A. Bjo¨rklund

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

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

Formation of cholinergic synapses by intrahippocampal septal grafts as revealed by choline acetyltransferase immunocytochemistry

The ultrastructural features of the contacts established by intrahippocampal grafts of foetal septal/diagonal band neurones in the dentate gyrus and the CA1 region of the previously denervated host hippocampus have been analysed with electron microscopic immunocytochemistry using a monoclonal antibody to choline acetyltransferase (ChAT). The results show that the grafted ChAT-positive neurones are capable of forming extensive synaptic contacts with neuronal targets in areas of the dentate gyrus and CA1 which normally receive such innervation. While all types of contacts normally found in association with the granule and pyramidal cell layers were also present in the graft-reinnervated specimens, the quantitative relationship between somatic and dendritic synapses was abnormal. Thus, the ChAT-immunoreactive synapses on cell bodies, which amounted to only a few percent in the normal animal, constituted over 60% in the grafted animals. Conversely, synapses on dendrites which constituted over 90% in the normal dentate were reduced to less than 40% in the grafted animals. The postsynaptic targets of the graft-derived cholinergic synapses included dendrites and cell bodies of dentate granule cells and CA1 pyramidal cells. This supports previous electrophysiological studies and indicates that the septal grafts may be able to modulate host hippocampal function via direct efferent connections onto the granule and pyramidal neurons in the host hippocampal formation.

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.

Astroglial response in the excitotoxically lesioned neostriatum and its projection areas in the rat

The anatomical distribution of the astrocytic glial reactions, following ibotenic acid-induced neuronal degeneration of the neostriatum in the rat, has been studied immunohistochemically using an antibody directed against the astrocytic marker, glial fibrillary acidic protein. The acute astroglial response to the excitotoxic lesion, determined 7 days post lesion, was compared with a sham-operated group and a chronic group that had received the excitotoxic lesion 6 months prior to histological evaluation. Total doses of 16-20 micrograms ibotenic acid injected unilaterally into the head of the neostriatum caused not only a marked neuronal cell loss but was also accompanied by a large increase in the number and size (about 5 times) of glial fibrillary acidic protein-stained astrocytes throughout the neostriatum by 7 days after lesion. Reactive astrocytes were also observed in the major neostriatal projection areas, the globus pallidus and the substantia nigra pars reticulata, at 7 days post lesion, although no neuronal cell loss could be detected in these regions using regular Cresyl Violet staining. Previous studies of lesions identical to the ones used here have shown that globus pallidus and substantia nigra are deafferented as a result of the neostriatal neuronal degeneration. The reactive astrocytes in the striatal projection areas had a 3-5 times larger size than control astrocytes from the same anatomical region. In animals that received a larger dose of ibotenic acid into the neostriatum (25 micrograms), neuronal cell loss was also observed in the neocortex and reactive glial fibrillary acidic-stained astrocytes were found in the entire neocortex of the injected hemisphere. In the chronic group, 6 months after the excitotoxic lesion, the astroglial response was clearly diminished or absent in the major neostriatal projection areas, but was still present within the lesioned neostriatum. The results suggest that focal neuronal destruction can result in widespread astrocytic glial reactions which follow the anatomical connectivity of the lesioned area. This may have implications for the understanding of the multifocal distribution of glial reactions seen in patients with striatal degeneration as a result of Huntingtons disease.

Axonal degeneration and regeneration of the bulbo-spinal indolamine neurons after 5,6-dihydroxytryptamine treatment

