Dopamine metabolism in the striatum of hemiparkinsonian model rats with dopaminergic grafts

Neuroscience Research 30 (1998) 43 – 52

Dopamine metabolism in the striatum of hemiparkinsonian model rats with dopaminergic grafts Takeshi Hashitani a,*, Kiminao Mizukawa b, Michiko Kumazaki a, Hitoo Nishino a a

Department of Physiology, Nagoya City Uni6ersity Medical School, Mizuho-cho Mizuho-ku, Nagoya 467, Japan b Department of Anatomy, Okayama Uni6ersity, Medical School, Shikata-cho, Okayama 700, Japan Received 8 September 1997; accepted 27 October 1997

Abstract To investigate dopamine (DA) levels as well as DA metabolism by which the striatal DAergic grafts may bring the functional recovery to hemiparkinsonian model rats, a microdialysis study was performed in the striatum, and an autoradiographic analysis for DA transpoter was made. In hemiparkinsonian model rats, the concentrations of DA, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in striatal perfusates, decreased considerably (less than 5% of control levels). In grafted rats that showed motor recovery, the concentration of DA recovered to almost control level, and DOPAC and HVA to about 20% of controls’ suggesting that the rate of DA metabolism is low. L-DOPA loading to grafted rats induced a big release of DOPAC and HVA, thus the DOPAC/DA ratio was close to that of the controls’. Methamphetamine loading increased the concentration of DA but did not change the level of DOPAC and HVA. Haloperidol loading increased DA, DOPAC and HVA. [3H]mazindol binding that reflects the activity of the DA transpoter decreased considerably in hemiparkinsonian model rats, but it reappeared more or less in grafted rats. Data indicated that in grafted striatum, the extracellular DA level is almost normal level while the rate of DA metabolism is low. By L-DOPA loading, the grafts show the capacity to synthesize, release and metabolize DA and then the DOPAC/DA ratio is normalized. Responses to methamphetamine and haloperidol, as well as the results of the autoradiographic study suggest that the grafts are under a good feedback regulation of DA metabolism. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neural transplantation; Parkinson’s disease; Dopamine; Microdialysis; Autoradiography; Transporter; Striatum; Rat

1. Introduction Fetal mesencephalic grafts in the striatum of hemiparkinsonian model rats ameliorate motor imbalance (Bjo¨rklund and Stenevi, 1979; Freed et al., 1980; Bjo¨rklund et al., 1981; Dunnett et al., 1981a,b; Bolam et al., 1987; Nishino et al., 1990). The mechanisms of the amelioration have been reported as follows; (1) grafted DAergic neurons survive and develop in the host striatum (Nishino et al., 1986a) (2) grafts synthesize and release dopamine (DA) (Zetterstro¨m et al., 1986; Nishino et al., 1990) (3) upregulation of DA * Corresponding author. Tel.: + 81 52 853 8134; fax: +81 52 842 3069.

receptor activity or destruction of DA transport-machinery would be restored (Blunt et al., 1991; Dawson et al., 1991; Nishino, 1993); (4) neural networks between grafted neurons and host brain would be restored (Freund et al., 1985; Nishino et al., 1986b; Bolam et al., 1987; Nishino et al., 1990). On the other hand, the possibility that the grafts release trophic substances and promote regeneration or sprouting of host neurons has been reported especially in the case of adrenal medullary grafts in MPTP intoxication models (Date et al., 1990). However, only a small amount of data dealt with the dynamic aspects of the grafts (Schmidt et al., 1982; Hattori et al., 1993, 1994, 1996), thus there still remains a question about the dynamic potential of the grafts that induces functional restoration. To get a clue

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for this question, in the present study, we first made DAergic grafts in DA denervated striatum and investigated the recovery of motor imbalance (rotational behavior). Secondly, we measured the concentration of striatal DA, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) using in vivo microdialysis, and their responses to loading of L-DOPA, methamphetamine and haloperidol. Thirdly, using autoradiography, we evaluated the variation of DA transporter activity in relation to functional restoration (Javitch et al., 1984). Dynamic response of striatal DA and its matabolism to these modulations may provide an important clue to understanding the activity level of the grafts and their potential to ameliorate motor function.

