Klas Wictorin

Mechanisms of action of intracerebral neural implants: studies on nigral and striatal grafts to the lesioned striatum

Abstract The ability of intracerebrally implanted grafts of neural tissues to promote functional recovery in brain-damaged recipient animals has raised the question of how such implants exert their functional effects. Non-specific, diffuse release of active compounds may be sufficient to restore defective neurotransmission in a denervated brain region, for example, or to provide trophic support for the survival and regeneration of damaged host neurons. The positive therapeutic effects of adrenal medullary grafts, recently reported in patients with Parkinsons disease, are likely to reflect such nonspecific hormonal or neurohumoral mechanisms. Morphological and electrophysiological studies, on the other hand, have shown that grafted fetal neurons can establish extensive efferent synaptic connections with previously denervated or neuron-depleted host brain regions and become at least partially integrated into the host neuronal circuitry. In the damaged nigrostriatal system, grafts of fetal nigral or striatal neurons can restore normal synaptic transmitter release and can also participate in a partial reconstruction of functional neural circuits in the host brain. This indicates that the potential of intracerebral grafts to induce or improve behavioral recovery in brain-damaged recipients rests on the multitude of trophic, neurohumoral and synaptic mechanisms that may allow the implanted tissue to promote host brain function and repair.

Anatomy and connectivity of intrastriatal striatal transplants

Abbreviations

Grafted neural stem cells develop into functional pyramidal neurons and integrate into host cortical circuitry.

In vitro expanded neural stem/progenitor cells can undergo region-specific differentiation after transplantation to the developing or adult brain, and display morphologies and markers characteristic of mature neurons. Here we have used patch-clamp techniques to explore whether grafted stem cells also can develop physiological properties of mature neurons and become functionally integrated within host neural circuitry. The immortalized neural progenitor cell line, RN33B, prelabeled with GFP by using a lentiviral vector, was transplanted into the cortex or hippocampus of neonatal rats. We found that the grafted GFP-positive cells differentiated into cells with morphological features of cortical or hippocampal pyramidal neurons, and that many of them had established appropriate cortico-thalamic and contralateral hippocampal connections, respectively, as revealed by retrograde tracing. Whole-cell patch-clamp recordings from grafted cells with morphological characteristics of pyramidal neurons showed that they were able to generate action potentials, and received functional excitatory and inhibitory synaptic inputs from neighboring cells. These data provide evidence that grafted neural progenitors can differentiate into morphologically mature pyramidal projection neurons, establish appropriate long-distance axonal projections, exhibit normal electrophysiological properties, and become functionally integrated into host cortical circuitry.

Transplantation of human neural progenitor cells into the neonatal rat brain: extensive migration and differentiation with long-distance axonal projections.

Here we examined the ability of human neural progenitors from the embryonic forebrain, expanded for up to a year in culture in the presence of growth factors, to respond to environmental signals provided by the developing rat brain. After survival times of up to more than a year after transplantation into the striatum, the hippocampus, and the subventricular zone, the cells were analyzed using human-specific antisera and the reporter gene green fluorescent protein (GFP). From grafts implanted in the striatum, the cells migrated extensively, especially within white matter structures. Neuronal differentiation was most pronounced at the striatal graft core, with axonal projections extending caudally along the internal capsule into mesencephalon. In the hippocampus, cells migrated throughout the entire hippocampal formation and into adjacent white matter tracts, with differentiation into neurons both in the dentate gyrus and in the CA1-3 regions. Directed migration along the rostral migratory stream to the olfactory bulb and differentiation into granule cells were observed after implantation into the subventricular zone. Glial differentiation occurred at all three graft sites, predominantly at the injection sites, but also among the migrating cells. A lentiviral vector was used to transduce the cells with the GFP gene prior to grafting. The reporter gene was expressed for at least 15 weeks and the distribution of the gene product throughout the entire cytoplasmic compartment of the expressing cells allowed for a detailed morphological analysis of a portion of the grafted cells. The extensive integration and differentiation of in vitro-expanded human neural progenitor cells indicate that multipotent progenitors are capable of responding in a regionally specific manner to cues present in the developing rat brain.

Connectivity of striatal grafts implanted into the ibotenic acid-lesioned striatum--I. Subcortical afferents.

