Dopaminergic responses to striatal damage

JOURNAL

OF THE

NEUROLOGICAL SCIENCES

ELSEVIER

Journal of the Neurological Sciences 139( 1996) 125- 130

Dopaminergic responses to striatal damage David W. Howells *, Gabriel T. Liberatore, John Y.F. Wong, Geoffrey A. Donnan Departments

of Neurology

and Medicine,

University

qf Melbourne,

Austin and Repatriation

Medkal

Cerztre, Heidelberg,

Victoria

3084. Australia

Received 22 May 1995;revised 30 January 1996;accepted 18 February 1996

Abstract The improvements obtained by grafting dopamine-rich tissues into the striatum of patients with Parkinson’s disease are generally attributed to production and release of dopamine by the graft. However, it is becoming increasingly clear that grafting also stimulates the host dopaminergic system. We provide evidence in a mouse model of striatal damage that surgical cavitation induces a concerted response from the dopaminergic system with proliferation of striatal presynaptic dopamine uptake sites, increased tyrosine hydroxylase activity, increased concentrations of dopamine, dihydroxyphenylacetic acid and homovanillic acid. The response increases with time and ultimately includes contralateral stimulation of striatal tyrosine hydroxylase activity and elevation of dihydroxyphenylacetic acid and homovanillic acid concentrations. The time course and extent of the host dopaminergic response suggests that it may make a significant contribution to observed clinical improvements after intrastriatal transplantation in human parkinsonism. Keywords: Striatum; Dopamine; Injury; Host; Regeneration

1. Introduction

Re-establishment of the nigrostriatal dopamine pathway by intracerebral implantation of tissues rich in dopamine was first reported as a potential therapy for Parkinson’s disease in 1979 (Bjorklund and Stenevi, 1979). Although adrenal medullary grafts have proved disappointing (Freed et al., 1990), grafts of foetal mesencephalic tissues do improve some features of parkinsonism in dopamine-depleted rodents (Rose et al., 1985; Sirinathsinghji et al., 1990; Moukhles et al., 19941, non-human primates (Bankiewicz et al., 1990), patients with MPTP-induced parkinsonism (Widner et al., 1992) and idiopathic Parkinson’s disease (Freed et al., 1992; Lindval et al., 1992; Spencer et al., 1992). The reported beneficial effect of reduced reliance on anti-parkinsonian drugs (with concomitant reduction of unwanted side effects), prolonged duration of action of L-DOPA, increased time in the mobile ‘ON’ phase, and reduced rigidity and hypokinesia during the ‘OFF’ phase (Freed et al., 1992; Lindval et al., 1992) offers some hope for future treatment of Parkinson’s * Correspondingauthor. Department of Neurology, Austin and Repatriation Medical Centre, Heidelberg, Victoria 3084, Australia. Tel.: (+ 6 l-3) 9496-3789; Fax: (+61-3) 9496-3805.

disease, particularly if grafted tissues do not succumb to the ravages of the disease itself. If further progress in grafting techniques is to be made, we must fully understand the processes that lead to clinical benefit. Reinnervation of the denervated host striatum, formation of synaptic contacts with host striatal neurones, restoration of striatal dopamine synthesis and dopamine release by the graft (Bjorklund et al., 1983; Freund et al., 1985; Nishino et al., 1990; Moukhles et al., 1994) are likely to play a role. However, evidence is accumulating which suggests that grafting also stimulates the activity of residual host dopaminergic neurones (Jaeger et al., 1983; Fiandaca et al., 1988; Bankiewicz et al., 1990; Hirsch et al., 1990; Plunkett et al., 1990; Przedborski et al., 1991; Howells et al., 1993). Animal models of parkinsonism with surgically Iesioned striata, but no grafts, have shown clinical improvement (Fiandaca et al., 1988; Plunkett et al., 1990; Przedborski et al., l991), but detailed neurochemical correlates are lacking. Isolated reports have shown increases in striatal presynaptic dopamine uptake site density in rodents (Przedborski et al., 1991; Howells et al., 1993) and increased host tyrosine hydroxylase immunoreactivity (TH-IR) after grafting (Bankiewicz et al., 1990) or striatal damage in primates (Fiandaca et al., 1988). However, a careful examination of the nature, extent and time course

