Functional integration of striatal allografts in a primate model of Huntington's disease.

Huntington's disease is an autosomal dominant, inherited disorder that results in progressive degeneration of the basal ganglia (especially the neostriatal caudate nucleus and putamen) and other forebrain structures and is associated with a clinical profile of movement, cognitive and psychiatric impairments for which there is at present no effective therapy. Neuropathological, neurochemical and behavioral features of the disease can all be reproduced in experimental animals by local injection of excitotoxic or metabolic toxins into the neostriatum. All these features of the disease can be alleviated, at least in rats, by transplantation of embryonic striatal tissue into the degenerated striatum, which was the basis for commencing the first clinical trials of striatal transplantation in Huntington's patients. However, although rat striatal xenografts may temporarily reduce apomorphine-induced dyskinesias in monkeys, there has been no demonstration that allograft techniques that work well in rats translate effectively to the much larger differentiated striatum of primates. Here we demonstrate good survival, differentiation and integration of striatal allografts in the primate neostriatum, and recovery in a test of skilled motor performance. Long-term graft survival in primates indicates probable success for clinical transplants in Huntington's disease; in addition, our data suggest that graft placement has a direct influence on the pattern and extent of functional recovery.

Can fetal neural transplants restore function in monkeys with lesion-induced behavioural deficits?

Experiments are now being conducted in monkeys to see whether the transplantation of fetal neural tissue, rich in certain neurotransmitter-producing cells, can restore behaviour in animals with movement or learning impairments induced by lesions that have destroyed important neurotransmitter pathways. Transplantation of dopamine neurons in humans may prove to be a useful therapy in Parkinson's disease, in which a severe movement disorder is associated with degeneration of the dopamine system. Transplantation of cholinergic neurones in monkeys can overcome a severe learning impairment induced by lesion of the cholinergic system. Cholinergic transplantation may eventually be of use in a variety of neurodegenerative dementing illnesses.

The effects of metoclopramide, sulpiride, and the stereoisomers of baclofen on amphetamine-induced behavior in the marmoset.

We have studied the effects on amphetamine-induced behavior in a primate species of drugs (other than the classic neuroleptics) sometimes used in the treatment of psychosis. Marmosets were treated with either saline (control animals) or amphetamine followed after 18 min by doses of either metoclopramide, sulpiride, or the d- or l-isomers of baclofen. Metoclopramide antagonized amphetamine-induced behavior at low doses while also causing sedation at higher doses in control animals. Neither sulpiride nor baclofen specifically antagonized amphetamine-induced behavior.

The time course of the behavioral effects of amphetamine and their reversal by haloperidol in a primate species.

A group of six marmosets was administered amphetamine (Phase I), amphetamine plus haloperidol (Phase II), and then amphetamine alone (Phase III) over consecutive periods of 27, 51, and 33 days after which drug treatment was terminated (Phase IV). The animals' behavior was monitored during these periods and during a predrug treatment control period. Five mutually exclusive categories of behavior were assessed during the experiment. Social contact between animals was significantly suppressed and inactivity was increased throughout Phases I--III but both measures returned to normal values during Phase IV. Locomotion was significantly decreased towards the end of Phase I and initially during Phase II. Rapid head movements (termed checking) were significantly increased at the beginning of Phase I and again when the haloperidol was withdrawn at the beginning of Phase III. Towards the end of Phase I the animals developed destructive self-grooming habits. The time course of the effects of amphetamine and haloperidol on the different behavioral categories suggests that different mechanisms may be involved in each case. Viewed as a model of schizophrenia, the time course of haloperidol in reversing amphetamine-induced suppression of locomotion most closely resembles the time course of the antipsychotic effect of neuroleptics in man. Some effects of amphetamine (e.g., suppression of social interaction) are not reversed by haloperidol, and some effects of withdrawal of haloperidol (e.g., precipitation of checking movements not present when haloperidol was commenced) do not have an obvious counterpart in the clinical situation.

Induction of beta (A4)-amyloid in primates by injection of Alzheimer's disease brain homogenate. Comparison with transmission of spongiform encephalopathy.

