Co-transplantation of Fetal Lateral Ganglionic Eminence and Ventral Mesencephalon Can Augment Function and Development of Intrastriatal Transplants

EXPERIMENTAL NEUROLOGY ARTICLE NO.

145, 214–227 (1997)

EN976477

Co-transplantation of Fetal Lateral Ganglionic Eminence and Ventral Mesencephalon Can Augment Function and Development of Intrastriatal Transplants Lauren C. Costantini1 and Abigail Snyder-Keller Wadsworth Center for Laboratories and Research, University at Albany School of Public Health, New York State Department of Health, Albany, New York 12201-0509

Methods to increase the development and sustained function of embryonic mesencephalic dopamine cells after transplantation into dopamine (DA)-depleted striatum are currently under investigation. Elements that are crucial for the maturation and connectivity of neurons during normal development of the brain may also play a role in the development and integration of grafted embryonic tissue. Based on in vitro and in vivo observations of the enhancing effects of striatal tissue on nigral dopaminergic cell development and survival, we demonstrate that inclusion of embryonic striatal cells, specifically from the lateral ganglionic eminence (LGE), produces dopaminergic transplants with augmented functional effects. Rats neonatally DA-depleted and co-transplanted with embryonic nigral and LGE cells developed improved functional outcome when compared with animals receiving only nigral cells, and they required the transplantation of fewer nigral cells to produce a strong behavioral effect. Anatomically, the inclusion of LGE cells produced increased DA cell survival, a higher density of reinnervation into the DA-depleted host striatum, and patches of DA fibers within the co-transplants. There were also an increased number of host striatal cells which induced the immediate-early gene c-fos in co-transplanted animals compared to animals receiving nigral cells alone, indicating a higher degree of host-cell activation. The ability to enhance function, cell survival, reinnervation, and host activation with nigral-striatal co-transplants in the presence of fewer nigral cells supports the hypothesis of a trophic influence of striatal cells on nigral DA cells. r 1997 Academic Press

INTRODUCTION

Alleviation of the symptoms of idiopathic Parkinson’s disease (PD) can be accomplished with strategies to

1 To whom correspondence should be addressed at current address: Neuroregeneration Laboratory, Harvard Medical School, McLean Hospital, MRC 119, 115 Mill Street, Belmont, MA 02178-9106. Fax: (617) 855-3284. E-mail: [email protected].

0014-4886/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

restore the level of striatal dopamine (DA), and data obtained in rodent and nonhuman primate models have demonstrated the usefulness of transplanting fetal ventral mesencephalic DA cells into the DA-depleted striatum (7, 15, 60). Drug-induced rotational analyses in these animal models have demonstrated the capacity of these dopaminergic cells to affect postsynaptic and presynaptic mechanisms (7), while more complex movements, analyzed in stair case tests, food pellet retrieval tests, paw reaching tests, and stepping tests, have exhibited limited responses to dopaminergic transplants (2, 26, 46). As a means of replacing deteriorating and degenerating cells within the human brain, the transplantation of fetal DA neurons is now being employed as a treatment for PD: the reversal of motor deficits in parkinsonian patients has been moderately successful after transplantation of fetal mesencephalic cells (29, 36, 42). Functional effects of dopaminergic transplants are often correlated with DA cell viability, yet with current grafting techniques the survival rate of grafted DA neuroblasts and young postmitotic DA neurons is in the range of only 1–5% (52). In order to optimize the function of transplanted DA neurons in DA-depleted brain, maximal survival and reinnervation capacity of these neurons must be attained. Supplementation with normal target substrates may be necessary for maximal development, activity, and survival of DA neurons. In vitro studies demonstrate that survival of DA cells is increased in the presence of striatal cells, membrane, and extract (18, 51), yet others report enhanced development with no increase in DA cell number (22, 63). Mesencephalic DA neurons cultured alone form dendritic arborizations but lack axons; the addition of striatal cells induces the development of a dense axonal plexus (32) and increases the capacity of cultured nigral cells to synthesize and take up DA (22, 50, 51), suggesting that the maturation and activity of DA neurons depend on the presence of appropriate target tissue. Based on the above in vitro studies, it is reasonable to speculate that striatal cells may have a trophic effect on embryonic DA cells in vivo: target-derived factors that

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may be present in embryonic striatal tissue during the development of the nigrostriatal pathway may provide a more stable environment for development and activity of the heterotopically transplanted DA cells into DA-depleted adult striatum. Previous studies have suggested that the presence of striatal target tissue in dopaminergic transplants into lesioned animals gives rise to a greater area of innervation (55) and greater behavioral effects; however, no increases in DA cell survival were observed (10, 67). Most transplantation studies in animal models utilize unilateral denervation of the striatum, with subsequent amphetamine (AMPH)-induced rotation correlating with percentage of DA terminal loss on the lesioned side (typically 8–10 rotations per minute). After transplantation of DA cells into the lesioned striatum, a decrease in rotation is measured, permitting only a small range over which to measure transplant efficacy. Our animal model consists of bilaterally lesioned striata produced by intraventricular 6-HDA injection in infancy and unilaterally transplanting DA cells into striatum (16, 56). Rotational behavior in response to AMPH develops as a result of successful reinnervation of the transplanted side of the brain, allowing quantification of an increase, rather than a decrease, in behavior. Utilizing the above model, we investigated the behavioral and anatomical influences of striatal lateral ganglionic eminence (LGE) cells (shown to give rise to exclusively striatal cells; 45, 49) on nigral DA cells when co-transplanted into DA-depleted striatum. Behavioral improvements seen in lesioned rats and monkeys have been related to several aspects of dopaminergic transplants, including the number of surviving neurons, extent of DA fiber reinnervation, topographic distribution of the graft in the host striatum, and reestablishment of synaptic connections with host striatum (7). We anatomically analyzed these parameters in nigral versus nigral-LGE co-transplants (Co-Tps) and correlated these findings with behavioral effects. Striatal tissue, remaining actively mitotic for a longer period of time than substantia nigra (64), can be dissected from rat embryos ranging from Embryonic Day (E)14 to E18. The enhancing effects of striatal cells on the development of DA cells have been observed at various ages in vitro (3, 22, 48, 50, 51); however, in previous co-transplant studies, a limited range of striatal donor ages has been employed, and thus the possibility that cells from younger or older donors might produce still greater effects has not yet been determined (10, 55, 67). The present study also investigated the effects of various ages of LGE tissue in Co-Tps. We previously found that inclusion of embryonic LGE with embryonic nigral tissue enhanced the functional effects of the dopaminergic transplants when transplanted into bilaterally DA-depleted striatum (16).

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These animals exhibited more robust and long-lasting turning behavior in response to both pharmacological and physiological (mild tail pinch) stimuli when equal volumes of nigral and LGE cells were co-transplanted. Toward the goal of allowing a more reasonable number of cells to be utilized while still producing a fully functional transplant, the exact number of nigral and LGE cells transplanted was controlled, and the number of cells transplanted was reduced in order to approximate the ideal clinical situation. The issue of whether transplants are incorporated into host circuitry has been repeatedly addressed in the literature (7, 8, 14, 16, 25, 57). Although extensive evidence of afferent and efferent contacts between host and nigral or striatal transplant cells has been demonstrated (8, 12, 25, 38, 39, 43, 44, 65, 68), regulated functional incorporation into host circuits is still a matter of debate. Grafts into denervated striatum restore expression of the immediate-early gene c-fos in response to AMPH in host striatal cells which have been functionally activated (14, 16, 57), indicating not only that fetal nigral grafts release DA in a defined area within the striatum, but also that the released DA binds appropriate receptors and invokes intracellular responses. To study the extent of the transplants’ influence in the host striatum, we also mapped cells which induced c-fos to demonstrate whether Co-Tps are better able to activate the host brain. METHODS Lesions

