dopamine released from mesencephalic transplants restores modulation of striatal acetylcholine...

EXPERIMENTALNEUROLOGY 105,251-259 (1989)

Dopamine Released from Mesencephalic Transplants Restores Modulation of Striatal Acetylcholine Release after Neonatal

6-Hydroxydopamine: An in Vitro Analysis

RENEE K. CARDER, DENISE JACKSON,* HOLLY J. MORRIS,* RAYMOND D. LUND, AND MICHAEL J. ZIGMOND*

Department of Neurobiology, Anatomy, and Cell Sciences, *Department of Behavioral Neurosciences, and Center for Neurosciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

After chemical lesions which destroy the nigrostriatal dopamine pathway, transplants rich in dopamine neurons innervate the striatum and, with appropriate stimulation, drive host motor behaviors normally medi- ated by dopamine. We wished to determine whether dopamine released from the transplant also reinstated dopaminergic inhibition of striatal acetylcholine release. Three-day-old rat pups received bilateral intraventricular injections of 6-hydroxydopamine. Three days later cell suspensions prepared from embryonic ventral mesencephalon were injected unilaterally into the stria- turn. Tail pinch and amphetamine were able to elicit contralateral turning in many of these animals. Only those animals which rotated 25 turns/min were included for further analysis. Subsequent assays indicated that 6-hydroxydopamine had depleted striatal dopamine to 4% of control and that the transplant had increased dopamine levels to 11% of control. Super- fused striatal slices were stimulated (8 Hz, 1 min) and then exposed to amphetamine (10 pM, 3 min). The slice released dopamine, as measured by HPLC, and acetylcholine, as measured by tritium efflux after preincubation with [3H]choline. Moreover, the release of acetylcholine was inhibited by endogenous dopamine as indicated by the ability of sulpiride (1 PM) to increase tritium efbux. Striatal slices prepared from lesioned animals showed a reduction in dopamine overflow in response to both electrical stimulation (0.6% of control) and amphetamine (1% of control), and a decrease in the ability of sulpiride to increase electrically evoked acetylcholine overflow (12% of control). Transplantation partially restored the dopaminergic response to electrical stimulation (2 1% of control), and amphetamine (15% of control) and fully restored the sulpiride-induced increase in acetylcholine overflow (98% of control). We conclude that transplanted dopaminergic neurons are able to release dopamine and to restore inhibitory control over the release of striatal acetylcholine. 0 1989 Academic Press, Inc.

INTRODUCTION

Ventral mesencephalic transplants placed into the striatum of neonatal rats with near-total lesions of their

dopamine (DA)-containing nigrostriatal afferents survive and innervate the host striatum as determined by tyrosine hydroxylase (TH) immunocytochemistry. Un- like adult animals similarly depleted, these rats survive with only minor motor deficits (21). This makes it possible to deplete both striata of dopamine, place a transplant on one side, and observe the effects of the transplant on promoting turning responses. In contrast, the most commonly used preparation in adult animals in- volves a unilateral lesion which produces ipsilateral turning itself. As a DA-rich transplant reinnervates the denervated striatum, a reduction in turning is observed (8). In rats receiving bilateral lesions and unilateral ventral mesencephalic transplants as neonates, tail pinch and amphetamine cause contralateral rotation. Because the two stimuli have been shown to release DA in normal rats (15,28), these results suggest that neurons from the transplant also may release DA and thereby influence host striatal function. To examine this hypothesis we have employed two approaches. First, we have measured endogenous DA release using striatal slices prepared from normal rats and DA-depleted rats with or without a ventral mesencephalic graft. The slices were superfused with a modified Krebs buffer and exposed to electrical field depolarization. Using the same preparation, we have also examined the impact of a dopaminergic receptor antagonist on the depolarization-induced release of acetylcholine (ACh), a procedure that has been employed previously to provide a measure of the influence of endogenous DA on cholinergic activity in striatum (11, 26).

METHODS AND MATERIALS

Chemicals. All chemicals used in these experiments were of the highest available purity and were obtained from Fisher Scientific Co. (Pittsburgh, PA) with the fol- lowing exceptions: Sephadex G-25 (coarse, 30-100 pm in diameter; Pharmacia, Piscataway, NJ); ascorbic acid, hemicholinium-3, L-tyrosine HCl, dopamine HCl, 3,4- dihydroxyphenylacetic acid, and 6-hydroxydopamine (Sigma Chemical Co., St. Louis, MO); desmethylimipra-

251 0014-48&x/89 $3.00 Copyright 0 1989 by Academic Press, Inc.

