation was visualized on semiserial frontal sections of the whole anterior hemisphere of the brain according to a procedure recently described (BERCSER& GLOWINSKI,1978). Thirty micron thick vibratome sections obtained from a-methylparatyrosine pretreated rats (to deplete the endogenous stores of catecholamines) were incubated in the presence of desipramine (5 x low6 M) and dopamine or noradrenaline (1OMhM\ to

Modifications of cortical dopaminergic neurons after depletion of noradrenaline visualize the dopaminergic fibres, or in the presence of benztropine (lo- s M) and noradrenaline ( low6 M) to visualize the noradrenergic fibres. Sections were then processed for fluorescence microscopy according to the glyoxylic acid/formaNehyde method (BEWER, TIUEWW,TASSIN & MOW, 1976). They were observed with a L&z Huorescence microscope equip@ with the Plam system and coupled to an XY plotter. The outline and the main struo tures of the sections, including the dopaminergic innervated areas, were drawn to allow an easier comparison between rats with lesions and control animals. Four controls and five rats with bilateral single 6-OHDA lesions (survival time 5-6 months) were used.

I

I

1

I

I

1571 I

I

I

Terminology

For the presentation of the data, a topographical description of the parts of the brain under investigation was used (LINDVALL, BJ~~RKLIJND & DIVAC,1978). This seemed more appropriate since the precise delineation of the frontal versus the cingular cortices in the rat brain is still open to discussion. Consequently, the pregenual cortex corresponds to areas punched out from the 12,000 to 9800~ slices whereas the supragenual cortex refers to areas punched out in the following slim. Statistical analysis Results were compared with their respective control values by the Student’s t-test (SNEDE~~~ & COCHRAN, 1967). RESULTS Increase of cortical dopamine levels after destruction of the ascending noradrenergic pathways.

In a first experiment, rats received bilateral multiple (4) microinjections of 6-OHDA (4 x 2 pg) laterally to the pedunculus superior and were killed 12 weeks later. Microdissections were made, as indicated in Fig. 1, on frontal slices to measure catecholamines in the rich

FIQ. 1. Schematic representation of tissue sampling in the anteromedial cortex. Localization of the samples dissected at - 10°C for the estimation of dopamine, DOPAC and noradrenaline levels. Protein values from the samples obtained from the 12,000 to the 9SOOq Q slice were respectively in ~g: 306 f 14, 330 f 15; 400 & 27, 450 f 25,420 f 26,328 f 17. Drawings are from the atlas of K&JIG & KLIPPEL(1963).

am0

ll6a llaaolomoDQI)amo Diama

lpmb

FIG. 2. Increased dopamine levels in various areas of the anteromedial cortex after degeneration of ascending noradrenergic fibres. Rats received bilateral multiple microinjections of BOHDA (4 x 2 pg) laterally to the pedunculus superior and were killed 12 weeks later. Tissues were dissected out from the 12,000 to the 9500 m coronal slices as illustrated in Fig. 1. The arrow indicates the beginning of the striatum. Hemicortices of 6-OHDA treated rats were arbitrarily divided into two groups depending on the changes in dopamine levels. Values are the mean f WM. of data obtained in five-six corresponding coronal slices. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test) when compared to respective values obtained in control rats.

dopaminergic areas of the pregenual and anterior supragenual cortices (anteromedial cortex) in the rats with lesions and in control rats. In the animals with lesions, noradrenaline levels were reduced by at least 75% in each of the cortical areas examined. Dopamine levels were higher than in controls, the overall mean value of dopamine levels of all cortical areas studied being increased by 34 f 8% (P < 0.001). A more detailed analysis revealed that dopamine levels were higher in the anteromedial cortices of about half of the lesioned ,rats but not in that of others. When it occurred, this increase varied between 40 and 75% in the various areas dissected in successive frontal slices (Fig. 2). A second experiment was made to ensure that the changes in dopamine levels were related to a specific degeneration of the ascending noradrenergic neurons and not to a non-specific e&ct of 6-OHDA on other neurons. Rats injected with desipramine (20 mg/kg, i.p.) or saline received 30min later bilateral single microinjections of 6-OHDA (4pg) laterally to the pedunculus superior. They were killed 14 weeks later and noradrenaline and dopamine levels were estimated in tissues punched out from the anteromedial cortex and from the ventral tegmental area. In rats injected with saline, the 6-OHDA microinjections induced a severe degeneration of the noradrencrgic neurons innervating the anteromedial cortex and the ventral @mental area (decrease in noradrenaline levels: 97 and 81”/, respectively). These

