171
Brain Research, 551 (1991) 171-177 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 A DONIS 000689939116663Q BRES 16663
Serotonergic sprouting in the neostriatum after intrastriatal transplantation of fetal ventral mesencephalon Y. Takeuchi L3, T. Sawada 3, S. Blunt L2, P. Jenner ~ and C.D. Marsden 2 l Parkinson's Disease Society Experimental Research Laboratories, Pharmacology Group, Biomedical Sciences Division, King's College London, London (U. K.), 2University Department of Clinical Neurology, Institute of Neurology, National Hospital, London (U. K.) and 3Department of Pediatrics, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto (Japan) (Accepted 8 January 1991)
Key words: Neuronal transplantation; Neostriatum; Dopamine; Serotonin; Immunohistoehemistry; Parkinson's disease
The sprouting of serotonergic fibers into the neostriatum of 6-OHDA-lesioned rats should be considered, when assessing the functional aspects of ventral mesencephalic grafts. The degree of serotonergic fiber growth into the neostriatum of rats subjected to unilateral 6-OHDA lesions followed by fetal ventral mesencephalic grafts was examined immunohistochemically. As a measure of serotonergic innervation density, the total length of immunoreactive fibers was quantified as percent area. Four weeks after transplantation, there was complete recovery of (+)-amphetamine-induced rotation. All of the ventral mesencephalic grafts contained serotonin-immunoreactive cells, but much fewer in number than the tyrosine hydroxylase-positive cells found in the same grafts. However, there was significant serotonergic hyperinnervation throughout the transplanted neostriatum compared with the control side. The hyperinnervation derived from the grafted neurons was most marked in the dorsal and lateral sectors of the rostral neostriatum. At 24 weeks after transplantation, the implanted neostriatum still showed a dense serotonergic innervation. INTRODUCTION The replacement of degenerating dopamine ( D A ) cells by fetal mesencephalic tissue is a new approach to the t r e a t m e n t of Parkinson's disease and the experimental background is based mainly on transplantation studies in 6-hydroxydopamine ( 6 - O H D A ) - l e s i o n e d rats 3'4'7"8"13'14. It has recently been shown that some serotonin (5H T ) - i m m u n o r e a c t i v e cells are always present in DA-rich ventral mesencephalic grafts using standard transplantation techniques 7, but 5-HT-immunoreactive fiber growth into the neostriatum from the grafts has not been demonstrated. In assessing the function of DA-rich grafts in the 6 - O H D A - l e s i o n e d rat model of Parkinson's disease, the extent of 5-HT-immunoreactive fiber growth is important since 5-HT can reduce drug-induced rotation in 6 - O H D A - l e s i o n e d animals and L-dihydroxyphenylalanine (L-DOPA) may be decarboxylated in striatal serotonergic fibers 14. In addition, serotonergic neurons have a presynaptic interaction with nigrostriatal dopaminergic neurons, through which they may modulate dopaminergic transmission6. We have therefore examined immunohistochemically the degree of serotonergic fiber growth into the neostriatum of rats subjected to unilateral
6 - O H D A lesions followed by fetal ventral mesencephalic grafts.
MATERIALS AND METHODS
6-OHDA-induced nigrostriatal lesions Female Wistar rats (Bantin and Kingman, U.K.) weighing 150-200 g were housed in groups of 6 rats per cage and maintained on a 12:12 h light-dark cycle with ad libitum access to food and water. Rats were anesthetized with pentobarbital (50 mg/kg) intraperitoneally (i.p.), and injected with 8 /zg as the base of 6-OHDA-HBr (Sigma, U.S.A.) in 4 #1 of 0.9% saline containing 0.2 mg/ml ascorbate into the left nigrostriatal bundle. Stereotaxic coordinates for the site were: A = 2.2 mm posterior to bregma, L = 1.5 mm on the left side lateral to the midline, V = 8.0 mm below the dura, with the incisor bar set +4.5 mm above the interaural line. Injections (4/~l) were made with Hamilton syringe, at the rate of 1 /~l/min with a further 4 min allowed for diffusion before retraction of the syringe.
Assessment of rotational behavior One to 2 weeks after lesion surgery, (+)-amphetamine-induced rotation was assessed over a 1 h period following intraperitoneal injection of (+)-amphetamine sulphate (5 mg/kg; Sigma, U.S.A.) dissolved in 0.9% saline. Rats were placed in individual plastic boxes and the number of complete turns made during a l-rain period was recorded every 10 rain. Only rats showing ipsilateral turning rates of >7/min were selected for further study3. (+)-Amphetamine-induced rotation was also counted 4 weeks after transplantation surgery (see below).
Correspondence: Y. Takeuchi, Department of Pediatrics, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602, Japan.
172 Amphetamine-induced rotation
Apomorphine-induced rotation was assessed 2-3 days following each (+)-amphetamine-induced rotation test. Contralateral rotation was monitored over a 1 h period following subcutaneous injection of apomorphine-HCl (0.5 mg/kg; Sigma, U.S.A.) dissolved in 0.9f/~ saline containing 0.2% ascorbate.
20-
1816 1412-
Transplantation of fetal ventral mesencephalon Graft tissue was taken from rat embryos of c r o w n - r u m p length (CRL) 12-14 mm. DA-rich tissue was dissected from the floor of the mesencephalic flexure, containing the developing cells of the substantia nigra and ventral tegmental area according to Bj6rklund et al.~. The tissue pieces of 10 embryos from one pregnant mother were collected in sterile 0.6% glucose-rich 0.9% saline, then incubated for 20 min at room temperature in a solution containing 0.1% trypsin (Sigma, grade 2 crude) in glucose-saline. Following incubation, the pieces were washed by repeated replacement of glucose-saline to a final volume of 10 embryonic pieces per 100 pl, then dissociated by repeated aspiration through a polished Pasteur pipette to form a suspension of dissociated cells. The cell suspension was drawn up into a 10 /tl glass microsyringe (Scientific Glass Engineering, Australia). Aliquots of graft tissue were stereotaxically implanted into the left neostriatum (caudate-putamen complex) in two deposits at the following sites: A = 1.8 mm, L = 2.5 mm, V = 5.0 mm; and A = 1.0 mm, L = 3.5 mm, V = 5.0 mm. T h r e e p l was deposited at each site, injected over 3 rain with a further 4 rain allowed for diffusion before retraction of the syringe needle (n = 9). Sham-transplants were performed using vehicle only (n = 3).