Abstract The cellular events induced by intraventricular injection of 5,6-dihydroxytryptamine (5,6-DHT) were studied with fluorescence histochemistry in the bulbo-spinal indolamine neuron systems, 1 day to 3 months after injection. Signs of acute axonal damage were observed at day 1 as abundant strongly fluorescent axonal dilatations in the bulbo-spinal pathway, mainly in the caudal medulla oblongata and at the junction to the spinal cord. Concomitantly, there was a loss of histochemically detectable, non-terminal axons in the white matter of the spinal cord. The disappearance of indolamine-containing terminals in the grey matter of the spinal cord occurred mainly between 2 and 10 days after 5,6-DHT treatment, and after 10 days or more, the thoracic and lumbar spinal cord appeared almost completely devoid of indolamine innervation, whereas the bulbo-spinal catecholamine neuron system appeared entirely unaffected. No acute damage by 5,6-DHT was detected in the indolamine-containing cell bodies in the medulla oblongata, but in the 30–90-day animals there was a partial disappearance of superficially located indolamine cell bodies, probably as a result of retrograde degeneration after severe axonal damage. The results indicate that, in the bulbo-spinal systems, 5,6-DHT has a direct neurotoxic action primarily on the superficially located non-terminal parts of the indolamine-containing axons, and that terminal degeneration largely occurs as an anterograde degeneration of the lesioned axons. The indolamine cell bodies are less susceptible to the direct action of 5,6-DHT. Ten days, or more, after 5,6-DHT treatment, new, delicate indolamine-containing fibers, probably identical with new axonal sprouts, gradually developed. Three months after injection, these newly appearing fibers occurred abundantly in large areas of the medulla oblongata and in the most cranial part of the spinal cord. These phenomena are interpreted as an extensive re-growth of the lesioned indolamine axons, and in the grey matter of the most cranial spinal cord the regenerating fibers appeared to re-establish their normal connections.

Sequential intrastriatal grafting of allogeneic embryonic dopamine-rich neuronal tissue in adult rats: Will the second graft be rejected?

An important issue in clinical neural grafting is whether a second instriatial allograft can survive well in a patient who has received an allograft before. In this study, the survival, immunogenicity and function of intrastriatal grafts of allogeneic or syngeneic embryonic dopamine-rich tissue in rats which had previously received either an intrastriatal allo- or syn-graft or sham injections were examined. The first graft tissue was taken from inbred Lewis or Sprague-Dawley rat embryos and grafted into an intact striatum of adult Sprague-Dawley rats subjected to a unilateral 6-hydroxydopamine lesion on the contralateral side. Eight weeks after the first transplantation, either allogeneic or syngeneic tissue was grafted as dissociated tissue into the dopamine depleted striatum. The function of the second grafts was assessed by rotational asymmetry at two different time points, i.e. eight and 14 weeks after the second transplantation. There were significant reductions of rotational asymmetry in all groups over time, but no significant difference between groups. Tyrosine hydroxylase immunocytochemistry was used to assess dopamine cell survival and graft size. Statistical analysis revealed no significant differnce in the mean number of tyrosine hydroxylase immunoreactive cells or the mean volume of the second grafts placed on the right side (lesioned side) between groups. Monoclonal antibodies were used to evaluate cellular immune reactions and the major histocompatibility complex class I and class II expression in and around grafts. No major histocompatibility complex class I expression was seen in any of the graft combinations. The expression of the major histocompatibility complex class II antigens was generally higher in patches in and around the second allograft of rats which had previously received an allograft than that in and around any other type of grafts. However, the expression of the major histocompatibility complex class II antigens was low throughout the grafts and did not appear as marked perivascular infiltrates. All the major histocompatibility complex class II positive cells displayed a microglia-like morphology, supported by the parallel microglia and macrophage-specific OX-42 immunostaining. The results show that there is no marked on-going immune reactions in or around the implantation site in any group fourteen weeks after a second transplantation. It may be concluded, therefore, that sequential allografting, using stereotaxic implantation of dissociated embryonic neural tissue into the striatal parenchyma, is possible to perform without a major risk of graft rejection, provided that an atraumatic technique is used.