Sprague–Dawley rats (originally derived from Griffin Laboratory stock, New York State Department of Health) were used in these experiments, and all procedures were approved by the Institutional Animal Care and Use Committee. Lesioning of the nigrostriatal pathway of 3-day-old rat pups was accomplished by injection of 6-hydroxydopamine (6-HDA; Sigma) into the lateral ventricles. The pups were pretreated with desmethylimiprimine (25 mg/kg) to protect noradrenergic neurons from the destructive effects of lesioning. Thirty minutes later, injections of 6-HDA (60 µg base in 5 µl) or vehicle (0.1% ascorbic acid in 0.9% NaCl) were made into each lateral ventricle (coordinates: A 0.5 from bregma; L 1.0; V 3.0). Lesioning was verified during anatomical analyses by the absence of DA fibers in the untransplanted striatum. Transplants

Females were time-mated and vaginal smears taken to verify the date of insemination, with the day of sperm positivity considered E0. Fetuses were removed from the pregnant female under ether anesthesia and placed into ice-cold HAMS-F10. Nigral tissue from E14

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embryos was dissected from the ventral mesencephalic flexure; striatal tissue from E14 or E16 embryos consisted of the lateral ganglionic eminence. The tissue was incubated for 30 min at 37°C in calcium- and magnesium-free Tyrode’s solution and then dissociated through repeated pipetting with fire-polished Pasteur pipettes. The tissue was centrifuged at 1000 rpm for 5 min, and the pellet was resuspended in 50–100 µl, depending on the size of the pellet. The suspension was diluted to the desired final concentration via hemocytometer quantification, and viability was determined by dye-exclusion techniques (acridine orange/ethidium bromide; 9). Each batch (one transplant session) contained animals receiving substantia nigra transplants (SN-Tps) and nigral-LGE Co-Tps. Cell suspensions for Co-Tps were prepared by combining and resuspending equal volumes of nigral suspensions and LGE suspensions, which had previously been adjusted to the desired concentration (either 280,000 or 500,000 cells per 5 µl; see Results). Five microliters of suspension was transplanted into the right striatum of previously lesioned 2- to 3-monthold rats. Stereotaxic coordinates for injection of cell suspensions were A 2.0, L 2.2, V 4.0. Cells were injected through a glass micropipette over a 3-min period, with a further 3-min period allowed for diffusion prior to slow removal of the pipette. Control animals received sham transplants of Ca21- and Mg21-free Tyrode’s or cells from embryonic LGE alone. Behavioral Analyses

Testing of turning behavior began at 15–25 days posttransplantation (post-Tp). On each day of testing, animals were injected with AMPH (2 mg/kg i.p.) and placed in a cylindrical container, and the number of rotations made in 1 min was counted at 10- and 20-min intervals, with the final AMPH score being the average of the two scores. The testing container was cleaned between animals. Results are expressed as means 6 SEM of the different transplant groups. Rotation scores were subjected to analysis of variance (ANOVA) for repeated measures and post hoc comparisons at specified time points. Lesioned animals receiving sham transplants (Ca21- and Mg21-free Tyrode’s solution) or LGE alone never developed turning in response to AMPH. Anatomical Analysis

Immunohistochemistry Two hours after a final stimulation by AMPH, rats were anesthetized with tribromoethanol and perfused transcardially through the ascending aorta, first with warm phosphate-buffered saline (PBS), followed by cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.3). After 15 min, brains were removed,

postfixed for 6 h, and then cryoprotected overnight in 15% sucrose in 0.1 M PB. Thirty-micrometer (µm) frozen sections through the striatum were cut coronally on a sliding microtome and collected in 0.1 M PB. Sections taken at regular intervals through the transplanted striatum were preincubated free-floating for 1–2 h in PBS with 0.2% Triton X-100 and 5% normal goat serum for tyrosine hydroxylase (TH) or 5% normal rabbit serum for Fos and then incubated for 48 h at 4°C in primary antibodies against TH (Chemicon, 1:600) and Fos (Cambridge Research Biochemicals, 1:3000). Sections were rinsed several times in PBS and then incubated for 1 h in biotinylated secondary antibody: goat anti-rabbit for TH and rabbit anti-sheep for Fos. After several more rinses in PBS, sections were incubated for 1 h in avidin–biotin complex (ABC; Vector Labs), and antibody binding was visualized with reaction in 0.05% diaminobenzidine (DAB) with 0.005% H2O2 and 0.25% nickel ammonium sulfate in PBS for approximately 3 min. Sections were mounted onto slides coated with chrom-alum gelatin, dehydrated through ascending alcohols and xylene, and then coverslipped with Histoclad (Clay Adams) or Permount (Fisher Scientific). Quantification of the surviving DA cells within each transplant was obtained by manually counting the number of TH-immunoreactive (TH-IR) cells in every fourth 30-µm section through the rostral-to-caudal extent of the transplant and adjusting this number according to Abercrombie (1). Image Analyses The extent and density of both TH-IR fiber outgrowth and Fos-IR cells in host striatum were quantified via image analysis. Images were taken from an Olympus AH-2 photomicroscope linked to a Macintosh Quadra 840AV, operating the NIH Image program, versions 1.54 and 1.60. TH. One 30-µm brain section at the level of maximal graft extent from each animal was analyzed at 503 magnification. After the microscope image was digitized, a 200 3 200 µm box was calibrated and utilized for all subsequent measurements. An optical density (OD) measurement obtained from the contralateral, lesioned-only striatum (taken as assay background) was subtracted from the transplanted striatum. Using 200 3 200 µm boxes beginning immediately adjacent to the graft perimeter and extending in horizontal bands medially and laterally to the edges of the striatum, OD measurements were taken at progressively greater distances from the graft into host. Fos. The density and extent of Fos-IR nuclei within host striatum were also quantified as the number of Fos-IR nuclei within a 200 3 200 µm box at progressively greater distances from the graft periphery into host striatum. Images were converted to binary images

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by thresholding, such that only cells with unequivocally positive black-stained nuclei were counted using the ‘‘Analyze Particles’’ function. Results are expressed as means 6 SEM of the different transplant groups. Unpaired t tests for independent measures were used to statistically compare cell counts and fiber outgrowth from transplants. RESULTS AMPH-Induced Rotation

After equalizing transplants for 500,000 nigral cells (injecting 500,000 nigral cells per SN-Tp and 500,000 nigral plus 500,000 LGE cells per Co-Tp), similar increases in AMPH-induced rotation were obtained over time in all groups (Fig. 1A), regardless of the age of LGE tissue (E14 or E16). Peak turning scores in most cases were obtained by Day 45 and then plateaued. Although Co-Tps turned slightly more on the average than SN-Tps at both Day 75 and Day 105 post-Tp, there were no significant group differences in rotational behavior in these animals receiving 500,000 nigral cells. Further utilizing our protocol for equal cell numbers, we decreased the number of each cell type by diluting the cell suspensions. The injection volume for each transplant remained at 5 µl, but SN-Tp animals now received suspensions containing 280,000 nigral cells, whereas the Co-Tp animals received suspensions containing 280,000 nigral cells plus 280,000 LGE cells. More obvious differences between SN-Tps and Co-Tps were observed when the total number of cells injected was decreased: SN-Tp animals demonstrated significantly lower turning scores than Co-Tp animals (Fig. 1B). Co-Tps exhibited an inverse relationship between age of LGE tissue and functional enhancement: 14/14 Co-Tp animals retained higher turning scores than 14/16 Co-Tps. Post hoc analyses revealed significant differences between 14/14 and SN-Tps on Day 75 and Day 105 (P , 0.05) and just failed to reach significance on Day 45 (P 5 0.055). There were no differences in functional effects between 14/16 Co-Tps and SN-Tps. Functional Effects of Different Ratios of Nigral and LGE Cells

