Transplantation of fetal CNS tissue into the peripheral nervous system: a model to study aberrant changes in the neuronal cytoskeleton.

Defined regions (septum, substantia nigra) of the embryonic central nervous system (CNS) were transplanted into the sciatic nerves of young adult rats. Immunocytochemical techniques were used to examine the expression of neurotransmitter related enzymes and neuronal cytoskeletal proteins in the grafts. The origin of the septal grafts was confirmed by immunoreactivity in neurons to choline acetyltransferase and the beta-nerve growth factor receptor (192-IgG). In substantia nigra grafts, neuronal perikarya and processes were identified with an antibody directed against tyrosine hydroxylase. Typical spatial distributions of phosphorylated (Mr 200,000) and non-phosphorylated (Mr 168,000 & 200,000) neurofilaments were observed in the short term (1-2 months) grafts with the monoclonal antibodies RT97 and SMI-32 respectively. Dense dendrite arbors and neuronal cell bodies were immunostained with an antibody that recognizes a high molecular weight microtubule associated protein (MAP2). In the long term (1 year) transplants, prominent cytoskeletal changes in the somata, axons and dendrites of neurons were evident. The cells showed a shift in phosphorylated neurofilament staining from the axon to the soma accompanied by a reduction in axonal immunoreactivity in the adjacent neuropil. Other abnormal features included swollen perikarya, hypertrophied axonal segments and short segments of kinked axons. Regression of the dendrite trees in the long standing grafts was also apparent when sections were reacted with the MAP2 antibody. These experiments indicate that grafted fetal neurons, isolated in the peripheral nervous system, differentiate and express markers like their counterparts in situ. After extended time periods under these circumstances, cytoskeletal modifications become apparent in the neurons. These aberrant changes are similar to morphological characteristics associated with aging and neurodegenerative disorders. This experimental paradigm offers a new approach to study cytoskeletal disturbances in neurons and provides a unique opportunity to examine conditions that may modulate the abnormal changes.     

Loss of non-phosphorylated neurofilament immunoreactivity, with preservation of tyrosine hydroxylase, in surviving substantia nigra neurons in Parkinson''s disease.

The distribution of neurofilament immunoreactivity in the substantia nigra was examined by immunohistochemistry in five patients dying with Parkinson's disease and six control patients dying without neurological disease. In controls, pigmented neurons in the substantia nigra were intensively labelled by SMI32, a monoclonal antibody to non-phosphorylated neurofilament protein. In the substantia nigra from patients who had Parkinson's disease, there was a pronounced reduction of SMI32 labelling intensity in surviving pigmented neurons. By contrast, tyrosine hydroxylase immunoreactivity in surviving pigmented neurons was normal. SMI32 labelling was normal in regions of the brainstem not affected by the neuropathological process of Parkinson's disease. Findings with either antibodies to phosphorylated neurofilament, or enzymatic dephosphorylation followed by SMI32 labelling, indicated that loss of SMI32 immunostaining in Parkinson's disease was not due to masking of the neurofilament epitopes by phosphorylation. Our results indicate that neurofilament proteins are particularly likely to be disrupted or destroyed by the neuropathological process of Parkinson's disease. Nevertheless, the normal appearance of tyrosine hydroxylase indicates that protein synthesising systems may be intact in surviving neurons. Loss of neurofilament immunoreactivity may prove a sensitive neuropathological marker for characterisation of degenerating neurons in Parkinson's disease.     

Direct demonstration of interactions between substance P immunoreactive terminals and tyrosine hydroxylase immunoreactive neurons in the substantia nigra of the rat: an ultrastructural study

The results of many anatomical, physiological, and pharmacological studies suggest that substance P-containing neurons of the striatum project to the substantia nigra, and that substance P influences the activity of dopaminergic nigrostriatal neurons. The purpose of the present ultrastructural study was to employ dual immunocytochemical labeling to determine the morphological basis for the observed actions of substance P on nigral dopaminergic neurons. Substance P-like and tyrosine hydroxylase-like immunoreactivities were localized simultaneously at the ultrastructural level in the substantia nigra of the rat. A double label method was utilized which relied on a combination of the peroxidase-antiperoxidase method (Sternberger, 1979) for substance P, and immunogold or silver enhanced immunogold labeling for tyrosine hydroxylase. The present results indicate that tyrosine hydroxylase immunoreactive (THLI) dendrites in the substantia nigra receive synaptic input from terminals exhibiting substance P-like immunoreactivity. These findings support the idea that substance P is a major neurotransmitter in the striatonigral loop, and suggest that striatal substance P neurons act directly upon nigral dopaminergic cells.     