All riehts of renmrh~rtinn in ~lnv fnrm vnanr.r~I

252 CARDER ET AL.

mine (Merrell-National Laboratories, Cincinatti, OH); dihydroxybenzylamine and tribromoethanol (Aldrich Chemical Co., Milwaukee, WI); sodium bisulfite (J. T. Baker Chemical Co., Phillipsburg, NJ); octyl sodium sulfate (Eastman Kodak Co., Rochester, NY); Triton X- 100, TS-1, and 3A20 (Research Products International Corp., Mount Prospect, IL); [3H]choline (80 Ci/mmol; New England Nuclear, Boston, MA); antibody to tyrosine hydroxylase (Eugene Tech International, Allen- dale, NJ); rabbit avidin-biotin complex (Vector Labora- tories, Inc., Burlingame, CA); diaminobenzidine (Fluka AG; Buchs, Switzerland); and Histoclad (VWR Scien- tific, San Francisco, CA). Sulpiride was a gift of Ravizza Pharmaceutical Co. (Milan, Italy). Water was glass dis- tilled and filtered (Type GS, 0.22-pm filters, Millipore Corp., Bedford, MA).

Animals. Rats derived from a Sprague-Dawley strain (Zivic-Miller, Allison Park, PA) were used in the experiments. All animals were housed in a temperature- controlled room (21-23°C) and maintained on Rodent Laboratory Chow (Ralston-Purina Co., St. Louis, MO) and tap water ad l&turn. Lights were on daily between 8 AM and 8 PM. Pups were housed in groups of 10 age- mates with an adult female until 1 month of age, when they were weaned and housed two per cage.

Lesion. Rat pups were anesthetized by hypothermia on Postnatal Day 2-3 and injected intraventricularly with either 10 ~1 of a solution containing 110 pug of 6- HDA in 0.1% ascorbic acid and 0.9% NaCl or the vehicle solution alone. Thirty minutes prior to the injection, pups were given desmethylimipramine (25 mg/kg, SC) to protect noradrenergic neurons from the toxic effects of 6-HDA (2).

Transplantation. A cell suspension rich in DA-containing neurons was injected unilaterally into the rostra1 striatum 2-3 days after rats had received the neurotoxic lesion. The donor cells were obtained from ventral mesencephalon of rat embryos of 14-day gestation and disso- ciated by trituration in a Ca’+- and Mg+-free Tyrode’s solution (3,21), The extent to which the transplantation was successful was determined 1 to 3 months later by administering amphetamine (2.0 mg/kg, ip) and tail pinch (1 s). Only those animals turning at least five times per min in response to each of these stimuli were included for further analyses because previous studies have shown that this amount of turning is associated with a medium-to-high innervation of the host striatum by TH-immunoreactive neurons of graft origin (3). The neurochemical impact of the transplant was examined using the procedures described below and was compared with results obtained from the side contralateral to the transplant, as well as from slices taken from lesioned and unlesioned rats that did not receive a transplant. Some animals were processed for TH immunocytochemistry to verify the extent of both the lesion and the innervation of the host striatum by the transplant.

Histology. Animals were anesthetized with tribromoethanol, placed on ice, and perfused through the as- cending aorta with 100 ml of 0.1 M phosphate-buffered saline containing 0.5% sodium nitrite, followed immedi- ately with 500 ml of cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were postfixed for 2- 4 h in 4% paraformaldehyde and then transferred to a 15% sucrose solution in 0.1 M phosphate buffer over- night. Thirty-micrometer frozen sections were cut in the coronal plane and stained with an antibody against tyrosine hydroxylase (19), using a modified procedure in- volving a diaminobenzidine/nickel intensification.