lmmunohistochemistry Four and 24 weeks following transplantation, rats were anesthetized with pentobarbital (50 mg/kg) i.p. and perfused transcardially with phosphate-buffered saline (PBS; 0.9% NaCI in 0.l M sodium phosphate buffer) and subsequently with a fixative containing 4% paraformaldehyde in 0.1 M sodium phosphate buffer. The brain blocks were immersed in the fixative solution for 48 h at 4 °C. The blocks were stored in 15% sucrose overnight and then sectioned in the frontal plane with a cryostat (20 pm, -20 °C), and 4 parallel series were collected through the area of the neostriatum and the substantia nigra in PBS. Two series were mounted onto glass slides and stained with Cresyl violet. Two other series of sections were rinsed in PBS containing 0.3% Triton X-100 and incubated in 10% normal goat serum for 1 h at room temperature. Free-floating sections in PBS containing 0.3% Triton X-100 were incubated for 48 h at 4 °C in primary antibodies to 5-HT (1:12 000; Takeuchi et al. ]9) or tyrosine hydroxylase (TH, 1:2000; Eugene Tec, U.S.A.). All sections were taken through the Vector A B C immunohistochemical method with diaminobenzidine as chromogen. Sections were mounted onto gelatin-coated slides, dehydrated and coverslipped. The specificity of 5-HT immunohistochemical staining was as described previously 19.
E
I0~
420-
..o-. . . . . -o- . . . . . *o 6 0 H D A + g r a n .
110
20
310
410
~0
dO minutes
Fig. 1. (+)-Amphetamine-induced rotation: each point shows ipsilateral turns per minute (mean + S.E.M.) following injection of 5 mg/kg i.p. Negative scores signify contralateral turns. Closed circles = 6-OHDA-lesioned rats, one week after lesioning (n = 12); open circles = 6-OHDA-lesioned and implanted rats, 4 weeks after transplantation (n = 9).
RESULTS
Rotation tests
When 6-OHDA-lesioned rats received (+)-amphetamine (5 mg/kg, i.p.), they began to sniff and after 10 rain a steady rotation towards the left or lesioned side had developed. The peak rotational rate was 18.0 + 2.5 per minute (mean _+ S.E.M., n = 12, Fig. 1). These selected rats received apomorphine-sulfate (0.5 mg/kg, s.c.) and they demonstrated a contralateral rotation (away from the lesioned side). The peak rotational rate was 11.7 + 2.0 per minute (n = 12, Fig. 2). After receiving grafts of DA-rich fetal tissue, these rats showed an almost complete recovery of (+)-amphetamine-induced rotational Apomorphine-induced
rotation
18-
Quantitative analysis The animals were sacrificed for quantitative immunohistochemistry 4 weeks following transplantation (n = 6). Quantitative analysis of 5-HT innervation density was made in two frontal sections per animal representing the rostral (at +2.6 level according to the atlas of K6nig and Klippe111) and caudal neostriatum (at -0.8 level). The rostral and caudal sections of the neostriatum were divided into 4 (dorsal, ventral, lateral and medial) and two sectors (dorso-medial and ventro-lateral), respectively. As a measure of 5-HT innervation density, the total or cumulative length of 5-HT-immunoreactive fibers were used according to the previous studies 18"2°. Using a Kontron IBAS 2000, 5-HT-immunoreactive fibers per unit area in both hemispheres was quantified as percent area after 'skeletonization'. In addition, the number of TH and 5-HT-immunoreactive cells in the grafts were counted per section at +2.6 level according to the atlas four weeks (n = 6) and 24 weeks (n - 3) following transplantation.
60HDA
1614-
.~ 12~ 10-
~
6-OHDA
L.
~m
4
...... "~ ...... "~...... X~...... ~ ...... "~ 6--OHDA+ graft 0 i
i
i
I
I
10
20
50
40
50
i
~0minutes
Fig, 2, Apomorphine-induced rotation: each point shows contralateral turns per minute (mean + S.E.M.) following injection of 0.5 mg/kg i.p. Closed circles = 6-OHDA-lesioned rats, one week after Icsioning (n = 12); open circles = 6-OHDA-lesioned and implanted rats, 4 weeks after transplantation (n = 9).
173
Fig. 3. TH immunohistochemical staining of the neostriatum 4 weeks after transplantation. A number of TH-positive grafted neurons were observed mainly at the periphery of the transplant and their TH-positive fibers were seen to project into the host striatal tissue. A xl00; B x 200.
behaviors (n = 9, Fig. 1). Some of the animals showed contralateral rotation (away from the left side) for 30 min following administration of (+)-amphetamine indicating an overcompensation by the grafts, but the majority of animals showed no (+)-amphetamine-induced rotation. Additionally, these rats revealed low levels of apomorphine-induced rotation, the peak rotational score being only 3.0 + 1.2 turns per minute (n = 9, Fig. 2). On the other hand, sham-transplanted rats showed no recovery of drug-induced rotational behaviors.
TH imrnunohistochemistry Four weeks after transplantation, virtually no THimmunoreactive cells were visible in the substantia nigra ipsilateral to 6 - O H D A lesion. All implanted rats were
found to have grafts containing multipolar or irregularshaped TH-positive cells (Fig. 3). The number of THimmunoreactive cells in the grafts was 71.7 + 5.8 (mean + S.E.M., n = 6) per section of the rostral striatum at +2.6 level. These cells were seen to give rise to TH-positive fibers growing into the host neostriatum. Neighboring the grafts, the density of TH-positive fibers approached that of the contralateral non DA-denervated neostriatum (Fig. 3). Although TH-positive fibers from the grafts were distributed throughout the neostriatum, there was a difference in the innervation density of TH-immunoreactive fibers in the peripheral portions of the neostriatum between the control and implanted sides (Fig. 5). In sham-transplanted rats, there was no difference in the innervation density of TH-immunoreactive
Fig. 4. 5-HT immunohistochemical staining of the neostriatum 4 weeks after transplantation. Some 5-HT-immunoreactive cells were present in the graft and a high density of 5-HT-immunoreactive fibers was observed both in the graft and the host neostriatum. A xl00, B x200.