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of these host dopaminergic changes in a single model has not been performed. We now provide evidence, in the mouse, of a slowly developing, but vigorous, dopaminergic response to striatal damage which includes proliferation of presynaptic dopamine uptake sites with bilateral stimulation of tyrosine hydroxylase activity and dopamine turnover. If similar changes occur after human striatal grafting procedures for Parkinson’s disease, the magnitude of the response may be sufficient to contribute to the beneficial effects of grafting.

2. Materials

and methods

2.1. Experimentul design Five groups of at least 6 male C-57 Black/6J mice (5-6 weeks old, 17-25 g) were subjected to a cavitation procedure (see below). Five age-matched groups of mice, which had no surgery, acted as controls. Three groups of test and control mice were killed 1, 4 and 12 weeks postcavitation and then prepared for presynaptic dopamine uptake site autoradiography. The remaining groups were killed 4 and 8 weeks postcavitation and the brains prepared for measurement of dopamine metabolites and tyrosine hydroxylase activity. 2.2. Surgery All mice were anaesthetised with Nembutal (70 mg/kg, i.p.), given atropine (0.5 mg/kg, i.p.> to keep their airway free of secretions, and placed in a rat stereotaxic frame with the incisor bar set at 0.00 mm. After opening the scalp, a l-mm burr hole was created using a dental drill over the left hemisphere (AP:lO, L:2.0). A 3-mm-deep cavity (measured from the surface of the skull) was then made in the striatal parenchyma by aspiration via an 18-gauge needle passed vertically through the burr hole. The wound was closed using 4.0 silk thread and an antibiotic cream containing polymyxin. bacitracin and neomycin was applied to the operation site to prevent infection. Surgically damaged and age-matched control mice were killed by Nembutal overdose (600 mg/kg, i.p.). 2.3. Dopamine metabolite and tyrosine hydroxylase quantitation For measurement of dopamine metabolites and tyrosine hydroxylase activity, mice were killed and brains were removed within 2 min of death. Two razor blades (Gillette, USA) fixed 2 mm apart were used to dissect a 2-mm-thick coronal slice straddling the site of striatal injury and from the same region (5.5-7.5 mm dorsal to the occipital lobe) in the controls. Operated and contralateral striata were separated on an ice-chilled glass plate and the surrounding