In Vitro preparation. Animals were killed by decapi- tation at 1 to 3 months of age. Brains were removed rap- idly and placed on ice, and the area containing the cor- pus striatum was dissected out. Rostra1 coronal slices (350 pm thick, approximately 2.5 mg each) were prepared using a McIlwain tissue chopper. Slices were superfused and stimulated as previously described (12,20). Briefly, slices were placed into a superfusion chamber (200 ~1) equipped with two platinum ring electrodes. Slices were separated from one another with layers of Sephadex G-25 resin and from the electrodes with nylon mesh. Tissue was superfused (100 pl/min) with 37°C Krebs bicarbonate buffer supplemented with 75 &f tyrosine and 0.86 mA4 ascorbic acid and bubbled with gas consisting of 5% CO, and 95% OZ. After an equilibration period of 45-60 min, 5-min fractions were collected throughout the experiment. After another 15 min to es- tablish a baseline, slices were stimulated at 8 Hz for 1 min with bipolar, rectangular pulses (18 mA, 2-2.5 ms/ pulse, 0.5 ms interpulse delay). Where indicated, a second stimulation was carried out 45 min later.

Dopamine release. To measure the release of endogenous DA, five slices were loaded into each chamber and fractions were collected in tubes containing 50 ~1 of 1.1 N perchloric acid and 2.2 mM NaHSO,. After 1 h of superfusion, slices were stimulated as described above. Forty minutes after stimulation, slices were exposed to amphetamine (10 PM) for 3 min. At the end of the experiment, superfused slices were retrieved and homogenized in 200 ~1 of 0.1 M HClO, containing 2.2 mM NaHS03. DA concentrations in the superfusate and tissue were analyzed by HPLC with electrochemical detection as previously described (14, 29). Fractional DA efflux was defined as the amount of DA collected in the superfusate expressed as a percentage of the DA content of the slices. Spontaneous efflux was defined as the average amount of eiIlux that occurred during the 15 min period prior to stimulation; overflow, as the total amount of eftlux above this spontaneous level that occurred during and after stimulation. Typically, electrical or pharmacological stimulation caused an overflow that lasted for 25-30 min.

Acetylcholine release. ACh release from superfused slices was estimated by measuring tritium efflux after

NIGRAL TRANSPLANTS RESTORE DA MODULATION OF ACh 253

B -:, FIG. 1. Tyrosine hydroxylase (TH)-stained coronal sections through the rostra1 striatum (S) of a control rat that had received a vehicle

injection (A) and a rat that was depleted of dopamine as a neonate and received a subsequent transplant (B). Note in A the dense terminal staining throughout the striatum, and in B the almost complete loss of terminal staining in the striatum contralateral to the transplant. Bar signifies 1 mm.

preincubation of slices with 0.1 palm [3H]choline accord- ing to methods previously described (12, 22). Briefly, striatal slices (one per chamber) were preincubated with [3H]choline and superfused as described above except that hemicholinium (10 puM) was added to prevent the reuptake of [3H]choline. The slices were stimulated as described above beginning 80 min (S,) and 125 min (S,) after onset of superfusion. Sulpiride (1 PM), a D2 receptor antagonist, was added to the superfusion buffer beginning 25 minutes prior to the S2. At the conclusion of the experiment, slices were removed from the chambers and homogenized in 100 ~1 of 0.1 mM HClO, and 2.2 mM NaHS03. Two lo-p1 aliquots of the homogenate were added to vials containing 5 ml of scintillation cocktail (2 ml 3A20/ml Triton X-100), and total tritium content was determined using liquid scintillation spectroscopy. These values were used to calculate the fractional release on the basis of the estimated amount of radioactiv- ity in the tissue at the beginning of each fraction. The remaining homogenate (80 ~1) was used to determine DA content.

Statistical analysis. All values are reported as means + SEM. The relationship between sulpiride-induced increase in tritium overflow in the striatum ipsilateral to the transplant and the amount of DA present in the tissue was assessed using a correlation analysis.

RESULTS

The impact of 6-hydroxydopamine and transplantation on dopaminergic innervation of striatum. Histological

examination of the striatum contralateral to that receiving the transplant showed a near-complete loss of TH- immunoreactive terminals as compared to unhandled or vehicle-treated rats (Fig. 1). In conjunction, there was also a loss of TH-immunoreactive cell bodies in the substantia nigra pars compacta as a result of the 6-HDA lesion (Fig. 2). This is in accord with previous studies of 6-HDA lesions in neonatal rats (19). On the other side, a graft was identified that most often was situated in the lateral ventricle, attached to the dorsomedial surface of the striatum (Fig. 3). TH-positive cell bodies and fiber plexus usually were located around the periphery of the transplant. These fibers exited the transplant and formed a dense plexus of fine fibers predominantly in the dorsomedial quadrant of the host striatum.