Relationship between the presence of dopaminergic neurons and GABA receptors in substantia nigra: Effects of lesions

Submaximal destruction of nigrostriatal dopaminergic projections resulted in a significant (25%) decrease in specific GABA binding in substantia nigra; under these conditions, striatal tyrosine hydroxylase activity was 15–44% of control. In rats with lesions which caused maximal destruction of nigrostriatal dopamine neurons (striatal tyrosine hydroxylase was less than 15% of control), specific GABA binding in substantia nigra was apparently not different from that obtained in intact controls. Two distinct processes may be occurring in response to the destruction of dopamine neurons: (1) the loss of GABA binding sites physically associated with nigral dopamine neurons; and (2) an increase in nigral GABA receptors associated with non-dopaminergic neurons. The latter process may result from a decrease in nigral GABA transmission secondary to the complete loss of dopaminergic synaptic activity in striatum.     

Appropriate target interactions prevent abnormal cytoskeletal changes in neurons: a study with intra-sciatic grafts of the septum and the hippocampus.

Transplantation of embryonic CNS regions into the PNS provides an opportunity to study temporal and spatial changes in the cytoskeleton that are associated with aging and neurodegenerative diseases. In this study, the fetal septum was transplanted alone or with the hippocampus into the sciatic nerves of young adult rats to determine whether the proper central neural target could prevent the expression of abnormal cytoskeletal changes. The substantia nigra, a non-target area of the septum, served as control co-grafts. After 1, 3, 6, 12, and 18 months of survival, the grafts were examined by immunocytochemistry with antibodies to phosphorylated and nonphosphorylated neurofilaments, microtubule-associated proteins (MAPs), and glial fibrillary acidic protein (GFAP). Subpopulations of neurons in the septal transplants expressed CAT and the NGF receptor (192-IgG). Long-term (12-18 months) expression of these two markers was only observed when the septum was combined with the hippocampus. Although isolated single grafts of septum survived within the PNS substratum, significant neuronal loss, extensive graft shrinkage, and aberrant cytoskeletal immunoreactivity were prominent in the long-term group. Changes that reflected an aging process included the ectopic expression of phosphorylated neurofilaments in neuronal perikarya, swollen axons, and a loss of MAP2 immunoreactivity that paralleled dendrite regression. In addition, abnormal "curly" fibers in the neuropil were also immunolabeled with an antibody directed against tau (5E2). Introduction of hippocampal co-grafts increased the final size of the septal transplants and prevented the cytoskeletal changes that accompanied the degeneration in the single septal grafts. The degree of GFAP immunostaining in the septum corresponded with advancing graft age and was minimized when grafted with the hippocampal formation. When the septum was combined with the substantia nigra, the grafts also underwent shrinkage and no protective influence from aberrant cytoskeletal staining was observed. These experiments exemplify the importance of an appropriate CNS neural target on the maintenance of long-term cholinergic neuron survival and normal morphology at the cytoskeletal level and illustrate the usefulness of these CNS-PNS constructs to examine conditions that influence the cytoskeleton.     

Peripheral nerve segments promote consistent long-term survival of adrenal medulla transplants in the brain.

