Search

Huntington's disease (HD) is a debilitating, genetically inherited neurodegenerative disorder that results in early loss of medium spiny neurons from the striatum and subsequent degeneration of cortical and other subcortical brain regions. Behavioral changes manifest as a range of motor, cognitive, and neuropsychiatric impairments. It has been established that replacement of the degenerated medium spiny neurons with rat-derived fetal whole ganglionic eminence (rWGE) tissue can alleviate motor and cognitive deficits in preclinical rodent models of HD. However, clinical application of this cell replacement therapy requires the use of human-derived (hWGE), not rWGE, tissue. Despite this, little is currently known about the functional efficacy of hWGE. The aim of this study was to directly compare the ability of the gold standard rWGE grafts, against the clinically relevant hWGE grafts, on a range of behavioral tests of motor function. Lister hooded rats either remained as unoperated controls or received unilateral excitotoxic lesions of the lateral neostriatum. Subsets of lesioned rats then received transplants of either rWGE or hWGE primary fetal tissue into the lateral striatum. All rats were tested postlesion and postgraft on the following tests of motor function: staircase test, apomorphine-induced rotation, cylinder test, adjusting steps test, and vibrissae-evoked touch test. At 21 weeks postgraft, brain tissue was taken for histological analysis. The results revealed comparable improvements in apomorphine-induced rotational bias and the vibrissae test, despite larger graft volumes in the hWGE cohort. hWGE grafts, but not rWGE grafts, stabilized behavioral performance on the adjusting steps test. These results have implications for clinical application of cell replacement therapies, as well as providing a foundation for the development of stem cell-derived cell therapy products.

The extracellular matrix glycoprotein tenascin-C is widely expressed during development and repair, making it surprising that few abnormalities have been found in transgenic mice lacking this molecule. We have therefore re-examined the transgenic mice described by Saga et al. [Saga, Y., Yagi, T., Ikawa, Y., Sakakura, T. & Aizawa, S. (1992) Genes Dev., 6 1821-1831] in which tenascin-C was knocked-out by homologous recombination, focusing on two aspects of the nervous system likely to reveal any abnormalities that might follow the loss of tenascin-C. First, we have determined the pattern of myelin and distribution of oligodendrocyte precursor cells in those areas, such as the optic nerve and retina where local concentrations of tenascin-C have been proposed to act as barriers to oligodendrocyte precursor migration and so prevent inappropriate myelination. Secondly, we have examined the behaviour of the mice in a number of well-characterized tests, e.g. beam-walking, passive avoidance and the Morris water maze. We find no abnormalities of myelination or oligodendrocyte precursor distribution in adult mice, showing that local concentrations of tenascin-C are not the sole mechanism responsible for the pattern of myelination in these regions of CNS. However, we do find a number of behavioural abnormalities in these mice and show that hyperlocomotion and deficits in coordination during beam walking can be ascribed to tenascin-C deficiency. The effects on coordination are, however, not seen on a 129 genetic background. Taken together, these results significantly extend the phenotype associated with tenascin-C deficiency but argue against a role in myelination.

Striatal grafts have been proposed as a potential strategy for striatal repair in Huntington's disease, but it is unknown whether the diseased brain will compromise graft survival. A transgenic mouse line has recently been described in which hemizygotes with an expanded CAG repeat in exon 1 of the HD gene exhibit a progressive neurological phenotype similar to the motor symptoms of Huntington's disease. We have therefore evaluated the effects of the transgenic brain environment on the survival, differentiation, and function of intrastriatal striatal grafts and undertaken a preliminary analysis of the effects of the grafts on the development of neurological deficits in the host mice. Hemizygote transgenic and wild-type littermate female mice received striatal grafts at 10 weeks of age and were allowed to survive 6 weeks. Normal healthy grafts were seen to survive and differentiate within the striatum of transgenic mice in a manner comparable to that seen in control mice. The transgenic mice exhibited a progressive decline in body weight from 9 weeks of age and a progressive hypoactivity in an open field test of general locomotor behavior. Although striatal grafts exerted a statistically significant influence on several indices of this impairment, all behavioral effects were small and did not exert any clinically relevant effect on the profound neurological deficiency of the transgenic mice.