Amyloid plaques, associated with argyrophilic dystrophic neurites, and cerebral amyloid angiopathy (CAA), but no neurofibrillary tangles, were found in the brains of three middle-aged marmoset monkeys that had been injected intracerebrally (ic) 6-7 yr earlier with brain tissue from a patient with early-onset Alzheimer's disease. Such changes were not found in the brains of three age-matched control marmosets. Immunochemically the amyloid plaques and CAA stained with antibody to beta (A4)-protein. The plaques and CAA displayed dichroic birefringence when stained with Congo red and viewed under polarized light. beta (A4)-amyloid plaques and CAA were also found in the brain of one of two marmosets injected ic 6 yr previously with brain tissue from a patient with prion disease with concomitant beta (A4)-amyloid plaques and CAA. An occasional beta (A4)-amyloid plaque was found in the brains of two of four marmosets injected ic > 4.5 yr previously with brain tissue from three elderly patients, two of whom had suspected (but untransmitted) CJD. No beta (A4)-amyloid plaques or CAA were found in six marmosets who were older than the injected animals, in four marmosets that had not developed spongiform encephalopathy (SE) having been injected several years previously with human brain tissue from three younger patients with suspected or atypical prion disease, or in 10 younger marmosets who had undergone various neurosurgical procedures. Seventeen marmosets injected in the same way with brain tissue from patients or animals with SE developed SE 17-49 mo after injection. These results suggest that beta (A4)-amyloidosis is a transmissible process comparable to the transmissibility of SE.

Behavioral assessment of the ability of intracerebral embryonic neural tissue grafts to ameliorate the effects of brain damage in marmosets.

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.

Tyrosine hydroxylase-immunoreactive neurons in the nucleus basalis of the common marmoset (Callithrix jacchus).

In the course of characterizing the distribution of putative catecholaminergic neurons in the brain of the common marmoset, we encountered a population of such cells in the basal forebrain. Tyrosine hydroxylase-immunoreactive neurons are abundant within the nucleus basalis magnocellularis throughout its entire rostrocaudal extent, but not in other cholinergic basal forebrain nuclei. Most tyrosine hydroxylase-immunoreactive cells are large and multipolar. Double staining with antibodies to choline acetyltransferase or nerve growth factor receptor confirmed that these tyrosine hydroxylase-immunoreactive neurons are cholinergic, and compose at least 40% of the nucleus basalis cholinergic cells. The presence of a catecholamine-synthesizing enzyme in the neurons that provide the major cholinergic input to the neocortex may have important consequences for cortical function, and may be relevant to the vulnerability of the nucleus basalis in certain neurodegenerative disorders.

An involvement of acetylcholine in object discrimination learning and memory in the marmoset.

Five marmosets (Callithrix jacchus) were tested, using a Wisconsin General Test Apparatus, on a series of junk object visual discrimination tasks, including new learning, 24-hr reversal and 24-hr retention. The effects of administering the cholinergic receptor blocking agent, scopolamine either just before or immediately after the new learning task, or just before the 24-hr reversal and retention tasks, were assessed. Results suggest that scopolamine impairs new learning and impairs the encoding of new information in long term memory. Some evidence of a mild retrieval deficit under scopolamine was also seen, while state-dependent effects were not apparent.

Topographical memory impairments after unilateral lesions of the anterior thalamus and contralateral inferotemporal cortex.

Monkeys with crossed unilateral excitotoxic lesions of the anterior thalamus and unilateral inferotemporal cortex ablation were severely impaired at learning two tasks which required the integration of information about the appearance of objects and their positions in space. The lesioned monkeys were also impaired at learning a spatial task and a task which required the integration of information about the appearance of objects and the background on which the objects were situated. Monkeys with only one of the unilateral lesions were not impaired and previous work has shown that monkeys with bilateral lesions of the anterior thalamus were not impaired on these tasks. These results indicate that the whole of the inferotemporal cortex-anterior thalamic circuit, which passes via the hippocampus, fornix, mamillary bodies and mamillothalamic tract, is essential for the topographical analysis of information about specific objects in different positions in space. Together with previous work, the results show that a unilateral lesion may affect cognition in the presence of other brain damage when an equivalent bilateral lesion alone does not. The tasks required the slow acquisition of information into long term memory and therefore assessed semantic knowledge although other research has shown impairment on topographical processing within working or episodic memory following lesions of the hippocampal-diencephalic circuit. It is argued that the hippocampal-diencephalic circuit does not have a role in a specific form of memory such as episodic memory but rather is involved in topographical analysis of the environment in perception and across all types of declarative memory.