Patients with Parkinson's disease have received intracerebral transplants of autologous adrenal medulla in the attempt to counteract their severe motor dysfunctions. Unfortunately, in the majority of cases, clinical improvement has not persisted and there has been extremely poor survival of the grafts. Based on the recent observations of long-term viability of adrenal medulla grafts in the interior of transected peripheral nerves, adrenal medulla/peripheral nerve complexes were constructed in the brain to promote extended viability of chromaffin cells. A three-step, time-dependent transplantation procedure is described that results in a 100% survival rate of the adrenal medulla graft. The grafts consist of a stable population of approximately 2.0 x 10(3) chromaffin cells that survive for at least 6 months (longest time point studied): Immunoreactivity to catecholamine-related enzymes (tyrosine hydroxylase, dopamine beta-hydroxylase) and the low-affinity NGF receptor (192-IgG) are expressed by the chromaffin cells. The ultrastructural characteristics of the cells are normal and comparable to their in vivo counterparts. Construction of these peripheral nerve/adrenal medulla complexes evidently improves local conditions in and around the grafts, enabling the chromaffin cells to remain viable. This new methodology achieves the goal of reliable and extended survival of the adrenal medulla graft after intracerebral transplantation. The enhanced longevity now provides an opportunity to reevaluate the efficacy of the adrenal medulla transplant to ameliorate the functional disorders associated with striatal dopamine depletion, especially over long time periods.     

Decreased tyrosine hydroxylase content in the dopaminergic neurons of MPTP–intoxicated monkeys: Effect of levodopa and GM1 ganglioside therapy

Parkinson's disease is characterized by the degeneration of melanized dopaminergic neurons of the substantia nigra. The functional capacity of the surviving dopaminergic neurons is affected, as suggested by the subnormal levels of tyrosine hydroxylase messenger RNA and protein found in the remaining cells. The reduced expression of tyrosine hydroxylase may be due to either the evolving neurodegenerative process or its downregulation, possibly secondary to chronic levodopa treatment. The cellular content of tyrosine hydroxylase was determined in the mesencephalon from 16 Macaca fascicularis monkeys, using a semiquantitative immunocytochemical method. Thirteen monkeys were rendered parkinsonian by weekly intravenous injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 2 (subacute treatment) or 20 (chronic treatment) weeks. Three of the monkeys received levodopa and 3 others received GM1 ganglioside. The loss of dopaminergic neurons in the mesencephalon of the MPTP-intoxicated monkeys was severe in the substantia nigra, intermediate in cell groups A8 and A10, and almost undetectable in the central gray substance. After both subacute and chronic treatment, the cellular content of tyrosine hydroxylase was reduced by 40% in the surviving neurons of the lesioned substantia nigra, but by less in the other mesencephalic dopaminergic regions. Neuronal survival and tyrosine hydroxylase content in monkeys that had received levodopa were not significantly different. The cellular content of tyrosine hydroxylase was increased in the substantia nigra of the monkeys that received GM1 ganglioside injections. The results show that the decreased expression of tyrosine hydroxylase found in nigral dopaminergic neurons after partial degeneration of the mesostriatal dopaminergic system is not influenced by levodopa treatment and is partially reversed by GM1 ganglioside administration.     

Cytoarchitectonics of substantia nigra grafts: a light and electron microscopic study of immunocytochemically identified dopaminergic neurons and fibrous astrocytes

Maturation of dopaminergic (DA) neurons and astroglia was studied in transplants of the substantia nigra grown for up to 7 months in the brain of rats. The investigation had three specific aims. The first was to observe effects of different transplant positions on the longevity of DA neurons. Second, the grafts were examined for changes of synaptic interactions and associations between DA neurons and astroglia. Third, an answer was sought to the question whether transplanted DA neurons migrate into the adjacent host brain. The grafts were taken from the ventral mesencephalon of rat embryos of different ages (day 14 to 18 of gestation) and placed into the cerebral cortex, tectum, cerebellum, or ventricles of newborn host animals. Following different times of survival the immunocytochemical localization of tyrosine hydroxylase (TH) and of glia filament protein (GFA) in the transplants were observed. In all of the transplantation sites, except for one, neurons of different morphologies that contained TH were found in the grafts. The cerebellar white matter of the host brain failed to support the long-term survival of DA neurons. The overall structure of mature substantia nigra grafts had some resemblance to intact substantia nigra (SN). On the ultrastructural level, it was found that morphological expression of some immature features of DA neurons, such as glial sheaths, somatic spines, and lack of oligodendroglia, persisted in mature grafts. Specific associations of DA neurons and astroglia in the grafts suggested that the cytoarchitectonic appearance of a given brain region may be related to the existence of particular neuron glia relationships. In contrast to intact SN, transplants revealed deficiencies in unlabeled pleomorphic boutons and contained some TH-immunoreactive terminals. Migration of DA neurons and their processes into the adjacent host brain was rarely observed.     