Recent studies in non-human primates support a role for the subthalamic nucleus in the expression of parkinsonian symptomatology, and it has been proposed that subthalamic lesions may provide a surgical treatment for the symptoms of Parkinson's disease in humans. We have applied a broad range of behavioural tests to characterize the effects of lesions of the subthalamic nucleus on parkinsonian symptoms in the unilateral 6-hydroxydopamine (6-OHDA) lesioned marmoset (Callithrix jacchus). Thirteen marmosets were trained on a battery of behavioural tasks that were conducted at regular intervals before and after surgery. All received unilateral 6-OHDA lesions to the medial forebrain bundle. Seven animals were then given an additional N-methyl-D-aspartate lesion of the ipsilateral subthalamic nucleus, whereas the remaining six animals received a variety of control or sham lesions to the nucleus. The 6-OHDA lesions induced a strong ipsilateral bias in head position; mild-moderate ipsilateral rotation spontaneously and after injection of saline or amphetamine; and contralateral rotation after injection of apomorphine. Hemineglect was evident as delayed initiation of reaches on the contralateral side on the staircase reaching task. Additional subthalamic lesions significantly reversed the bias in head position from ipsilateral to contralateral and decreased neglect as evidenced by improved latencies to initiate reaching on the contralateral side at the staircase. However, deficits in skilled movements persisted in the subthalamic nucleus lesion group in that they did not complete the staircase task any faster than the control group and remained impaired on another task which required reaching into tubes. These behavioural effects demonstrate that excitotoxic lesioning of the subthalamic nucleus can ameliorate some, but not all, parkinsonian-like deficits in the unilateral 6-OHDA lesioned marmoset.

Grafts of embryonic striatal primordia are able to elicit behavioural recovery in rats which have received an excitotoxic lesion to the striatum, and it is believed that the P zones or striatal-like tissue within the transplants play a crucial role in these functional effects. We performed this study to compare the effects of different donor stage of embryonic tissue on both the morphology (see accompanying paper) and function of striatal transplants. Both the medial and lateral ganglionic eminence was dissected from rat embryos of either 10 mm, 15 mm, 19 mm, or 23 mm crown-rump length, and implanted as a cell suspension into adult rats which had received an ibotenic acid lesion 10 days prior to transplantation. After four months the animals were tested on the "staircase task" of skilled forelimb use. At 10-14 months rats from the groups which had received grafts from 10 mm or 15 mm donor embryos were taken for positron emission tomography scanning in a small diameter positron emission tomography scanner, using ligands to the dopamine D1 and D2 receptors, [11C]SCH 23390 and [11C]raclopride, respectively. A lesion-alone group was also scanned with the same ligands for comparison. Animals which had received transplants from the 10 mm donors showed a significant recovery with their contralateral paw on the "staircase test". No other groups showed recovery on this task. Similarly, the animals with grafts from the youngest donors showed a significant increase in D1 and D2 receptor binding when compared to the lesion-alone group. No increase in signal was observed with either ligand in the group which had received grafts from 15 mm donors. Success in paw reaching showed a strong correlation to both the positron emission tomography signal obtained and the P zone volume of the grafts. These results suggest that striatal grafts from younger donors (10 mm CRL) give greater behavioural recovery than grafts prepared from older embryos. This recovery is due to both the increased proportion of striatal-like tissue within the grafts and an increase in functional D1 and D2 dopamine receptors measured by positron emission tomography, i.e. a more extensive integration of the graft with the host brain.

The behaviour of marmosets with unilateral 6-hydroxydopamine lesions of the nigrostriatal bundle and grafts of embryonic mesencephalon in either the caudate nucleus or the putamen was compared with that of lesion-alone and unoperated controls. The grafts comprised injections of cell suspensions prepared from marmoset ventral mesencephalon (i.e. allografts) targeted at four sites either entirely within the caudate nucleus or entirely within the putamen. Behavioural tests, including measures of amphetamine-induced rotation, neglect and use of each arm to retrieve food from inside tubes, were given before and after the 6-hydroxydopamine lesion and at regular intervals for 6 months after transplantation surgery. Grafts in the caudate nucleus reduced the ipsilateral rotation induced by amphetamine, whereas grafts in the putamen did not. Despite the absence of an effect on rotation, the putamen grafts were effective in reducing lesion-induced deficits on the task in which the marmosets were required to reach into tubes. In this latter task, the caudate grafts were also effective when the monkeys were given a free choice of which hand to use. However, when constrained to use the hand contralateral to the lesion and graft, the performance of the marmosets with caudate grafts was not significantly improved compared with that of lesion-alone controls. Neither the grafts in the caudate nucleus nor the grafts in the putamen abolished the contralateral somatosensory neglect induced by the lesion, although there was a trend for the marmosets with putamen grafts to contact the label on the contralateral side more quickly than those with caudate grafts or the lesion-alone controls. These results demonstrate that the location of embryonic nigral grafts within the primate striatum influences the profile of functional recovery.