Dopamine and cholecystokinin immunoreactive neurones in mesencephalic grafts reinnervating the neostriatum: Evidence for selective growth regulation

Pieces of embryonic mesencephalic tissue rich in dopamine and cholecystokinin immunoreactive neurones were grafted to the dorsal surface of the caudate-putamen of adult host rats subjected to unilateral dopamine depleting lesions. After 3 months, neuronal survival in the graft and fibre outgrowth into the host brain were studied by tyrosine hydroxylase and cholecystokinin immunohistochemistry, both in serial sections and by elution and restaining of the same sections. Both dopamine- and cholecystokinin-containing neurones as well as neurons containing both compounds survived the transplantation process. The ratio of neurones in which dopamine and cholecystokinin-like immunoreactivity occurred independently and in coexistence was similar in the grafts to that seen in the intact ventral mesencephalon. This suggests that the grafted cells maintain and express at least some of their normal chemical characteristics in the ectopic cortical location. Only those fibres which contained tyrosine hydroxylase but apparently lacked the cholecystokinin-like peptide showed extensive reinnervation of the host neostriatum. The cholecystokinin-positive fibres were found in a narrow zone immediately adjoining the graft. These results indicate that the dopaminergic reinnervation of the denervated neostriatum is preferentially carried out by the population of grafted mesencephalic dopamine neurones apparently lacking the cholecystokinin-like peptide. This suggests the presence of growth regulating mechanisms in the denervated neostriatum which selectively favour the ingrowth of fibres from the appropriate dopaminergic neuronal subset. The transplantation technique may therefore provide a powerful tool for the study of neurone-target interactions in the establishment of neuronal connections, and of the possible role of peptidergic coexistence in the development and organization of monoaminergic pathways and their innervation patterns.

Neuropeptide messenger RNA expression in the 6-hydroxydopamine-lesioned rat striatum reinnervated by fetal dopaminergic transplants: Differential effects of the grafts on preproenkephalin, preprotachykinin and prodynorphin messenger RNA levels

In situ hybridization histochemistry was used to analyse the expression of the messenger RNAs encoding for enkephalin, substance P and dynorphin in the striatum of normal rats, rats subjected to a unilateral 6-hydroxydopamine lesion of the mesostriatal dopamine pathway and lesioned rats bearing intrastriatal transplants of fetal nigral neurons. About half of the rats in each group received twice-daily subcutaneous injections of 5 mg/kg apomorphine and the other half received control injections of saline, for nine days. Three hours after the last injection, the rats were killed by decapitation. Cryostat sections through the striatum were incubated with, 35S-labeled oligodeoxyribonucleotide probes hybridizing with preproenkephalin, preprotachykinin or prodynorphin messenger RNA. One additional series of sections was incubated with [3H]GBR 12935 in order to label dopamine uptake sites. Quantitative evaluation of the hybridization signal was performed both at the macroscopic level (autoradiographic film analysis) and at the cellular level (optical density of silver grains over identified cells). The grafted nigral neurons reversed the lesion-induced up-regulation of preproenkephalin messenger RNA in the whole striatal complex. By contrast, the graft-induced effect on the lesion-induced down-regulation of preprotachykinin messenger RNA was restricted to the region of the host striatum where the graft-derived dopamine fibers exhibited their densest distribution (up to 0.5 mm from the border of the grafts). However, following chronic treatment with apomorphine, preprotachykinin messenger RNA expression approached control levels in a wider portion of the grafted striata (up to 1 mm from the border of the grafts). Basal prodynorphin messenger RNA expression, which was also down-regulated in the lesioned striata, was only partially restored by the transplants. Repeated injections of apomorphine enhanced prodynorphin messenger RNA in the lesioned striata to levels several fold higher than normal. This massive increase in prodynorphin messenger RNA expression was completely prevented by the transplants over a large volume of the host striatum (> 1 mm from the graft-host border), but a trend towards an abnormally high prodynorphin messenger RNA expression was still present in peripheral striatal areas that were not reached by graft-derived dopamine fibers. The present results indicate that fetal nigral neurons transplanted to the 6-hydroxydopamine-lesioned striatum have differential effects on the activity of enkephalin-containing (i.e. mainly striatopallidal) and substance P- or dynorphin-containing (i.e. mainly striatonigral) neurons. An inhibitory control over the activity of striatopallidal neurons is completely restored by the grafts, even in non-reinnervated striatal regions, suggesting that neurohumoral mechanisms underlie this effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Tyrosine hydroxylase and dopa decarboxylase activities in the guinea-pig uterus: Further evidence for functional adrenergic denervation in association with pregnancy

