Survival and function of dissociated rat dopamine neurones grafted at different developmental stages or after being cultured in vitro

The in vitro culture approach was combined with the cell suspension grafting technique to examine whether the maturation of dopamine (DA) neurones in vitro imposed similar limitations on their ability to survive grafting as when they are allowed to develop in situ in the fetus. The functional capacity, survival and growth of DA neurones from 2.5- and 7-day-old cultures, grafted to rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway, was compared with similar grafts freshly prepared from fetal donors of embryonic days 14, 16 and 20. Grafts of freshly dissociated mesencephalic DA neurones, taken from embryonic day 14-16 donors and 2.5-day-old cultures, generally survived well and markedly reduced amphetamine-induced rotational asymmetry in the recipient rats. However, when cultured for 7 days prior to grafting, or when taken from 20-day-old fetuses, the mesencephalic DA neurones survived very poorly and the grafts did not have any functional effects. Plating of aliquots of cell suspension used for grafting indicated that the survival rate of dissociated DA neurones is in the same order of magnitude when grown in vitro (about 2 DA neurones per 1000 cells) as when grafted in vivo to the rat striatum (about 1-5 DA neurones per 1000 cells). When the number of surviving grafted DA neurones was plotted against the behavioural effects of the grafts, a threshold number of around 100-200 DA neurones was found necessary to obtain a marked reduction (greater than 50%) in amphetamine-induced rotational asymmetry. Moreover, the survival of 300-500 DA neurones seemed to produce a 'ceiling effect' beyond which additional surviving DA neurones gave rise to little or no further effect on the amphetamine-induced rotational behaviour.     

Partial Restoration of Striatal GABAA Receptor Balance by Functional Mesencephalic Dopaminergic Grafts in Mice with Hereditary Parkinsonism

Levels of inhibitory amino acid receptors were studied in the weaver (wv/wv) mouse model of dopamine (DA) deficiency after unilateral intrastriatal transplantation of fetal mesencephalic cell suspensions. Graft integration was verified by turning behavior tests and from the topographical levels of the DA transporter, tagged autoradiographically with 3 nM [3H]GBR 12935. The average increase in [3H]GBR 12935 binding in grafted dorsal striatum compared to nongrafted wv/wv striatum was 60% 3 months after grafting. Autoradiography of 8 nM [3H]flunitrazepam and 12 nM [3H]muscimol binding was carried out to visualize the distribution of GABAA receptors in +/+ mice and in recipient weaver mutants. A 17% increase in [3H]flunitrazepam binding and a 20% increase in [3H]muscimol binding was found in the nongrafted dorsal striatum of weaver mutants compared to +/+. The functional mesencephalic grafts had a partial normalizing effect on both [3H]flunitrazepam and [3H]muscimol binding in the dorsal striatum of the weaver recipients. The normalization brought about by the grafts was around 20% for [3H]flunitrazepam binding and more than 40% for [3H]muscimol binding. The results are discussed in the context of the important interaction between the converging glutamatergic corticostriatal and DAergic nigrostriatal pathways in controlling the functional GABAergic output of the basal ganglia in Parkinson's disease and in experimental models of DA deficiency.     

Functional compensation afforded by grafts of foetal neurones

1. Adult neurones grafted to the adult nervous system show limited survival and growth potential. By contrast, certain foetal neurones of a defined age, when grafted to the damaged adult nervous system survive, and form a dense innervation of the host brain with morphologically defined synapses. Monamine containing neurones in the rat have these properties. In a number of model systems their ability to survive grafting to the adult brain has been demonstrated, the optimal conditions for their survival and the importance of endogenous growth factors continue to be investigated. 2. Lesions of the ascending dopamine pathways to the dorsal and ventral striatum result in profound motor disorders. Dopamine-rich foetal neurones from the mesencephalon grafted as blocks of tissue or as cell suspensions provide a rich innervation to the dopamine-depleted target areas and reverse many of these motor impairments. Similar models have been developed for the cholinergic innervation from the basal forebrain to the hippocampus and cortex. Cholinergic depletion of these forebrain structures impair performance on spatial memory tasks, which are reversed by grafts of foetal basal forebrain neurones to cortex or hippocampus. 3. The grafting model is a powerful tool with which to define the functional role of neurotransmitters in brain function. The neurological properties of foetal monoamine and cholinergic neurones which underlie their ability to afford functional compensation to the damaged nervous system and their vulnerability in certain degenerative diseases of the CNS in man remain obscure.     