The effectiveness of a recently developed immunotoxin, 192 IgG-saporin, was evaluated for making selective lesions of subgroups of basal forebrain cholinergic neurons. Following a pilot series of injections into the nucleus basalis magnocellularis to establish the effective dose for intraparenchymal lesions, separate groups of rats received injections of the immunotoxin into the septum, into the diagonal band of Broca or into the nucleus basalis magnocellularis. The lesions produced extensive and effective loss of cholinergic neurons in the discrete areas of the basal forebrain, as identified by loss of cells staining for acetylcholinesterase and p75NGFr, with a parallel loss of acetylcholinesterase staining and choline acetyltransferase activity in the target areas associated with each injection site in the dorsolateral neocortex, cingulate cortex and hippocampus. The selectivity of the lesion for cholinergic neurons was supported by the lack of gliosis and sparing of small to medium-sized cells at the site of injection of the toxin, including the glutamate decarboxylase immunoreactive cells that contribute to the septohippocampal projection. In spite of the extensive disturbance in the cholinergic innervation of the neocortex and hippocampus, immunotoxin lesions produced no detectable deficit in the Morris water maze task in any of the lesion sites within the basal forebrain. By contrast small but significant deficits were seen on tests of nocturnal activity (septal and nucleus basalis magnocellularis lesions), open field activity (septal and diagonal band lesions), passive avoidance (nucleus basalis magnocellularis lesions) and delayed non-matching to position (septal lesions). The results indicate that the 192 IgG-saporin provides a powerful tool for making effective lesions of the basal forebrain cholinergic neurons, and that the behavioural sequelae of such lesions warrant further detailed investigation.

The transplantation of neuronal tissue into the brains of patients with Parkinson's disease is already being assessed as an experimental treatment for the symptoms of this disease, and the possibility of using similar graft tissue to ameliorate the symptoms of other neurodegenerative diseases is being considered. In this context, a small number of transplant experiments have been carried out in monkeys with lesions of the central dopamine and cholinergic systems. These experiments make it possible to determine the optimum methods of transplantation in an animal whose brain is structurally more closely related to the human than that of the rat and to assess the behavioral consequences of transplantation on symptoms that either resemble very closely the symptoms seen in patients, or are of a complex cognitive nature and are therefore more difficult to measure in the rat. It is intended that these experiments will contribute to the development of better treatments for the neurodegenerative diseases, either by the use of transplantation as a clinical treatment, or by contributing to a better understanding of the mechanisms that normally maintain neuronal function and that fail in these diseases.

We have examined whether dopaminergic mesencephalic grafts implanted into neonates can provide more extensive protection against deficits induced by a subsequent unilateral lesion of the mesotelencephalic dopaminergic pathway than when the grafts are implanted in adulthood. A dopamine-rich neuronal cell suspension obtained from embryonic day 14 mesencephali was injected unilaterally into the neostriatum of otherwise intact neonatal or adult rats at one day or two months of age, respectively. Two months later, the ipsilateral mesotelencephalic dopaminergic pathway was destroyed by unilateral injection of 6-hydroxydopamine. The behavioural effects of the grafts were evaluated in tests of drug-induced rotation and skilled paw reaching. After completion of the behavioural testing, animals were killed and brains were processed for tyrosine hydroxylase immunohistochemistry. In rats receiving transplants as adults, grafts were compact and located in the neostriatum. In contrast, in rats receiving transplants neonatally, fewer dopaminergic neurons survived and they were dispersed over a large area of the host neostriatum and nucleus accumbens. After lesioning, all animals manifested strong rotation in response to amphetamine: this was not initially prevented by the grafts, made at either age, up to three months following the lesion, but was reduced in both groups of grafted rats by seven months after lesioning. This prolonged period for the development of recovery contrasts markedly with the rapid recovery obtained when similar grafts are implanted into the denervated neostriatum of adult rats that had received a prior 6-hydroxydopamine lesion. The development of apomorphine rotation, thought to reflect the development of receptor supersensitivity following lesions, was partially blocked to a similar extent by the grafts in both age groups. In contrast to their effects in the rotation tests, the dopaminergic grafts had no detectable effect on the profound contralateral deficit induced by the lesions in the paw-reaching test, whether implanted into neonatal or adult brains. Thus, whereas the age of the host at the time of implantation can markedly influence the gross morphological organization of dopaminergic grafts implanted into the neostriatum, the functional effects were similar, whether the grafts were implanted into neonatal or adult hosts.

The aim of this study was to compare the functional effects of neonatal, weanling and adult lesions of the dopaminergic (DA) mesencephalic neurones on paw-reaching behaviour. The mesotelencephalic DA pathway was destroyed unilaterally in neonatal (3 and 7 day), weanling (21 day) and adult (2 months) rats by local injection of 6-hydroxydopamine into the medial forebrain bundle at the level of the lateral hypothalamus, followed by behavioural studies conducted 2 months later. Amphetamine and apomorphine induced similar rates of rotation irrespective of the age of the lesion. By contrast skilled reaching with the contralateral paw was profoundly disrupted by lesions made in adult or weanling rats, but a much reduced deficit was observed in neonatally lesioned rats. Tyrosine hydroxylase immunohistochemistry indicated a similar degree of dopamine cell loss from the substantia nigra in all groups. These observations suggest that the host brain undergoes developmental changes 1-3 weeks postnatally that influence the long-term effects of lesions in the nigrostriatal dopamine system.