2. Materials and methods

2.1. Animals Young female Wistar rats (weighing 80 – 90 g at the start of the experiment) were used. They were housed in cages (four to five rats together) with ad libitum access to food and water in a room (25°C) under 12-h light/ 12-h dark condition. Midbrain donor tissues were obtained from fetuses (E 15 – 16) of the same inbred strain. Animal care and handling were carried out according to the guidelines of the Institute for Experimental Animal Sciences, Nagoya City University Medical School.

2.2. 6 -OHDA lesion Under pentobarbital anesthesia (40 mg/kg, i.p.) with atropine sulfate (0.01 mg/kg, i.p.), 4 ml of 6-OHDA solution (8 mg as free base in 4 ml of saline containing 0.5 mg/ml ascorbic acid) was injected stereotaxically into the left substantia nigra (SN, 2 mm anterior to interaural line; 1.6 mm lateral to midline and 7.2 mm below the dura) over 4 min (Ko¨nig and Klippel, 1963; Nishino et al., 1990). The injection cannula remained in position for 5 min after the injection and then removed slowly.

2.3. Methamphetamine-induced rotations For the detection of motor imbalance and its improvement after grafting, methamphetamine (3 mg/kg, i.p.), induced rotation (Ungerstedt and Arbuthnott, 1970) was assessed by counting rotations per min at every 10 min for 1 h. Rats that made more than eight full turns per min in two tests at 1 and 2 weeks after 6-OHDA lesion, were regarded as lesioned rats. The rotations were evaluated at 2, 4, 6, 8, 12 and 16 weeks after transplantation to investigate the motor improvement.

2.4. Transplantation Under deep anesthesia, fetuses were obtained from pregnant rats (gestational day 15–16). Ventral mesencephalic tissue was dissected out from the embryos and collected in a basic medium (0.6% glucose in saline). After removing dura and vessels, the tissues were chopped into small pieces and were incubated in a solution containing 0.05% trypsin (Sigma, type II) and 0.001% collagenase (Sigma, type IV) for 30 min at 37°C. Trypsin action was stopped by adding soybean trypsin inhibitor (0.1 mg/ml). DNase (0.1 mg/ml) was added to prevent cell aggregation. Tissues were then dissociated into cell suspensions by gentle trituration using a fire-polished Pasteur pipette and washed three times in the basic medium. Quantities of 5 ml of the suspension (cell density; about 5× 107 cells/ml) were stereotaxically injected into two separate sites of the striatum ipsilateral to the lesion (A, 0.5 mm anterior to the bregma; L, 2.5 mm; V, 5 mm below the dura; and A, 0.5 mm posterior to the bregma; L, 3.2 mm; V, 5 mm below the dura) at 3 weeks after the lesion (Nishino et al., 1990).

2.5. Tyrosine hydroxylase immunocytochemistry At 16 weeks after the transplantation, animals were perfusion-fixed with a fixative. Brains were removed and cryosections (50 mm thickness) were processed for immunocytochemistry for tyrosine hydroxylase (TH) to detect the growth of implanted catecholaminergic cells and also to detect the extent of 6-OHDA lesion in the SN. The procedure was the same as reported previously (Nishino et al., 1986a, 1990).