Diminished survival of mesencephalic dopamine neurons grafted into aged hosts occurs during the immediate postgrafting interval

The survival rate of dopamine (DA) neurons in mesencephalic grafts to young adult rats is poor, estimated at 5-20%, and even poorer in grafts to the aged striatum. Grafted cells die in young adult rats during the first 4 days after implantation. The present study was undertaken to determine whether the decreased survival of DA neurons in grafts to aged rats is (1) due to additional cell death during the immediate postgrafting interval or (2) due to protracted cell loss during longer postgrafting intervals. We compared survival rates of tyrosine hydroxylase-immunoreactive (THir) neurons in cell suspension grafts to young adult (3 months) and aged (24 months) male Fischer 344 rats at 4 days and 2 weeks after transplantation. At 4 days after grafting, mesencephalic grafts within the aged rat striatum contain approximately 25% of the number of THir neurons in the same mesencephalic cell suspension grafted to young adult rats. This corroborates the decreased survival of grafted DA neurons we have demonstrated previously at 10 weeks postgrafting. THir neurons in grafts to the intact striatum possessed a significantly shorter "long axis" than their counterparts on the lesioned side. No significant differences in the number of apoptotic nuclear profiles or total alkaline phosphatase staining between mesencephalic grafts to young and aged rats were detectable at 4 days postgrafting. In summary, the present study indicates that the exaggerated cell death of grafted DA neurons that occurs following implantation to the aged striatum occurs during the immediate postgrafting interval, timing identical to that documented for young adult hosts.     

Pattern of synaptophysin immunoreactivity within mesencephalic grafts following transplantation in a parkinsonian primate model

The majority of investigations into the degree of restoration of neural circuitry following transplantation of the embryonic ventral mesencephalon to the striatum have focused upon the particular neurochemical subtypes of the fibers exchanged between graft and host. Visualization of neurites of specific neurotransmitter type while informative regarding the specificity of graft–host interactions, vastly underrepresents overall synaptogenesis as it may occur in the grafting situation. The present approach of using a molecular marker characteristic of all normal, functional synapses provides broader information about the synaptic remodeling that occurs after tissue grafting. Synaptophysin (SY), an integral membrane protein of the synaptic vesicle, is a reliable marker of nerve terminal differentiation. Immunohistochemical staining with antibodies directed against SY and the dopamine synthetic enzyme tyrosine hydroxylase (TH) was used to assess overall synaptic differentiation as well as the relationship between SY immunoreactivity and the distribution of grafted dopamine (DA) neurons and processes in mesencephalic grafts and mesencephalic-striatal co-grafts implanted in the striatum of MPTP-treated African green monkeys. Grafted embryonic cerebellar tissue was used as a comparison graft type that does not normally exchange prominent direct projections with striatum. Dense pericellular arrays of SY-positive terminals were associated with TH-positive neurons in mesencephalic grafts. In mixed mesencephalic-striatal co-grafts, TH-positive fiber patches within the striatal portion of the graft demonstrated a high degree of correspondence with SY immunoreactivity. In contrast, grafts of cerebellar tissue did not display the same pattern of prominent pericellular arrays of SY staining. These observations suggest that functional synapses are abundantly present within grafted mesencephalon, and that these contacts are enriched in areas of the graft occupied by DA neurons. Implantation of an inappropriate striatal target, the cerebellum, results in visibly diminished innervation. The pattern of SY labeling observed suggests that tissue grafts are extensively innervated, probably both from extrinsic and intrinsic sources, and that the pattern and density of this innervation corresponds to the appropriateness of the graft–host interaction.     

Limited recovery of striatal dopaminergic fibers by adrenal medullary grafts in MPTP-treated aging mice

