Ola G. Nilsson

Human fetal dopamine neurons grafted in a rat model of Parkinson's disease : immunological aspects, spontaneous and drug-induced behaviour, and dopamine release

SummaryWe have used a rat model of Parkinsons disease (PD) to address issues of importance for a future clinical application of dopamine (DA) neuron grafting in patients with PD. Human mesencephalic DA neurons, obtained from 6.5–8 week old fetuses, were found to survive intracerebral cell suspension xenografting to the striatum of rats immunosup-pressed with Cyclosporin A. The grafts produced an extensive new DA-containing terminal network in the previously denervated caudate-putamen, and they normalized amphetamine-induced, apomorphine-induced and spontaneous motor asymmetry in rats with unilateral lesions of the mesostriatal DA pathway. Grafts from an 11.5-week old donor exhibited a lower survival rate and smaller functional effects. As assessed with the intracerebral dialysis technique the grafted DA neurons were found to restore spontaneous DA release in the reinnervated host striatum to normal levels. The neurons responded with large increases in extracellular striatal DA levels after the intrastriatal administration of the DA-releasing agent d-amphetamine and the DA-reuptake blocker nomifensine, although not to the same extent as seen in striata with an intact mesostriatal DA system. DA fiber outgrowth from the grafts was dependent on the localization of the graft tissue. Thus, grafts located within the striatum gave rise to an extensive axonal network throughout the whole host striatum, whereas grafted DA neurons localized in the neocortex had their outgrowing fibers confined within the grafts themselves. In contrast to the good graft survival and behavioural effects obtained in immunosuppressed rats, there was no survival, or behavioural effects, of human DA neurons implanted in rats that did not receive immunosuppression. In addition, we found that all the graft recipients were immunized, having formed antibodies against antigens present on human T-cells. This supports the notion that the human neurons grafted to the non-immunosuppressed rats underwent immunological rejection. Based on an estimation of the survival rate and extent of fiber outgrowth from the grafted human fetal DA neurons, we suggest that DA neurons that can be obtained from one fetus may be sufficient to restore significant DA neurotransmission unilaterally, in one putamen, in an immunosuppressed PD patient.

Behavioural effects of human fetal dopamine neurons grafted in a rat model of Parkinson's disease

SummaryThe ventral mesencephalon, containing the developing dopaminergic neurons of the substantia nigra-ventral tegmental region, was obtained from aborted human fetuses of 9–19 weeks of gestation. The tissue was grafted into the striatum of rats previously subjected to a 6-hydroxydopamine lesion of the mesostriatal dopamine pathway. The graft recipients were immunosuppressed by daily injections of Cyclosporin A. Amphetamine-induced motor asymmetry was reduced, and finally totally reversed, only in rats receiving grafts from the 9-week old fetal donor. The fluorescence microscopic analysis revealed large numbers of surviving dopamine neurons, and extensive fiber outgrowth into the host striatum, in these rats. By contrast, rats receiving grafts from 11–19 week old donors had at most only few surviving dopamine neurons. These results indicate that human fetal mesencephalic tissue may be an efficient source of dopamine neurons for functional intracerebral grafting in patients with Parkinsons disease.

Spatial learning impairments in rats with selective immunolesion of the forebrain cholinergic system

A monoclonal antibody to the low-affinity NGF receptor, 192 IgG, coupled to a cytotoxin, saporin, was recently introduced as an efficient selective neurotoxin for the NGFr-bearing cholinergic neurones in the rat basal forebrain. In the present study we report that an intracerebroventricular injection of this 192 IgG-saporin conjugate induces a severe, long-lasting spatial learning impairment, as assessed in the Morris water-maze task. This behavioural impairment was associated with 65-90% depletion of choline acetyltransferase activity (ChAT) in the hippocampus and cortex. ChAT activity associated with other cholinergic neurone systems in the brain (striatum, mesencephalon, spinal cord), was left virtually unaffected. This new immunotoxin holds great promise as a tool for selective and efficient lesions of the forebrain cholinergic system in functional and behavioural studies.