To explore the potency of the observed effects of LGE cells on dopaminergic transplants, and toward the goal of allowing a more reasonable number of cells to be utilized, various ranges of cell numbers were analyzed. We focused on the use of E14 LGE cells in Co-Tps with E14 nigral cells, since these Co-Tps seemed to produce most enhanced functional effects. Animals received E14 nigral cells plus E14 LGE cells in ratios of 1:1, 3:1, 6:1, and 10:1; in all cases 280,000 nigral cells were utilized. After observing that, over the course of time after transplantation, the most significant differences be-

FIG. 1. Development of AMPH-induced rotation in transplant animals. (A) Development of AMPH (2 mg/kg)-induced turning in transplant animals receiving 500,000 nigral cells; SN-Tps (closed circles; n 5 8), Co-Tps receiving E14 LGE (14/14; open circles; n 5 8), or Co-Tps receiving E16 LGE (14/16; open boxes; n 5 14); data are expressed as mean number of turns per minute and error bars represent SEM. Repeated-measures ANOVA indicated no significant differences. (B) Development of AMPH (2 mg/kg)-induced turning in transplant animals receiving 280,000 nigral cells; SN-Tps (closed circles; n 5 7 on Day 25 and Days 45–105; n 5 4 on Days 15, 20, 30, 35, and 40), Co-Tps receiving E14 LGE (14/14; open circles; n 5 11 on Day 25 and Days 45–105; n 5 12 on Days 15, 20, 30, 35, and 40), or Co-Tps receiving E16 LGE (14/16; open boxes; n 5 6); data are expressed as mean number of turns per minute and error bars represent SEM. An asterisk indicates a statistically significant difference in rotation scores between SN-Tp animals and 14/14 Co-Tp animals as revealed by ANOVA (P , 0.05).

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FIG. 2. Development of AMPH-induced turning in co-transplant animals receiving different ratios of nigral-to-LGE cells. Development of turning in response to AMPH (2 mg/kg) of SN-Tps (closed circles; n 5 3), 1:1 Co-Tps (open circles; n 5 9), 3:1 Co-Tps (open squares; n 5 3), 6:1 Co-Tps (open triangles; n 5 5), or 10:1 Co-Tps (open diamonds; n 5 4); data are expressed as mean number of turns per minute and error bars represent SEM. Post hoc analyses revealed significant differences between 1:1 and all other groups on Day 75 and Day 105 (*P , 0.05) and consistently lower turning scores between 10:1 and all other groups on Days 45, 75, and 105 (**P , 0.05).

tween groups appeared at approximately Day 45, the time course of behavioral testing was altered: animals were subsequently tested on Days 25, 45, 75, and 105. Animals receiving nigral and LGE cells in a 1:1 ratio developed turning behavior which was elevated by Day 45 in response to AMPH and showed significantly higher turning scores than SN-Tps, and all other Co-Tps, on Days 75 (P , 0.02) and 105 (P , 0.05) post-Tp (Fig. 2). Co-Tps consisting of 3:1 and 6:1 ratios were no different than SN-Tps, while post hoc analyses revealed that Co-Tps in a 10:1 ratio were consistently inferior to 1:1 Co-Tps as well as to SN-Tps (P , 0.05). Anatomical Organization and Expanse With the observation that embryonic LGE cells produce enhanced function of transplanted embryonic nigral cells, we directed our attention to anatomical correlates of the enhanced function, in order to specifically determine the effect that LGE cells are furnishing. Immunocytochemical staining for tyrosine hydroxylase (TH) revealed transplants consisting of large aggregates (Fig. 3) that spanned at least 500 µm in the rostral–caudal direction. In SN-Tps (Figs. 3A and 3C) TH-immunoreactive (TH-IR) cell bodies were predomi-

nantly located along the perimeter of the grafts, in close proximity to the denervated host striatum, leaving the core of the transplants fairly devoid of DA cell bodies. Whereas some TH-IR fibers were observed coursing through the core of SN-Tps, the majority of DA innervation projected out into the DA-denervated host striatum. Immunocytochemical analyses revealed distinct differences in the internal organization between SNTps and Co-Tps (Figs. 3B and 3D): Co-Tps consisted of DA cell bodies which were scattered rather than confined to the perimeter. TH-IR fibers clustered into densely stained patches within Co-Tps, and DA cell bodies within the transplant were often clustered around these patches of fibers. In order to quantify differences in expanse between the two transplant types, the rostral-to-caudal extent of each transplant was analyzed. When 500,000 nigral cells were utilized in each transplant type, there were no significant differences in longitudinal extent between SN-Tps and Co-Tps (Fig. 4A, first set of bars). However, 14/14 Co-Tps and 14/16 Co-Tps receiving 280,000 nigral cells and 280,000 LGE cells were significantly more extensive than SN-Tps receiving 280,000 nigral cells (Fig. 4A, second set of bars; P , 0.001 and P , 0.05, respectively). In this parameter, SN-Tps again were sensitive to a decrease in the number of nigral cells transplanted, since the extent of the SN-Tps receiving 280,000 nigral cells was significantly lower than those SN-Tps receiving 500,000 nigral cells (P , 0.01), whereas Co-Tps did not decrease appreciably in size. DA Cell Survival

Inclusion of LGE cells enhanced the survival of TH-IR cells in Co-Tps when compared with SN-Tps: there was a significant difference between survival of DA cells when 500,000 nigral cells were injected in SN-Tps and 14/14 Co-Tps (Fig. 4B, first set of bars; P , 0.01). This effect was also observed when fewer cells were injected (Fig. 4B, second set of bars): both 14/14 and 14/16 Co-Tps had significantly higher DA cell survival when compared with SN-Tps receiving 280,000 nigral cells (P , 0.002). Furthermore, Co-Tps receiving only 280,000 nigral cells even showed a trend toward enhanced survival when compared with SN-Tps receiving 500,000 nigral cells (P 5 0.0756, with 17 df). Reinnervation of Host Striatum

Reinnervation of the DA-denervated striatum in all transplant animals was densest in the areas adjacent to the graft–host interface and declined as distance from the transplanted cells increased (see Fig. 3). When equating for number of nigral cells transplanted, CoTps were able to more densely reinnervate the host denervated striatum at all distances from the host–

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FIG. 3. Anatomy of transplants. Photomicrographs of brain sections through the transplanted striatum; sections are in the coronal plane and stained for TH. (A) Low-power view of 6-HDA-lesioned striatum after transplantation of E14 nigral cells (SN-Tp). TH immunoreactivity is largely confined to neuronal cell bodies and processes derived from the transplanted DA cells. (B) Low-power view of lesioned striatum after transplantation of E14 nigral cells plus E14 LGE cells (Co-Tp). Note the differences in intensity and extent of the TH-IR fiber network around the transplants and extending into the denervated host striatum. (C) High-power view of (A) reveals surviving DA neurons preferentially located along the transplant periphery, while the core is relatively free of DA cells. (D) High-power view of (B) illustrates patches of TH-immunoreactive fibers, with DA cells often clustered around these patches.

transplant interface when compared with SN-Tps (Fig. 5). Co-Tps utilizing E14 or E16 LGE produced a significantly higher degree of reinnervation than SN-Tps when both groups received 500,000 nigral cells (P , 0.01). Once again SN-Tps were more sensitive to a decrease in nigral cell number to 280,000, while Co-Tps (utilizing E14 or E16 LGE) obtained a significantly higher density of reinnervation. Co-Tps seemed to reach a maximal reinnervation capacity regardless of the number of cells injected (280,000 vs 500,000);

Co-Tps receiving only 280,000 nigral cells exhibited significantly denser reinnervation when compared with 280,000 SN-Tps by repeated measures ANOVA (P , 0.001). The OD measurement from unlesioned control animals was 200. Activation of Host Striatal Cells

Baseline levels of Fos were negligible in unstimulated striatal cells; 2 h after AMPH, Fos-IR cells in