Spontaneous dopamine eflux. After an initial equilibration period, DA was released spontaneously from control slices into the superfusion fluid at a stable rate of 0.34 ng/mgprotein/5 min, a value equivalent to a fractional rate of 0.4% of the tissue content per 5 min. In slices prepared from the lesioned striatum contralateral to the transplant, spontaneous efllux was 0.07 ng/mg protein/5 min. Although this represented a marked reduction in the absolute rate of DA efllux, subsequent bio- chemical analyses of the slices revealed that 6-HDA had produced an even greater reduction in the DA content of striatal tissue. Thus, the rate of fractional efflux of DA (2.4% of the tissue content) was greater than the control rate (Table 1).

254 CARDER ET AL.

FIG. 2. TH-stained coronal sections through the midbrain of a control rat (A) and a rat that was depleted of dopamine as a neonate and received a subsequent transplant (B). Note in A the dense neuronal staining in the substantia nigra pars compacta (S) and the ventral tegmental area (V). In B there is almost a complete loss of neuronal staining in the substantia nigra pars compacta, while many of the TH-positive neurons are still present in the ventral tegmental area. Bar signifies 1 mm.

DA efflux from striatal slices ipsilateral to the transplant was 0.67 ng/mg protein/5 min. This rate was nine- fold greater than that observed contralateral to the transplant and even somewhat greater than that observed in intact, control tissue. Nonetheless, subsequent analysis indicated that the DA content of these slices was increased only slightly above that observed contralateral to the transplant such that the rate of fractional efflux was 7.9% of the tissue content (Table 1).

Electrically evoked dopamine efflux. Field depolarization for 1 min resulted in a rapid rise of DA efflux in control slices, which reached a peak of 3.52 ng/mg protein/5 min during the first 5 min and then decayed to near-basal rates during the next 10 min. Total DA overflow during this period represented 6.4% of tissue content. No detectable increase in DA efflux was observed when slices contralateral to the transplant were stimulated. However, DA efflux from slices ipsilateral to the transplant increased, reaching a peak of 0.3 ng/mg pro-

tein/5 min above baseline which then gradually declined to prestimulation levels during the next 25 min. Total DA overflow during this period was 12.7% of tissue content (Table 1; Fig. 4).

Amphetamine-induced dopamine efflux. Exposure of slices to amphetamine (10 PM) for 3 min caused a large increase in DA efflux from slices prepared from control animals; the peak rate of DA overflow was 2.8 ng/mg protein/5 min and the total was 15% of tissue contents. The drug had no impact on DA efllux from lesioned slices contralateral to the transplant. In contrast, amphetamine increased DA efflux from slices ipsilateral to the transplant. Peak overflow was 0.28 ng/mg protein/5 min and total overflow represented 21.4% of tissue DA (Table 1; Fig. 4).

Dopaminergic inhibition of acetylcholine efflux. Control slices showed a low, stable rate of spontaneous ACh efllux which was greatly enhanced by electrical stimulation (Table 2). A second stimulation produced a


FIG. 3. Photomontage of a TH-stained coronal section through the rostal striatum (S) ipsilateral to the transplant illustrating an intraventricular transplant (Tp) with extensive TH-positive fiber outgrowth into the host striatum. V, lateral ventricle. Bar signifies 100 pm.

comparable increase in efflux. Similar results were obtained using slices prepared from lesioned animals, both ipsilateral and contralateral to the transplant (Table 2).

Sulpiride (1 p*M) increased ACh efflux from striatal slices prepared from unlesioned rats by 81% of paired controls (Table 3). In comparison, slices prepared from lesioned striatum increased ACh release by 9% of paired controls in response to sulpiride. However, slices ipsilat- era1 to a transplant showed an increase in ACh release which was comparable to that observed from control

slices (Table 2). Although this response was quite vari- able (-27 to 416% of paired controls), subsequent analyses of tissue DA indicated that the response to sulpiride was related to the amount of the DA present (r = 0.849, P < 0.001; Fig. 5). Indeed, it was possible to demonstrate a normal response to sulpiride from some slices containing less than 10% of the normal DA content. Moreover, in three cases of transplants with somewhat higher levels of DA, the response to sulpiride was much greater than that observed with control slices.