cortical tissue removed before weighing and freezing on dry ice prior to storage at - 80°C. Frozen striatum was thawed in 0.32 M sucrose (2 vols.), homogenised (Sonifier cell disruptor, Mode1 B-l5P, 60 W with 40% pulsed power for 5 min) then split into two equal volumes, one for the measurement of dopamine and its metabolites, the other for the determination of tyrosine hydroxylase activity. For the analysis of dopamine and its metabolites (Murai et al., 19SS), 2.5 ng of 3,4-dihydroxybenzylamine (DHBA) in 0.32 M sucrose was added to the homogenate as an internal standard. Protein was precipitated and removed by shaking in the presence of 0.4 M perchloric acid followed by centrifugation at 12 000 g for 15 min (Beckman Microfuge II, 4°C). An aliquot of 50 ~1 of the clear supernatant was injected directly onto the HPLC system which was calibrated by injection of 50 ~1 of authentic dopamine metabolites (1.25 ng dopamine, DOPAC, HVA and 2.5 ng DHBA) after every 10 samples. The HPLC system consisted of a 100 X 3 mm Phase II ODS column (Bioanalytical systems (BAS)), a BAS dual piston pump (Model PM-go), a CMA/microdialysis AB CMA/200 microsampier, an electrochemical detector (BAS LC44), and a BAS RE-4 flow cell with a glassy carbon working electrode. A filtration degassed (0.45 km type HVLP, Millipore) 50 mM sodium dihydrogen phosphate, 20 mM Tris-sodium citrate mobile phase (pH 4.0), containing 2 mM heptane sulphonic acid, 0.1 mM EDTA and 9% v/v methanol delivered at a flow rate of 0.6 ml/min was used to elute the dopamine metabolites. For measurement of tyrosine hydroxylase activity (Hendry and Iversen, 19711, 10 l.~l of freshly prepared substrate/cofactor mixture (5 mM Tris-HCl, 2.4 mM Ltyrosine, 80 mM L-[2,6-‘Hltyrosine (100 mCi, spec. act. 48 Ci/mM), 1 mM 2-amino-4-hydroxy-6,7-dimethyl5,6,7,%tetrahydropteridine hydrochloride (DMPH,), 72 PM P-mercaptoethanol. and 1 mM 3-hydroxy-4-bromobenzyloxyamine dihydrogen phosphate (NSD 105.5) were added to 10 ~1 of striatal homogenate and the tubes immediately transferred to a water bath and incubated at 37°C for 20 min. The reaction was stopped by the addition of 200 ~1 of 0.4 M perchloric acid containing an excess of unlabelled L-DOPA (2 mg/ml) and cooling to 4°C. If not assayed immediately, the samples were stored at -20°C and thawed prior to assay. The reaction product ["H]LDOPA, was separated from [“Hltyrosine on a 2-cm high column containing approximately 400 mg of washed alumina (Sigma WN-3: neutral) resuspended in 0.5 M potassium phosphate buffer. Just prior to chromatography, 3.8 ml of ‘neutralising solution’ (2: I : 1 of 0.1 M Tris buffer pH 7.4, 0.3 M sodium hydroxide and 0.2 M EDTA, respectively) was added to each sample which was then immediately applied to the column and washed 6 times with 5 mM Tris-HCI buffer, pH 8.6. After the final wash, 1.5 ml of I M acetic acid was used to elute the [‘H]L-DOPA directly into a scintillation vial to which 12 ml of scintil-

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2.4. Presynuptic dopamine uptake site density

400

For autoradiographic determination of presynaptic dopamine uptake site density (Howells et al., 19931, mice were killed and the brains snap frozen in isopentane cooled with dry-ice before being stored at - 80°C. In the 12-week postcavitation group, 3 mice died, leaving 3 for autoradiographic quantitation. A series of 20-pm coronal sections spanning the graft site (or equivalent region in controls) were then cut from each brain using a cryostat (Bright Instruments, chamber and blade maintained at -20°C). These sections were thaw-mounted onto gelatin chromealum-coated glass slides then desiccated and stored at - 80°C. [“HlMazindol binding was measured after preincubation for 5 min at 4°C in 50 mM Tris buffer (pH 7.4, containing 300 mM NaCl, 5 mM KC1 and 0.2% bovine serum albumin), followed by incubation for 60 min in the same buffer containing 4 nM [‘Hlmazindol (spec. act. 24 Ci/mmol, New England Nuclear). To determine the extent of non-specific binding, alternate sections were incubated as above, but in the presence of 1 FM unlabelled mazindol. Excess mazindol was washed off with two consecutive washes in 50 mM Tris buffer at 0°C and the sections dried before exposure to tritium-sensitive Hyperfilm (Amersham) for 3 weeks. After autoradiography, all sections were stained with thionin for anatomical alignment. A minimum of 3 coronal sections from each animal, spanning the damaged site, were used for quantitation. [3H]Mazindol binding density was measured in an approximately 2-mm-

350

control

Cavitation Weeks Post Surgery

Fig. 1. Striatal [3H]mazindol binding (fmol/mg protein) 1, 3 and 12 weeks after cavitation compared with age-matched control mice. Hatched panels, ipsilateral striatum; open panels, contralateral striatum. Mean + SEM. * p I 0.05, f-test comparing ipsilateral striatum 12 weeks after cavitation with controls.

lant (Ecolite (+), ICN Biomedicals) was added. Vials were shaken and left overnight to reduce chemiluminesence before counting (Packard, Tri-carb 4530). 8 s .E