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unlesioned animals appeared as dense, black nuclear staining scattered throughout the striatum, while a DA-depleting lesion blocked this induction (data not shown). In transplanted rats, Fos-IR cells were numerous in the surrounding host striatum (Fig. 6A), while few Fos-IR cells were found in the opposite, denervated striatum that did not receive transplanted cells. The distribution of Fos-IR cells in the host striatum after AMPH often extended throughout a larger area of the host striatum than that covered by the densest transplant-derived DA outgrowth (Fig. 6B). In order to compare the degree of host-cell activation between SN-Tps and Co-Tps, we quantified the density and extent of host striatal cells exhibiting Fos expression after AMPH in both groups of animals. In transplants containing 500,000 nigral cells, Co-Tps utilizing E14 or E16 LGE exhibited a higher degree of c-fos induction at all distances from the graft (Fig. 6C), which reached significance at points from 400 µm to 1.2 mm from the graft–host interface (P , 0.05). In comparisons of transplants containing 280,000 nigral cells, Co-Tps (E14 or E16 LGE) were again able to activate a significantly higher number of striatal host cells per

FIG. 4. Extent and survival of transplants. (A) Longitudinal extent of transplants. The first set of three bars depicts transplants containing 500,000 nigral cells: SN-Tp (n 5 8); 14/14 Co-Tp (n 5 10), 14/16 Co-Tp (n 5 14). The second set of three bars depicts transplants containing 280,000 nigral cells: SN-Tp (n 5 7), 14/14 Co-Tp (n 5 11), 14/16 Co-Tp (n 5 6). Error bars represent SEM. An asterisk indicates a statistically significant difference in transplant volume from SNTps in that group (P , 0.05). (B) Survival of DA cells in transplants. Counts of TH-IR cells within transplants were made at 1003 and adjusted according to Abercrombie (see Methods). The first set of three bars depicts transplants containing 500,000 nigral cells: SN-Tp (n 5 8), 14/14 Co-Tp (n 5 10), 14/16 Co-Tp (n 5 14). The second set of three bars depicts transplants containing 280,000 nigral cells: SN-Tp (n 5 7), 14/14 Co-Tp (n 5 11), 14/16 Co-Tp (n 5 6). Error bars represent SEM. An asterisk indicates a statistically significant difference in survival of DA cells within the transplants between SN-Tp animals and Co-Tp animals (P , 0.05).

FIG. 5. Reinnervation of host striatum by transplants. The density and extent of outgrowth of TH-IR fibers into the previously lesioned host striatum from transplants containing 500,000 (SN-Tp, closed circles, n 5 8; Co-Tp, open circles, n 5 24) or 280,000 (SN-Tp, closed boxes, n 5 7; Co-Tp, open boxes, n 5 17) nigral cells. The density and extent of reinnervation were quantified by subjecting coronal brain sections to image analysis. Data are expressed as optical density (OD) of TH immunoreactivity within a calibrated 200 3 200 µm box and extending out at 200-µm intervals from the graft–host interface to the edge of the striatum; error bars represent SEM. Repeated-measures ANOVA revealed a statistically significant difference between Co-Tps (utilizing E14 or E16 LGE) and SN-Tps (P , 0.001). The OD measurement from unlesioned control animals was 200.

FIG. 6. Host cell activation after AMPH. (A) Numerous Fos-IR cells are seen in the striatum of a transplanted animal 2 h after AMPH (2 mg/kg), extending beyond the limits of the densest transplant-derived DA innervation, as seen in an adjacent section processed for TH-IR (B). (C) The number and extent of host striatal cells expressing Fos-IR 2 h after AMPH in animals receiving SN-Tps and Co-Tps were quantified by subjecting brain sections to image analysis. Data are expressed as number of Fos-IR cells within a calibrated 200 3 200 µm box and extending out at 200-µm intervals from the graft–host interface to the edge of the striatum. Transplants containing 500,000 (SN-Tp, closed circles, n 5 6; Co-Tp, open circles, n 5 19) or 280,000 (SN-Tp, closed boxes, n 5 3; Co-Tp, open boxes, n 5 9) nigral cells. Error bars are SEM. Mean number of Fos-IR cells in unlesioned control animals 2 h after AMPH was 14. Error bars represent SEM. A single asterisk indicates statistically significant differences between SN-Tps and 14/14 Co-Tps receiving 500,000 SN cells, and double asterisks indicate differences between transplants receiving 280,000 nigral cells as revealed by post hoc analyses (P , 0.05).

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unit area (P , 0.05, except at 400 µm from the transplant periphery: P 5 0.06). DISCUSSION

The present investigation revealed the capacity of embryonic dopaminergic cells to respond to an enhancing influence of their normal target embryonic striatal tissue when co-transplanted into an ectopic environment, the lesioned adult striatum. Inclusion of embryonic LGE cells with nigral cell suspensions attained the desired goals of enhanced function, viability, and reinnervation and has also resulted in the requirement of fewer embryonic nigral cells. Behavioral Effects

The advantage of our unilateral transplant model into bilaterally lesioned striatum is that rotational behavior contralateral to the transplant in response to AMPH (requiring only 2 mg/kg) develops as a result of successful transplant reinnervation, allowing the measurement of an increase rather than a decrease in behavior (16, 56). This model allows a generous range over which to measure development and efficacy of the transplant, with some animals reaching over 60 turns per minute in response to AMPH after transplantation. Co-Tp animals exhibited higher turning scores when compared with SN-Tp animals when only 280,000 nigral cells (and an equal number of E14 LGE cells) were utilized. In many cases, the enhanced effects of the Co-Tps were most apparent at later time points, an important aim for long-term functional effects in the clinical setting. Our dose of AMPH is the lowest in the literature (2 mg/kg as opposed to the typical 5 mg/kg), the lesion is anatomically assessed for completeness in each animal, and behavioral tests occur in such a time frame and frequency as to reflect the complete development and innervation of the transplanted cells. Other co-grafting studies in unilaterally lesioned rodents resulted in a slightly more rapid decrease in AMPHinduced ipsilateral turning (10). When separate cell suspensions of mesencephalic and striatal cells were transplanted into unilaterally lesioned rats, there was a significantly greater decrease in AMPH-induced rotations 5 weeks after transplant when compared with SN grafts (67). An alternative bridging approach, utilizing intranigral dopaminergic transplants with embryonic striatal tissue implanted along a column extending from the nigra to the striatum, exhibited only fair functional effects in a few animals (27). Our findings confirm and expand upon previous nigral-striatal cotransplant studies and also reveal the basis for the enhancing effects of co-transplanted striatal tissue. After equalizing the numbers of each cell type within the transplants to 280,000, responses to behavioral stimuli were still significantly elevated in Co-Tp ani-

mals when compared with SN-Tps, demonstrating that exceptional function can be obtained in the presence of a small number of nigral cells when LGE cells are included. Due to the obstacles involved in obtaining fetal tissue, the present requirement of several fetuses to obtain even mild clinical improvements presents another hurdle in a clinical context. In clinical trials thus far, at least four fetuses are required for transplantation into one patient (29, 36, 42) since approximately 10% of DA cells survive transplantation (11, 37, 46). Co-Tp animals did contain twice the total number of cells in their grafts, but the fact that this total-cellnumber difference did not affect the functional outcome in previous experiments (16) supports the hypothesis that functional effects are dependent upon the growth, development, physiological activity, and incorporation of the SN cells within the transplant and are not simply a function of total cell number within the transplant. On the other hand, in the process of determining the significance of the proportion of this LGE tissue relative to nigral tissue (a 1:1 ratio of nigral-to-LGE cells produced the greatest functional results in response to AMPH), the fact that 10:1 Co-Tps produced a functional outcome inferior to even the SN-Tps (although containing the same number of nigral cells) suggests that the presence of only a small number of LGE cells may actually diminish functional effects. Reports of the site-directed DA fiber growth to embryonic striatal cells in preference to adult striatum (20), as well as growth from transplanted nigral cells in one striatum toward embryonic striatal cells transplanted in the opposite striatum (55), illustrate a strong interaction between these cell types when matched for developmental stage. Thus in the presence of a low proportion of LGE cells (10:1), SN cells may compete to innervate this relatively small amount of trophic striatal tissue, potentially limiting outgrowth into host striatum and thus blunting functional effects. This competition would not exist in SN-Tps alone, explaining the fact that SN-Tps are able to sustain better behavioral responses in comparison to 10:1 Co-Tps. In the presence of an equal amount of LGE cells (1:1), however, nigral cells are less likely to be in competition for trophic effects. Tissue from early developmental stages is required for dissociated cell grafts, as the disruption that occurs during dissection is damaging to more differentiated tissue. Nigral cells produce most reliable graft function when removed from E14 embryos (9); however, because neurons that form the striatum have a longer range of development, these cells become postmitotic from E13/14 through birth. The present data suggest that E14 LGE cells are better able to enhance the function of nigral cells when compared with E16 (and E18) (16) LGE cells and are supported by anatomical effects (below) as well as in vitro results (3).