174
CC
Fig. 5. Immunohistochemicai staining for TH in the dorsal sector at the rostral level (+2.6). A: control (right) side; B: lesioned and transplanted (left) side. ×200.
fibers in the neostriatum between the control and sham-transplanted sides. Twenty-four weeks after transplantation, surviving TH-positive cells were visible in the grafts, the n u m b e r of TH-positive cells being 82.3 ___ 10.6 (mean + S . E . M . , n = 3) per section of the rostral striatum at +2.6 level. TH-positive fibers were distributed throughout the neostriatum as observed 4 weeks after transplantation.
5-HT immunohistochemistry Four weeks after transplantation, all of the ventral mesencephalic grafts also contained 5-HT-immunoreactive cells. The n u m b e r of them was 17.2 __+ 1.4 (mean + S . E . M . , n = 6) per section of the rostral striatum at + 2 . 6 level, but much fewer than that of TH-positive cells
in the same grafts (Fig. 4). H o w e v e r , there was a m a r k e d 5-HT hyperinnervation throughout the implanted neostriatum c o m p a r e d with the control side. The morphological characteristics of 5-HT-immunoreactive fibers were much the same as those of the control side. 5-HT-positive fibers c o m p o s e d an intricate network of thin, filiform profiles, irregularly p u n c t u a t e d by small, round or ovoid dilatations, i.e. varicosities. On the other hand, relatively straight fibers of larger size, which were observed among the m y e l i n a t e d axons on the control side, were rarely seen. In the grafts, the innervation density of 5-HT-immunoreactive fibers was so high that neither intervaricose segments nor proximal portions of 5-HT axons could be observed (Figs. 4, 6). The 5-HT hyperinnervation o b s e r v e d throughout the neostriatum
Fig. 6. Immunohistochemical staining for 5-HT in the dorsal sector at the rostral level (+2.6) A: control (right) side. B: lesioned and transplanted (left) side. The innervation of 5-HT-immunoreactive fibers was much denser on the transplanted side (B) compared to the control side (A). × 200.
175
Fig. 7. Immunohistochemical staining for 5-HT in the neostriatum at the rostral (+2.6, A,B) and caudal (-0.8, C,D) levels, 24 weeks after transplantation. On the transplanted side, serotonergic hyperinnervation remained to be unchanged throughout the neostriatum. B: the dorsolateral sector of the rostral neostriatum. D: the ventrolateral sector of the caudal neostriatum. On the control side, a moderate density of 5-HT-immunoreactive fibers was observed. A: the dorsolateral portion of the rostral neostriatum. C: the ventrolateral sector of the caudal neostriatum, x 200.
was most marked in the dorso-lateral portion of the rostral neostriatum, where the innervation density of 5-HT fibers was almost as high as that of the D A innervation density on the control side (Figs. 5, 6). In sham-transplanted rats, no marked difference was observed in the innervation density of striatal serotonergic fibers between the two sides. Twenty-four weeks after transplantation, the serotonergic hyperinnervation was still present in the neostriatum of the implanted rats and the number of 5HT-positive cells was 15.8 + 2.7 (mean + S.E.M., n = 3) per section of the rostral striatum at 2.6 level. The quantitative analysis also revealed that 5-HTimmunoreactive fibers were unevenly distributed, with the percent area of immunostained fibers ranging from 2.65 to 6.53 per unit area (Table I). The innervation density was greatest ventrally and caudally on the control
side. On the implanted side, the hyperinnervation was greatest in the lateral and dorsal sectors of the rostral neostriatum which showed 12.7 and 11.1, respectively, as a percent area of 5-HT-immunostained fibers. The quantitative analysis revealed a significant hyperinnervation of 5-HT-immunoreactive fibers throughout the sectors of both levels. On the other hand, there was no difference in the innervation density of 5-HT-immunoreactive fibers in the cerebral cortex between implanted and control sides (Table I). DISCUSSION The main finding of this study is that significant serotonergic sprouting occurs in the neostriatum after intrastriatal transplantation of fetal ventral mesencephaIon using the standard transplantation technique.
176 TABLE I
Percent area of 5-HT-immunoreactive fibers was listed with standard deviation in the rostral (+2.6) and caudal (-0.8) levels of the neostriaturn, according to the atlas of KOnig and Klippel Evaluation of the gradients was carried out by comparing the data between the control (right) and transplanted (left) sides using the Student's t-test. Asterisks indicate values which are significantly different: *P < 0.01; **P < 0.001 ; NS: not significant (n = 6).