Synergistic effects of unilateral immunolesions of the cholinergic projections from the basal forebrain and contralateral ablations of the inferotemporal cortex and hippocampus in monkeys.

Monkeys, with unilateral immunotoxic lesions of the basal nucleus of Meynert that remove cholinergic innervation of the ipsilesional neocortex, and ablations of the contralateral inferotemporal neocortex, were impaired on retention of visual discriminations learnt before surgery and on acquisition of new discriminations. This demonstrates that the cholinergic projection from the basal nucleus supports the functions of its cortical target area. Our previous studies have shown that the impairment on discrimination performance following bilateral lesions of the basal nucleus is transient and that bilateral lesions of the diagonal band of Broca, that remove cholinergic innervation of the hippocampus, are without effect on these tasks. However, the impairment resulting from bilateral lesions of the basal nucleus plus the diagonal band, or from bilateral inferotemporal cortex ablations, is severe and persistent. Bilateral inferotemporal ablations deprive the hippocampus of much of its visual input by producing a discontinuity in cortico-cortical transmission, whereas basal nucleus lesions may merely prevent the modification of visually-derived information in the inferotemporal cortex without depriving the hippocampus of visual input. In the monkeys with crossed unilateral basal nucleus plus inferotemporal cortex lesions, the addition of a diagonal band lesion to the basal nucleus lesion produced an impairment on retention of visual discriminations and sustained the acquisition impairment. This confirms the previous finding that the basal nucleus and diagonal band act synergistically in producing a severe and permanent impairment. Further addition of an excitotoxic hippocampal lesion to the hemisphere with the inferotemporal cortex ablation did not add to the learning impairment. This supports the suggestion that the inferotemporal cortex ablation has deprived the hippocampus of its visual input.Overall, these experiments demonstrate that the cholinergic projections from the basal nucleus and diagonal band participate in the learning and memory functions of the temporal lobes.

Non-spatial acquisition and retention deficits following small excitotoxic lesions within the hippocampus in monkeys.

Marmoset monkeys with excitotoxic lesions confined to cornu ammonis subfields 1-3, subiculum and pre-subiculum, but sparing the entorhinal cortex, were impaired on retention and learning of conditional object-choice discriminations. For each of these discriminations, the monkeys were required to choose one of two objects depending on which of two patterned backgrounds was used on each trial. Two styles of order of trial presentation were used: 'random' presentation which maximised the degree of interference between trials, and 'runs' presentation which was intended to encourage the monkeys to learn each component of the discrimination separately. Before surgery monkeys found the discriminations more difficult to learn when the trials were presented in the 'runs' style than when presented in the 'random' style suggesting that the task is best learnt by applying a conditional rule. After surgery a significant 'group x style' interaction indicated that the 'runs' style was especially difficult for the lesioned monkeys. From these results we suggest that the hippocampus is involved in learning about and remembering non-spatial, conditional relations between objects.

Conditional learning and memory impairments following neurotoxic lesion of the CA1 field of the hippocampus.

Monkeys with bilateral lesions of the CA1 field of the hippocampus produced by the injection of neurotoxin diagonally along the length of the hippocampus were found to have a severe impairment on the retention of a conditional task learnt prior to surgery and on the new acquisition of several types of this task. They were equally impaired on conditional tasks that required a spatial response or an object choice in response to either visual or spatial cues. They were not impaired on simple visual discrimination tasks, simple spatial discrimination tasks or reversal learning of these tasks. This patterns of impairment resembles that seen in the same species with neurotoxic lesions within the vertical limb of the diagonal band of Broca or transection of the fornix. Monkeys with subtotal lesions of the adjacent medial temporal area were not consistently impaired on any of these tasks. The results suggest that hippocampal lesions produce anterograde and retrograde amnesia for information other than reward association.