Systemic injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) damages the dopaminergic (DA) nigrostriatal system in C57BL/6 mice. We have investigated the effect of MPTP neurotoxicity and subsequent adrenal medullary grafts into the striatum of young (2-3 months) and aging (12 months) mice. MPTP treatment (4 X 20 mg/kg ip given 3 or 12 h apart in young mice and 12 h apart in aging mice) resulted in 80-90% depletion of striatal DA and virtual disappearance of tyrosine hydroxylase (TH)-immunoreactive (IR) fibers in both young and aging mice 1 week following treatment. Only partial recovery of TH-IR fibers was seen 5 weeks after MPTP treatment in young mice, while virtually no recovery was seen in aging mice. Adrenal medullary minced pieces were grafted into the striatum of young and aging mice 1 week after MPTP treatment. In young mice, dense TH-IR fibers were observed in the striatum on the grafted side 4 weeks later, far denser than those in sham-operated striatum. Although this staining was most prominent around the grafts, many TH-IR fibers also were found in the ventral striatum close to the nucleus accumbens. No such increase in TH-IR fibers was found on the nongrafted side. DA concentration on the grafted side recovered to 45% of the control level. In aging mice receiving similar grafts, TH-IR fibers also were observed in the grafted striatum, but were less dense and more restricted around the site of the graft compared with young mice. DA concentration on the grafted side was 29% of the control level. We conclude that the MPTP-depleted nigrostriatal DA system in aging mouse brain can recover partially following adrenal medullary grafts, but the degree of recovery is more limited compared with that in young brain.     

An in vitro interval before transplantation of mesencephalic reaggregates does not compromise survival or functionality

Grafts of primary ventral mesencephalic tissue and cell suspensions to the denervated striatum are currently utilized as a treatment strategy for Parkinson's disease. Survival rates of grafted dopamine (DA) neurons are extremely poor (5-20%) and is even poorer in grafts to the aged striatum. Short pretreatment of grafted cells with various survival-promoting agents has elicited 2- to 3-fold improvements in these survival rates. However, the duration of pretreatment is limited by the necessity of implanting the embryonic cells within a critical period after tissue harvest, potentially limiting the beneficial effects of these interventions. This study details the use of a modified mesencephalic reaggregate culture system combined with striatal-derived trophic factor support to provide an extended ex vivo cell culture interval before grafting. Mesencephalic cell suspension grafts implanted immediately following dissociation were compared to grafts of an equivalent number of cells reaggregated in the presence of striatal oligodendrocyte-type-2 astrocyte (SO2A) conditioned medium for 3 or 7 days. All grafts were placed in the denervated striatum of young adult male Fischer 344 rats. Rotational assessment of amphetamine-induced rotations indicates that aggregates maintained for 3 days in culture present statistically similar functional recovery profiles as compared to cell suspension grafts. Grafts of mesencephalic reaggregates maintained in vitro for 7 days did not display significant improvements in functional recovery. Immunohistochemical analysis for tyrosine hydroxylase immunoreactive (THir) neurons conducted at 10 weeks post-grafting revealed equivalent survival rates of THir neurons in grafts of fresh cell suspensions and aggregates held in culture for 3 days. Grafts of reaggregates held in culture for 7 days possessed significantly fewer THir neurons, about 25% of the cell suspension or 3-day aggregate grafts. This ex vivo reaggregate system allows for extended pretreatment (3 days) of mesencephalic cells with survival-promoting agents and immunological screening of tissue before transplantation.     

The Importance of Graft Placement and Task Complexity for Transplant-Induced Recovery of Simple and Complex Sensorimotor Deficits in Dopamine Denervated Rats

The present study examined the role of graft placement and behavioural task complexity in determining the functional efficacy of intrastriatal grafts of dopamine-rich fetal ventral mesencephalon (VM) placed in the dopamine (DA) depleted striatum. The functional effects of two different striatal placements of VM grafts were evaluated using tests of drug-induced motor asymmetry, simple sensorimotor orienting response, and a more complex sensorimotor integrative task (disengage behaviour), in which the rat has to perform the orienting response while in the act of eating. Rats with complete unilateral 6-hydroxydopamine (6-OHDA) lesions of the mesostriatal DA pathway, received either implants of dissociated fetal VM in the central or ventrolateral portions of the denervated striatum. Nongrafted lesioned rats served as controls. Nine weeks after grafting, the rats were tested on separate days for disengage behaviour, sensorimotor orientation, and amphetamine-induced rotational behaviour. Consistent with previous findings, the two graft placements had differential effects on drug-induced motor asymmetry and sensorimotor responses: the centrally placed VM grafts reversed amphetamine-induced rotational asymmetry but had little effect on the sensorimotor deficit, whereas the ventrolaterally placed grafts reversed the sensorimotor orientation deficits without any effect on the drug-induced rotation. In contrast, fetal VM grafts, regardless of their placement, did not ameliorate the observed deficits in disengage behaviour; that is the grafted rats that had recovered their sensorimotor response in the absence of food were unable to perform the same orienting response while eating. These results provide evidence that functional intrastriatal VM grafts which are capable of restoring sensorimotor responses or motor asymmetry fail to affect lesion-induced deficits in a task that requires more complex sensorimotor integration. It is suggested that the degree of anatomical integration of the grafted DA neurons into the host circuitry will determine the efficacy of the grafts to influence more complex sensorimotor integrative deficits in the DA lesion model.     