Abstract The capacity for the synthesis of neuronal noradrenaline was studied during pregnancy and post partum in the guinea-pig uterus, using animals with bilateral or unilateral pregnancies. The activities of the noradrenaline-synthesizing enzymes, tyrosine hydroxylase and in some experiments also l -dopa decarboxylase were measured in various parts of the uterus and the adjacent paracervical region, which contains adrenergic ganglion cells contributing to the uterine adrenergic innervation. The submandibular gland was used for comparison. During advancing pregnancy the tyrosine hydroxylase activity in the fetus-containing uterine horn was progressively reduced, to reach undetectable levels at term. This change was not seen in the cervix and the submandibular gland. By analogy with previous fluorescence microscopic observations on the uterine adrenergic innervation, it is suggested that the very pronounced reduction in tyrosine hydroxylase activity in the fetus-containing uterine horn during advancing pregnancy is due to degeneration of the adrenergic terminal network. In the contralateral empty uterine horn of a unilateral pregnancy the tyrosine hydroxylase activity was decreased by about 90% at term despite evidence for a relatively intact adrenergic nerve plexus. In the post partum period the tyrosine hydroxylase activity increased very slowly in both the previously empty and fetus-containing horns. In the latter tissue, even 6 months after delivery, the activity was only 14% of that in uterine horns of virgin animals. In view of the marked regional heterogeneity in the changes, also related to the position and size of the onceptus, it is assumed that they are caused mainly by local humoral and probably also mechanical factors within the uterus.

Serotonin neurons grafted to the adult rat hippocampus. II. 5-HT release as studied by intracerebral microdialysis.

Extracellular levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were monitored by microdialysis in the hippocampal formation previously denervated of its serotonergic input by an intraventricular injection of 5,7-dihydroxytryptamine (5,7-DHT), and in 5,7-DHT denervated hippocampi reinnervated by grafted fetal rat serotonin neurons. Two weeks after 5,7-DHT lesion, baseline 5-HT release was reduced to levels below detection, and KCl- and p-chloro-amphetamine-evoked release was reduced by 90-95%. In the chronically denervated hippocampus (3 months after lesion), baseline 5-HT release had recovered to near-normal levels, but KCl- and p-chloroamphetamine-evoked release remained severely impaired. Addition of the 5-HT re-uptake blocker indalpine to the perfusion medium induced a 5-6-fold increase in serotonin overflow in the normal hippocampus, while the serotonin overflow in the 5,7-DHT denervated hippocampus remained unaffected. The intrahippocampal fetal raphe transplants restored 5-HT release to near-normal levels, not only under baseline conditions but also in the presence of re-uptake blockade. Both KCl- and p-chloroamphetamine-induced release had recovered in the grafted hippocampus and the responses were even greater than those seen in normal animals. In both normal and grafted hippocampus addition of the sodium channel blocker tetrodotoxin reduced 5-HT overflow to the level seen in the denervated hippocampus. The new hippocampal serotonin innervation, established by the grafts, was markedly denser than normal, and the tissue 5-HT and 5-HIAA levels were 3-4-fold higher than normal in the grafted hippocampi. The 5-HIAA level in the perfusate collected from the grafted hippocampi showed a similar increase above normal, whereas 5-HT release was maintained within the normal range, both under baseline conditions and in the presence of re-uptake blockade. The results indicate that the grafted serotonergic raphe neurons are spontaneously active at the synaptic level, despite their ectopic location. The ability of the grafted neurons to maintain 5-HT release within the normal range suggests that local regulatory mechanisms at the terminal level can compensate for abnormalities in the graft-derived innervation density.