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Fig. 2. Dopaminergic activity 2 months after striatal cavitation (Cav) compared with age-matched control mice (Con). A: tyrosine hydroxylase activity ([‘HIDOPA formed (cpm)/20 min). B: DOPAC concentration (pg/mg wet tissue). C: HVA concentration (pg/mg wet tissue). D: dopamine concentration (pg/mg wet tissue). Hatched panels, ipsilateral striatum; open panels. contralateral striatum. Mean 5 SEM. * p I 0.05, * 1 p I 0.005, t-test comparing ipsdateral and contralateral striatum after cavitation with ‘ipsilateral’ control striatum. For dopamine, the comparison is between ipsilateral and contralateral striatum after cavitation.

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thick crescent of striatum immediately adjacent to the site of damage using a Microcomputer Imaging Device (Image Research Inc., Brock University, St. Catherine’s, Ont., Canada). All values are expressed as fmol/mg tissue of ligand binding after calibration with a tritium microscale (Amersham) exposed with each sheet of film. Student’s t-test was used to compare treatment groups with age-matched untreated controls while a paired t-test was used to compare data from operated and non-operated sides of the striatum within each group.

3. Results Binding of [‘Hlmazindol to presynaptic dopamine uptake sites was not altered 1 week after surgery, but showed a steady increase subsequently, with the greatest increase in the ipsilateral striatum 12 weeks after surgery ( p I 0.05, Fig. 1). In age-matched controls, [ “Hlmazindol binding did not change with time. One (p I 0.05, not shown) and 2 months postsurgery, tyrosine hydroxylase activity was increased in both the damaged left striatum and the undamaged contralateral striatum (Fig. 2A, p 5 0.05). DOPAC concentrations were not altered 1 month after surgery, but after 2 months, there was a bilateral doubling of DOPAC concentration (Fig. 2B, p I 0.005). Similarly, there was no significant change in HVA concentration after the first month, but after 2 months, HVA was increased (Fig. 2C, p 5 0.005). For dopamine, the variability was much greater than for either DOPAC or HVA. In the first month after surgery, the dopamine concentration did not change, but after two months, the dopamine content in the ipsilateral striatum was significantly greater than in the contralateral striatum (Fig. 2D, p I 0.05). Although not statistically significant, a similar ipsilateral vs contralateral trend was seen in the unoperated controls.

4. Discussion Evidence that intrastriatal grafting stimulates the activity of residual host dopaminergic neurones (Jaeger et al., 1983; Fiandaca et al., 1988; Bankiewicz et al., 1990; Hirsch et al., 1990; Plunkett et al., 1990; Przedborski et al., 1991; Howells et al., 1993) is crucial to our understanding of the mechanisms involved in the successes of grafting in Parkinson’s disease. Nevertheless, few have attempted a comprehensive assessment of which elements of the dopaminergic system are stimulated by striatal damage. In this study, we demonstrate that striatal damage induces an increasing and extensive stimulation of dopaminergic activity. As in our previous study (Howells et al., 1993) striatal cavitation stimulated binding of the ligand [ ‘Hlmazindol to presynaptic dopamine uptake sites (Fig. I). This finding, in