256 CARDER ET AL.

TABLE 1

Dopamine Overflow in Response to Electrical and Amphetamine Stimulation”

Tissue DA content (ng DA/mg protein)

Spontaneous efflux (ng DA/mg protein/5 min)

DA overflow (ng DA/mg protein)

Electrical stimulation Amphetamine stimulation

Control striatum n=4 79.8 5~ 0.8 0.34 + 0.09 5.12 + 0.79 11.97 + 1.23

Contralateral to transplant n=4 2.9 f 0.1 0.07 f 0.01 0.03 f 0.30 0.13 + 0.04

Ipsilateral to transplant n=4 8.5 f 0.1 0.67 + 0.03 1.08 + 0.30 1.82 f 0.35

n Slices were stimulated at 8 Hz for 1 min, followed 40 min later by exposure to amphetamine (10 &kf) for 3 min. Means f SEM are shown.

DISCUSSION

These results show that after severe nigrostriatal DA depletions in neonatal rats, the transplantation of ventral mesencephalic tissue into the striatum increases the capacity of this region to release endogenous DA. This increased capacity for DA release was evident under basal conditions, in response to electrical stimulation,

Eke 4.0 Stim

3.5

I -

6

3.0 I\

and during exposure to amphetamine. Moreover, the DA released by depolarization appeared to exert inhibitory control over cholinergic interneurons.

Histological examination of tissue from comparably treated animals indicated the presence of a large number of TH-immunoreactive cells and fibers within the stria- turn of animals that had received the transplant. In contrast, TH-positive cells were not seen in the depleted

Afwh

0.5 -0-o -0-d 0.0 t

0 IO 20 30 40 50 60 70 60 90

0.01 0 10 20 30 40 50 60 70 60 90

Time (min)

FIG. 4. Mean + SEM ng DA/mg protein released over time in response to electrical stimulation (8 Hz, 18 mA, 1 min) and exposure to amphetamine (10 pM, 3 min). Slices from control striatum are represented by open circles (n = 4). Slices from striatum ipsilateral to the transplant are represented by closed circles (n = 4). Slices from striatum contralateral to the transplant are represented by closed triangles (n = 4).


TABLE 2

Basal and Electrically Stimulated Acetylcholine Release’

Basal fractional

release Sl fractional

release S2 fractional

release

Control striatum n=7 1.44 + 0.17 10.90 f 1.98 8.0 + 0.99

Lesion striatum n=8

Lesion and 1.22 f 0.19 9.28 f 1.04 8.44 + 1.25

transplant striatum

n = 21 1.67 f 0.10 9.26 + 0.85 9.32 + 0.88

’ Slices were preincubated with [SH]choline (0.1 pLM) and stimulated at 8 Hz for 1 min beginning 80 min (Sl) and 125 min (S2) after the onset of superfusion. Total tritium efflux was used as a measure of ACh release. Means + SEM are shown.

striatum and only occasional stained fibers were evident. Other studies in which mouse ventral mesencephalon was used as the donor tissue confirm that the greatly enhanced TH-positive staining is indeed due to the transplant (4). Although it was not possible to determine the source of the additional DA release that was observed, it is presumed to have been derived from these transplanted dopaminergic cells.

Several distinctions can be made between the responses of striatal slices from animals receiving transplants and those from intact animals. First, although the slices prepared from striatum ipsilateral to a transplant contained only 11% of the DA present in control tissue, the spontaneous rate of DA efflux from these slices was actually greater than that from control slices. Second, whereas electrical depolarization did increase DA efflux from slices ipsilateral to a transplant, the peak overflow in response to the stimulation was not significantly greater than would have been expected from the DA content of the tissue; i.e., there was no increase above control in peak fractional release. As a result, the ratio of electrically evoked peak overflow to spontaneous release

was much smaller from slices ipsilateral to a transplant than from control slices. Similar results were obtained in response to amphetamine. Third, the time required for DA efflux to return to baseline after stimulation was considerably longer for slices ipsilateral to a transplant than for control slices. Finally, it was possible to demonstrate normal or even above-normal dopaminergic inhibition of ACh release from slices containing a much reduced DA innervation.