Cografts of adrenal medulla with peripheral nerve enhance the survivability of transplanted adrenal chromaffin cells and recovery of the host nigrostriatal dopaminergic system in MPTP-treated young adult mice

Schwann cells from transected peripheral nerve segments are known to produce nerve growth factor (NGF). We performed adrenal medullary grafts or cografts of adrenal medulla and sciatic nerve into the striatum of MPTP-treated young adult mice, and compared the survivability of grafted chromaffin cells and the recovery of intrinsic host DA fibers using computerized image analysis of tyrosine hydroxylase (TH)-immunoreactive (IR) fibers and neurochemical analysis with high performance liquid chromatography (HPLC). Adrenal medullary chromaffin cells cografted with sciatic nerve survived better than those in adrenal grafts alone; host DA fiber recovery was more prominent in mice with cografts than in mice with adrenal grafts alone. A large number of TH-IR surviving cells in cografted mice showed long neuronal processes which were rarely seen in the mice receiving adrenal graft alone. We conclude that cograft of adrenal medulla and sciatic nerve promotes intrinsic host DA fiber recovery better than adrenal medulla grafts alone, and that survivability of grafted chromaffin cell may promote host DA fiber recovery. Adrenal medullary autografts have been used in patients with Parkinson's disease; we suggest that if this approach is to be used in the future, methods to increase the survivability of grafted chromaffin cells, such as co-grafting with pieces of peripheral nerve, be considered to enhance the survivability of the chromaffin cells, which might be closely related to the functional recovery of the patients by this grafting procedure. Of course, such strategies as the present cografting approach must be demonstrated to work in older animals using older donor tissue before proceeding to this next step in humans.     

In vivo measurement of spontaneous release and metabolism of dopamine from intrastriatal nigral grafts using intracerebral dialysis

Spontaneous release and metabolism of dopamine (DA) from intrastriatal grafts of fetal mesencephalic DA neurons was measured by intracerebral dialysis. Mesencephalic DA cell suspensions were implanted into the head of the caudate-putamen in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the mesostriatal DA pathway. Four months later, when tests for amphetamine-induced turning behaviour showed that the grafts had become functional, loops of dialysis tubing were implanted into the striatum on the grafted side and the contralateral non-lesioned side of the grafted rats, and in a similar position in the denervated caudate-putamen of 6-OHDA lesioned control rats. Dialysis perfusates collected from the 6-OHDA lesioned striata showed a reduction of about 95-98% in DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). In the grafted animals these levels had recovered to about 40% of control for DA and to 12-16% of control for HVA and DOPAC. In addition, the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) was increased in the grafted striata compared to both the lesioned and non-lesioned controls. Amphetamine had little or no effect on DA release in the 6-OHDA lesioned rats, but caused a marked increase in DA release in the grafted rats, this response being proportional to that seen in intact striata. Since the subsequent histochemical analysis showed that the dialysis probe had been located in the transplant-reinnervated part of the caudate-putamen, the results provide additional evidence that the grafted DA neurons exert their functional effects through a continuous active transmitter release from their newly-established terminals in the reinnervated host target.     

Effect of Prior Dopamine Denervation on Survival and Fiber Outgrowth from Intrastriatal Fetal Mesencephalic Grafts