2.6. Autoradiography The activity of DA transporter was evaluated by the binding of [3H]mazindol (15.5 Ci/nmol) using autoradiography (Ogawa et al., 1997). Rats were sacrificed by decapitation and brains were quickly removed and frozen on crushed dry ice. Frozen sections were made on a cryostat (20 mm thickness), thaw-mounted on slide glass pretreated with gelatin, air dried at room temperature and stored at − 70°C. Prior to the binding assay the sections were gradually brought to room temperature and incubated twice with a buffer (containing 300 mM NaCl, 5 mM KCl, 50 mM desipramine and Tris– HCl at pH 7.9) for 2 min. Then, the sections were incubated with the ligand (1.5 nM [3H]mazindol) for 40 min at 4°C, washed with the buffer and distilled water, and dried. Dried slides were apposed to tritium-sensitive Hyperfilm for 3–4 weeks. The apposed Hyperfilms were developed, and optical densities were subjected to the quantification of receptor autography using an image analysis system (Amersham).

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2.7. Microdialysis The striatal extracellular concentration of DA, DOPAC and HVA at rest and after loading of drugs affect DA transmission and metabolism (methamphetamine 3 mg/kg, i.p., haloperidol 0.5 and 1 mg/kg, i.p., L-DOPA 100 and 200 mg/kg, i.p.) in control, 6-OHDA lesioned and transplanted rats were investigated using in vivo microdialysis and HPLC assays(Nakahara et al., 1989; Nishino et al., 1990). Microdialysis was performed over 16 – 20 weeks after 6-OHDA lesion in lesioned rats and over 12–16 weeks after transplantation in grafted rats. Under deep anesthesia, a guide cannula oriented to the striatum (A, at the level of bregma; L, 2.85 mm; V, 2.5 mm) was implanted. The cannula was fixed to the skull with three anchor screws and dental acrylic. After 1 week recovery, a dialysis probe with cellulose acetate tubing (4 mm length, 0.25 mm diameter, molecular weight cutoff 5000 Da) was inserted into the striatum through the guide cannula and fixed by sticky wax. The membrane portion of the probe was oriented to locate in the median of two transplantation sites. The probe was perfused with Ringer’s solution (NaCl 147 mM, CaCl2 2.3 mM, KCl 4 mM, pH 6.0) at a rate of 2 ml/min. After 3 h stabilization, dialysate was collected automatically every 20 min. Before each drug administration three samples were collected (as a base-line control) and additional samples were collected over 3–4 h. Concentration of DA, DOPAC and HVA in the dialysates was assayed by a high performance liquid chromatography with an electrochemical detector (HPLC-ECD-100, EICOM, Japan) and an Eicompack MA-ODS column (7 mm, 4.6 × 250 mm, EICOM). The mobile phase contained 14.7 g/l of citric acid, 13.6 g/l of sodium acetate, 200 mg/l of sodium 1-octanesulfonate, 10 mg/l of EDTA, and 17% methanol at pH 4.0. The recovery rate of the system was about 20%. 3. Results

3.1. Methamphetamine rotations By injection of methamphetamine, 6-OHDA lesioned rats made ipsilateral rotations even 19 weeks after the lesion. However, 30 of 40 grafted rats showed a decrease in rotations(Fig. 1). In this report, we dealt with these 30 grafted rats that showed a decrease in rotations after transplantation. In lesioned rats, the number of rotations were 13.09 0.4 and 13.79 0.3 turns/min (mean 9S.E.M., n= 30) at 1 and 2 weeks after the lesion, respectively and 17.491.2 at 19 weeks after the lesion. In grafted rats, the number of rotations were 13.2 9 0.6 and 13.19 0.6 turns/min at 1 and 2 weeks after the lesion (n=30), and 5.190.8 turns/min at 2 weeks and −0.790.3 turns/min at 16 weeks after the transplantation.

Fig. 1. Methamphetamine-induced rotations before and after DAergic transplantation. After transplantation, the number of rotations reduced below zero at the 4th week and remained stable thereafter (open circles; n =30). In 6-OHDA lesioned rats without transplantation, rotations did not change significantly (closed circles; n=30). Ordinate, rotations (mean 9S.E.M.) per min over 1 h after methamphetamine loading (3 mg/kg, i.p.).+ , −; ipsi and contralateral to the lesion. Abscissa, weeks before and after the transplantation (TP).