It seems probable that several factors are jointly responsible for these observations. First, the relative spar- sity of high affinity DA uptake sites within the lesioned slices appears to result in an increase in the diffusion of DA from dopaminergic terminals into the superfusion fluid (20,23) and may be expected to increase the access of DA to cholinergic neurons as well (13, 17). Such a mechanism is consistent with our observation of an ex- tended decay time for the DA overflow occurring in response to electrical and pharmacological challenges. A decrease in the number of available DA terminals can also lead to an increase in the amount of DA released from those residual terminals (7, 20, 23). However, this probably was not an important factor in our results since no increase in the peak fractional overflow from slices ipsilateral to a transplant was observed. Second, in contrast to intact striatum where only the terminal axonal segment of DA cells are present in the slice, many of the transplanted dopaminergic neurons may be present in slices from transplanted tissue. It is not clear what effect this may have on DA release. It is possible, however, that the very high rate of spontaneous efflux and the reduced ratio of electrically evoked overflow to spontaneous efflux are related in some way to the presence of dopaminergic soma and/or dendrites. Differences between normal DA terminals and those formed by the transplanted DA neurons may also be of significance.

A third factor that may be operating is an increase in the responsiveness of cholinergic neurons to DA. A proliferation of striatal DA receptors after nigrostriatal bundle lesions is suggested by increases in DA binding sites and behavioral supersensitivity to DA agonists (5,

TABLE 3

Effect of Sulpiride on Acetylcholine Release”

DA content

(Ptdd S2/Sl control S2/Sl sulpiride* % Change relative to paired control

Control striatum n=7

Lesion striatum n=8

Lesion and transplant striatum n = 21

11.51 f 0.55 0.79 t 0.08 1.46 k 0.22 181+ 13

0.28 + 0.06 0.92 t 0.08 1.01 2 0.11 109f 2

0.76 k 0.12 1.06 k 0.07 1.82 +- 0.27 180 3~ 24

’ Slices were preincubated with [SH]choline (0.1 r&f) and stimulated at 8 Hz for 1 min beginning 80 min (Sl) and 125 min (S2) after the onset of superfusion. Total tritium efflux was used as a measure of ACh release. Means k SEM are shown.

* Sulpiride (l&4) was added 25 min prior to the onset of S2.

258 CARDER ET AL.

l-

-

0.5 1.0 1.5 2.0 2.5

pg Dopaminelg Tissue

FIG. 5. Correlation of pg DA/g tissue with the sulpiride-induced increase in tritium overflow expressed as a percentage of paired controls (striatal slices from the same animal which do not receive sulpiride). Each point represents data from a paired set of striatal slices (striatum ipsilateral to the transplant is represented by closed circles, n = 21, P = 0.849, P < 0.001; lesioned striatum is represented by open triangles, n = 8).

l Transplant A Lesion

l

6,X$27,30). Moreover, we have observed an increase in

the capacity of apomorphine to inhibit depolarization- induced ACh efflux (13).

Finally, in adult rats with unilateral 6-HDA-induced lesions of the nigrostriatal pathway, TH-immunoreactive axons from grafts of embryonic ventral mesencephalon have been reported to make symmetric synapses with cell bodies of giant neurons, presumably cholinergic interneurons (10). These synapses constituted 6% of all TH-positive synapses found and were characterized by dense pericellular fiber arrangements around the giant cell perikarya. The fiber baskets were seen only in asso- ciation with the giant cholinergic cells and these baskets were never seen in the normal striatum. It is not known whether in neonatally lesioned rats graft-derived dopaminergic fibers also exhibit this abnormal hyperinnerva- tion of cholinergic interneurons. However, it is interest- ing to speculate that the direct synaptic connections of DA axons onto the perikarya of cholinergic neurons could be an important mode of dopaminergic regulation of cholinergic transmission. Such connections also could be responsible for some of the behavioral effects of the transplant.

In conclusion, these experiments suggest that although ventral mesencephalic transplants only elevate striatal DA content slightly, transplanted DA cells are capable of releasing a sufficient quantity of extracellular DA to exert a substantial effect upon postsynaptic striatal cholinergic neurons. These results should be consid- ered in light of the apparent role of abnormalities in dopaminergic and cholinergic function in extrapyramidal motor disturbances (1, 9, 16, 25). Thus, these observations extend previous findings on the functional impact of mesencephalic transplants (3,8,21) by indicating that this important effect of DA also can be restored.