[3H]Dopamine (DA) uptake radioautography and tyrosine hydroxylase (TH) immunocytochemistry were used to assess quantitatively the effects of the presence or absence of host mesostriatal DA afferents on the survival and fiber outgrowth from fetal ventral mesencephalic DA neurons grafted into the neostriatum of adult recipient rats. Rats received bilateral intrastriatal transplants of fetal ventral mesencephalic tissue 1 month after a unilateral injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle (denervated side). Five to six months later, some of the grafted rats received a second 6-OHDA injection in the left nigrostriatal bundle (acutely denervated or 'intact' side). After a further 7 days, slices of each hemisphere from the latter rats were incubated with [3H]DA and processed for film and high resolution radioautography. The density of the film radioautographs was measured with a computerized image analysis system and calibrated by silver grain cluster (i.e. DA terminal) counting over selected areas of the same sections in light microscope radioautographs. The brains of the remaining grafted rats were processed for TH immunoreactivity 6 - 12 months after graft surgery. Neither the size of the grafts, nor the number of surviving TH-positive graft neurons showed any significant difference between the nondenervated and the denervated sides. However, the size of the TH-positive cell bodies was significantly greater in the grafts on the denervated side. In the [3H]DA uptake radioautographs, considerable outgrowth of DA fibers was evident in the neostriatum on the 'intact' side in spite of the presence of an intact host DA innervation until 7 days before sacrifice. The overall DA fiber outgrowth was nevertheless almost two-fold greater on the denervated side, and extended deeper into the host neostriatum than on the 'intact' side; only 7% of the total neostriatal area, on average, was at background level compared to 30% on the 'intact' side, and the overall density of neostriatal DA innervation amounted to 36% of normal as compared to 20% on the 'intact' side. The correlation between the overall density of graft-derived DA innervation and the size of the grafts was linear on the 'intact' side, but reached a plateau with relatively small grafts on the denervated side. However, the ventral striatum on both sides was very poorly innervated by these grafts. These findings demonstrate that the mature neostriatal tissue can support axonal growth and innervation from grafted fetal DA neurons even in the presence of a normal complement of endogenous DA fibers. Prior removal of the host striatal DA innervation does not influence the overall size of the grafts nor the number of surviving DA neurons, but induces an increase in the cell body size and fiber outgrowth of the grafted DA neurons.     

Bilateral recovery of striatal dopamine after unilateral adrenal grafting into the striatum of the 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydropyridine (2'CH3-MPTP)-treated mouse

A rodent model of Parkinson's disease, the 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydropyridine treated mouse, was used to determine whether striatal dopamine levels recover following grafting adrenal medulla into the striatum. Four types of grafts were performed: 1) adult mouse adrenal medulla, 2) adult adrenal medulla that had been freeze-thawed to kill viable cells, 3) postnatal day 7 adrenal medulla, and 4) sham grafts lacking tissue. At 1 month after grafting, only postnatal day 7 grafts contained surviving cells. However, all three types of tissue grafts promoted a unilateral recovery of host dopaminergic fibers on the side of the graft. In striking contrast to the unilateral recovery of the dopaminergic fibers, striatal dopamine levels were increased bilaterally in all tissue grafted mice. These observations suggest that adrenal tissue grafted into the striatum, whether it remains viable or not, has more widespread biochemical effects on the host dopaminergic system than previously recognized. Moreover, these observations bear on mechanisms that may underlie the general recovery of motor disturbances reported in human Parkinson's disease patients who have received a striatal graft of adrenal tissue.     

Co-grafted embryonic striatum increases the survival of grafted embryonic dopamine neurons

To enhance the current therapeutic benefit of dopamine (DA) neuron grafts in Parkinson's disease, strategies must be developed that increase both DA neuron survival and fiber outgrowth into the denervated striatum. Previous work in our laboratory has demonstrated that dopaminergic neurons grow to greater size when co-grafted with striatal cell suspensions and display extensive tyrosine hydroxylase-positive (TH+) projections, but no conclusion could be reached concerning enhancement of survival of grafted DA neurons. The aim of the present study was to characterize further the potential trophic effects of striatal co-grafts on grafted mesencephalic DA neuron survival. Unilaterally lesioned male Fischer 344 rats were grafted with either a suspension of mesencephalic cells or with both mesencephalic and striatal cell suspensions. Co-grafts were either mixed together or placed separately into the striatum. Lesioned rats receiving no graft served as controls. Rotational behavior was assessed following amphetamine challenge at 2 weeks prior to grafting and at 4 and 8 weeks following grafting. Only rats receiving co-grafts of nigral and striatal suspensions separated by a distance of 1 mm showed significant behavioral recovery from baseline rotational asymmetry. Both mixed and separate striatal co-grafts were associated with a doubling of DA neuron survival compared with solo mesencephalic grafts. In the mixed co-graft experiment, DA neurite branching appeared enhanced and TH-rich patches were observed, whereas with co-grafts that were separated, TH+ innervation of the intervening host striatum was increased significantly. These results provide the first evidence suggesting that nigral-striatal co-grafts, particularly those placed separately and in proximity to each other, increase both DA neuron survival and neurite extension from the mesencephalic component of the grafts. J. Comp. Neurol. 399:530–540, 1998. © 1998 Wiley-Liss, Inc.     