Human fetal dopamine neurons grafted in a rat model of Parkinson's disease: ultrastructural evidence for synapse formation using tyrosine hydroxylase immunocytochemistry

SummaryHuman fetal mesencephalic dopamine (DA) neurons, obtained from 6.5–9 week old aborted fetuses, were grafted to the striatum of immunosuppressed rats with 6-hydroxydopamine lesions of the ascending mesostriatal DA pathway. The effects on amphetamine-induced motor asymmetry were studied at various timepoints after grafting. At eight weeks, functional graft effects were not evident but after 11 weeks small effects on motor asymmetry could be monitored and rats tested 19–21 weeks after grafting exhibited full reversal of the lesion-induced rotational behaviour. Four rats were sacrificed at different timepoints between 8 and 20 weeks and the grafted DA neurons were studied in tyrosine hydroxylase (TH) immunocytochemically stained sections at the light and electronmicroscopic level. The grafts contained a total of 500–700 TH-positive neurons in each rat. In one rat sacrificed 8 weeks after grafting the grafted neurons were TH-positive but exhibited virtually no fiber outgrowth. In another rat, sacrificed after 11 weeks, a sparse TH-positive fiber plexus was seen to extend into the adjacent host neostriatum. Two rats sacrificed after 20 weeks both contained TH-positive neurons that gave rise to a rich fiber network throughout the entire host neostriatum, and this fiber network was also seen to extend into the globus pallidus and nucleus accumbens. Very coarse TH-positive processes, identified as dendrites in the electron microscope, projected up to 1.5–2.0 mm from the graft into the host striatum. Ultrastructural analysis revealed that the grafted neurons had formed no TH-positive synaptic contacts with host striatal neurons after 8 weeks, and at 11 weeks some few TH-positive synapses were identified. Twenty weeks after transplantation, abundant TH-positive synaptic contacts with host neurons were seen throughout the neostriatum, and such contacts were identified in the globus pallidus as well. Thus, the present study provides tentative evidence for a time-link between the development of synaptic contacts and the appearance of functional graft effects. Similar to the normal mesostriatal DA pathway, ingrowing TH-positive axons formed symmetric synapses and were mainly seen to contact dendritic shafts and spines. However, in comparison to the normal rat striatum there was a higher incidence of TH-immunoreactive boutons forming synapses onto neuronal perikarya. The TH-positive dendrites that extended into the host striatum were seen to receive non-TH-immunoreactive synaptic contacts, presumably arising from the host neurons. These results suggest that human fetal DA neurons are able to develop a reciprocal synaptic connectivity with the host rat when grafted to the adult brain. Grafting of human fetal DA neurons may therefore be expected to provide a means of restoring regulated synaptic DA release in patients with Parkinsons disease.

Cyclosporin A increases survival of cross-species intrastriatal grafts of embryonic dopamine-containing neurons

SummaryThe survival and function of cross-species (mouse-to-rat) grafts of fetal mesencephalic dopamine (DA) neurons, implanted as a cell suspension in the striatum of rats with lesions of the mesostriatal DA system, have been studied in animals with and without immunosuppression induced by Cyclosporin A (CyA). At 6 weeks after grafting 3 out of 7 non-CyA treated animals showed some degree of graft survival and variable functional compensation. In those three animals an average of 92 DA neurons per graft was counted. In the grafted animals treated with daily CyA injections, all grafts survived and produced partial or complete functional compensation, and they had an average of 557 DA neurons per graft. It is concluded that intracerebral graft survival and function can be greatly improved by CyA treatment and that the immunological protection of neural transplants in the brain is only partial.

Selective lesioning of the basal forebrain cholinergic system by intraventricular 192 IgG-saporin: Behavioural,biochemical and stereological studies in the rat.

The elucidation of the functional role of the basal forebrain cholinergic system will require access to a highly specific and efficient cholinergic neurotoxin. Recently, selective depletion of the nerve growth factor (NGF) receptor‐bearing cholinergic neurons in the rat basal forebrain and a dramatic loss of cholinergic innervation in the related cortical regions have been obtained following intraventricular injection of a newly introduced immunotoxin, 192 IgG‐saporin. Here we extend these initial findings and report that administration of increasing doses (1.25, 2.5, 5.0 or 10 μg) of the 192 IgG‐saporin conjugate into the lateral ventricles of adult rats induced dose‐dependent impairments in the water maze task and passive avoidance retention, but only weak and inconsistent effects on locomotor activity. These behavioural changes were paralleled by a reduction in choline acetyltransferase activity in hippocampus and several cortical areas (up to 97%) and selective depletions of NGF receptor‐positive cholinergic neurons in the septal‐diagonal band area and nucleus basalis magnocellularis (up to 99%). By contrast, the non‐cholinergic parvalbumin‐containing neurons in the septum were completely spared, and other cholinergic projection systems (such as in the striatum, thalamus, brainstem and spinal cord) were unaffected even at the highest dose. The observed changes in the water maze and passive avoidance tasks, as well as the cholinergic cell loss, were maintained up to at least 8 months following the intraventricular injection of a single dose (5 μg) of the immunotoxin. The results confirm the usefulness of the 192 IgG‐saporin toxin for selective and profound lesions of the basal forebrain cholinergic neurons and provide further support for a role of the basal forebrain cholinergic system in cognitive functions.