Substance P-dopamine relationship in the rat substantia nigra: a light and electron microscopy study of double immunocytochemically labeled materials

The synaptic relationships between substance P-containing terminals and dopaminergic neurons (immunoreactive for tyrosine hydroxylase) in the substantia nigra were studied at both light and electron microscopic levels using a pre-embedding double-labeling immunocytochemical method. Many substance P-containing terminals were found to form synapses directly with dendrites and somata of nigral dopaminergic neurons. Since most of the substance P-containing axon terminals arise from the striatum, this result suggests that striatal substance P neurons can have monosynaptic influence on nigral dopaminergic neurons.     

Effects of adrenal medulla grafts in neonatal rat hosts on subsequent bilateral substantia nigra lesions

Bilateral lesioning of rat substantia nigra results in a syndrome of aphagia, adipsia, and akinesia. Rats can be protected from the development of this syndrome if they have previously received intraventricular fetal substantia nigra grafts as neonates. In this experiment, we determined whether neonatal adrenal medulla grafts would offer similar protection. One day old Sprague-Dawley rat pups received bilateral intraventricular allografts of either adrenal medulla or sciatic nerve from adult donors. At 3-4 months of age the rats received bilateral lesions of the substantia nigra with 6-hydroxydopamine. After lesioning, food and water consumption were greater in the rats receiving adrenal medulla grafts, as compared to the sciatic nerve control group. Nevertheless, food and water consumption was markedly decreased in both groups, and activity levels did not differ between the two groups. Histology showed consistently surviving grafts with large numbers of surviving catecholamine-containing cells. Thus adrenal medulla grafts in neonatal hosts offer partial protection against the syndrome of adipsia and aphagia produced by subsequent substantia nigra lesions, but this protection is much less than that produced by substantia nigra grafts.     

Adrènal medulla grafts survice and exhibit catecholamine-specific fluorescence in the primate brain

Parkinson's disease most consistently involves pathologic changes in the substantia nigra, which is the major source of dopamine to the striatum. It has been shown that either fetal substantia nigra or adrenal medulla tissue implanted into the rat brain survives, produces dopamine, and improves behavioral abnormalities induced by deprivation of the caudate nucleus of its dopaminergic innervation. Thus, catecholaminecontaining grafts could be potential replacements for destroyed or damaged dopaminergic neurons in patients with Parkinson's disease. To explore the potential of this therapeutic approach, fetal substantia nigra or host adrenal medulla were grafted to the denervated caudate nucleus of the rhesus monkey. Under the specific conditions of our experiment, fetal substantia nigra did not survive in either of two animals tested. On the other hand, some tissue from adrenal medulla grafts survived in all four animals tested. These grafted cells contained catecholamines, as indicated by the presence of specific glyoxylic acid-induced catecholamine fluorescence. In two of the four animals, however, the grafts contained fewer than 10 surviving cells, and in the other two animals, about 190 and 300 cells were found, respectively. Despite the small numbers of cells, this is the first demonstration that peripheral tissue autografts can survive implantation into the nonhuman primate central nervous system.     

Diminution of dopaminergic neurons in the substantia nigra of sporadic amyotrophic lateral sclerosis

The substantia nigra was examined immunohistochemically using the antibody to tyrosine hydroxylase in 15 patients with sporadic amyotrophic lateral sclerosis (ALS). The number of dopaminergic neurons was diminished in the substantia nigra of seven cases. The diminution was not related to the age, duration of the illness or use of respirators. Supranuclear ophthalmoplegia developed in four and dementia in three out of seven patients with reduction of nigral dopaminergic neurons. In addition, five out of the seven patients developed respiratory failure within 2 years after the onset of the illness. The nigral dopaminergic system may be involved in rapidly progressive ALS patients with supranuclear ophthalmoplegia and/or dementia.     