J. P. TASSINet u/.

1572

mJnM*aM

Reduction

of‘

the dihydroxyphenyiucrtic

ratio in the anteromedial ascendiny

FIG. 3. Prevention by desipramine pretreatment of the increase in cortical dopamine levels and the degeneration of ascending noradrenergic fibres in 6-OHDA treated rats. Desipramine (20 m&kg) was injected intraperitoneally 30 min before the bilateral single 6-OHDA microinjection (4 pg) made laterally to the pedunculus superior and rats were killed 14 weeks later. The anteromedial cortex (10,000 and 9500 pm slices) was punched out with a tube presenting an equilateral triangular shape (3.7 mm side) at its extremity. The caudal part of the ventral tegmental area was dissected by hand from the slices corresponding to the 2100 and 1600 pm planes of the atlas of KOENIG & KLIPPEL (1963). Protein quantities were (in mg) 0.68 + 0.03 and 0.22 + 0.02 for the anteromedial cortex and the ventral tegmental area respectively. Both dopamine and noradrenaline levels were estimated in tissues. Results are the mean k S.E.M. of data obtained from six to ten samples obtained with control rats and 6-OHDA treated animals which were either treated or not with desipramine. The dopamine and noradrenaline levels in the anteromedial cortex and the ventral tegmental area of control rats were 0.71 + 0.01 and 6.9 + 0.6 (dopamine) and 3.1 + 0.15 and 5.6 + 0.5 (noradrenaline) ng/mg protein respectively. effects were associated with a significant increase in cortical dopamine levels (43%). The levels of dopamine in the ventral @mental area were not affected (Fig. 3). As observed in the first experiment the increase in cortical dopamine levels occurred on several but not in all rats with 6-OHDA-induced lesions. Desipramine pretreatment completely prevented the degeneration of noradrenergic neurons as well as the increase in cortical dopamine levels (Fig. 3). The total number of rats with 6-OHDA-induced lesions used in the overall study was 28. Dopamine levels were increased by more than 30y0 (when compared with respective controls) in 37 out of the 56 hemicortices analysed. In 14 rats, the levels of dopamine were enhanced on both sides, no effect was seen in 5 animals whichever hemicortex was analysed. Finally, dopamine levels were only increased (by up to 30%) on one side in the remaining rats. In all these cases (56 samples), noradrenaline levels were reduced by more than 75%.

norudrenergic

cortex

cijkr

ucididopumine dqenerution

of

neurons

The increase in the cortical dopamine ~evcis observed in the rats injected with 6-OHDA could be related to a reduced release of dopdmine from dopaminergic nerve terminals. Since we habe previously shown that the DOPAC/dopamine ratio provides a valid index of changes in the rate of dopamine utilization (LAVIELLEet al., 1979). this ratio was estimated in tissues of rats which had received multiple injections of 6-OHDA (experiment I described previously) More precisely. DOPAC and dopamine were simultaneously estimated in tissues dissected from each frontal section (Fig. I). and the average mean DOPAC/dopamine ratio in each anteromedial cortex was calculated in lesioned and control animals. The DOPAC/dopamine ratio was significantly lower (-42”;,, P < 0.001) in the rats Injected with 6-OHDA than in the control animals, suggesting a reduced rate of utilization of the transmitter. Moreover. as illustrated in Fig. 4. this effect was seen in ail hemicortices (except one) and was not related to the magnitude of the changes in dopamine le~ls. It was even observed when cortical dopamine levels wcrc only slightly affected by the degeneration at’ the noradrenergic neurons (dopamine leveiz up to l?o”,, ,)i the control value). I---l----l

DAin the

mtero-ineUiai cortex (in % of controls)

FIG. 4. Individual changes in the dihydroxyphenylacetic acid/dopamine ratio versus the increase in dopamine levels in anteromedial cortices of 6-OHDA treated rats. Dopamine and DOPAC levels were estimated in tissues dissected from six successive coronal slices of anteromedial cortices of control (open circles) and 6-OHDA treated (solid circles) rats as illustrated in Fig. 1. Lesions were placed as described in the caption to Fig. 2. The DOPAC; dopamine ratio was estimated in each sample examined. Each point corresponds to the mean value of dopamine Ievek and of the mean v&e of the DOPAC/dopamine ratio estimated in the six successive coronal slices. Each individual result is expressed in percent of the mean of the control values. The mean control values for the six anteromedial cortices examined were 0.62 + 0.05 for the DOPAC/dopamine ratio and 0.7 + 0.03 ng/mg -protein for the dopamine levels.