Addition of fresh blood to intrastriatal grafts of embryonic mesencephalon into the hemiparkinsonian rat does not impair the survival of grafted dopaminergic neurones

Cell transplantation therapy for Parkinson's patients, although seen to bring benefit to some patients during first clinical trials, remains impracticable on a large scale, in part because of the poor survival of the dopaminergic neurones transplanted. The loss of dopaminergic neurones occurs rapidly over the first 1-2 days after transplantation, in response to factors intrinsic to the host brain. Here we investigated whether contamination of the grafted cell suspension with blood during the transplantation procedure may be one factor responsible for the poor survival of DA neurons within the graft, possibly through factors such as free iron or complement. 6-Hydroxydopamine lesioned rats were grafted with 2 microl suspension of dissociated E14 ventral mesencephalon to which 1 microl blood or 1 microl grafting medium was added. After 6 weeks, there was no significant difference in the number of surviving DA neurones in the two groups. We conclude that contamination of grafts with blood is not a major factor responsible for the extensive death of dopaminergic neurones within them.     

联合培养胚鼠的黑质及纹状体LGE细胞脑内移植治疗帕金森病大鼠的实验研究

目的　观察并检测胚鼠纹状体外侧节突 (LGE)对多巴胺能 (DA)细胞存活性的促进和营养导向作用。方法　将帕金森病 (PD)模型随机分成四组 :Co -culture组 (n =12 ) ;Cograft组 (n =12 ) ;Solo -VM组 (n =12 ) ;Con trol组 (n =8)。将胚鼠LGE细胞和腹侧中脑组织 (VM )制成细胞悬液 ,植入Control组外的其他各组动物的尾壳核。 2周后进行PD鼠行为学检测 ,连续观察 2 4周 ,继之将各组大鼠处死 ,进行免疫组化染色。结果　Co -culture组和Co - graft组大鼠移植后旋转行为较Solo -VM组大鼠明显减少。CO -culture组和CO - graft组之间大鼠的旋转行为比较 ,无统计学差异。免疫组化观察证实LGE和VM离体培养移植和新鲜移植均能提高DA细胞的存活性 ,增加宿主纹状体内DA纤维重新支配的密度 ,并形成明显的DA细胞团。结论　LGE细胞对VM移植物有明显的营养导向作用 ,并可增强DA细胞的存活 ,促进移值后DA细胞功能持久维持 ,并增加DA细胞再支配的密度     

Target‐specific forebrain projections and appropriate synaptic inputs of hESC‐derived dopamine neurons grafted to the midbrain of parkinsonian rats

Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are a promising unlimited source of cells for cell replacement therapy in Parkinson's disease (PD). A number of studies have demonstrated functionality of DA neurons originating from hESCs when grafted to the striatum of rodent and non‐human primate models of PD. However, several questions remain in regard to their axonal outgrowth potential and capacity to integrate into host circuitry. Here, ventral midbrain (VM) patterned hESC‐derived progenitors were grafted into the midbrain of 6‐hydroxydopamine‐lesioned rats, and analyzed at 6, 18, and 24 weeks for a time‐course evaluation of specificity and extent of graft‐derived fiber outgrowth as well as potential for functional recovery. To investigate synaptic integration of the transplanted cells, we used rabies‐based monosynaptic tracing to reveal the origin and extent of host presynaptic inputs to grafts at 6 weeks. The results reveal the capacity of grafted neurons to extend axonal projections toward appropriate forebrain target structures progressively over 24 weeks. The timing and extent of graft‐derived dopaminergic fibers innervating the dorsolateral striatum matched reduction in amphetamine‐induced rotational asymmetry in the animals where recovery could be observed. Monosynaptic tracing demonstrated that grafted cells integrate with host circuitry 6 weeks after transplantation, in a manner that is comparable with endogenous midbrain connectivity. Thus, we demonstrate that VM patterned hESC‐derived progenitors grafted to midbrain have the capacity to extensively innervate appropriate forebrain targets, integrate into the host circuitry and that functional recovery can be achieved when grafting fetal or hESC‐derived DA neurons to the midbrain.     