~ _
+ 2.6
c,~
Right
Left
4.43±0.17
NS
4.89±0.46
2.65+0.12 3.60--+0.46
** **
12.74_+1.07 11.07-+0.20
2.97 ± 0.28 5.61±0.34
* **
4.30-+ 0.73 7.60±0.63
6.07-+0.50
**
9.90-+1.03
6.53±0.80
**
10.43-+1.15
- 0.8
F
o
Intrastriatal grafts of fetal mesencephalic DA-rich tissue can compensate for motor deficits resulting from lesions of the nigrostriatal pathway s. In the rat, the functional recovery is believed to depend on the DA fiber growth, into the DA-denervated striatum. There is evidence that the grafts are metabolically, physiologically and biochemically active as they reveal transmitter synthesis, normal firing patterns, and spontaneous DA release s. The functional effects produced by these DA grafts may be due to a controlled synaptic release of D A and not to a simple diffusion of DA 4. Recently, host afferents into fetal ventral mesencephalic tissue grafted to the neostriatum of adult rats were also described 7. Although a large number of studies have been made concerning the D A fiber growth after intrastriatal transplantation of fetal ventral mesencephalon 2~7~9, few studies emphasize the 5-HT fiber growth into the neostriatum. As pointed out by a recent study, some 5-HT-immunoreactive cell bodies are always present in grafts dissected and prepared according to the standard transplantation technique 1. By carefully dissecting only the rostral part of the fetal mesencephalon, the frequency of these cells can be reduced, but it is only by treating the cell suspension with 5,7-dihydroxytryptamine (5,7-DHT) that 5-HT cells can be eliminated in DA-rich cell suspension 7. Thus it is important to consider the exis-
tence of 5-HT-immunoreactive cells and fibers in the neostriatum following intrastriatal transplantation of fetal ventral mesencephalon. Dunnett et al. reported that some rats with mesencephalic DA-rich graft showed an increase in serotonin level in the neostriatum '~. However, the possibility of serotonergic innervation derived from fetal mesencephalon was thought unlikely and it was suggested that the graft-derived 5-HT-immunoreactive fibers soon disappear among the intact serotonergic innervation intrinsic to the host neostriatum surrounding the graft 9. The relation of mesencephalic grafts and host 5-HT density in the striatum was addressed in animals 6-OHDA-treated and transplanted as infants, but the occasional graft 5-HT cells were not considered to contribute to the 5-HT hyperinnervation ~7. The discrepancy between their results and the present study may be attributed to the sensitivity of immunohistochemical methods for 5-HT. However, it should be stressed that in our studies, pretreatments employing tryptophan and monoamine oxidase inhibitor (MAOI) are not used to enhance the visualization of 5-HT, because such pharmacological treatments increase the level of cross-reactive indoleamines other than 5-HT. We advocate that immunohistochemical studies for 5-HT should be carried out in the absence of M A O I and/or tryptophan, in order to compare the data under different experimental conditions more precisely, as pointed out by Ishimura et al. ~°. The 6-OHDA lesion can reduce the 5-HT innervation of the lesioned hemisphere compared to the control hemisphere or to the normal brain 21, and the majority of 5-HT hyperinnervation in the neostriatum is attributed to extrinsic serotonergic sprouting from grafted ventral mesencephalon. It is noteworthy that the serotonergic hyperinnervation does not prevent the replacement of DA innervation derived from the grafts. The fact that TH-positive cells grew and showed fiber outgrowth in the presence of 5-HT hyperinnervation suggests that 5-HT-immunoreactive fibers do not occupy synaptic sites vacated by the 6-OHDA lesion. It is of interest that removal of DA-afferents may lead to an enlargement of the intrinsic serotonergic projection to the neostriatum during the neonatal period, while such hyperinnervation after 6-OHDA lesioning was never observed in adult rats 16'2]. Serotonergic neurons are considered to have a high capacity for axonal sprouting, but further research is required to clarify factors inducing extrinsic or intrinsic 5-HT sprouting in DA-denervated lesions. Considering the fact that 5-HT can reduce druginduced rotation in 6-OHDA-iesioned animals and that modulation of D A transmission by 5-HT afferents seems to be possible, the occurrence of 5-HT hyperinnervation may be related to the functional effect produced by the grafts.
177 Acknowledgements. The authors thank the Parkinson's Disease Society and the Medical Research Council (MRC) for support. We are also grateful to Dr. Luthert, Department of Neuropathology, Institute of Psychiatry, for useful suggestions in quantitative
analysis. Y.T. was sponsored by Anglo-Japanese Scientific Exchange Program between the Royal Society and the Japan Society for Promotion of Science during 1989-90. S.B. is receiving a MRC Training Fellowship.
ABBREVIATIONS
CP F TO V
CA CC CI
anterior commissure corpus callosum internal capsule
REFERENCES 1 Bj6rklund, A., Stenevi, U., Schmidt, R.H., Dunnett, S.B. and Gage, F.H., Intracerebrai grafting of neuronal cell suspensions. I, Introduction and general methods of preparation, Acta Physiol. Scand., Suppl. 522 (1983) 1-7. 2 Bj6rklund, A., Stenevi, U., Schmidt, R.H., Dunnett, S.B. and Gage, F.H., Intracerebral grafting of neuronal cell suspensions. II. Survival and growth of nigral cell suspensions implanted in different brain sites, Acta Physiol. Scand., Suppl. 522 (1983) 9-19. 3 Blunt, S., Jenner, P. and Marsden, C.D., The effect of chronic L-DOPA treatment on the recovery of motor function in 6-OHDA-lesioned rats receiving ventral mesencephalic grafts, Neuroscience, submitted. 4 Brown, V.J. and Dunnett, S.B., Comparison of adrenal and foetal nigral grafts on drug-induced rotation in rats with 6-OHDA lesions, Exp. Brain Res., 78 (1989) 214-218. 5 Clarke, D.J., Brundin, P., Stecker, R.E., Nilsson, O.G., BjOrklund, A. and Lindval, O., Human fetal dopamine neurons grafted in a rat model of Parkinson's disease: ultrastructural evidence for synapse formation using tyrosine hydroxylase immunocytochemistry, Exp. Brain Res., 73 (1988) 115-126. 6 De Simoni, M.G., Dal Toso, G., Fodritto, E, Sokola, A. and Algeri, S., Modulation of striatat dopamine metabolism by the activity of dorsal raphe serotonergic afferences, Brain Research, 411 (1987) 81-88. 7 Doucet, G., Murata, Y., Brundin, P., Bosler, O., Mons, N., Geffard, M., Ouimet, C.C. and Bj6rklund, A., Host afferents into intrastrial transplants of fetal ventral mesencephalon, Exp. Neurol., 106 (1989) 1-19. 8 Dunnett, S.B., Bj6rklund, A., Schmidt, R.H., Stenevi, U. and Iversen, S.D., Intracerebral grafting of neuronal cell suspensions. IV. Behavioral recovery in rats with unilateral 6-OHDA lesions following implantation of nigral cell suspensions in different forebrain sites, Acta Physiol. Scand., Suppl. 522 (1983) 29-37. 9 Dunnett, S.B., Hernandez, T.D., Summerfield, A., Jones, G.H. and Arbuthnott, G., Graft-derived recovery from 6-OHDA lesions: specificity of ventral mesencephalic graft tissues, Exp. Brain Res., 71 (1988) 411-424. 10 lshimura, K., Takeuchi, Y., Fujiwara, K., Tominaga, M.,
caudate-putamen (neostriatum) fornix optic tract lateral ventricle.