Restoration of learning ability in fornix-transected monkeys after fetal basal forebrain but not fetal hippocampal tissue transplantation.

Monkeys with bilateral transection of the fornix were severely but selectively impaired on learning and retention of visuospatial conditional discriminations, visual conditional discriminations and non-conditional spatial-response tasks. Bilateral transplantation of cholinergic-rich fetal basal forebrain tissue into the hippocampus abolished significant learning impairments on all those tasks impaired by fornix lesions when tested three to nine months after transplantation whereas bilateral transplants of non-cholinergic fetal hippocampal tissue into hippocampus showed no such beneficial effect. Acetylcholinesterase staining was severely depleted throughout the dentate gyrus and hippocampus in fornix-transected monkeys compared with animals with control corpus callosum ablations. Staining was largely restored to normal in the host hippocampus and dentate gyrus in monkeys with cholinergic transplants, whereas acetylcholinesterase staining was abnormal in those with non-cholinergic grafts. These experiments suggest that where a "higher order" cognitive function, in this case the acquisition of specific types of information into long-term memory, is disturbed by a neuropharmacologically simple lesion, cognitive function can be restored by transplantation of neurons containing appropriate neurotransmitters.

Learning impairments following injection of a selective cholinergic immunotoxin, ME20.4 IgG-saporin, into the basal nucleus of Meynert in monkeys.

Four groups of monkeys (Callithrix jacchus) were injected with saline or increasing amounts of the immunotoxin, ME20.4 IgG-saporin, directly into the basal nucleus of Meynert via a frontal trajectory which avoided damage to the overlying basal ganglia. ME20.4 IgG binds to the primate p75 low-affinity neurotrophin receptor, when the saporin derivitized antibody is injected into the basal forebrain, it selectively destroys the magnocellular neurons of the basal nucleus of Meynert which are the cells of origin of the cholinergic projection to the neocortex. The highest dose of ME20.4 IgG-saporin produced a significant impairment on acquisition of a perceptually difficult visual discrimination. There was no significant effect on retention of tasks learnt before or after surgery, nor on concurrent acquisition of several perceptually easy discriminations or serial reversal of an easy discrimination. These results suggest that the impairment is not due to visual, motor or motivational difficulties and does not consist of difficulties with the formation of reward associations. Rather the impairment is largely confined to acquisition of perceptual discriminations. There was a significant correlation between the density of ME20.4 immunostaining in the basal nucleus of Meynert and the density of acetylcholinesterase histochemical staining in the frontal and temporal cortex and an inverse correlation between both of these and the degree of learning impairment in the animals. Lesioned animals also showed significant impairment on acquisition and reversal of perceptually easy discriminations when treated with a dose of scopolamine which did not impair performance in control animals. These results provide further evidence that cortical cholinergic neurotransmission contributes to certain forms of learning. The availability of a selective cholinergic immunotoxin effective in primates provides an important new tool for the study of cholinergic function and its involvement in ageing, Alzheimer's disease and other pathological states.

Stimulus-bound perseveration after frontal ablations in marmosets.

Marmosets with bilateral ablations of either the lateral or ventral surface of the frontal lobes were found to perseverate on object but not spatial serial reversal. They also perseverated on reversal of a visuospatial task where different stimuli required different spatial responses. No differences were found between the two lesion groups. Since the control animals showed mild perseveration on spatial but not object serial reversal it is argued that frontal ablations do not lead to perseveration of a natural tendency but rather that object and visuospatial perseveration are forms of stimulus-bound behaviour which do not occur when the animal is performing a spatial task in which stimulus position is irrelevant. Perseveration was reduced by pretreatment with a dopamine-blocking drug (haloperidol). It is suggested that information from temporal lobe mechanisms, involved in long-term memory, and from frontal lobe mechanisms, which exert shorter acting influences on behaviour, compete within the basal ganglia to determine stimulus choice. The ascending dopaminergic pathway may modulate the balance between these competing factors.

Restoration of cognitive abilities by cholinergic grafts in cortex of monkeys with lesions of the basal nucleus of Meynert.