Modifications of cortical dopaminergic neurons after depletion of noradrenaline

1573

when compared with control rats. This effect was associated with a parallel change in the number of [‘Hldopamine uptake sites that increased (26 f 4% P < 0.001). The individual changes in dopamine levels and C3H]dopamine uptake sites observed in the rats with 6-OHDA-induced lesions are illustrated in Fig. 6. Fluorescence histochemistry. In complementary experiments, as described in Experimental Procedures, the network of dopaminergic nerve terminals was visualized histochemically by incubating frontal brain sections of a-methylparatyrosine pretreated rats in the presence of desipramine and dopamine or noradrenaline. In addition, the incubation of other sections in EMwGENaJS M @ k-DA UPTAKE the presence of benztropine and noradrenaline made it possible to see the extent of the noradrenergic FIG. 5. Schematic representation of tissue sampling for the denervation. This denervation was almost complete in simultaneous estimation of [‘H]dopamine uptake and the five 6-OHDA-treated animals examined. amine levels in anteromedial hemicortices. Five hundred Two types of dopaminergic fibres could be visuamicron thick brain slices were obtained with a microtome refrigerated at -7°C. Microdiscs (1.4 mm diameter) were lized by these uptake experiments: thin fibres (Fig. 7), homogenized in 0.25 M sucrose in order to determine the similar in their morphological aspect and topography number of high affinity C3H]dopamine uptake sites while to those observed after glyoxylic acid perfusion, i.e. the triangular samples (3.7 mm side) were used for the under visualization of the endogenous stores of cateradioenzymatic determination of the endogenous amine cholamines (LINDVALL, BJ~~RKLUNLJMOORE St levels. Protein quantities were respectively 0.078 f 0.008 SIENEVI, 1974; BERGER et al., 1976). They were and 0.36 _+0.03 mg for the microdiscs and the triangular samples. Drawings are from the atlas of K~NIG & KLIPPEL observed in the deeper layers of the pregenual part of the anteromedial cortex, around the white matter and (1963). in the rhinal area. A second type of fibre which The reduction of the DOPAC/dopamine ratio was related to the degeneration of the noradrenergic neurons and not to a non-specific effect of 6-OHDA.

This was confirmed in an experiment made in rats which had received bilateral single microinjections of 6-OHDA, since this effect was no longer seen any more in desipramine pretreated rats (DOPAC/dopamine ratios were respectively 110 f 12% and 61 f 6%; P < 0.02 of the control value for rats which had been pretreated, or had not been, with desipramine). Extension of the cortical innervation after degeneration of the ascending noradrenergic neurons Uptake of C3H]dopamine. An increased density of the network of dopaminergic nerve terminals could be responsible for the changes in cortical dopamine levels seen in the rats with 6-OmA-induced lesions. In order to test this hypothesis dopamine levels and C3H]dopamine uptake were estimated in alternate successive frontal brain sections 14 weeks after the degeneration of ascending noradrenergic neurons induced by bilateral multiple microinjections of 6-OHDA made laterally to the pedunculus superior (see Fig. 5). The mean changes in dopamine levels and in the number of specific [“Hldopamine uptake sites estimated on sucrose homogenates in the presence of desipramine (5 x lo-’ M) were calculated in each anteromedial hemicortex analyscd. Confirming previous experiments, the mean increase in dopamine levels was 36 f 4% (P < 0.001)

FIG. 6.

Increase. in the [3H]dopamine uptake activity versus the increase in dopamine kvels in anteromedial cortices of COHDA treated rats. Rats received bilateral multiple microinjections of COHDA (4 x 2 B) laterally to the pedunculus superior and were killed 14 weeks later. [311JDopamine uptake activities and dopamine levels were estimated in tissues dissected from 6 serial coronal slices of the anteromedial cortias of control (open circles) and COHDA lesioned (solid circles) rats as described in Fig. 5. Bach point corresponds to the mean of [3H]dopamine uptake activity estimated in five microdiscs and to the mean of dopamine levels &imated on three triangular samples in each anteromedial. h&cortex. Individual results are expressed in percent of the mean of control values which were 16 f 1.7nCi/5min/mg protein and 1.2 f 0.08 ng/mg protein for [3H]dopamine uptake activity and dopamine levels respectively.

I574

J. P.

TASSIN et ul.