Yoshioka, H. and Sawada, T., Quantitative analysis of the distribution of serotonin-immunoreactive cell bodies in the mouse brain, Neurosci. Lett., 91 (1988) 265-270. 11 KOnig, J.ER. and Klippel, R.A., The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Stem, Williams and Wilkins, Baltimore, 1963. 12 Milson, J.A. and Pycoek, C.J., Effects of drugs acting on cerebral 5-hydroxytryptamine mechanisms on dopamine-dependent turning behavior in mice, Br. J. Pharmacol., 56 (1976) 77-85. 13 Perese, D.A., Ulman, J., Viola, J., Ewing, S.E. and Bankiewicz, K.S., A 6-hydroxydopamine-induced selective parkinsonian rat model, Brain Research, 494 (1989) 285-293. 14 Pycock, C.J., Turning behavior in animals, Neuroscience, 5 (1980) 461-514. 15 Segovia, J., Meloni, R. and Gale, K., Effect of dopaminergic denervation and transplant-derived reinnervation on a marker of striatal GABAergic function, Brain Research, 493 (1989) 185189. 16 Snyder, A.M., Zigmond, M.J. and Lund, R.D., Sprouting of serotoninergic afferents into striatum after dopamine-depleting lesions in infant rats: a retrograde transport and immunocytochemical study, J. Comp. Neurol., 245 (1986) 274-281. 17 Snyder-Keller, A.M., Carder, R.K. and Lund, R.D., Development of dopamine innervation and turning behavior in dopamine-depleted infant rats receiving unilateral nigral transplants, Neuroscience, 30 (1989) 779-794. 18 Takeuchi, Y. and Sano, Y., Serotonin nerve fibers in the primary visual cortex of the monkey, quantitative and immunoelectron microscopical analysis, Anat. Embryol., 169 (1984) 1-8. 19 Takeuchi, Y., Kimura H. and Sano, Y., Immunohistochemical demonstration of the distribution of serotonin neurons in the brainstem of the rat and cat, Cell Tiss. Res., 224 (1982) 247-267. 20 Takeuchi, Y., Fujiwara, K., Sato, N., Tominaga, M., Hasegawa, K., Osamura, T. and Sawada, T., Further confirmation of serotonin reduction in the neostriatum during hyperthermiainduced convulsions: a quantitative immunohistochemical study, Acta Neuropathol., 77 (1989) 254-257. 21 Takeuchi, Y., Sawada, T., Blunt, S., Jenner, P. and Marsden, C.D., Effects of 6-OHDA lesions of the nigrostriatal pathway on striatal serotonin innervation revealed by immunohistochemistry, in preparation.
Brain Research, 551 (1991) 171-177 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 A DONIS 000689939116663Q BRES 16663
Serotonergic sprouting in the neostriatum after intrastriatal transplantation of fetal ventral mesencephalon Y. Takeuchi L3, T. Sawada 3, S. Blunt L2, P. Jenner ~ and C.D. Marsden 2 l Parkinson's Disease Society Experimental Research Laboratories, Pharmacology Group, Biomedical Sciences Division, King's College London, London (U. K.), 2University Department of Clinical Neurology, Institute of Neurology, National Hospital, London (U. K.) and 3Department of Pediatrics, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto (Japan) (Accepted 8 January 1991)
Key words: Neuronal transplantation; Neostriatum; Dopamine; Serotonin; Immunohistoehemistry; Parkinson's disease
The sprouting of serotonergic fibers into the neostriatum of 6-OHDA-lesioned rats should be considered, when assessing the functional aspects of ventral mesencephalic grafts. The degree of serotonergic fiber growth into the neostriatum of rats subjected to unilateral 6-OHDA lesions followed by fetal ventral mesencephalic grafts was examined immunohistochemically. As a measure of serotonergic innervation density, the total length of immunoreactive fibers was quantified as percent area. Four weeks after transplantation, there was complete recovery of (+)-amphetamine-induced rotation. All of the ventral mesencephalic grafts contained serotonin-immunoreactive cells, but much fewer in number than the tyrosine hydroxylase-positive cells found in the same grafts. However, there was significant serotonergic hyperinnervation throughout the transplanted neostriatum compared with the control side. The hyperinnervation derived from the grafted neurons was most marked in the dorsal and lateral sectors of the rostral neostriatum. At 24 weeks after transplantation, the implanted neostriatum still showed a dense serotonergic innervation. INTRODUCTION The replacement of degenerating dopamine ( D A ) cells by fetal mesencephalic tissue is a new approach to the t r e a t m e n t of Parkinson's disease and the experimental background is based mainly on transplantation studies in 6-hydroxydopamine ( 6 - O H D A ) - l e s i o n e d rats 3'4'7"8"13'14. It has recently been shown that some serotonin (5H T ) - i m m u n o r e a c t i v e cells are always present in DA-rich ventral mesencephalic grafts using standard transplantation techniques 7, but 5-HT-immunoreactive fiber growth into the neostriatum from the grafts has not been demonstrated. In assessing the function of DA-rich grafts in the 6 - O H D A - l e s i o n e d rat model of Parkinson's disease, the extent of 5-HT-immunoreactive fiber growth is important since 5-HT can reduce drug-induced rotation in 6 - O H D A - l e s i o n e d animals and L-dihydroxyphenylalanine (L-DOPA) may be decarboxylated in striatal serotonergic fibers 14. In addition, serotonergic neurons have a presynaptic interaction with nigrostriatal dopaminergic neurons, through which they may modulate dopaminergic transmission6. We have therefore examined immunohistochemically the degree of serotonergic fiber growth into the neostriatum of rats subjected to unilateral
6 - O H D A lesions followed by fetal ventral mesencephalic grafts.