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Anatomical Organization

The target-specific interactions between embryonic nigral and striatal cells were apparent anatomically: the presence of TH-IR fibers clustered into densely stained patches within the Co-Tps indicates that the nigral cells innervated the co-transplanted embryonic LGE as well as the host striatum. The presence of these anatomically defined ‘‘patch zones’’ have been implicated in increased integration into host circuitry and enhanced functional effects of striatal transplants. The DA cell bodies within Co-Tps were often clustered around these patches of fibers, and in fact, DeBeaurepaire (20) observed that embryonic nigral cells innervate embryonic striatal cells in preference to the mature, denervated host striatum when co-transplanted into the adjacent ventricle, and transplanted nigral cells show fiber outgrowth toward transplanted striatal cells in the opposite striatum (55). Although there is currently no direct evidence of synaptic contacts between nigral and LGE cells within Co-Tps, it can be surmised that the nigral cells will establish synaptic connections with the co-transplanted embryonic striatal cells. The internal organization and local release of DA may yield a more regulated environment for the co-transplanted DA neurons to execute their functional effects. Studies of striatal transplants into excitotoxically lesioned striatum have revealed a patchy distribution of certain markers, including AChE, DARPP-32, calbindin-D, and TH, all of which closely co-localize within the graft (34). Until recently, researchers have harvested cells from both medial and lateral ganglionic eminences for striatal cells used in neural transplantation. It was recently established that selective dissection of the LGE resulted in a higher proportion of patch zones (80–90% patch zones) and produced a higher degree of morphological incorporation of these grafts into host striatum (49). Anatomical tracing demonstrated efferents from these grafts to the globus pallidus, but not the substantia nigra (19). The fact that our striatal dissections were of the LGE in all cases, and the anatomical evidence of an extremely patchy organization within our functionally enhanced Co-Tps, supports the possibility that afferents and efferents which develop to and from striatal transplants may also contribute to the circuitry established in these Co-Tps. The apparent differences in longitudinal extent between transplant types may be attributed to an enhanced development of the cells within Co-Tps, or the fact that Co-Tps did initially consist of twice as many total cells as SN-Tps. The fact that the survival of DA cells was enhanced in Co-Tps does support the hypothesis that these cells, equal numbers of which were initially injected into SN-Tps and Co-Tps, may be partly responsible for the larger volume in Co-Tps. On the other hand, this larger volume may be due to

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continued or renewed stimulation of cell division after transplantation (Costantini et al., in preparation). Dopamine Cell Survival and Reinnervation Capacity

One of the main goals of successful transplant function is obtaining high viability and survival of the grafted cells; however, the number of surviving fetal DA neurons in neuronal grafts placed into striatum of lesioned rats or monkeys varies widely, and the relationship between the number of DA neurons in the graft and the degree of restoration of DA levels remains questionable. Previous in vitro and in vivo studies report discrepancies in the effects of striatal cells on DA cell survival versus differentiation, which may in part be attributed to different methodologies (cultures prepared from different-age embryos, growth conditions, detection of DA neurons). Prochiantz and colleagues demonstrated increases in DA uptake and synthesis, in the absence of increases in the number of DA neurons, in co-cultures of E13 nigra and E15 striatum under serum-containing (50), serum-free (22), and striatal membrane (51) conditions. Although they were able to demonstrate this effect in the presence or in the absence of nonneuronal cells, other studies have emphasized the influence of both nigral (62) and striatal (21, 23, 40) astrocytes on survival, development, and rescue of DA cells. Dong and co-workers (23) demonstrated that striatal glia promoted the survival of nigral DA cells, but striatal neurons were more important for their maturation. Both of these factors may be contributing to the effects observed in our Co-Tps. Our data reveal that embryonic LGE cells do impart a survival-enhancing effect on DA neurons in Co-Tps, even when a small number of nigral cells were included in the transplant, which is relevant in clinical circumstances when the availability of fetal cells is limited. The degree of survival was highest in Co-Tps with E14 LGE cells, reflecting the same pattern as observed with functional analyses: an inverse relationship of striatal donor age and enhancement of DA cell survival. Our previous in vitro means of assessing this interaction confirmed our transplant results, as co-cultures (specifically 14/14) showed a higher number of DA cells initially after plating, as well as a higher number maintained over time (seen with all ages of LGE cells) (3). Both effects are relevant in transplants, since the first outcome suggests an enhanced attraction between the cell types, which may impart a stabilizing force during the time when the transplanted cells are most ‘‘vulnerable.’’ The latter result illustrates an enduring influence that results in enhanced survival over time, which is instrumental to the sustained function of transplants. Fiber outgrowth from grafted neurons is a key issue in neural grafting experiments. It allows a larger area of the striatum to be influenced by DA release and also

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represents the basic element necessary for circuit reconstruction: graft-derived axons forming functional synapses (15). Turning behavior in adult transplant animals is often related to dopaminergic innervation (7), and turning in neonates given bilateral lesions and unilateral transplants increased with the development of innervation (56). The incomplete transplant-induced reversal of DA-denervated symptoms in primates and in patients may be due, in large part, to the relatively large volume of the striatum that is thought to require reafferentation compared to the volume of tissue grafts. Even though the graft-induced elevations in tissue DA concentrations are substantial (28, 52), values taken distant from the graft suggest that reinnervation of the entire caudate does not occur (24). Attempts to enhance this expanse have been made, such as with multipledeposit microtransplants (47). In addition to enhancing the number of DA cells within the transplants, the co-transplanted LGE cells also influenced the development of fiber outgrowth into the denervated host striatum. A more complete reinnervation of the host, reflected here by a higher density and extent of TH-IR fibers in Co-Tp animals, allows the transplanted cells to influence the host to a greater degree in one of two ways: tonic release of DA simply reaching a higher number of host striatal cells due to the increased innervation, or increased synaptic contacts within this area contributing to more comprehensive circuit reconstruction. The fact that the enhanced reinnervation in our Co-Tps was still several times lower than in the normal striatum, even though they exhibited enhanced functional responses, supports the notion that complete reinnervation may be unnecessary. The nigrostriatal system has an enormous capacity to compensate for very low levels of DA, as evidenced by the lack of parkinsonian symptomology until 80% of DA is lost (33). The plateau observed in the ability of Co-Tps to reinnervate the striatum regardless of the number of nigral cells (500,000 vs 280,000) may represent a ‘‘maximal reinnervation capacity’’ of a denervated adult host striatum. Therefore, a complete reinnervation of the entire area of the denervated striatum at the same density as that in an unlesioned striatum may not be mandatory in order to achieve functional effects. With the analytical methods used in this study, we were unable to determine whether the number of TH-IR fibers was increased or whether the expression of TH per fiber was upregulated. The influence of striatal cells on dopaminergic fiber outgrowth in vivo has been reported previously; in addition to the functional effects observed by Yurek et al. (67) and Brundin et al. (10), these groups observed a preference of dopaminergic neurite outgrowth for embryonic striatal tissue and appearance of DA-rich patches (55). Embryonic striatal cells grafted alone also

have the capacity to influence host DA cells, as displayed by dense TH-IR fiber innervation of these grafts when placed in animals with intact nigrostriatal pathways (66). This may be relevant in the clinical application where some portion of nigral DA cells may still be intact and able to respond to the trophic striatal graft. Host Cell Activation