3.2. In 6i6o microdialysis 3.2.1. Basal le6el Concentrations of DA, DOPAC and HVA in the perfusates (basal levels) are shown in Table 1. In control rats, the concentrations of DA, DOPAC and HVA were 9.39 0.6, 8649 42 and 4449 36 fmol/ml, respectively. In lesioned rats, DA was undetectable (below 0.1 fmol/ml). DOPAC and HVA were reduced to 2 and 4% of controls’. In grafted rats, DA recovered to almost control levels but DOPAC and HVA recovered to only 14 and 20% of controls’, respectively. 3.2.2. Effect of L -DOPA loading L-DOPA treatment (200 mg/kg, i.p.) increased DA to about ten times that of basal level in control rats. The maximal increase was observed over 40–60 min after the treatment and the increase continued for 2 h (Fig. 2). In grafted rats, DA increased to four times that of basal level with a similar time course as controls’. In lesioned rats DA increased but the response was much smaller than the other two groups. DOPAC increased to more than seven times that of the basal level in Table 1 Basal levels of the concentration of DA, DOPAC and HVA in perfusates from striatum of control, 6-OHDA lesioned and grafted rats fmol/ml

Control

Grafted

Lesioned

DA DOPAC HVA

9.3 90.6 864 9 42 444 9 36

8.1 91.5 c c 126 913** c 89 9 11** c

u.d.** 19 93** 21 95**

Data (mean 9S.E.M.) were the mean of three samples (n=20) (paired t-test; ** PB0.01 compared to basal level of control. c c PB0.01, c PB0.05 compared to basal level of lesioned rats).

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3.2.3. Effect of methamphetamine treatment Fig. 4 shows the effect of methamphetamine treatment (3 mg/kg, i.p.) on concentrations of DA, DOPAC and HVA in three groups (n= 5). In control rats, methamphetamine induced a significant increase in DA. The maximal level was more than 150 fmol/ml (15 times that of the basal level) over 20–40 min after the administration. In grafted rats, DA increased to 30 fmol/ml (five times that of the basal level). The time course of the increase was similar to that of controls’. In two of the five lesioned rats, DA increased to a detectable level (over 0.1 fmol/ml), but undetectable in the other three rats.

Fig. 2. Effect of L-DOPA loading on DA, DOPAC and HVA in control (open circles), 6-OHDA lesioned (open triangles) and grafted rats (closed circles). Time courses of concentration of DA, DOPAC and HVA (mean9S.E.M., n= 4) are shown before and after LDOPA (200 mg/kg, i.p.) injection. The basal level (at time 0) was calculated as the mean of three samples taken before drug injection. Ordinates; concentration. Abscissas; time (min).

control rats. The maximal increase was 80 min after the treatment and then gradually returned to the basal level. DOPAC increased to about 15 times that of the basal level in both grafted and lesioned rats with a similar time course as controls’. The level was significantly higher in grafted than lesioned rats (P B0.05). HVA also increased significantly (control rats, seven times; grafted and lesioned rats, more than 15 times that of basal levels) over 120 – 160 min after L-DOPA treatment. The increase continued for 3 – 4 h. Again, the level was significantly higher in grafted than lesioned rats. Fig. 3 shows dose-dependent maximal increases of DA, DOPAC and HVA after L-DOPA loading.

Fig. 3. Peak values of DA, DOPAC and HVA following L-DOPA treatment (100, 200 mg/kg, i.p.) in control, 6-OHDA lesioned and grafted rats. Ordinates; peak values. Abscissas; no treatment (0), 100 and 200 mg L-DOPA treatment, respectively. Peak values increased dose-dependently after L-DOPA treatment in all three groups (PB 0.01). Peak values in control and grafted groups were sigificantly higher than those in lesioned group in all situations (PB0.01).