Catecholamine content of intracerebral adrenal medulla grafts

The rotational behavior which is produced by substantia nigra lesions can be decreased by adrenal medulla grafts adjacent to the denervated striatum. Perhaps these grafts secrete dopamine that diffuses into the striatum. In the present study, we measured concentrations of catecholamines in adrenal medulla grafts as compared with the normal adrenal medulla. The grafts were found to have high but extremely variable concentrations of dopamine. In hosts with substantia nigra lesions, concentrations of dopamine in the adrenal medulla grafts were decreased. Substantia nigra lesions, however, tended to increase concentrations of epinephrine in the grafts, while norepinephrine and total catecholamine concentrations were not significantly affected. It is concluded that at least some adrenal medulla grafts contain concentrations of dopamine sufficient to account for their behavioral effects.     

Substance P synaptic interactions with GABAergic and dopaminergic neurons in rat substantia nigra: An ultrastructural doublelabeling immunocytochemical study

The distribution of substance P (SP), tyrosine hydroxylase (TH), and glutamic acid decarboxylase (GAD) immunoreactivity in the substantia nigra of the rat was studied by means of an ultrastructural double-labeling immunocytochemical method. Direct synaptic contact between SP-immunoreactive terminals and GAD-positive nigral neurons was more often observed in the pars lateralis than the pars reticularis and was rarely observed in the pars compacta. Substance P-positive terminals also formed synapses with cell bodies and dendrites of TH-positive, dopaminergic neurons in the pars compacta and pars reticulata. Multiple SP-immunoreactive terminals were often observed with symmetrical and, less frequently, asymmetrical synapses on individual TH-containing dendrites. Evidence of SP-containing terminals contacting both GABAergic and dopaminergic neurons in the substantia nigra suggests a direct excitatory action upon nigral projection neurons.     

Effects of adrenal medulla grafts on plasma catecholamines and rotational behavior.

The mechanisms by which adrenal medulla grafts influence the function of host brains in animal models of Parkinson's disease are unclear. To explore this issue, fragments of adrenal medulla or sciatic nerve were transplanted into the lateral ventricle of bilaterally adrenalectomized (ADX) or sham-ADX rats with unilateral 6-hydroxydopamine lesions of the substantia nigra. Additional control group received sham-transplantation surgery. Behavioral effects of these procedures were tested following administration of apomorphine, amphetamine, or nicotine. Plasma catecholamines were measured before and after transplantation surgery. In both ADX and sham-ADX rats, adrenal medulla grafts produced greater decreases in apomorphine-induced rotational behavior than did sciatic nerve grafts or sham-transplanted groups. Decreases in rotation were smaller in ADX than in sham-ADX animals, regardless of graft treatment. Plasma catecholamines increased after transplantation surgery in each of the sham-ADX groups, regardless of graft type. Increases in plasma dopamine concentrations were associated with decreases in rotational behavior. Five months after transplantation, grafted chromaffin cells demonstrated catecholamine fluorescence, tyrosine hydroxylase (TH) and chromogranin A immunoreactivities, and expression of TH mRNA. It is concluded that adrenal medulla grafts produce decreases in apomorphine-induced rotation through a combination of two independent effects. One is a specific effect of adrenal medulla grafts. The second is a nonspecific effect that requires an intact adrenal gland and may be related to increases in plasma catecholamine concentrations.     

Effects of adrenal medulla graft on recovery of GABAergic and dopaminergic neuron deficits in mice: behavioural,pharmacological and immunohistochemical study

We studied the capacity of adrenal medullary transplant to restore the deficits of GABAergic and dopaminergic neurons in mice injected with quinolinic acid (QA), using an open field test as well as pharmacological and immunohistochemical techniques. We analysed behavioural traits—total locomotor activity, peripheral and central activities, grooming, leaning and rearing in the QA-lesioned mice and mice that had undergone adrenal medulla (AM) transplantation. We found that the adrenal transplant recovered a loss of GABAergic neurons. It reduced QA-induced hyperactivity in locomotion and improved emotional indices. In addition, immunohistochemical studies of catecholaminergic markers—tyrosine hydroxylase (TH), dopamine (DA) and neuronal vesicular monoamine transporter type 2- and a single post-trial injection of tetrabenazine (TBZ; 5 mg/kg) indicated that catecholamines-synthesising chromaffin cells in the AM grafts were also involved in the beneficial effects. A likely interpretation of this behavioural pattern of results is that adrenal medullary transplants set into play an interaction between GABAergic and DAergic factors. Our results may contribute to the clarification of the beneficial effects of AM transplants in striatal function.     