Evidence for a high degree of influence on host cells by Co-Tps when compared with SN-Tps was provided by expression of the immediate-early gene c-fos in host striatum. The induction of the immediate early gene c-fos in striatum resulted in increased levels of Fos protein which was revealed by immunocytochemistry, providing an anatomical map of activated cells (53). In striatal neurons, an abrupt increase in extracellular DA causes a transient induction of c-fos demonstrated to be mediated by D1 receptor activation (30, 57). Lesioning of the nigrostriatal pathway leads to a decrease in AMPH-induced Fos expression, and transplantation of dopaminergic cells normalizes this decrease in striatum (16, 57) as well as in the globus pallidus (14). Both SN-Tps and Co-Tps promoted c-fos expression in host striatum after AMPH (peaking at 2 h after AMPH) over a more extensive area than that densely innervated by the grafted DA neurons, due to DA diffusion, or reflecting the fact that c-fos can be detected in cells activated trans-synaptically (14). Co-Tps produced a higher magnitude of host cell activation when compared with SN-Tps, indicating that the inclusion of LGE cells in Co-Tps may increase amounts of DA synthesized and released from Co-Tps, reduce reuptake of DA, or generate changes postsynaptically at the receptor level. However, DA metabolism has been shown to occur in proportion to the amount of reinnervation after transplantation, suggesting that regulatory mechanisms in the reinnervated areas of the striatum are active (61). These data suggest that increased activation of postsynaptic receptors in host striatum, brought about by enhanced cell survival and increased host reinnervation, may account in part for the enhanced behavioral effects with Co-Tps. Whether the c-fos induction in our paradigm does produce long-term changes in neuronal activity remains to be seen. One study utilized antisense oligonucleotides to c-fos to alter agonist-induced rotation in intact animals (59), suggesting that induction of c-fos by AMPH is directly involved in this rotational response. Yet the rotation response appears within minutes after AMPH administration, whereas Fos protein peaks 2 h later and may thus play a role in adaptation of these neurons to DA stimulation rather than modifying the behavioral output itself. Incorporation of our Co-Tps may occur to a greater degree due to the presence of afferents and efferents to the transplanted striatal cells. Grafts of fetal striatal

CO-TRANSPLANTATION OF FETAL LATERAL GANGLIONIC EMINENCE

neurons into excitotoxin-lesioned striatum restore functional recovery in this lesion model, such as reduced hyperactivity, motor asymmetries, and apomorphineinduced dyskinesias (5, 31). Tracing and immunocytochemical studies have demonstrated that such transplants receive cortical, nigral, raphe, and thalamic afferents, which form synaptic contacts on mediumsized spiny neurons, and then send efferents to the globus pallidus (39, 66). The ability of these intrastriatal striatal grafts to counteract the lesion-induced behavioral and anatomical deficits depends on some degree of functional integration with host striatal circuitry, including input from the host DA system. GABA release from striatal transplants has been demonstrated to be under the normal regulatory control from the intact host dopaminergic system (12). The presence of both striatal and nigral cells in our Co-Tps would suggest that this GABA release is also occurring in a somewhat regulated manner, contributing to functionality. Campbell and Bjorklund (13) demonstrated Fos expression in striatal grafts after cortical stimulation (predominantly in DARPP-32 patches), as well as alterations in neuropeptide expression within these grafts after knife transection of the corticostriatal pathway. The possibility that the striatal cells of our Co-Tps are also receiving some, if not all, of these afferents is reasonable and may be one mechanism for the enhanced functional effects observed. Summary

The behavioral and anatomical results demonstrate an ability of embryonic LGE cells to magnify the functional effects and augment the anatomical development and integration of dopaminergic transplants; however, the basis for these enhancing effects seems multifaceted. One feature of developing striatum that is enriched in E14 striatum is the proportion of patch neurons relative to matrix neurons, defined in part by their distinct birthdates: patch neurons are generated at E13–E15 in the rat, while E15 designates the genesis of matrix neurons (64). We have previously shown that E14 LGE tissue consists almost exclusively of patch neurons, whereas E16 LGE tissue contains fewer patch neurons as a proportion of the total number of cells (3). The expression of certain receptors, growth factors, or adhesion molecules which are developmentally regulated may contribute to this effect. Early in development, the enrichment of GluR1 receptors in patch cells which co-localize with TH-IR fibers and substance P has been demonstrated, as has the subsequent patchy expression of GluR2/3, NMDAR1, and NMDAR2A/2B (58). Evidence from this laboratory has also revealed an enrichment in trkB, the high-affinity receptor for BDNF, in striatal patches during development (17), as well as a patchy distribution within Co-Tps (Costantini and Snyder-Keller, unpublished observations).

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Several growth factors have been isolated and characterized, such as BDNF and GDNF, which influence the survival, development, and morphological differentiation of dopaminergic cells in vitro (4, 41). DalToso (18) isolated an active fraction of bovine striatal extract which increases DA uptake in mouse nigral cells in culture, suggesting that a soluble factor may be produced by striatum. Conversely, embryonic cells may rely upon cell contacts during early development for enhanced function. Studies have revealed reaggregation of E14 DA cells and extensive neuritic outgrowth with E14 striatal cells in co-culture (54), as well as in co-grafts (35). Human mesencephalic cells cultured on striatal glia display a severalfold increase in DA cell survival and proportion of DA cells with differentiated morphology (23). The finding that target neurons influence DA differentiation, and target glia influence their survival, indicates multiple target cell type-specific regulation of the development of innervating neurons (23). The present study illustrates the capacity of embryonic striatal cells to potentiate the functional effects and anatomical development of nigral dopaminergic transplants in the bilaterally lesioned rat. Targettissue dependency is a consistent mechanism employed by the developing nervous system to attain full growth and differentiation of cell types. The immature striatum provides a target for axons of the developing substantia nigra as they form the dopaminergic nigrostriatal pathway, and the above data demonstrate that these striatal cells also stimulate the development and activity of transplanted DA neurons by providing a trophic influence and/or a permissive milieu. Not only do these studies demonstrate the potential of augmenting the function and anatomical development of transplanted cells into DA-denervated brain, but they also explore the mechanisms which are crucial to the normal development and integration of these DA cells in the brain. ACKNOWLEDGMENTS We are grateful to Ms. Yili Lin for expert technical assistance. This work was supported by NIH Grant MH46577.

REFERENCES 1.

Abercrombie, M. 1946. Estimation of nuclear populations from microtome sections. Anat. Rec. 94: 239–247. 2. Abrous, D., A. Shaltot, E. Torres, and S. Dunnett. 1993. Dopamine-rich grafts into the neostriatum and/or nucleus accumbens: Effects on drug-induced behaviors and skilled paw reaching. Neuroscience 53: 187–197. 3. Aronica, E., L. Costantini, and A. Snyder-Keller. 1996. Reciprocal influences of nigral cells and striatal patch neurons in dissociated co-cultures. J. Neurosci. Res. 44: 540–550. 4. Beck, K., B. Knusel, and F. Hefti. 1993. The nature of the trophic