FIG. 7. Thin type of dopaminergic fibres in the medial deep pregenual cortex. x960. Control rat I-methylparatyrosine pretreatment. Section incubated in the presence of pargyline (IO-“ M), desipramine (5.10e6 M) and dopamine (10e6 M). FIG. 8. Thick type of dopaminergic fibres with closely packed varicosities in the superficial supragenual cortex. x 960. Same animal as in Fig. 7. Dopamine was replaced by noradrenaline ( 10d6 M). FIG. 9. Area of dopaminergic nerve terminals in the very anterior part of the pregenual cortex. x 312. Normal rat. Same treatment as in Fig. 7. FIG. 10. Same area as in Fig. 9. Higher density of dopaminergic axons. x 312. 6OHDA suppression of the cortical noradrenergic innervation.

treated animal:

FIG. 1 I. Dopaminergic innervation of the anterior supragenual cortex (200 pm caudal to the genu of the corpus callosum). Brain surface at the top. The white vertical bar represents the thickness of the molecular layer. x 312. Same treatment as Fig. 8. FIG. 12. Same area as Fig. I 1. Higher density of the dopaminergic axons in the second and third cortical layers. x 3 12. 6-OHDA treated rats.

Modifications of co&a+ dopaminergic~neurons after depletion of noradrenahne

1579

and wit%&0ytf, sim the rats were k&d a fozq time after ti CIOS&paeked varicosities (Fig. S), was seen in the k&m, the gmss&Iepresence of dopamine BCCU~Uin degenerating nomdmergic neurons was supragenuaf cortex, predominating in the seumd and la@ eliminated. Although the average increase in cortical third layer and extending int5 the molecular layer. They were ah chserv~ in the d&al Sea and Were dopamine Lvels was 41% in the overall population of better +Gua&ed after imxrbatiorr in the presence of the 2%MlHlDA treated rats studied, the intensity of appeared thiiker~ miwe izztendy ilttmmw

nora~~~ En the rats with ~O~~A-~nd~~ lesions, a hi@ density of dopaminergic fibres was easily detected in areas such as the anterior frontal lobe (Figs 9-10) and the anterior supragenual cortex (Figs 11-Q, which exhibit a lower density of ~5~~r~ innervation in contrf animals, On the c5ntrary, it wz+smofe difilcult t63sex:whether a c5Werd spr5utkg of the dopamine&c fibres had oc431rred in WMS such as the WD tral part of the pregenual medial area and the rhinal area which are more richly irmervated in control rats+ In the posterior part of the supragenual area, at the fey& of the anterior #bilge (84tX)m) the normai begs ~~~~ation is so dense that it was not possible to identify sprouting With the histochemical method. The dopaminergic fibres Were slightly more munerous in the maleeular layer after the disappearance of the ~~ra~e~~ innervatian. ELoWever~they did not tend to prugferate extensively in this layer. The collateral sprouting of the d5paminergie fibm seemed to be mainly restricted to the areas particularly innervated by these fibres, i.e. deeper layers in the ventral pregenual area and more super%ial layers in the ~~a~n~~ cortex.

Striking modifications of the properties of the mesocortical dopaminergic neur5ns projecting to the scorns cerebr& cortex of the rat were seen &er the apron ofthe amending noradrenergjc pathways. Endoed, the density of&f3 c5rhtI dopamiacrgic innervation was increased 3-8 months after the bilateral 6-OHDA lesions of the noradrenergic neurons. This suggested the oc5urrence 5f a collateral sprouting of the dopaminergic 8bres. Moreover, as retie& by the decreased r&e of uti%m&n of dopamine, the activity of these dopaminergic neur5ns ~8s reduced. Increased density

of dopaminwgic

innervation of the

COHeU Three ~~p~~~ approach were used to demmztrate the extensim of the dopamhrergic innervation in the armzromedial cortex. Endogenous kwels of dopamiw. First, endogenous doparnine h&s were e&mat& in discrete m areas. As de&bed irr ExperimmtaJ Pmxdurea, to av5id turjist&ats3&

diflffatce in tf5pssjm

kw&

between rats w&b lesions and eontr5L rats it was veri&xi that in the conditions used noradrenaline present in tissues of control rats did not interfere at all with the radioenzymatic estimation of dopamine. More