Functional organization of striatum as studied with neural grafts

The function of the striatum has proved elusive. A structure which, at the gross microscopic level, appears homogeneous is now revealed to be heterogeneous in terms of its afferent and efferent relationships with cortex, limbic system and mid brain. Cerebral cortex projects topographically to caudate/putamen. Lesions to different cortical areas result in different behavioural impairments which are mirrored by selective neuronal or neurochemical lesions to the sectors of striatum receiving input from the cortex. Foetal neurones prepared from substantia nigra or striatum grafted to a damaged area of adult striatum reverse the lesion-induced behavioural impairments. Within different sectors of striatum the neurones and their afferent and efferent connections are defined to striosomes and matrix representing a finer grain of intrastriatal organization, the functional significance of which is unclear. It remains a challenge within such complex anatomical circuitry to discover the full extent of anatomical reintegration and functional compensation that can be achieved with grafts of foetal neurones.     

Embryonic ventral mesencephalic grafts to the substantia nigra of MPTP-treated monkeys: feasibility relevant to multiple-target grafting as a therapy for Parkinson's disease.

Transplantation of embryonic dopamine (DA) neurons is being studied as an experimental replacement therapy for the DA-deficiency characteristic of Parkinson's disease. Some studies suggest that one of the limitations of this approach is that intrastriatal placement of implants fails to consistently restore completely normal movement. One potential cause of this suboptimal therapeutic outcome is that changes in the neural activity of several structures in the basal ganglia circuitry resulting from striatal DA depletion is not adequately normalized by graft-derived DA replacement in striatum alone. In the present study, we assessed the feasibility of grafting embryonic DA neurons into the substantia nigra (SN) of adult parkinsonian monkeys as an approach to restoration of the DA modulation of striatal-nigral afferents that is lost after degeneration of SN neurons. Sixteen St. Kitts African green monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) received implants of embryonic monkey ventral mesencephalon (VM), or sham implants, aimed at the rostral SN. At 6 months after grafting, staining for tyrosine hydroxylase (TH) indicated that grafted DA neurons survived at this site, albeit often in reduced numbers compared with VM grafts to striatum. Grafted neurons extended neurites into the parenchyma of the SN, but there was no evidence of lengthy extension of graft-derived neurites rostrally along the trajectory of the mesostriatal fiber system. A region-specific, modest increase in DA levels and TH-positive fiber density in the ventral-medial putamen was detected, accompanied by modest but significant decreases in parkinsonian behaviors at 5-6 months after grafting. Our findings support the view that grafting embryonic tissue to the SN is a feasible procedure in nonhuman primates that provides a modest but detectable benefit of its own. These results encourage the further development of multiple-target grafting strategies as a means of restoring modulation of anatomically widespread basal ganglia structures relevant to treatment of Parkinson's disease.     

Non-dopaminergic neurons in ventral mesencephalic transplants make widespread axonal connections in the host brain

Motor dysfunction in Parkinson's disease (PD) can be effectively alleviated through intra-striatal transplantation of fetal ventral mesencephalic tissue. The success of this approach is dependent on the survival, axonal outgrowth and synaptic integration of newly grafted dopamine neurons with the host striatum. The functional outcome of transplantation therapy has, however, been highly variable, particularly in PD patients, but also in animal models of PD, and thus there is a need for a deeper understanding of possible mechanisms underlying this variability such as graft composition and the resulting graft–host connectivity. Here we describe a series of transplantation experiments whereby mouse VM tissue has been grafted into the striatum of 6-hydroxydopamine lesioned rats. Six weeks after grafting immunohistochemical analysis using the mouse specific ‘M2M6’ antibodies revealed both dopaminergic and non-dopaminergic components of graft-derived fibre outgrowth into the host brain. We report here that while dopaminergic outgrowth was predominately confined to the striatum, there was also a significant degree of non-dopaminergic outgrowth to extra-striatal structures including the thalamus, cortex and midbrain. Retrograde tracing experiments showed that grafted neurons of GABAergic identity contribute to this non-dopaminergic outgrowth. In line with our recent findings on the function of serotonergic neurons in fetal VM grafts, these results further underscore the potential impact that non-dopaminergic neurons may have on the functional outcome of intrastriatal fetal VM grafts.