Combined cholinergic and serotonergic denervation of the forebrain produces severe deficits in a spatial learning task in the rat

The purpose of the present experiments was to study the effects of a combined cholinergic and serotonergic denervation of the rat forebrain on spatial learning using the Morris water maze task. Experiment 1 compared the acute effects of a radiofrequency lesion of the septum, an intraventricular 5,7-dihydroxytryptamine (5,7-DHT) lesion, and a combined septal plus 5,7-DHT lesion. Although the 5,7-DHT lesion alone did not produce any significant deficits in the water maze task, the lesion greatly potentiated the learning impairments produced by the septal lesion. Thus, the rats with both lesions combined showed severe difficulties in finding the platform and they did not develop any place navigational search strategy. This effect was not dependent on any effect on swimming ability or locomotor activity. The long-term effects of the combined septal and 5,7-DHT lesion was investigated in experiment 2, where the rats were tested in the water maze both 5 and 24-25 weeks after surgery. In this experiment, the rats showed the same severe deficits in spatial learning in both tests, showing that the impairments remain for long periods and after extended training. The results show that a combination of a cholinergic and a serotonergic denervation of the rat forebrain produces pronounced impairments in spatial learning in the Morris water maze task, and that this effect is long-lasting. This indicates that the recently proposed serotonergic deficit in patients with Alzheimers disease may contribute importantly to the cognitive disabilities in these patients.

Acetylcholine release in the rat hippocampus as studied by microdialysis is dependent on axonal impulse flow and increases during behavioural activation

Changes in extracellular levels of acetylcholine and choline in the hippocampal formation were measured using intracerebral microdialysis coupled to high performance liquid chromatography with post-column enzyme reaction and electrochemical detection. Various pharmacological and physiological manipulations were applied to awake unrestrained normal rats and rats subjected to a cholinergic denervation of the hippocampus by a complete fimbria-fornix lesion (1-2 weeks previously). Low baseline levels of acetylcholine (about 0.3 pmol/15 min sample) could be detected in the absence of acetylcholinesterase inhibition in all animals. However, in order to obtain stable and more readily detectable levels, the acetylcholinesterase inhibitor neostigmine was added to the perfusion medium at a concentration of 5 or 10 microM and was used during all subsequent manipulations. Addition of neostigmine increased acetylcholine levels approximately 10-fold (to 3.7 pmol 15 min) in the normal rats, which was about 4-fold higher than the levels recovered from the denervated hippocampi. Depolarization by adding KCl (100 mM) to the perfusion fluid produced a 3-fold increase in the extracellular acetylcholine levels, and the muscarinic antagonist atropine (3 microM) resulted in a 4-fold increase in the normal rats, whereas these drugs induced only small responses in the denervated rats. Neuronal impulse blockade by tetrodotoxin (1 microM) resulted, in normal rats, in a 70% reduction in extracellular acetylcholine levels. Sensory stimulation by handling increased acetylcholine levels by 94% in the normal rats, whereas this response was almost totally abolished in the denervated hippocampi. Behavioural activation by electrical stimulation of the lateral habenula resulted in a 4-fold increase in acetylcholine release in normal animals, and this response was totally blocked by a transection of the lateral habenular efferents running in the fasciculus retroflexus. The levels obtained by lateral habenula stimulation were reduced by about 95% in the rats with fimbria-fornix lesions. Following an acute knife transection of the fimbria-fornix performed during ongoing dialysis, acetylcholine levels dropped instantaneously by 70%, indicating that the extracellular acetylcholine levels in the hippocampus are maintained by a tonic impulse flow in the septohippocampal pathway. The extracellular levels of choline were reduced by about 30% after the addition of neostigmine in the normal rats, and increased by about 50% in both normal and denervated rats after addition of KCl to the perfusion fluid. No changes could be detected after atropine, handling, lateral habenula stimulation, or acute fimbria-fornix or fasciculus retroflexus transection.(ABSTRACT TRUNCATED AT 400 WORDS)