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the basal level). The increase continued for more than 3 h. In grafted rats, DA increased to 15 fmol/ml (twice that of the basal level). DA was undetectable in lesioned rats. DOPAC and HVA also increased to about twice that of the basal levels in control rats. The increase of DOPAC and HVA in grafted and lesioned rats was similar in ratio to that of the controls’ though the absolute values were smaller. The increased levels of DOPAC and HVA were significantly higher in grafted than lesioned rats (PB0.05). Fig. 6 shows the maximal levels of DA, DOPAC and HVA after haloperidol treatment in three groups. Data shows dose-dependent increases in all groups.

Fig. 4. Effect of methamphetamine on DA, DOPAC and HVA in control (open circles), 6-OHDA lesioned (open triangles) and grafted rats (closed circles). Time courses of concentration of DA, DOPAC and HVA (mean 9S.E.M., n= 4) are shown before and after methamphetamine (3 mg/kg, i.p.) injection.

In contrast to DA, concentrations of DOPAC and HVA decreased to 30 and 40% of basal levels, respectively in control rats. However, in grafted rats, concentrations of DOPAC and HVA did not decrease after methamphetamine treatment. Thus, DOPAC/DA ratio was about 80–100 and 20 – 30 in control and grafted rats, respectively before the treatment and 5 – 10 in both control and grafted rats after the treatment.

3.2.4. Effect of haloperidol Fig. 5 shows the effect of haloperidol treatment (1 mg/kg, i.p.) on concentration of DA, DOPAC and HVA in three groups (n = 5). In control rats, DA increased to more than 40 fmol/ml (four times that of

Fig. 5. Effect of haloperidol on DA, DOPAC and HVA in control (open circles), 6-OHDA lesioned (open triangles) and grafed rats (closed circles). Time courses of the concentration of DA, DOPAC and HVA (mean 9S.E.M., n= 4) are shown before and after haloperidol (1 mg/kg, i.p.) injection.

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Haloperidol treatment: In control rats the DOPAC/ DA ratio remained constant during the first 1 h then decreased to about half. The ratio in grafted rats did not change significantly though DA increased twice. Methamphetamine treatment: In control rats the DOPAC/DA ratio decreased to ten to five (1/10–1/20 of the pretreatment level) and it decreased to ten–five (1/2–1/3 of the pretreatment level) in grafted rats also.

3.3. TH immunocytochemistry Striatal and midbrain sections from grafted rats that showed amelioration in motor imbalance were processed for TH immunocytochemistry at 16 weeks after transplantation to detect implanted DAergic cells and the extent of 6-OHDA lesions. TH positive cells sur-

Fig. 6. Peak values of DA, DOPAC and HVA following haloperidol treatment (0, 0.5, 1.0 mg/kg, i.p.) in control, 6-OHDA lesioned and grafted rats. DA, DOPAC and HVA increased dose-dependently in all groups (paired t-test ** PB 0.01, * PB 0.05 compared to each basal level).

3.2.5. DOPAC/DA ratio Fig. 7 shows the variations in DOPAC/DA ratio before and after treatment of L-DOPA, haloperidol and methamphetamine in control and grafted rats. L-DOPA treatment: In control rats the DOPAC/DA ratio remained constant during the first 1 h though DA increased (up to ten times). In grafted rats, the DOPAC/DA ratio increased almost in a linear fashion with the increase of DA (up to four times). The ratio increased to over three times that of the basal ratio over 3 – 4 h in control rats, and to seven to eight times that of the basal ratio over 1 – 3 h in grafted rats. Ratios over 40 min–1 h in grafted rats were close to those of controls’ at resting stage.

Fig. 7. DOPAC/DA ratios before and after L-DOPA, haloperidol and methamphetamine loading in control (open circles) and grafted (closed circles) rats.