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conjunction with observations that host tyrosine hydroxylase immunoreactive positive fibres are increased after striatal grafting (Bankiewicz et al., 1990) or cavitation (Fiandaca et al., 1988) suggests that striatal damage causes proliferation of dopaminergic terminals. In our model, the change in dopamine presynaptic uptake site density was accompanied by increased tyrosine hydroxylase activity 1 (not shown) and 2 months postcavitation (p 5 0.05, Fig. 2A), consistent with an increased capacity for dopamine synthesis. While tyrosine hydroxylase activity can be stimulated by phosphorylation of the enzyme and increased affinity for its tetrahydrobiopterin cofactor (Kettler et al., 1974) the combination of increased enzyme activity (Fig. 2A) and immunoreactivity (Fiandaca et al., 1988; Bankiewicz et al., 1990; Hirsch et al., 1990) suggest increased expression of tyrosine hydroxylase. One month after cavitation, no changes in striatal dopamine, DOPAC or HVA concentrations were noted. However, after 2 months, cavitation caused a significant bilateral increase in both DOPAC and HVA concentration (p I 0.005, Fig. 2B, C) and an ipsilateral increase in dopamine content (p 5 0.05, Fig. 2D). Because of the wide variation in the dopamine data and the presence of an increasing trend in the 2-month control group, the dopamine data must be interpreted with caution. For example, as dopamine concentration depends on synthesis, release, reuptake and metabolism, it has been shown that dopamine concentration within nerve terminals does not correlate well with activity of dopaminergic neurones (Camp and Robinson, 1992) and that tyrosine administration increases the striatal concentration of DOPAC, but not dopamine (Milner and Wurtman, 1986). It is reasonable to suggest that, in our model, dopamine turnover was increased by striatal damage, but the homeostatic mechanisms in the striatum and, in particular, the metabolism of dopamine to DOPAC and HVA (which accumulate, Fig. 2B, C), minimise dopamine accumulation. Together, these findings confirm that striatal damage stimulates a concerted upregulation of dopaminergic activity, even in the absence of grafted dopaminergic tissue. Since the multiple stereotaxic injections (often as many as 10, using l-3 mm cannulae) used in many intrastriatal transplantation protocols undoubtedly cause significant structural damage, the host response we have documented could readily account for the ‘sprouting’ responses seen around adrenal medulla and foetal mesencephalic grafts in monkeys (Fiandaca et al., 1988; Bankiewicz et al., 1990) and patients with Parkinson’s disease (Hirsch et al., 1990). Importantly, the very presence of these host sprouting responses after grafting in patients with Parkinson’s disease demonstrates the remarkable plasticity of the nigrostriatal dopaminergic system, even late in the course of the disease. Twelve weeks after striatal cavitation, presynaptic dopamine uptake site density was increased by 30% (Fig. 1). As shown previously, 10 months after striatal implants

D. W. Howells

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of gel foam, presynaptic dopamine uptake site density adjacent to the implant is increased by 73% (Howells et al., 1993). Within 2 months of striatal cavitation, DOPAC, tyrosine hydroxylase activity, and HVA were increased by 97, 58 and 47%, respectively (Fig. 2). These findings indicate a slow maturation of a vigorous dopaminergic response, the magnitude of which may be sufficient to contribute significantly to the benefits of intrastriatal grafting in man. The timing of the host response is certainly consistent with the abolition of amphetamine-induced rotation 33 days after sham implants in 6-OHDA-lesioned rats (Przedborski et al., 1991), 76-88% reduction in apomorphine induced rotation 6 months after striatal cavitation in MPTP-lesioned monkeys (Plunkett et al., 1990) and partial amelioration of disabling symptoms in patients with Parkinson’s disease after resection of large parts of the caudate (Meyers, 195 I). The time course of these changes also correlates well with “a gradual and significant amelioration of parkinsonian symptoms starting at 6 and 12 weeks after grafting, respectively, reaching maximum stability at approximately 4 to 5 months” after putaminal implantation of foetal mesencephalic tissue in two patients with Parkinson’s disease (Lindval et al., 1992) and slow, but persistent, improvements in Unified Parkinson’s Disease Rating Scale Scores, Activities of Daily Living Scores and Video-Rating Scores for body bradykinesia and postural control during the 6 months after foetal mesencephalic implantation in 7 patients with Parkinson’s disease (Freed et al., 1992). To control for variability in harvesting, handling and processing of tissue, these experiments were designed with an ‘internal control’, in which the surgically damaged striatum was compared with the contralateral, homotypic region on the same coronal slice of tissue. This comparison showed that although the greatest increase in tyrosine hydroxylase activity 1 month after cavitation was seen ipsilaterally, by 2 months, both the operated and unoperated striata exhibited similar increases in tyrosine hydroxylase activity (Fig. 2A). DOPAC and HVA concentrations also showed a similar bilateral increase 2 months after cavitation (Fig. 2B, C) indicating slow development of a bilateral response. Although not statistically significant, presynaptic dopamine uptake density was greater ipsilaterally than contralaterally at each time point (Fig. 1). Ten months after gel foam implantation this difference is fully established with doubling of ipsilateral presynaptic dopamine uptake density and only a modest increase in the unoperated contralateral striatum (Howells et al., 1993). It is of interest that application of basic fibroblast growth factor to the striatum of MPTP-lesioned mice in gel foam implants produces similar bilateral changes of tyrosine hydroxylase activity and dopamine metabolite concentrations with ipsilateral changes of in tyrosine hydroxylase immunoreactivity (Otto and Unsicker, 1990). These contralateral changes may well occur because of crossed catecholaminergic nigrostriatal projections connecting the