Incidence of intracerebral and subarachnoid haemorrhage in southern Sweden

OBJECTIVES Spontaneuos intracranial haemorrhage—that is, mainly subarachnoid haemorrhage (SAH) and primary intracerebral haemorrhage (PICH)—constitutes an important part of all strokes. As previous epidemiological studies have demonstrated highly variable incidence rates, we conducted a large prospective investigation of all haemorrhagic strokes during a 1 year period. METHODS Twelve hospitals serving a defined population of 1.14 million in southern Sweden registered all cases with spontaneous intracranial haemorrhage, including those found dead outside hospitals, during 1996. All patients were examined with CT of the brain or underwent necropsy. Incidence rates adjusted to the Swedish population for age and sex, as well as location of haematoma and prevalence of risk factors were calculated. RESULTS A total of 106 patients with SAH and 341 patients with PICH were identified. The annual incidence/100 000 was 10.0 (6.4 for men and 13.5 for women) for SAH and 28.4 (32.2 for men and 24.7 for women) for PICH when adjusted to the Swedish population. Subarachnoid haemorrhage affected twice as many women as men. The incidence of both types of haemorrhage increased with advancing age, but in particular, this was the case for supratentorial PICH. Lobar haematomas were the most common (51.6%) type of PICH. Among patients with PICH, 37% had hypertension, 41% other vascular disease, and 12% were on oral anticoagulation. Among patients with SAH, 28% had hypertension and 18% vascular disease before the haemorrhage but no one was on treatment with oral anticoagulation. CONCLUSIONS The incidence of PICH was high, especially for the older age groups. PICH was, on average, three times as common as SAH. The study underscores the importance of PICH and SAH as significant stroke subgroups.

Amelioration of spatial memory impairment by intrahippocampal grafts of mixed septal and raphe tissue in rats with combined cholinergic and serotonergic denervation of the forebrain.

Previous studies in the rat have shown that a serotonergic depletion greatly potentiates the learning and memory impairments produced by pharmacological or lesion-induced cholinergic blockade in the forebrain. The impairment produced by combined serotonergic-cholinergic lesions is reminiscent of that seen in memory-impaired aged rats. In the present experiment, we investigated whether grafts of cholinergic septal tissue and serotonergic mesencephalic raphe tissue, placed in the hippocampus, could reverse the severe memory impairment produced by combined cholinergic-serotonergic lesions. Adult rats were given an intraventricular injection of 5,7-dihydroxytryptamine followed by a radiofrequency lesion of the septum 1-2 weeks later. Three weeks after lesion surgery, the rats were given bilateral intrahippocampal cell suspension grafts of either fetal septal or mesencephalic raphe tissue, or both. The rats were tested for spatial learning and memory in the Morris water maze task at 4 and 10 months after grafting. At 4 months, lesioned and grafted groups were all impaired compared to the normal controls in their swim time and distance swum to find the platform, and they did not show any spatially focussed search strategy in the spatial probe trial when the platform was removed from the tank. At 10 months, the rats with mixed cholinergic and serotonergic grafts were no longer impaired compared to normals in their swim time and distance to find the platform, and they were significantly improved compared to the other grafted groups. Moreover, in the spatial probe trial, the rats with mixed cholinergic and serotonergic grafts displayed a spatially focussed search behaviour over the previous platform site, which was not seen in the lesioned control rats or in the other graft groups. Morphological analysis of the hippocampus revealed that the septal grafts produced an acetylcholinesterase-positive innervation but were totally devoid of serotonin innervation. The raphe grafts produced mainly a serotonin innervation, of both acetylcholinesterase- and serotonin-positive fibres. The results suggest that a mixture of septal and raphe tissue is required when grafted to the hippocampal formation in order to ameliorate the severe spatial learning and memory impairments produced by a combined cholinergic and serotonergic denervation, and that each of these graft types separately are not sufficient to ameliorate such deficits.