ACKNOWLEDGMENTS

Support for this research was provided by NIH Grants EY05283 and NS19608. Preliminary reports of some aspects of the research were presented at the Eighteenth Annual Meeting of the Society for Neuro- science, Toronto, Canada, November, 1966. We are grateful to Dr. Jeffrey Radel for assistance with many of the figures and to Ms. Fran Shagas for her photographic assistance.

REFERENCES

1. BARTHOLINI,G.,H.STADLER,ANDK.G.LLOYD. 1975.Choliner- gic-dopaminergic interregulations within the extrapyramidal


2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

system. Pages 411-418 in P. G. WASSER, Ed., Cholinergic Mecha- nisms. Raven Press, New York.

BREESE, G. R., AND T. D. TRAYLOR. 1971. Depletion of brain nor- adrenaline and dopamine by 6-hydroxydopamine. Brit. J. Phar- macol. 42: 88-99.

CARDER, R. K., A. M. SNYDER-KELLER, AND R. D. LUND. 1987. Amphetamine- and stress-induced turning after nigral transplants in neonatally dopamine-depleted rats. Deu. Bruin Res. 33: 315-318.

CARDER, R. K., A. M. SNYDER-KELLER, AND R. D. LUND. 1988. Behavioral and anatomical correlates of immunologically induced rejection of nigral xenografts. J. Comp. Neural. 277: 391- 402.

CREESE, I., D. R. BURT, AND S. H. SNYDER. 1977. Dopamine receptor binding enhancement accompanies lesion-induced behavioral supersensitivity. Science 197: 596-598.

CREESE, I., AND S. H. SNYDER. 1979. Nigrostriatal lesions en- hance striatal [3H]apomorphine and [“Hlspiroperidol binding. Eur. J. Pharmacol. 56: 277-281.

CUBEDDU, L. X., AND J. S. HOFFMANN. 1982. Operational charac- teristics of the inhibitory feedback mechanism for regulation of dopamine release via presynaptic receptors. J. Pharmacol. Exp. Ther. 223: 497-501.

DUNNETT, S. B., A. BJBRKLUND, F. H. GAGE, AND U. STENEVI. 1985. Transplantation of mesencephalic dopamine neurons to the striatum of adult rats. Pages 451-469 in A. BJ~~RKLUND AND U. STENEVI, Eds., Neural Grafting in the Mammalian CNS. Elsevier, Amsterdam.

DUVOISIN, R. C. 1967. Cholinergic-anticholinergic antagonism in parkinsonism. Arch. Neural. 17: 124-136.

FREUND, T. F., J. P. BOLAM, A. BJBRKLUND, U. STENEVI, S. B. DUNNETT, J. F. POWELL, AND A. D. SMITH. 1985. Efferent synaptic connections of grafted dopaminergic neurons reinnervating the host neostriatum: A tyrosine hydroxylase immunocytochem- ical study. J. Neurosci. 5: 603-616.

HERTTING, G., A. ZUMSTEIN, R. JACKISCH, I. HOFFMAN, AND K. STARKE. 1980. Modulation by endogenous dopamine of the release of acetylcholine in the caudate nucleus of the rabbit. Nau- nyn Schmiedebergs Arch. Pharmacol. 315: 111-117.

JACKSON, D., M. K. STACHOWIAK, J. P. BRUNO, AND M. J. ZIG- MOND. 1988. Inhibition of striatal acetylcholine release by endogenous serotonin. Brain Res. 457: 267-273.

JACKSON, D., J. P. BRUNO, M. K. STACHOWIAK, AND M. J. ZIG- MOND. 1988. Inhibition of striatal acetylcholine release by serotonin and dopamine after the intracerebral administration of 6- hydroxydopamine to neonatal rats. Brain Res. 457: 267-273.

KELLER, R. W., JR., A. OKE, I. MEFFORD, AND R. ADAMS. 1976. Liquid chromatographic analysis of catecholamines: Routine as- say for regional brain mapping. Life Sci. 19: 995-1004.

KELLER, R. W., JR., E. M. STRICKER, AND M. J. ZIGMOND. 1983. Environmental stimuli but not homeostatic challenges produce apparent increases in dopaminergic activity in the striatum: An analysis by in viva voltametry. Bruin Res. 279: 159-170.