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two sides of the striatum (Gerfen et al., 1982; Consolazione et al., 1985) transferring sensorimotor information from one hemisphere to the other and resulting in direct reciprocal control of striatal activities (Consolazione et al., 1985). The bilateral nature of the increase in tyrosine hydroxylase activity and dopamine turnover is particularly relevant in view of repeated observations of bilateral clinical improvement after unilateral striatal implantation of dopaminergic tissue (Freed et al., 1992; Lindval et al., 1992; Spencer et al., 1992). In summary, we have demonstrated that striatal damage stimulates a gradual, concerted activation of striatal dopaminergic activity with stimulation of tyrosine hydroxylase activity, increased concentrations of DOPAC, HVA and dopamine and proliferation of presynaptic dopamine uptake sites. This process appears to involve a bilateral stimulation of tyrosine hydroxylase activity and dopamine turnover, superimposed upon ipsilateral sprouting of dopamine terminals. The changes and their time course appear extensive enough to explain motor responses in animal models and similar changes may contribute to observed clinical improvements in Parkinson’s disease patients with intrastriatal grafts.

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Nishino, H., Hashitani, T., Kumazaki, M. et al. (1990) Long-term survival of grafted cells, dopamine synthesis/release, synaptic connections, and functional recovery after transplantation of foetal nigral cells in rats with unilateral 6-OHDA lesions in the nigrostriatal dopamine pathway. Brain Res., 534: 83-93. Otto, D. and Unsicker, K. (1990) Basic FGF reverses chemical and morphological deficits in the nigrostriatal system of MPTP-treated mice. J. Neurosci., 10: 1912-1921. Plunkett, R.J., Bankiewicz, K.S., Cummins, A.C. et al. (1990) Long-term evaluation of hemiparkinsonian monkeys after adrenal autografting or cavitation alone. J. Neurosurg., 73: 918-926. Przedborski, S., Levivier, M., Kostic, V. et al. (1991) Sham transplantation protects 6-hydroxydopamine-induced dopaminergic toxicity in rats: behavioural and morphological evidence. Brain Res., 550: 23l238. Rose, G., Gerhardt, G., Stromberg, I., Olson, L. and Hoffer, B. (1985) Monoamine release from dopamine-depleted rat caudate nucleus reinnervated by substantia nigra transplants: an in vivo electrochemical study. Brain Res., 341: 92-100. Sirinathsinghji, D.J.S., Dunnett, S.B., Northrop, A.J. and Morris, B.J. (1990) Experimental hemiparkinsonism in the rat following chronic unilateral infusion of MPP+ into the nigrostriatal dopamine pathwayIII. Reversal by embryonic nigral dopamine grafts. Neuroscience, 37: 757-766. Spencer, D.D., Robbins, R.J., Naftolin, F. et al. (1992) Unilateral transplantations of human foetal mesencephalic tissue into the caudate of patients with Parkinson’s disease. New Engl. J. Med., 327: l5411548. Widner, H., Tetrud, J., Rehncrona, S., et al. (1992) Bilateral foetal mesencephalic grafting in two patients with parkinsonism induced by 1-methyl-Cphenyl- 1,2,3,6-tetrahydopyridine (MPTP). New Engl. J. Med., 327: 1556-1563.