Anatomical and functional reconstruction of the nigrostriatal pathway by intranigral transplants

The main transplantation strategy in Parkinson's disease has been to place dopaminergic grafts not in their ontogenic site, the substantia nigra, but in their target area, the striatum with contrasting results. Here we have used green fluorescent protein transgenic mouse embryos as donors of ventral mesencephalic cells for transplantation into the pre-lesioned substantia nigra of an adult wild-type host. This allows distinguishing the transplanted cells and their projections from those of the host. Grafted cells integrated within the host mesencephalon and expressed the dopaminergic markers tyrosine hydroxylase, vesicular monoamine transporter 2 and dopamine transporter. Most of the dopaminergic cells within the transplant expressed the substantia nigra marker Girk2 while a lesser proportion expressed the ventral tegmental area marker calbindin. Mesencephalic transplants developed projections through the medial forebrain bundle to the striatum, increased striatal dopamine levels and restored normal behavior. Interestingly, only mesencephalic transplants were able to restore the nigrostriatal projections as dopamine neurons originating from embryonic olfactory bulb transplants send projections only in the close vicinity of the transplantation site that did not reach the striatum. Our results show for the first time the ability of intranigral foetal dopaminergic neurons grafts to restore the damaged nigrostriatal pathway in adult mice. Together with our previous findings of efficient embryonic transplantation within the pre-lesioned adult motor cortex, these results demonstrate that the adult brain is permissive to specific and long distance axonal growth. They further open new avenues in cell transplantation therapies applied for the treatment of neurodegenerative disorders such as Parkinson's disease.     

Evidence for crossed catecholaminergic nigrostriatal projections by combining wheat germ agglutinin-horseradish peroxidase retrograde transport and tyrosine hydroxylase immunocytochemistry

After unilateral injections of wheat germ agglutinin-horseradish peroxidase into the rat caudate-putamen, a few retrogradely labeled neurons were found in the contralateral pars compacta of the substantia nigra. These contralaterally projecting nigral cell bodies also immunoreacted positively to a specific tyrosine hydroxylase antiserum. We conclude that crossed catecholaminergic nigrostriatal projections may contribute to the reciprocal regulation exerted by the two nigrostriatal dopaminergic systems.     

Dopaminergic innervation of substance P-containing striatal neurons by fetal nigral grafts: an ultrastructural double-labeling immunocytochemical study.

Evidence for survival and growth of fetal substantia nigra grafts in host striatum and partial reversal of behavioural and biochemical deficits in the host animal is well documented. Afferent synaptic connections arising from the graft and contacting host structures have also been reported; however, the properties of the neurons receiving this input is less clear. The purpose of this study was to determine if substance P-containing neostriatal neurons receive a dopaminergic input from nigral grafts. Fetal substantia nigra cell suspensions were stereotaxically implanted in the deafferented neostriatum of Wistar rats 2 weeks after a unilateral 6-hydroxydopamine (6-OHDA) lesion in the ipsilateral substantia nigra or medial forebrain bundle. The ultrastructural features of the graft-host synaptic interactions were analysed by employing an electron microscope immunocytochemical double-labeling technique. Tyrosine hydroxylase (TH) and substance P-immunoreactive structures were simultaneously demonstrated by means of the peroxidase-antiperoxidase method using two different chromogens with distinct reaction products easily differentiated at the light and electron microscope levels. TH-immunoreactive sites were first demonstrated using 3,3'-diaminobenzidine tetrahydrochloride (DAB); then substance P immunoreactivity was localized using benzidine dihydrochloride (BDHC). TH-immunoreactive terminals of axons originating from the graft made synaptic contacts with substance P-positive cell bodies and dendrites from the host. These results indicate that at least partial restoration of the normal nigrostriatal circuitry can be achieved following nigral grafts. The demonstration of specific synaptic input on host substance P neurons provides an anatomical basis for direct functional modulation of the deafferented host neostriatum by the nigral graft.