this e&&t varied among the truliorrs antis Indeed, in some rata, dopanke lev& were increased by as much as 81%; in others, they were slightly higher or identical to the mean value estimated in the popula* tion of control rats. Curiously, in some cases the levels of dopamine were markedly increased in one ~ter~e~~ ~~~rtex and much &rssin the other. ~pparentiy~ this variability was not finked to the extent of destruction of the noradtenergic innervation since cortical noradrenaline levels were reduced by more than 75% whatever was the cerebral hemicortex examined in all 6-OHDA treated rats. No sign&ant cctrref&i5~ could be obtained betrveen the ~piitud~ of the decrease in the environs cortical h&s of noradrenahue and the increase of dopamine levels when rest&s obtained in individual rats were analysed. However, this correlation can be questioned due to the: relative imprecision of the estimatian of residual nor&renal&e few& in rats With k%ions~ Lndeaxf, nu~~~~~ fevels Were on& ~u~~~t to give radioactivity doubb the blank vahte when they were reduced by 90% when compared to controla The topographical analysis of the rich dopaminergic areas diisef&d in the successive frontal slices revealed that the increase in dopamine levels occurred in ah areas ex~n~, The egret was SiightLy greater in the supragenual cortex which, in contrast to the pregenual cortex, is not mainly innervated by dopaminergic neurons originating from the ventral tegmental area, but to a larger extent by dopaminergic neurons located in the mediai part of the substantia n&a (Fl.rx& Ha=& ~~~~ &3E%X3N~ LIDBRlNK& LJtJNGDNE, 1974; &%TI%D, 1976; S&foq LEMOAL,GAUZY& CARDO,1976; FALLON& MOQRE, 1978; LMDV~L et al., 1978). Uptake al” ewemus dopamixre. it is generally agreed that the estimation of the hi& at&&y specific: upfake of f3H-jd5pamine in a given tissue provides a good index of the density of the dopaminergi~ innervation (CU%I.Q HORN, MACMY & Ivnts~~, 1973; SNVDW & COYLE, 1969; Thsst~ et al., 1974) As observed With dopamine levels, the number of specific E3H3dopamine uptake sites in the anter5medircl cartex w8s ~~~~t~y increased in the over&l populatirrn of 6-OkiDA treated rats+ but the intensity of this effect variexlfrom one animal to another. Histochrlrmlcalanalysis. Finally, the histochemf& analysis made with a technique which has previously been shoWn clearly to distinguish noradrenergic from tic nerve terminals in contr5l rats (~~ & ~~~~~ 1978f, e5nGrmed the presence of a denser network of do~rn~r~c nerve t~~~~~s in some cortical areas after the degeneration of the noradrenergic neurons. This phenomenon was still

1580

J. P. TAWN

observed 6 months after placement of the lesions. The sprouting process concerned both the thin (pregenuai) and thick (supragenual) fibres which are thought to Originate from the ventral tegmental area and the substantia nigra, respectively (LINDVALLrt al., 1978; FALLON & MOORE, 1978). As already suggested by the biochemical study, the collateral sprouting appeared more pronounced in areas such as the anterior frontal lobe and the anterior supragenual cortex.

Reduced acticity

of’ cortical dopaminergic

neurons

As just discussed, the increase in cortical dopamine levels can be attributed to the extension of the dopaminergic innervation. However, this effect may also be partially related to a reduced release of dopamine from dopaminergic nerve terminals. This appears to be the case, since in the 6-OHDA treated rats the DOPAC/dopamine ratio was significantly decreased in all cortical areas examined. This effect was of similar amplitude (about -40%) whatever the increase in dopamine levels. This suggests that it is independent of the collateral sprouting phenomenon. Changes in the activity of the dopaminergic neurons innervating the anteromedial cortex can validly be detected by measuring changes in the DOPAC/dopamine ratio in tissues. Indeed, the selective marked activation of the mesocortical dopaminergic neurons induced by electrical foot shocks was demonstrated by the acceleration in the decline of dopamine levels in a-methylparatyrosine injected rats (THERRY,TASSIN,BLANC& GLOWINSKI,1976), as well as by the increase in the DOPAC/dopamine ratio in animals not treated with the inhibitor of catecholamine synthesis (FADDA, ARGIOLA$ MELIS, T&SARI, ONALI & GESSA, 1978; LAVIELLEet al., 1979). Thus the decreased DOPAC; dopamine ratio observed in the 6-OHDA treated rats suggests a marked reduction of the activity of the dopaminergic neurons projecting to the anteromedial cortex. Both the collateral sprouting and the reduced activity of the dopaminergic neurons projecting to the anteromedial cortex in the 6-OHDA treated rats were specifically related to the degeneration of the ascending noradrenergic neurons and not to a non-specific local destruction of other types of neurons. Indeed, the increase in dopamine levels as well as the reduction of the DOPAC/dopamine ratio were no longer seen in 6-OHDA treated rats pretreated with desipramine to prevent the transport of this neurotoxin into noradrenergic fibres. Interestingly enough, recent experiments indicated that the destruction of the cortical serotoninergic innervation did not effect the dopamine levels or the DOPAC/dopamine ratio in the anteromedial cortex (D. HER& H. SIMON, G. BLANC,A. LKOPRAWSKI,J. GUIWINSKI,M. LEMOAL & J. P. TASSIN, unpublished observations). This further revealed that the lack of noradrenergic innervation plays a critical role in the effects observed.

et rd.