LLOYD, K. G. 1978. Neurotransmitter interactions related to cen- tral dopamine neurons. Pages 129-207 in M. B. Y. YOUDIM, W. LOVENBERG, D. F. SHARMAN, AND J. R. LAGNADO, Eds., Essays in Neurochemistrv and Neurooharmacolonv. Wilev. New York.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

MACKENZIE, R. G., M. K. STACHOWIAK, E. M. STRICKER, AND M. J. ZIGMOND. 1985. Restoration of dopaminergic inhibition of cholinergic function after partial damage to the nigrostriatal bundle. Sot. Neurosci. Abstr. 11: 891.

MACKENZIE, R. G., AND M. J. ZIGMOND. 1984. High- and low- affinity states of striatal D2 receptors are not affected by 6-hydroxydopamine or chronic haloperidol treatment. J. Neurochem. 43: 1310-1318.

SNYDER, A. M., M. J. ZIGMOND, AND R. D. LUND. 1986. Sprouting of serotoninergic afferents into striatum after dopamine-depleting lesions in infant rats: A retrograde transport and immunocy- tochemical study. J. Comp. Neurol. 245: 274-281.

SNYDER, G. L., M. K. STACHOWIAK, R. W. KELLER, JR., E. M. STRICKER, AND M. J. ZIGMOND. 1986. Release of endogenous DA and dopac from striatal slices after DA-depleting lesions: Effects of stimulation frequency and DA synthesis inhibition. Sot. Neu- rosci. Abstr. 12: 136.

SNYDER-KELLER, A. M., R. K. CARDER, AND R. D. LUND. 1989. Development of dopamine innervation and turning behavior in dopamine-depleted infant rats receiving unilateral nigral transplants. Neuroscience in press.

SOMOGYI, G. T., AND J. C. SZERB. 1972. Demonstration of acetylcholine release by measuring overflow of labelled choline from cerebral cortical slices. J. Neurochem. 19: 2667-2677. STACHOWIAK, M. K., R. W. KELLER, JR., E. M. STRICKER, AND M. J. ZIGMOND. 1987. Increased dopamine efflux from striatal slices during development and after nigrostriatal bundle damage. J. Neurosci. 7: 1648-1654.

STARKE, K., W. REIMANN, A. ZUMSTEIN, AND G. HERTTING. 1978. Effect of dopamine receptor agonist and antagonists on release of dopamine in the rabbit caudate nucleus in uitro. Nuunyn- Schmiedebergs Arch. Pharmacol. 305: 27-36.

TARSY, D. 1977. Dopamine-acetylcholine interactions in the basal ganglia. Pages 213-246 in W. S. FIELDS, Ed., Neurotrans- mitter Function: Basic and Clinical Aspects. Symposia Special- ists, Miami.

VIZI, E. S., L. G. HARSING, JR., AND J. KNOLL. 1977. Presynaptic inhibition leading to disinhibition of acetylcholine release from interneurons of the caudate nucleus: Effects of dopamine, p-en- dorphin, and D-ala’-pro’-enkephaliamide. Neuroscience 2: 953- 961.

WADDINGTON, J. L., A. J. CROSS, A. LONGDEN, F. OWEN, AND M. POULTER. 1979. Apomorphine-induced rotation in the unilateral 6-OHDA-lesioned rat: Relationship to changes in striatal ade- nylate cyclase activity and ‘H-spiperone binding. Neuropharma- cology l&643-645. ZETTERSTROM, T., M. HERRERA-MARSCHITZ, AND U. UNGER- STEDT. 1986. Simultaneous measurement of dopamine release and rotational behaviour in 6-hydroxydopamine denervated rats using intracerebral dialysis. Brain Res. 376: 1-7.

ZIGMOND, M. J., A. L. ACHESON, M. K. STACHOWIAK, AND E. M. STRICKER. 1984. Neurochemical compensation after nigrostriatal bundle injury in an animal model of preclinical parkinsonism. Arch. Neural. 41: 856-861.

ZIGMOND, M. J., AND E. M. STRICKER. 1980. Supersensitivity after intraventricular 6-hydroxydopamine: Relation to dopamine depletion. Experimentia 36: 436-437.

dopamine released from mesencephalic transplants restores modulation of striatal acetylcholine...

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