226

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

COSTANTINI AND SNYDER-KELLER action of BDNF, IGF-1, and bFGF on mesencephalic dopaminergic neurons developing in culture. Neuroscience 52: 855–966. Bjorklund, A., K. Campbell, D. Sirinathsinghji, R. Fricker, and S. Dunnett. 1994. Functional capacity of striatal transplants in the rat Huntington model. In Functional Neural Transplantation (S. Dunnett and A. Bjorklund, Eds.), pp. 157–195. Raven Press, New York. Bjorklund, A., and O. Lindvall. 1986. Catecholaminergic brain stem regulatory systems. In Handbook of Physiology: The Nervous System. Intrinsic Regulatory Systems in the Brain (F. E. Bloom, Ed.), pp. 155–235. Am. Physiol. Soc., Baltimore, MD. Bjorklund, A., O. Lindvall, O. Isacson, P. Brundin, K. Wictorin, R. Strecker, D. Clarke, and S. Dunnett. 1987. Mechanisms of action of intracerebral neural implants: Studies on nigral and striatal grafts to the lesioned striatum. Trends Neurosci. 10: 509–516. Bolam, J., T. Freund, A. Bjorklund, S. Dunnett, and A. Smith. 1987. Synaptic input and local output of dopaminergic neurons in grafts that functionally reinnervate the host striatum. Exp. Brain Res. 68: 131. Brundin, P., O. Isacson, and A. Bjorklund. 1985. Monitoring of cell viability in suspensions of embryonic CNS tissue and its use as a criterion for intracerebral graft survival. Brain Res. 331: 251–259. Brundin, P., O. Isacson, F. Gage, and A. Bjorklund. 1986. Intrastriatal grafting of dopamine-containing neuronal cell suspensions: Effects of mixing with target or non-target cells. Dev. Brain Res. 24: 77–84. Brundin, P., and A. Bjorklund. 1987. Survival, growth, and function of dopaminergic neurons grafted to the brain. Prog. Brain Res. 71: 293–308. Campbell, K., P. Kalen, K. Wictorin, C. Lundberg, R. Mandel, and A. Bjorklund. 1993. Characterization of GABA release from intrastriatal striatal transplants: Dependence on host-derived afferents. Neuroscience 53: 403–415. Campbell, K., and A. Bjorklund. 1995. Neurotransmitterrelated gene expression in intrastriatal striatal transplants. III. Regulation by host cortical and dopaminergic afferents. Mol. Brain Res. 29: 273–284. Cenci, M., P. Kalen, R. Mandel, K. Wictorin, and A. Bjorklund. 1992. Dopaminergic transplants normalize amphetamine- and apomorphine-induced FOS expression in the hydroxydopaminelesioned striatum. Neuroscience 46: 943–957. Clarke, D., P. Brundin, R. Strecker, O. Nilsson, A. Bjorklund, and O. Lindvall. 1988. 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: 115–126. Costantini, L., B. Vozza, and A. Snyder-Keller. 1994. Enhanced efficacy of nigral-striatal cotransplants in bilaterally dopaminedepleted rats: An anatomical and behavioral analysis. Exp. Neurol. 127: 219–231. Costantini, L., S. Feinstein, M. Radeke, and A. Snyder-Keller. 1996. TrkB receptors localize to patches in developing striatum. Soc. Neurosci. Abstr. 22: 1479. Dal Toso, R., O. Giorgi, C. Soranzo, G. Kirschner, G. Ferrari, M. Favaron, D. Benvegnu, D. Presti, S. Vicini, G. Toffano, G. Azzone, and A. Leon. 1988. Development and survival of neurons in dissociated fetal mesencephalic serum-free cell cultures: I. Effects of cell density and of an adult mammalian striatalderived neuronotrophic factor (SDNF). J. Neurosci. 8: 733–745. Deacon, T., P. Pakzaban, and O. Isacson. 1994. The lateral ganglionic eminence is the origin of cells committed to striatal phenotype: Neural transplantation and developmental evidence. Brain Res. 668: 211–219.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33. 34.

35. 36.

BeBeaurepaire, R., and W. Freed. 1987. Embryonic substantia nigra grafts innervate embryonic striatal co-grafts in preference to mature host striatum. Exp. Neurol. 95: 448–454. Denis-Donini, S., J. Glowinski, and A. Prochiantz. 1983. Specific influence of striatal target neurons on the in vitro outgrowth of mesencephalic dopaminergic neurites: A morphological quantitative study. J. Neurosci. 3: 2292–2299. Di Porzio, U., J. Daguet, J. Glowinski, and A. Prochiantz. 1980. Effect of striatal cells on in vitro maturation of mesencephalic dopaminergic neurons grown in serum-free conditions. Nature 288: 370–373. Dong, J., A. Detta, M. Bakker, and E. Hitchcock. 1993. Direct interaction with target-derived glia enhances survival but not differentiation of human fetal mesencephalic dopaminergic neurons. Neuroscience 56: 53–60. Doucet, G., P. Brundin, L. Descarries, and A. Bjorklund. 1990. Effect of prior dopamine denervation on survival and fiber outgrowth from intrastriatal fetal mesencephalic grafts. Eur. J. Neurosci. 2: 279–290. Doucet, G., Y. Murata, P. Brundin, O. Bosler, N. Mons, M. Geffard, C. Quimet, and A. Bjorklund. 1989. Host afferents into intrastriatal transplants of fetal ventral mesencephalon. Exp. Neurol. 106: 1–19. Dunnett, S., I. Wishaw, D. Rogers, and G. Jones. 1987. Dopaminerich grafts ameliorate whole body motor asymmetry and sensory neglect but not independent limb use in rats with 6-hydroxydopamine lesions. Brain Res. 415: 63–78. Dunnett, S., D. Rogers, and S. Richards. 1989. Nigrostriatal reconstruction after 6-OHDA lesions in rats: Combination of dopamine-rich nigral grafts and nigrostriatal ‘‘bridge’’ grafts. Exp. Brain Res. 75: 523–535. Elsworth, J., J. Sladek, J. Taylor, T. Collier, D. Redmond, and R. Roth. 1996. Early gestational mesencephalon grafts but not later gestational mesencephalon, cerebellum, or sham grafts, increase dopamine in caudate nucleus of MPTP-treated monkeys. Neuroscience 72: 477–484. Freeman, T., C. Olanow, R. Hauser, G. Nauert, D. Smith, C. Borlongan, P. Sanberg, D. Holt, J. Kordower, F. Vingerhoet, B. Snow, D. Calne, and L. Gauger. 1995. Bilateral fetal nigral transplantation into the postcommisural putamen in Parkinson’s disease. Ann. Neurol. 38: 379–388. Graybiel, A., R. Mortally, and H. Robertson. 1990. Amphetamine and cocaine induce drug-specific activation of the c-fos gene in striosome-matrix compartments and limbic subdivisions of the striatum. Proc. Natl. Acad. Sci. USA 87: 6912–6916. Hantraye, P., D. Riche, M. Maziere, and O. Isacson. 1992. Intrastriatal transplantation of cross-species fetal striatal cells reduces abnormal movements in a primate model of Huntington’s disease. Proc. Natl. Acad. Sci. USA 89: 4187–4191. Hemmendinger, L., B. Garber, P. Hoffman, and A. Heller. 1981. Target neuron-specific process formation by embryonic mesencephalic dopamine neurons in vitro. Proc. Natl. Acad. Sci. USA 78: 1264–1268. Hornykeiwicz, O. 1973. Parkinson’s disease: From brain homogenate to treatment. Fed. Proc. 32: 183–189. Isacson, O., D. Dawbarn, P. Brundin, F. Gage, P. Meson, and A. Bjorklund. 1987. Neural grafting in a rat model of Huntington’s disease: Striosomal-like organization of striatal grafts as revealed by acetylcholinesterase histochemistry, immunocytochemistry, and receptor autoradiography. Neuroscience 22: 481–497. Jaeger, C. 1986. Axon terminal clustering in nigrostriatal double grafts. Dev. Brain Res. 24: 309–314. Kordower, J., T. Freeman, B. Snow, F. Vingerhoets, E. Muffson, P. Sanberg, R. Hauser, D. Smith, G. Nauert, D. Perl, and C. Olanow. 1995. Neuropathological evidence of graft survival and