MATERIALS AND METHODS
6-OHDA-induced nigrostriatal lesions Female Wistar rats (Bantin and Kingman, U.K.) weighing 150-200 g were housed in groups of 6 rats per cage and maintained on a 12:12 h light-dark cycle with ad libitum access to food and water. Rats were anesthetized with pentobarbital (50 mg/kg) intraperitoneally (i.p.), and injected with 8 /zg as the base of 6-OHDA-HBr (Sigma, U.S.A.) in 4 #1 of 0.9% saline containing 0.2 mg/ml ascorbate into the left nigrostriatal bundle. Stereotaxic coordinates for the site were: A = 2.2 mm posterior to bregma, L = 1.5 mm on the left side lateral to the midline, V = 8.0 mm below the dura, with the incisor bar set +4.5 mm above the interaural line. Injections (4/~l) were made with Hamilton syringe, at the rate of 1 /~l/min with a further 4 min allowed for diffusion before retraction of the syringe.
Assessment of rotational behavior One to 2 weeks after lesion surgery, (+)-amphetamine-induced rotation was assessed over a 1 h period following intraperitoneal injection of (+)-amphetamine sulphate (5 mg/kg; Sigma, U.S.A.) dissolved in 0.9% saline. Rats were placed in individual plastic boxes and the number of complete turns made during a l-rain period was recorded every 10 rain. Only rats showing ipsilateral turning rates of >7/min were selected for further study3. (+)-Amphetamine-induced rotation was also counted 4 weeks after transplantation surgery (see below).
Correspondence: Y. Takeuchi, Department of Pediatrics, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602, Japan.
172 Amphetamine-induced rotation
Apomorphine-induced rotation was assessed 2-3 days following each (+)-amphetamine-induced rotation test. Contralateral rotation was monitored over a 1 h period following subcutaneous injection of apomorphine-HCl (0.5 mg/kg; Sigma, U.S.A.) dissolved in 0.9f/~ saline containing 0.2% ascorbate.
20-
1816 1412-
Transplantation of fetal ventral mesencephalon Graft tissue was taken from rat embryos of c r o w n - r u m p length (CRL) 12-14 mm. DA-rich tissue was dissected from the floor of the mesencephalic flexure, containing the developing cells of the substantia nigra and ventral tegmental area according to Bj6rklund et al.~. The tissue pieces of 10 embryos from one pregnant mother were collected in sterile 0.6% glucose-rich 0.9% saline, then incubated for 20 min at room temperature in a solution containing 0.1% trypsin (Sigma, grade 2 crude) in glucose-saline. Following incubation, the pieces were washed by repeated replacement of glucose-saline to a final volume of 10 embryonic pieces per 100 pl, then dissociated by repeated aspiration through a polished Pasteur pipette to form a suspension of dissociated cells. The cell suspension was drawn up into a 10 /tl glass microsyringe (Scientific Glass Engineering, Australia). Aliquots of graft tissue were stereotaxically implanted into the left neostriatum (caudate-putamen complex) in two deposits at the following sites: A = 1.8 mm, L = 2.5 mm, V = 5.0 mm; and A = 1.0 mm, L = 3.5 mm, V = 5.0 mm. T h r e e p l was deposited at each site, injected over 3 rain with a further 4 rain allowed for diffusion before retraction of the syringe needle (n = 9). Sham-transplants were performed using vehicle only (n = 3).
lmmunohistochemistry Four and 24 weeks following transplantation, rats were anesthetized with pentobarbital (50 mg/kg) i.p. and perfused transcardially with phosphate-buffered saline (PBS; 0.9% NaCI in 0.l M sodium phosphate buffer) and subsequently with a fixative containing 4% paraformaldehyde in 0.1 M sodium phosphate buffer. The brain blocks were immersed in the fixative solution for 48 h at 4 °C. The blocks were stored in 15% sucrose overnight and then sectioned in the frontal plane with a cryostat (20 pm, -20 °C), and 4 parallel series were collected through the area of the neostriatum and the substantia nigra in PBS. Two series were mounted onto glass slides and stained with Cresyl violet. Two other series of sections were rinsed in PBS containing 0.3% Triton X-100 and incubated in 10% normal goat serum for 1 h at room temperature. Free-floating sections in PBS containing 0.3% Triton X-100 were incubated for 48 h at 4 °C in primary antibodies to 5-HT (1:12 000; Takeuchi et al. ]9) or tyrosine hydroxylase (TH, 1:2000; Eugene Tec, U.S.A.). All sections were taken through the Vector A B C immunohistochemical method with diaminobenzidine as chromogen. Sections were mounted onto gelatin-coated slides, dehydrated and coverslipped. The specificity of 5-HT immunohistochemical staining was as described previously 19.
E
I0~
420-
..o-. . . . . -o- . . . . . *o 6 0 H D A + g r a n .
110
20
310
410
~0
dO minutes
Fig. 1. (+)-Amphetamine-induced rotation: each point shows ipsilateral turns per minute (mean + S.E.M.) following injection of 5 mg/kg i.p. Negative scores signify contralateral turns. Closed circles = 6-OHDA-lesioned rats, one week after lesioning (n = 12); open circles = 6-OHDA-lesioned and implanted rats, 4 weeks after transplantation (n = 9).
RESULTS
Rotation tests
When 6-OHDA-lesioned rats received (+)-amphetamine (5 mg/kg, i.p.), they began to sniff and after 10 rain a steady rotation towards the left or lesioned side had developed. The peak rotational rate was 18.0 + 2.5 per minute (mean _+ S.E.M., n = 12, Fig. 1). These selected rats received apomorphine-sulfate (0.5 mg/kg, s.c.) and they demonstrated a contralateral rotation (away from the lesioned side). The peak rotational rate was 11.7 + 2.0 per minute (n = 12, Fig. 2). After receiving grafts of DA-rich fetal tissue, these rats showed an almost complete recovery of (+)-amphetamine-induced rotational Apomorphine-induced
rotation
18-
Quantitative analysis The animals were sacrificed for quantitative immunohistochemistry 4 weeks following transplantation (n = 6). Quantitative analysis of 5-HT innervation density was made in two frontal sections per animal representing the rostral (at +2.6 level according to the atlas of K6nig and Klippe111) and caudal neostriatum (at -0.8 level). The rostral and caudal sections of the neostriatum were divided into 4 (dorsal, ventral, lateral and medial) and two sectors (dorso-medial and ventro-lateral), respectively. As a measure of 5-HT innervation density, the total or cumulative length of 5-HT-immunoreactive fibers were used according to the previous studies 18"2°. Using a Kontron IBAS 2000, 5-HT-immunoreactive fibers per unit area in both hemispheres was quantified as percent area after 'skeletonization'. In addition, the number of TH and 5-HT-immunoreactive cells in the grafts were counted per section at +2.6 level according to the atlas four weeks (n = 6) and 24 weeks (n - 3) following transplantation.