Subcortical afferents to transplants of fetal striatal tissue, implanted into the excitotoxically lesioned striatum of adult recipient rats, were studied with retrograde and anterograde axonal tracers and immunohistochemistry. One week after a striatal ibotenic acid lesion, involving most of the head of the caudate-putamen, a suspension of fetal striatal tissue (embryonic day 14-15) was injected into the lesioned area. In one group of rats, the ibotenic acid lesion was preceded (10 days) by large intrastriatal injections of True Blue, with injection sites matching the area to be lesioned. This was done to retrogradely pre-label the host brain afferents to the area of the striatum later to be lesioned and grafted. At 3 or 6 months post-transplantation, small injections (50 nl) of rhodamine-labelled latex beads were made into the striatal grafts. In animals where the injections were confined to the graft, retrogradely labelled host brain neurons were found in the thalamus, the substantia nigra, amygdala and dorsal raphe nucleus. Double-labelling analysis revealed that the vast majority of the rhodamine bead-labelled neurons also contained True Blue, which indicates that the host afferents to the graft, to a large extent, were derived from the neurons which normally project to the area of the caudate-putamen which was lesioned by the ibotenic acid injection. To further substantiate these observations a second group of lesioned and grafted animals received unilateral wheatgerm agglutinin-horseradish peroxidase injections into the ipsilateral host thalamus at 4 months post-transplantation in order to anterogradely label the host thalamostriatal axons. In a third group of animals serotonin immunocytochemistry was performed in order to detect possible afferents from the raphe nuclei. In contrast to the serotonin-containing fibers, which were fairly evenly distributed throughout the graft tissue, the peroxidase-labelled thalamic afferents were most prominent in the peripheral zones of the grafts and they were densely aggregated at the graft-host interface. The combined results provide evidence that the intrastriatal grafts receive afferents from the host substantia nigra, thalamus, amygdala and dorsal raphe nucleus, but with different distributions. The afferents from the substantia nigra, amygdala and raphe nuclei seem to distribute throughout the grafted tissue, although they are most dense in the peripheral parts, whereas the thalamic afferents are largely confined to the peripheral areas of the transplants and to the graft-host interface.

Migration patterns and phenotypic differentiation of long-term expanded human neural progenitor cells after transplantation into the adult rat brain.

We have examined long-term growth-factor expanded human neural progenitors following transplantation into the adult rat brain. Cells, obtained from the forebrain of a 9-week old fetus, propagated in the presence of epidermal growth factor, basic fibroblast growth factor, and leukemia inhibitory factor were transplanted into the striatum, subventricular zone (SVZ), and hippocampus. At 14 weeks, implanted cells were identified using antisera recognizing human nuclei and the reporter gene green fluorescent protein. Different migration patterns of the grafted cells were observed: (i) target-directed migration of doublecortin (DCX, a marker for migrating neuroblasts)-positive cells along the rostral migratory stream to the olfactory bulb and into the granular cell layer following transplantation into the SVZ and hippocampus, respectively; (ii) non-directed migration of DCX-positive cells in the grey matter in striatum and hippocampus, and (iii) extensive migration of above all nestin-positive/DCX-negative cells within white matter tracts. At the striatal implantation site, neuronal differentiation was most pronounced at the graft core with axonal projections extending along the internal capsule bundles. In the hippocampus, cells differentiated primarily into interneurons both in the dentate gyrus and in the CA1-3 regions as well as into granule-like neurons. In the striatum and hippocampus, a significant proportion of the grafted cells differentiated into glial cells, some with long processes extending along white matter tracts. Although the survival time was over 3 months in the present study a large fraction of the grafted cells remained undifferentiated in a stem or progenitor cell stage as revealed by the expression of nestin and/or GFAP.

Projection neurons in fetal striatal transplants are predominantly derived from the lateral ganglionic eminence

In the present study, we have characterized aspects of integration, growth and phenotypic differentiation of embryonic grafts derived from the selective dissection of either the lateral or medial portion of the ganglionic eminences of the rodent forebrain. Donor tissues were derived from embryonic day 15 rat, or embryonic day 14 mouse embryos, and injected, as single cell suspensions into the striatum or substantia nigra of adult rats previously subjected to an intrastriatal ibotenic acid lesion. Two to six weeks following grafting, immunocytochemical detection of DARPP-32, the 32,000 mol. wt dopamine- and cyclic AMP-regulated phosphoprotein, was used to identify areas with a striatum-like phenotype within both the intrastriatal and the intranigral grafts. It was thus revealed that all the lateral ganglionic eminence grafts, irrespective of their placement, were dominated by striatum-like tissue (up to 90% of the total graft volume), while the medial ganglionic eminence transplants were only sparsely positive (< 10% of the total graft volume). These striatum-like regions of the grafts were selectively innervated by tyrosine hydroxylase immunopositive fibres from the host substantia nigra. Furthermore, axons derived from the lateral ganglionic eminence mouse grafts placed in the striatum, as detected by the mouse-specific neuronal marker M6, showed a more extensive and directed outgrowth towards the globus pallidus when compared to fibres emanating from the medial ganglionic eminence grafts. Mouse lateral and medial ganglionic eminence grafts placed into the substantia nigra exhibited similar fibre outgrowth patterns; both types of grafts thus innervated the substantia nigra-pars reticulata and extended axons into the cerebral peduncle. These results show that DARPP-32-positive striatal projection neurons are derived, for the most part, from the lateral ganglionic eminence and that the restricted lateral ganglionic eminence dissection provides a more optimal source of striatal tissue for grafting in the rat Huntington model.