CO-TRANSPLANTATION OF FETAL LATERAL GANGLIONIC EMINENCE

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

striatal reinnervation after transplantation of fetal mesencephalic tissue in a patient with Parkinson’s disease. N. England J. Med. 332: 1118–1124. Kordower, J., J. Rosenstein, T. Collier, M. Burke, E. Chen, J. Li, L. Martel, A. Levey, E. Mufson, T. Freeman, and C. Olanow. 1996. Functional fetal nigral grafts in a patient with Parkinson’s disease: Chemoanatomic, ultrastructural, and metabolic studies. J. Comp. Neurol. 370: 203–230. Labandiera-Garcia, J., J. Tobio, and M. Guerra. 1994. Comparison between normal developing striatum and developing striatal grafts using drug-induced Fos expression and neuronspecific enolase immunohistochemistry. Neuroscience 60: 399– 415. Labandiera-Garcia, J., K. Wictorin, E. Cunningham, and A. Bjorklund. 1991. Development of intrastriatal striatal grafts and their afferent innervation from the host. Neuroscience 42: 407–426. Langeveld, C., C. Jongenelen, E. Schepens, J. Stoof, A. Bast, and B. Drukarch. 1995. Cultured rat striatal and cortical astrocytes protect mesencephalic dopaminergic neurons against hydrogen peroxide toxicity independent of their effect on neuronal development. Neurosci. Lett. 192: 13–16. Lin, L., D. Doherty, J. Lile, S. Bektesh, and F. Collins. 1993. (GDNF) a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 260: 1130–1132. Lindvall, O., P. Brundin, H. Widner, S. Rehncrona, B. Gustavii, R. Frackowiak, K. Leenders, G. Sawle, J. Rothwell, C. Marsden, and A. Bjorklund. 1990. Grafts of fetal dopamine neurons survive and improve motor function in Parkinson’s disease. Science 242: 574–577. Liu, F., S. Dunnett, H. Robertson, and A. Graybiel. 1991. Intrastriatal grafts derived from fetal striatal primordia. III. Induction of modular patterns of Fos-like immunoreactivity by cocaine. Exp. Brain Res. 85: 501–506. Mandel, R., K. Wictorin, M. Cenci, and A. Bjorklund. 1992. Fos expression in intrastriatal striatal grafts: Regulation by host dopaminergic afferents. Brain Res. 583: 207–215. Nakao, N., P. Odin, and P. Brundin. 1994. Selective subdissection of the striatal primordium for cultures affects the yield of DARPP-32 containing neurons. NeuroReport 5: 1081– 1084. Nikkhah, G., P. Odin, A. Smits, A. Tingstrom, A. Othberg, P. Brundin, K. Funa, and O. Lindvall. 1993. Platelet-derived growth factor promotes survival of rat and human mesencephalic dopaminergic neurons in culture. Exp. Brain Res. 92: 516–523. Nikkhah, G., M. Cunningham, A. Jodicke, U. Knappe, and A. Bjorklund. 1994. Improved graft survival and striatal reinnervation by microtransplantation of fetal nigral cell suspensions in the rat Parkinson model. Brain Res. 633: 133–143. Ostergaard, K., J. Schou, and J. Zimmer. 1990. Rat ventral mesencephalon grown as organotypic slice cultures and cocultured with striatum, hippocampus, and cerebellum. Exp. Brain Res. 82: 547–565. Pakzaban, P., T. Deacon, L. Burns, and O. Isacson. 1993. Increased proportion of acetylcholinesterase-rich zones and improved morphological integration into host striatum of fetal grafts derived from the lateral but not the medial ganglionic eminence. Exp. Brain Res. 97: 13–22. Prochiantz, A., U. DiPorzio, A. Kato, B. Berger, and J. Glowinski. 1979. In vitro maturation of mesencephalic dopaminergic neurons from mouse embryos is enhanced in presence of their striatal target cells. Proc. Natl. Acad. Sci. USA 76: 5387–5391. Prochiantz, A., M. Daguet, A. Herbert, and J. Glowinski. 1981. Specific stimulation of in vivo maturation of mesencephalic

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65. 66.

67.

68.

227

dopaminergic neurons by striatal membranes. Nature 293: 570–572. Rioux, L., D. Gaudin, L. Bui, L. Gregoire, T. DiPaolo, and P. Bedard. 1991. Correlation of functional recovery after a 6-hydroxydopamine lesion with survival of grafted fetal neurons and release of dopamine in the striatum of the rat. Neuroscience 40: 123–131. Sagar, S., F. Sharp, and T. Curran. 1988. Expression of c-fos protein in brain: Metabolic mapping at the cellular level. Science 240: 1328–1331. Shalaby, I., P. Hoffmann, and A. Heller. 1984. Release of dopamine from mesencephalic neurons in aggregate cultures: Influence of target and non-target cells. Brain Res. 307: 347– 350. Sladek, J., T. Collier, J. Elsworth, J. Taylor, R. Roth, and D. Redmond. 1993. Can graft-derived neurotrophic activity be used to direct axonal outgrowth of grafted dopamine neurons for circuit reconstruction in primates? Exp. Neurol. 124: 134–139. Snyder-Keller, A., R. Carder, and R. Lund. 1989. Development of dopamine innervation and turning behavior in dopaminedepleted infant rats receiving unilateral transplants. Neuroscience 30: 779–794. Snyder-Keller, A. 1991. Striatal c-fos induction by drugs and stress in neonatally dopamine-depleted rats given nigral transplants: Importance of NMDA activation and relevance to sensitization phenomena. Exp. Neurol. 114: 155–165. Snyder-Keller, A., and L. Costantini. 1996. Glutamate receptor subtypes localize to patches in the developing striatum. Dev. Brain Res. 94: 246–250. Sommer, W., B. Bjelke, D. Gante, and K. Fuxe. 1993. Antisense oligonucleotide to c-fos induces ipsilateral rotational behavior to d-amphetamine. NeuroReport 5: 277–220. Spencer, D., R. Robbins, F. Naftolin, D. Phil, J. Marek, T. Vollmer, C. Leranth, R. Roth, L. Price, A. Gjedde, B. Bunney, K. Sass, J. Elsworth, L. Kier, R. Makuch, P. Hoffer, and E. Redmond. 1992. Unilateral transplantation of human fetal mesencephalic tissue into the caudate nucleus of patients with Parkinson’s disease. N. Eng. J. Med. 327: 1541–1548. Strecker, R., T. Sharp, P. Brundin, T. Zetterstrom, U. Ungerstedt, and A. Bjorklund. 1987. Autoregulation of dopamine release and metabolism by intrastriatal nigral grafts as revealed by intracerebral dialysis. Neuroscience 22: 169–178. Takeshima, T., K. Shimoda, Y. Sauve, and J. Commissiong. 1994. Astrocyte-dependent and -independent phases of the development and survival of rat embryonic day 14 mesencephalic, dopaminergic neurons in culture. Neuroscience 60: 809– 823. Tomozawa, Y., and S. Appel. 1986. Soluble striatal extracts enhance development of mesencephalic dopaminergic neurons in vitro. Brain Res. 399: 111–124. van der Kooy, D., and G. Fishell. 1987. Neuronal birthdate underlies the development of striatal compartments. Brain Res. 401: 155–161. Wictorin, K. 1992. Anatomy and connectivity of intrastriatal striatal transplants. Prog. Neurobiol. 38: 611–639. Wictorin, K., C. Ouimet, and A. Bjorklund. 1989. Intrinsic organization and connectivity of intrastriatal striatal transplants in rats as revealed by DARPP-32 immunohistochemistry. Eur. J. Neurosci. 1: 690–701. Yurek, D., T. Collier, and J. Sladek. 1990. Embryonic mesencephalic and striatal co-grafts: Development of grafted dopamine neurons and functional recovery. Exp. Neurol. 109: 191–199. Zhou, F., and N. Buchwald. 1989. Connectivities of the striatal grafts in adult rat brain: a rich afference and scant striatonigral efference. Brain Res. 504: 15–30.