60HDA
1614-
.~ 12~ 10-
~
6-OHDA
L.
~m
4
...... "~ ...... "~...... X~...... ~ ...... "~ 6--OHDA+ graft 0 i
i
i
I
I
10
20
50
40
50
i
~0minutes
Fig, 2, Apomorphine-induced rotation: each point shows contralateral turns per minute (mean + S.E.M.) following injection of 0.5 mg/kg i.p. Closed circles = 6-OHDA-lesioned rats, one week after Icsioning (n = 12); open circles = 6-OHDA-lesioned and implanted rats, 4 weeks after transplantation (n = 9).
173
Fig. 3. TH immunohistochemical staining of the neostriatum 4 weeks after transplantation. A number of TH-positive grafted neurons were observed mainly at the periphery of the transplant and their TH-positive fibers were seen to project into the host striatal tissue. A xl00; B x 200.
behaviors (n = 9, Fig. 1). Some of the animals showed contralateral rotation (away from the left side) for 30 min following administration of (+)-amphetamine indicating an overcompensation by the grafts, but the majority of animals showed no (+)-amphetamine-induced rotation. Additionally, these rats revealed low levels of apomorphine-induced rotation, the peak rotational score being only 3.0 + 1.2 turns per minute (n = 9, Fig. 2). On the other hand, sham-transplanted rats showed no recovery of drug-induced rotational behaviors.
TH imrnunohistochemistry Four weeks after transplantation, virtually no THimmunoreactive cells were visible in the substantia nigra ipsilateral to 6 - O H D A lesion. All implanted rats were
found to have grafts containing multipolar or irregularshaped TH-positive cells (Fig. 3). The number of THimmunoreactive cells in the grafts was 71.7 + 5.8 (mean + S.E.M., n = 6) per section of the rostral striatum at +2.6 level. These cells were seen to give rise to TH-positive fibers growing into the host neostriatum. Neighboring the grafts, the density of TH-positive fibers approached that of the contralateral non DA-denervated neostriatum (Fig. 3). Although TH-positive fibers from the grafts were distributed throughout the neostriatum, there was a difference in the innervation density of TH-immunoreactive fibers in the peripheral portions of the neostriatum between the control and implanted sides (Fig. 5). In sham-transplanted rats, there was no difference in the innervation density of TH-immunoreactive
Fig. 4. 5-HT immunohistochemical staining of the neostriatum 4 weeks after transplantation. Some 5-HT-immunoreactive cells were present in the graft and a high density of 5-HT-immunoreactive fibers was observed both in the graft and the host neostriatum. A xl00, B x200.
174
CC
Fig. 5. Immunohistochemicai staining for TH in the dorsal sector at the rostral level (+2.6). A: control (right) side; B: lesioned and transplanted (left) side. ×200.
fibers in the neostriatum between the control and sham-transplanted sides. Twenty-four weeks after transplantation, surviving TH-positive cells were visible in the grafts, the n u m b e r of TH-positive cells being 82.3 ___ 10.6 (mean + S . E . M . , n = 3) per section of the rostral striatum at +2.6 level. TH-positive fibers were distributed throughout the neostriatum as observed 4 weeks after transplantation.
5-HT immunohistochemistry Four weeks after transplantation, all of the ventral mesencephalic grafts also contained 5-HT-immunoreactive cells. The n u m b e r of them was 17.2 __+ 1.4 (mean + S . E . M . , n = 6) per section of the rostral striatum at + 2 . 6 level, but much fewer than that of TH-positive cells
in the same grafts (Fig. 4). H o w e v e r , there was a m a r k e d 5-HT hyperinnervation throughout the implanted neostriatum c o m p a r e d with the control side. The morphological characteristics of 5-HT-immunoreactive fibers were much the same as those of the control side. 5-HT-positive fibers c o m p o s e d an intricate network of thin, filiform profiles, irregularly p u n c t u a t e d by small, round or ovoid dilatations, i.e. varicosities. On the other hand, relatively straight fibers of larger size, which were observed among the m y e l i n a t e d axons on the control side, were rarely seen. In the grafts, the innervation density of 5-HT-immunoreactive fibers was so high that neither intervaricose segments nor proximal portions of 5-HT axons could be observed (Figs. 4, 6). The 5-HT hyperinnervation o b s e r v e d throughout the neostriatum
Fig. 6. Immunohistochemical staining for 5-HT in the dorsal sector at the rostral level (+2.6) A: control (right) side. B: lesioned and transplanted (left) side. The innervation of 5-HT-immunoreactive fibers was much denser on the transplanted side (B) compared to the control side (A). × 200.
175
Fig. 7. Immunohistochemical staining for 5-HT in the neostriatum at the rostral (+2.6, A,B) and caudal (-0.8, C,D) levels, 24 weeks after transplantation. On the transplanted side, serotonergic hyperinnervation remained to be unchanged throughout the neostriatum. B: the dorsolateral sector of the rostral neostriatum. D: the ventrolateral sector of the caudal neostriatum. On the control side, a moderate density of 5-HT-immunoreactive fibers was observed. A: the dorsolateral portion of the rostral neostriatum. C: the ventrolateral sector of the caudal neostriatum, x 200.
was most marked in the dorso-lateral portion of the rostral neostriatum, where the innervation density of 5-HT fibers was almost as high as that of the D A innervation density on the control side (Figs. 5, 6). In sham-transplanted rats, no marked difference was observed in the innervation density of striatal serotonergic fibers between the two sides. Twenty-four weeks after transplantation, the serotonergic hyperinnervation was still present in the neostriatum of the implanted rats and the number of 5HT-positive cells was 15.8 + 2.7 (mean + S.E.M., n = 3) per section of the rostral striatum at 2.6 level. The quantitative analysis also revealed that 5-HTimmunoreactive fibers were unevenly distributed, with the percent area of immunostained fibers ranging from 2.65 to 6.53 per unit area (Table I). The innervation density was greatest ventrally and caudally on the control
side. On the implanted side, the hyperinnervation was greatest in the lateral and dorsal sectors of the rostral neostriatum which showed 12.7 and 11.1, respectively, as a percent area of 5-HT-immunostained fibers. The quantitative analysis revealed a significant hyperinnervation of 5-HT-immunoreactive fibers throughout the sectors of both levels. On the other hand, there was no difference in the innervation density of 5-HT-immunoreactive fibers in the cerebral cortex between implanted and control sides (Table I). DISCUSSION The main finding of this study is that significant serotonergic sprouting occurs in the neostriatum after intrastriatal transplantation of fetal ventral mesencephaIon using the standard transplantation technique.