Connectivity of striatal grafts implanted into the ibotenic acid-lesioned striatum—III. Efferent projecting graft neurons and their relation to host afferents within the grafts

Efferent projections of intrastriatally implanted striatal neurons have been studied using a combination of anterograde and retrograde axonal tracers. Adult rats subjected to a unilateral ibotenic acid lesion of the head of the caudate putamen received cell suspension grafts obtained from 14 15-day-old striatal primordia. Three and a half to 20 months after transplantation the rats received either intratransplant injections of the anterograde axonal tracer Phaseolus vulgaris leucoagglutinin or injections of fluorescent retrograde tracers. Fluoro-Gold and rhodamine-labelled latex beads, into the host globus pallidus and substantia nigra. Injections of Phaseolus vulgaris leucoagglutinin located entirely within the grafts labelled axons that ramified extensively within the tissue itself, as well as axons that extended caudally, across the graft host border, along the myelinated fascicles of the internal capsule to arborize in the medial parts of the host globus pallidus. A few axons also reached the entopeduncular nucleus. Injections of Fluoro-Gold into the host globus pallidus labelled large numbers of graft neurons, which had a prominent patchy distribution and were most abundant in the caudal portions of the grafts. Clear retrograde labelling was also seen after injection of Fluoro-Gold or rhodamine beads into the host substantia nigra, although the number of labelled graft neurons was 30-50 times lower than that seen after pallidal injections. Combined injections of Fluoro-Gold into the pallidus and rhodamine beads into the nigra showed that the vast majority of cells labelled from the nigra were also labelled by Fluoro-Gold from the pallidus. In some of the grafted and Fluoro-Gold-injected animals, the fetal donor tissue had been labelled with [3H]thymidine prior to transplantation. Many examples of neurons labelled with both [3H]thymidine and Fluoro-Gold were found after tracer injections into the host globus pallidus, and double-labelled neurons were identified also after Fluoro-Gold injections into the host substantia nigra. In several animals retrograde tracing was combined with labelling of host dopaminergic afferents (by tyrosine hydroxylase immunohistochemistry) and cortical afferents (by injections of Phaseolus vulgaris leucoagglutinin into the host frontal cortex). Comparison of adjacent sections revealed a striking overlap between the patches of Fluoro-Gold-labelled graft neurons (labelled from the host pallidum) and the dense patches of tyrosine hydroxylase-positive terminals. In addition, many of the Fluoro-Gold-labelled cell patches received a high density of cortical afferents labelled by Phaseolus vulgaris leucoagglutinin.(ABSTRACT TRUNCATED AT 400 WORDS)

Striatal c-fos Induction by Cocaine or Apomorphine Occurs Preferentially in Output Neurons Projecting to the Substantia Nigra in the Rat

Fluorogold or rhodamine‐labelled latex beads were injected in the substantia nigra (SN) or the globus pallidus (GP) in order retrogradely to label striatal output neurons that project to the two target structures. Ten days later, striatal c‐fos was induced by systemic administration of cocaine (five normal rats; 25 mg/kg cocaine i.p. 2 h before killing) or apomorphine (five unilaterally dopamine‐denervated rats; 0.25 mg/kg apomorphine s. c. 2 h before killing), and detection of the Fos protein in the striatum was achieved by immunofluorescence. Sections through the caudate‐putamen that displayed good labelling from both SN and GP were selected for a quantitative analysis: the number of retrogradely labelled cells that exhibited Fos immunoreactivity, as well as the total number of retrogradely labelled cells located within a grid (0.16 mm2 in size) were counted manually at 25 x magnification. Cocaine induced a proportionally higher c‐fos expression in striate‐nigral compared to striate‐pallidal neurons, whereas apomorphine activated Fos almost exclusively in striate‐nigral neurons. The present findings are consistent with the idea that striatal c‐fos induction by dopaminergic agents is primarily mediated by an interaction with D1 ‐receptors, which are thought to be selectively localized on neurons projecting to SN.

Intrinsic Organization and Connectivity of Intrastriatal Striatal Transplants in Rats as Revealed by DARPP-32 Immunohistochemistry: Specificity of Connections with the Lesioned Host Brain

Intrastriatal grafts of tissue obtained from the striatal or neocortical primordia of rat fetuses have been studied with respect to their intrinsic organization and connectivity using antibodies to DARPP‐32 in combination with acetylcholinesterase (AChE) histochemistry, tyrosine hydroxylase (TH) immunocytochemistry, and anterograde and retrograde axonal tracing techniques. The striatal grafts were characterized by distinct patches of DARPP‐32‐immunoreactive neurons, which were identical to the densely AChE‐positive patches stained in adjacent sections from the same specimens. The non‐patch areas possessed only few DARPP‐32‐positive neurons and contained only sparse AChE‐positive fibres. The cortical grafts, by contrast, contained no neurons with clear‐cut DARPP‐32‐positivity and they exhibited a sparse, evenly distributed AChE fibre network, similar to that seen in the non‐patch areas of the striatal grafts.