What are the respective contributions or the noradrenergic nerve terminals innerva&g the cerebral cortex and of those projecting to thr ventral tegmental area, since noradrenaline levels KCTI’markedly reduced in the two structures’! Central catecholaminergic neuron5 m;ty iorm ne~ sprouts in two situations, after their own injury (regeneration sprouting) (BJijRi;Ltjlr’DK: S-n.Ntq 1971. KATZMAN,BJ~~RKLUND. OWMAN. Srr~ VI 6i Wtn. 1971; BJ~RKLUND,KATZMA~:,STENEVI& WEST. 1971) or after degeneration of other ncuroIIs projectmp to similar target areas [collateral sprouting (M~KRE. BJ~~RKLUND& STENEVI, 1971; RAISMAX8 Fr~1.13 1973: GILAU & REIS, 197911. Thib l:rtter process was demonstrated in most cases after degeneration or rloncatecholaminergic systems. However. sprouts from undamaged noradrenergic fibres wcrt’ also observed in the cerebellum

after lesions of other noradrenergic

fibres innervating this structure (PI\ ht.1. %(;-\I. & Br.oo~, 1974). Our results suggest tha: the collateral sprouting of the cortical dopaminerglc fibrcs is related to the disappearance of the noradrenergic fibres innervating the same areas. However. the dopaminergic fibres did not invade all the sites innervated by noradrenergic fibres since they only prohferated from place to place in the molecular layer which IS particularly rich in noradrenergic nerve terminals. Since the cortical noradrenergic nerve termin& exhibit very few classical synapses (BEAUXI- 8 I)I:st.*\KRu.s. 137X) the presence of unoccupied noradrentirgie postsynaptic sites may not be the primary factor lu\olved in the collateral sprouting of dopaminergic !ibrcs. The reduced activity of the dopaminergic neurons observed in the 6-OHDA treated rat!, could be related to the extension of their field of rnnci-bation since. in the opposite situation, an increased !urllover of dopamine in remaining intact dopaminergic neurons has been previously seen after partial degeneration of the mesocortical dopaminergic pathway UASSIN, VEL.IXY, STINUS, BLANC:.GLOWINSKI& THIFi be the involved, since reduction in cortical DOPAC/dopamine ratid still detected 6-OHDA treated exhibiting very little in cortical dopamine I.e. wheu collateral process was pronounced it is possible the reduced activity the mesocortical dopaminergic is partly to the of noradrenergic nerve mnervating the ventral area, since pronounced decrease in noradrenaiine levels observed in area in 6-OHDA treated If this the case.

Modifications of cortical dopaxninergic neurons after depletion of noradrenaline present results would indite that the activity of the dopaminergic cells located in the ventral tegmental area is under the control of noradrenergic neurons projcctii to this area. It remains to be established if these neurons are those of the ventral noradrenergic pathway or those which originate from the locus coeruleus, since a pathway connecting the locus coeruleus and the ventral tegmental area has recently been described (JONES & MOORE, 1977; SIMON, LEMOAL, STINUS& CuAs$l979). Possiblefinctional

implications

An increase in dopamine levels has been observed in the @lateral posterior cerebrum of the rat several weeks after the unilateral electrolytic lesion of the locus coeruleus (WORTH, COLLINS KXTT & AUSTIN, 1976). Furthermore, it has also been shown that dopamine levels were higher in the globus pallidus, the septum and the preoptic area 12 months after the peripheral injection of 6-OHDA in new-born rats (OKE, KELLER& AD+, 1978). According to behavioural studies important interactions occur between

1581

noradtc&gic and dopaminergic systems within the CNS (see review of ANTELMAN& CAGGKJLA,1977). Consequently, the dopaminergic neurons innervating the anteromedial cortex may not be the only ones affected by the degeneration of ascending noradrenergic neurons in our experiments. In any case, the changes in the properties of the mesocortical dopaminergic neurons presently described could have an important functional significance. Particularly, they could be involved in some of the behavioural disturbances which have been attributed to the degeneration of noradrenergic neurons originating from the locus coeruleus (MASON & I~RSEN, 1975).

Acknowledgements-The authors wish to thank Drs P. LECO~ and E. DUBOIS(Neurophysiology laboratory, Gif/ Yvette) for having performed the ‘multiple 6-OHDA’ microinjections. Mrs M. MARYhas provided an excellent photographic assistance. This research was supported by grants from DRET (78/004),INSERM (77.510)and RhBne-Poulenc SA.