Three groups of marmosets were trained to perform a series of visual discrimination tasks in a Wisconsin General Test Apparatus. Two groups then received bilateral lesions of the basal nucleus of Meynert using the excitotoxin N-methyl-D-aspartate and were found to be severely impaired on relearning a visual discrimination first learnt prior to surgery. One lesioned group then received grafts of acetylcholine-rich tissue dissected from the basal forebrain of fetal marmosets. Three months later the marmosets with lesion alone remained impaired on a number of retention and reversal tasks whereas the transplanted animals were no longer significantly impaired. Histological examination of the brains indicated that all lesioned animals had sustained substantial loss of the cholinergic neurons of the basal nucleus of Meynert (assessed by nerve growth factor receptor immunoreactivity) and that the lesion-alone animals showed marked loss of the cholinergic marker acetylcholinesterase in the dorsolateral frontal and parietal cortex. All transplanted animals had surviving graft tissue (visualized by Cresyl Violet staining, dense acetylcholinesterase staining and the presence of a limited number of nerve growth factor receptor-immunoreactive neurons) in the neocortex and 5/6 transplanted animals showed near complete restitution of acetylcholinesterase staining in frontal and parietal cortex. Examination of individual animal data showed that the animal without this restitution performed very poorly. The performance of the remaining transplanted animals was significantly better than that of the animals with lesion alone. There was a significant positive correlation between the degree of acetylcholinesterase staining and good performance on tasks sensitive to frontal lobe damage. These results demonstrate that acetylcholine-rich tissue transplanted into the neocortex of primates with damage to the cholinergic projections to the neocortex can produce substantial restitution of function provided that an appropriate level of interaction between graft and host tissue is achieved.

Different effects on learning ability after injection of the cholinergic immunotoxin ME20.4IgG-saporin into the diagonal band of Broca, basal nucleus of Meynert, or both in monkeys.

Immunotoxic lesions of the diagonal band of Broca (VDB) in monkeys disrupted cholinergic input to the hippocampus, producing impaired learning of visuospatial conditional discriminations but not simple visual discriminations. Immunotoxic lesions of the basal nucleus of Meynert (NBM) deprived the cortex of most of its cholinergic input, producing impaired learning of simple visual discriminations but not visuospatial conditional discriminations. Combined lesions of the NBM + VDB resulted in impaired learning of both types of task. The impairment after NBM lesions ameliorated with time but could be reinstated by a low dose of the glutamate blocking drug MK801, which, at this dose, did not impair simple visual discrimination learning in normal monkeys. The cholinergic projections from the NBM and VDB may sustain the function of the glutamatergic pyramidal cell pathways within the cortex and hippocampus, respectively.

Effects of arecoline and pilocarpine on learning ability in marmosets pretreated with hemicholinium-3.

Common marmosets (Callithrix jacchus) were trained to perform serial reversal position discrimination tasks in a Wisconsin General Test Apparatus. Intraventricular injection of hemicholinium-3 4 h before testing resulted in a profound impairment of position discrimination learning which could be overcome by the intramuscular administration of low doses of the muscarinic agonists, arecoline or pilocarpine.

Cholinergic learning deficits in the marmoset produced by scopolamine and ICV hemicholinium.

Common marmosets (Callithrix jacchus) were trained to perform daily position discrimination learning tasks in a Wisconsin General Test Apparatus. Acetylcholine receptor blockade with scopolamine was found to impair position learning. Testing on the day after scopolamine treatment suggested that a task learnt under scopolamine was not encoded into long term memory. Acetylcholine depletion achieved by the intraventricular injection of hemicholinium 4 h before testing resulted in a profound impairment of position discrimination learning. It is suggested that central acetylcholine depletion in primates may provide a useful model of senile dementia.

Perseverative behaviour after amphetamine; dissociation of response tendency from reward association.

Low doses of amphetamine were found to alter the ability of marmosets to take account of changes in reward values of object stimuli in a visual discrimination task. Under amphetamine, animals changed their motor responses and stimulus choice in order to preserve the acquired reward value or meaning of certain stimuli. These results suggest that the perseverative effect of amphetamine on behaviour is due to impaired cognitive flexibility rather than to an enhancement of motor habit.