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Fig. 8. TH immunocytochemistry of striatal section at 16 weeks after transplantation of DAergic cells. TH positive cells survived and extended neurites in the host striatum. Scales, 500 (A) and 100 mm (B).

vived along the transplantation track in the striatum (500 –2500 cells per rat) and extended neurites extensively (Fig. 8). In lesioned rats, there were no TH positive neurons in the ipsilateral SN, and the number of TH positive neurons in the ipsilateral ventral tegmental area was also decreased (data not shown).

3.4. Autoradiograph of DA transporter Intense silver precipitation ([3H]mazindol binding) was observed in bilateral striata of control rats (Fig. 9A). In lesioned rats, [3H]mazindol binding in the striatum ipsilateral to the lesion was strongly reduced (Fig. 9B). In grafted rats, the binding recovered more or less in the striatal area not far from the graft track (Fig. 9C).

4. Discussion Fetal mesencephalic grafts ameliorated motor imbalance in 30 of 40 hemiparkinsonian model rats that had unilateral 6-OHDA lesion in the SN. Many TH positive cells survived and developed well in the striatum of these rats. By microdialysis analysis, striatal DA was undetectable (below 0.1 fmol/ml, the limit of our assay system) in lesioned rats, but it recovered to almost control level in grafted rats, as similar to previous studies (Nishino et al., 1990; Nishino, 1993; Hattori et al., 1993, 1994, 1996). It is reported that, the concentra-

tion of extracellular DA remains in normal range till the size of lesion exceeds 80%, and once the lesion exceeds 95%, a remarkable drop of DA is detected (Castan˜da et al., 1990). From this point of view, 6OHDA lesioned rats of the present study should have a sufficient lesion (over 95%), and the grafts restored the DA level at least up to those in animals with lesion smaller than 80%. This was verified by TH immunocytochemistry of the lesioned SN where no TH positive cells were found. DOPAC/DA ratio is a useful indicator that reflects DA turnover in tissue level. In the present study, DOPAC/DA ratio was 80–100 in control rats while it was at a lower level (20–30) in grafted animals even after motor imbalance was restored. The decrease of DOPAC/DA ratio in grafted rats might be reasonable as the decrease in metabolism may result in keeping extracellular DA level higher. After L-DOPA (200 mg/kg) loading, DA, DOPAC and HVA increased considerably in control rats as previously reported (Brannan et al., 1989). In grafted rats, they increased with a similar time course as those of controls but the peak value was 30–50% of controls’. In grafted rats, DOPAC/DA ratio recovered to almost control level after L-DOPA loading. This suggests that although the turnover rate of DA is set at a lower level at resting stage, the grafts have a capacity to increase the rate of metabolism to normal level when an adequate amount of substrate (DOPA) is supplied (Hefti et al., 1981).

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By methamphetamine treatment, the concentration of DA increased to 15 – 20 times that of the basal level in control rats and to about five times that in grafted rats. Methamphetamine stimulates the release and blocks the re-uptake of DA (Butcher et al., 1988). As the release of DA would depend on the amount of DA storage releasable at DAergic terminals, the smaller response to methamphetamine loading in grafted rats might be due to a smaller storage of DA. DAergic neurons that survived after 6-OHDA lesion have higher tyrosine hydroxylase activity and a higher rate of neural discharge for the normalization of extracellular DA (Schmidt et al., 1982; Zigmond et al., 1984; Wolf et al., 1989; Robinson et al., 1990). These data may suggest that, the rate of neural discharge of grafted DAergic neurons in DA depleted striatum might be in

Fig. 9. Autoradiographs showing [3H]mazindol binding in sections of the striatum from three rats. (A) control (intact rat). (B) lesioned rat 19 weeks after 6-OHDA injection in unilateral nigrostriatal pathway. (C) grafted rat 16 weeks after transplantation. Arrows indicate grafting track.