REFERENCES AGID Y., JAVGYF. L GL~WINSKIJ. (1973) Hyperactivity of remaining DA neurons after partial destruction of the nigrostriatal DA system in the rat. Nature, Land. 245, lSO-151. A~LMAN S. M. & CAGGIULAA. R. (1977) NorepinephrineDopamine interactions and behavior. Science, N.k: 195,

646-653. BEAUDET A. C DESCRIES L. (1978) The monoamine innervation of rat cerebral cortex: synaptic and nonsynaptic

axon terminals. Neuroscience 3,851-860. BECKSTEAD R. M. (1976) Convergent thalamic and mesencephalic projections to the antero-medial cortex in the rat. J. camp. Neural. 166,403-416. BERC#ZR B. & G~~SKI J. (1978) DA uptake in S-HT terminals in oitro: a valuable tool for the histochemical differentiation of catecholaminergic and serotoninergic terminals in rat cerebral structures. Brain Res. 147, 29-45. BERIXRB., Tlrsst~ J. P., BLANCG., MOYNEM. A. & THWRY A. M. (1974) Histochemical confirmation for dopaminergic innervation of the rat cerebral cortex after destruction of the noradrenergic ascending pathways. Brain Res. 81, 332-337.

BEROWB., THIEIRRY A. M., TASSINJ. P. & MOW M. A. (1976)Dopaminergic innervation of the rat prefrontal cortex: a fluorescence histochemical study. Brain Res. 106, 133-145. BI&KLUND A., KATZMANR., STENEVIU. & WESTK. A. (1971) Development and growth of axonal sprouts from NA and S-HT neurons in the rat spinal cord. Brain Res. 31, 21-33. BJ~~RKLUND A. & SWNEVIU. (1971) Growth of central catecholamine neurons into smooth muscle grafts in the rat mesencephalon. Brain Res. 31, l-20. CUEW A., HORNA. S, MACKAYA. V. P. & IWRSENL. L. (1973) Catecholamines in the median eminence: new evidence for a major noradrenergic input. Nature, Land. 243,465-467. EMS~NP. C. & Kooa G. (1978) The origin and distribution of dopamine-containing afferents to the frontal cortex. Brain Res. 142, 249-267. FADDA F., ARGIOLASA., MELIS M. E., TISSARIA. M., ONALI P. L. & GESSAG. L. (1978) Stress-induced increase in 3+dihydroxyphenylacetic acid (DOPAC) levels in the cerebral cortex and in N. Accumbens: Reversal by diazepam. ,5@ Sci. 23, 2219-2224. FALU~NJ. H. 8t MOIXE R. Y. (1978) Catecholamine innervation of the basal forebrain-IV. Topography of the DA projection to the basal forebrain and neostriatum. J. camp. Neural. 180, 545-580. FUXEK., H~KFELTT., JOIUNSSON O., JONWN G., LIDBRINKP. & LJUNGDXHL A. (1974) The origin of DA nerve terminals in limbic and frontal cortex. Evidence for mesa-cortical DA neurons. Brain Res. 82, 34%355. GAUCXIYC., TASSINJ. P., GL~WINSKIJ. & CHERAMY A. (1976) Isolation and radioenzymatic estimation of picogram quantities of DA and NE in biological samples. J. Neurochem. 26, 471480. GILADG. M. & REISD. J. (1979) Collateral sprouting in central mesolimbic DA neurons: biochemical and immunocytochemical evidence of changes in the activity and distribution of tyrosine hydroxylase in terminal fields and in cell bodies of A10 neurons. Brain Res. 160, 17-36. JO= B. & MIXXUZ R. Y. (1977) Ascending projections of the locus coeruleus in the rat-II. Autoradiographic study. Brain Res. 127, 23-53. KATZMANR., BJURKLUND A., OWMANC. H, STENEVIU. & WENTK. A. (1971) Evidence for regenerative axon sprouting of central catecholamine neurons in the rat mesencephalon following electrolytic lesions. Brain Res. 25, 579-596.

15x2

J. P. TANNIN et al

KbNlG J. F. R. 8~ KLIPPELR. A. (1963) The Rat Brain: a Stereotaxic Atlas qf the Forebrain and Lower Parts ,$ the Brain