176 TABLE I
Percent area of 5-HT-immunoreactive fibers was listed with standard deviation in the rostral (+2.6) and caudal (-0.8) levels of the neostriaturn, according to the atlas of KOnig and Klippel Evaluation of the gradients was carried out by comparing the data between the control (right) and transplanted (left) sides using the Student's t-test. Asterisks indicate values which are significantly different: *P < 0.01; **P < 0.001 ; NS: not significant (n = 6).
~ _
+ 2.6
c,~
Right
Left
4.43±0.17
NS
4.89±0.46
2.65+0.12 3.60--+0.46
** **
12.74_+1.07 11.07-+0.20
2.97 ± 0.28 5.61±0.34
* **
4.30-+ 0.73 7.60±0.63
6.07-+0.50
**
9.90-+1.03
6.53±0.80
**
10.43-+1.15
- 0.8
F
o
Intrastriatal grafts of fetal mesencephalic DA-rich tissue can compensate for motor deficits resulting from lesions of the nigrostriatal pathway s. In the rat, the functional recovery is believed to depend on the DA fiber growth, into the DA-denervated striatum. There is evidence that the grafts are metabolically, physiologically and biochemically active as they reveal transmitter synthesis, normal firing patterns, and spontaneous DA release s. The functional effects produced by these DA grafts may be due to a controlled synaptic release of D A and not to a simple diffusion of DA 4. Recently, host afferents into fetal ventral mesencephalic tissue grafted to the neostriatum of adult rats were also described 7. Although a large number of studies have been made concerning the D A fiber growth after intrastriatal transplantation of fetal ventral mesencephalon 2~7~9, few studies emphasize the 5-HT fiber growth into the neostriatum. As pointed out by a recent study, some 5-HT-immunoreactive cell bodies are always present in grafts dissected and prepared according to the standard transplantation technique 1. By carefully dissecting only the rostral part of the fetal mesencephalon, the frequency of these cells can be reduced, but it is only by treating the cell suspension with 5,7-dihydroxytryptamine (5,7-DHT) that 5-HT cells can be eliminated in DA-rich cell suspension 7. Thus it is important to consider the exis-
tence of 5-HT-immunoreactive cells and fibers in the neostriatum following intrastriatal transplantation of fetal ventral mesencephalon. Dunnett et al. reported that some rats with mesencephalic DA-rich graft showed an increase in serotonin level in the neostriatum '~. However, the possibility of serotonergic innervation derived from fetal mesencephalon was thought unlikely and it was suggested that the graft-derived 5-HT-immunoreactive fibers soon disappear among the intact serotonergic innervation intrinsic to the host neostriatum surrounding the graft 9. The relation of mesencephalic grafts and host 5-HT density in the striatum was addressed in animals 6-OHDA-treated and transplanted as infants, but the occasional graft 5-HT cells were not considered to contribute to the 5-HT hyperinnervation ~7. The discrepancy between their results and the present study may be attributed to the sensitivity of immunohistochemical methods for 5-HT. However, it should be stressed that in our studies, pretreatments employing tryptophan and monoamine oxidase inhibitor (MAOI) are not used to enhance the visualization of 5-HT, because such pharmacological treatments increase the level of cross-reactive indoleamines other than 5-HT. We advocate that immunohistochemical studies for 5-HT should be carried out in the absence of M A O I and/or tryptophan, in order to compare the data under different experimental conditions more precisely, as pointed out by Ishimura et al. ~°. The 6-OHDA lesion can reduce the 5-HT innervation of the lesioned hemisphere compared to the control hemisphere or to the normal brain 21, and the majority of 5-HT hyperinnervation in the neostriatum is attributed to extrinsic serotonergic sprouting from grafted ventral mesencephalon. It is noteworthy that the serotonergic hyperinnervation does not prevent the replacement of DA innervation derived from the grafts. The fact that TH-positive cells grew and showed fiber outgrowth in the presence of 5-HT hyperinnervation suggests that 5-HT-immunoreactive fibers do not occupy synaptic sites vacated by the 6-OHDA lesion. It is of interest that removal of DA-afferents may lead to an enlargement of the intrinsic serotonergic projection to the neostriatum during the neonatal period, while such hyperinnervation after 6-OHDA lesioning was never observed in adult rats 16'2]. Serotonergic neurons are considered to have a high capacity for axonal sprouting, but further research is required to clarify factors inducing extrinsic or intrinsic 5-HT sprouting in DA-denervated lesions. Considering the fact that 5-HT can reduce druginduced rotation in 6-OHDA-iesioned animals and that modulation of D A transmission by 5-HT afferents seems to be possible, the occurrence of 5-HT hyperinnervation may be related to the functional effect produced by the grafts.
177 Acknowledgements. The authors thank the Parkinson's Disease Society and the Medical Research Council (MRC) for support. We are also grateful to Dr. Luthert, Department of Neuropathology, Institute of Psychiatry, for useful suggestions in quantitative
analysis. Y.T. was sponsored by Anglo-Japanese Scientific Exchange Program between the Royal Society and the Japan Society for Promotion of Science during 1989-90. S.B. is receiving a MRC Training Fellowship.
ABBREVIATIONS
CP F TO V
CA CC CI
anterior commissure corpus callosum internal capsule
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caudate-putamen (neostriatum) fornix optic tract lateral ventricle.
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