higher levels than controls’. If it is the case, the facilitation may reduce the further release of DA by methamphetamine. However, the grafts had a capacity to respond to methamphetamine, suggesting that the grafted neurons might have the potential and a reservoir for DA release. By administration of methamphetamine, the concentration of DOPAC and HVA decreased considerably in control rats, while it did not in grafted and lesioned rats. Consequently, the DOPAC/DA ratios became quite close in control and grafted rats. This may suggest that, in grafted neurons, the DA re-uptake mechanism might be down-regulated more or less, thus no further reduction could be induced by methamphetamine loading. From this point of view the examination of transporter activity in grafted animals would be interesting. In lesioned rats, the [3H]mazindol binding decreased significantly in the whole striatum ipsilateral to the lesion as has been reported in Parkinson’s disease (Niznik et al., 1991; Ogawa et al., 1997). In grafted rats that showed motor improvement, the sites of [3H]mazindol binding recovered more or less in the striatal area not far from the graft track. The silver precipitation ([3H]mazindol binding) was intense not in and around but outside the graft track while the TH positive cells survived around the graft track. This discrepancy of distribution may reflect a stretch of neurites from grafted neurons. The facts that the DA transporter activity has been re-expressed and the concentration of DA is at almost control level suggest that, the grafted neurons have the capacity to release a fairly good amount of DA in the host striatum. In intact rats, haloperidol treatment increases the extracellular DA, DOPAC and HVA in the striatum through the feedback system (Zetterstro¨m et al., 1984; Herman et al., 1985; Meloni et al., 1988). In the present study, DA increased to four times and DOPAC and HVA to about twice that of basal levels after haloperidol loading in control rats, thus the DOPAC/DA ratio decreased to about half. In grafted rats, haloperidol increased DA to twice that of the basal level. DOPAC and HVA also increased at almost the same degree as DA. Thus DOPAC/DA ratio did not change by haloperidol treatment. The reason for the discrepancy is not clear, but it might be due to a rather smaller increase in DA and metabolites in grafted rats. Haloperidol stimulates DAergic cells directly or indirectly, and enhances DA release and turnover (Zetterstro¨m et al., 1984; Meloni et al., 1988). This might result in the release of DA and the metabolites in a similar extent. There are at least two feed back pathways for the action of haloperidol on DA neurons: one is an indirect pathway via GABAergic neurons with a striato-nigral long feedback loop, and the other is a direct effect via autoreceptors of DA neurons in the SN. Grafted DAergic neurons have synaptic connec-

T. Hashitani et al. / Neuroscience Research 30 (1998) 43–52

tions in the host brain (Freund et al., 1985; Nishino et al., 1986b; Bolam et al., 1987; Nishino et al., 1990), and a feedback system via these connections has been shown pharmacologically(Herman et al., 1985; Meloni et al., 1988). Therefore, it is reasonable to suppose that grafted neurons have feedback connections in the striatum and are under the control of the host neuronal elements. Combining the above data, the DA turnover in grafted animals could be summarized as follows. (1) At resting stage, DA turnover is set in a lower level, and extracellular DA is within normal levels. (2) L-DOPA loading increases the release of DA and metabolites, and increases the DA turnover. (3) Grafted neurons are able to respond to a DA releaser, methamphetamine. (4) The re-uptake mechanism of DA in the striatum (DA transporter activity) is restored partially. (5) Feedback regulatory controls are subserving. The evaluation of the activity of grafted neurons in the host brain is difficult since (1) the re-uptake mechanism of DA is not simple (2) the relation between the number of DAergic neurons and the concentration of extracellular DA is not linear, and (3) DOPAC/DA ratio might show an intricate movement after DA denervation. Thus, we cannot draw the final conclusions yet. However, based on the data presented here, we can propose a hypothesis that the grafted DAergic neurons are under the control of a regulatory mechanism to normalize the concentration of extracellular DA level, and are able to respond to various stimuli via feedforward and feedback regulations in the host brain.

Acknowledgements We thank Dr H. Hatanata for providing us with anti-TH antibody. This work was supported in part by the Ministry of Education, Science, Sports and Culture, Grant-in Aid for Developmental Scientific Research 04557005 (H.N.).

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