Stem. Williams & Wilkins, Baltimore. LAvIELLES., TAssIN J. P.. THIERRYA. M., BLANCG., HER* D., BARTHELEMY C. & GLOWINSKIJ. (1979) Blockade by benzodiazepines of the selective high increase in DA turnover induced by stress in mesocortical dopaminergic neurons of the rat. Brain Res. 168, 585-594. LrNDvALL0.. BJ~~RKLUND A. & DIVACI. (1978) Organization of catecholamine neurons projecting to thy irontai cortex in the rat. Brain Res. 142, l-24. LINDVALLO., BJBRKLUND A., MOORER. Y. & S~NEVI J. (1974) Mesencephalic DA neurons projectmg IO neocortex. BM~,~ Rrs. 81, 325-331. LOWRY 0. H.. ROSEBROLJC~H N. J., FARR A. L. & RANDALLR. J. (1951) Protem measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MALMFORS T. & SACHSC. (1968) Degeneration of adrenergic nerves produced by 6-OHDA. Eur. J. Ph~rmacoi. 3, X9-9?, MACONS. T. 8~ IVERSENS. D. (1975) Learning in the absence of forebrain NA. Nature, Lond. 258, 422 324 MOOKER. Y., BJ~RKLUNDA. & STENEVIU. (1971) Plastic changes in the adrenergic innervation of thr rat septal area in response to denervation. Brain Res. 33, 1335. OKE A., KELLERR. & ADAMSR. N. (1978) DA and NE enhancement in discrete rat brain regions following neonatal 6-OHDA treatment. Brain Res. 148, 245-250. PICKELV. M., SEGALM. & BLOOMF. E. (1974) Axonal proliferation following lesions of cerebellar peduncles. A combined fluorescence microscopic and radioautographic study. J. camp. Neurol. 155, 4360. RAISMAN G. & FIELDP. M. (1973) A quantitative investigation of the development of collateral reinnervation after partial deafferentation of the septal nuclei. Brain Res. 50, 241-264. SIMONH., LEMOAI.M.. STINTSL. & CALASA. (1979) Anatomical relationships between the ventral mesencephaiic tegmenturn A10 region and the locus coeruleus as demonstrated by anterograde and retrograde tracing techniques. J. Neural. Transm. 44, 77 -86. SNIDECORT. W. & C~CHRANW. G. (1967) Sratisrical Methods. Iowa State Univ. Press. Ames. SNYDER S. H. & COYLEJ. T. (1969) Regional differences in 3H-NA and 3H-DA uptake into rat bram homogenates. J. Pharmac. e.up. Ther. 165, 78-86. TASSIN J. P.. B~CKAERT J., BLANCG., STINUSL., THIERRY A. M., LAVIELLE S.. PREMONTJ. & GLO~INSKI J. (1978)

Topographical distribution of dopaminergic innervation and dopaminergic receptors of the anterior cerebral cortex of the rat. Brain Res. 154, 241-251. TANN J. P., LAVIELLES., BLANCG.. THLERRYA. M. & BERGERB. (1979) Decreased activity and increased arborizations 01 the rat mesocortical DA neurons after destruction of ascending NE bundies. In Catecholamines: Basic und CIinicul Frontiers (ed. USDIN E.), in press. Pergamon Press, New York. TASSINJ. P.. CHERAMYA., BLANC G., THIERRYA. M. & GLOWINSKIJ. (1976) Topographical distribution of dopaminergic innervation and dopaminergic receptors in the rat striatum-I. Microestimations of ‘H-DA uptake and DA content in microdiscs. Brain Res. 107, 291-301. TASIN J. P., THIERRYA. M., BLANCG. & GLOWINSKIJ. (1974) Evidence for a specific uptake of dopamme by dopaminergic terminals of the rat cerebral cortex. Naunyn-Schmiedebergs Arch. Pharmac. 282, 239-244. TASSINJ. P.. VELLEY L., STINUSL., BLANC G., GLDWINSKIJ. St THIERRYA. M. (1975) Development of cortical and nigro-neostriatal dopaminergic systems after destruction of central noradrenergic neurons in foetat or neonatal rats. Brain Res. 83, 93106. A. M.. TASSIKJ. P.. BLANC G. & GL~WINSKIJ. (1976). Selective activation of the mesocortkal

THIERRY

DA system by

stress. Nature, Lond. 263, 242-244. TH~ERRYA. M., BLANC G., SOBELA., STINUSL. & GLOW~NSKI J. (1973) Dopaminergic

terminals in the rat cortex. Sciencr,

N. Y 182, 499-501. LJUNGBERC; T., RANJE CH., SCHULTZ W. & TULL~~H 1. (1977) Neuronal transmission and animal behaviour. A search for correlations in the central DA pathways. In Proceedings of the 27th Inrernationa[ Union t!/ Physiological Sciences (ed. Comitt? National Frangais des Sciences Physiologiques), Vol. 12, p. 709. WORTHW. S., &LLINS J., KETTD. & AU~N J. H. (1976) Serial changes in NE and DA in rat brain after locus coeruteus lesions. Brain Res. 106, 198-203.

[‘NGERSED~ U..

(Accepted 16 June 1979)