Individual Peculiarities of the Development and Differentiation of Embryonic Neocortex Transplants in Intact Adult Mouse Brain.

We studied individual peculiarities of the development and differentiation of allogeneic transplants of neocortical cells isolated from embryos at different stages of development in intact brain of adult mice. Despite standard transplantation technique, intraparenchymal grafts considerably varied in size, morphology, and structural organization. The cells in the transplants developing inside the brain ventricles of the recipient formed histotypical structures resembling organoids. Transplants of each age group (12.5, 14.5, and 19.5 days) demonstrated individual peculiarities of cell migration, differentiation, and fiber growth. Only from cells of 12.5-day transplants formed spiny pyramidal neurons typical of V layer of the cerebral cortex. Differentiation of catecholaminergic neurons untypical of brain cortex was observed only in 14.5-day transplants. In few transplants of each age group, extensive cell migration from the transplant was observed. In some transplants, dense astrocyte accumulation was seen. In all cases (n=52), the response of the recipient's glia to the transplant was observed, but formation of an extensive glial barrier was noted only in one case. Our findings suggest that the entire range of the results determined by individual peculiarities of the transplant growth and recipient's response should be thoroughly realized when introducing the methods of neurotransplantation into regenerative medicine.

[Neurons with Different Neurotransmitters in Embryonic Neocortical Allografts in the Rat Sciatic Nerve].

Different subsets of interneurons in the Wistar rat neocortex and in neocortical transplants developing in a damaged nerve were identified by the following immunohistochemical markers: glutamate decarboxylase (GAD 67) for GABAergic nerve cells, NO-synthase (NOS) for NO-ergic neurons, choline acetyltransferase (ChAT) for cholinergic cells, and tyrosine hydroxylase for catecholaminergic structures. Twenty-eight days after surgery, individual GAD 67-ir, NO-ir, ChAT-ir, and very rarely TH-ir cells were detected in the graft. It was shown that the number of GAD 67-ir neurons per unit area in the grafts was less than in the rat neocortex P20.

[Phenotypic Differentiation of Neurons in Intraocular Transplants].

Neurochemical differentiation of neurons in transplants developing in rat anterior eye chamber was studied. Pieces of the somatosensory neocortex area, isolated from 17-day fetuses of Wistar rats, were used for the transplantation. The general cytological analysis and immunochemical identification of GABAergic neurons in neocortical transplants and in the appropriate brain area of the recipient rats (control) were carried out after 6 months. Cytoarchitectonics typical for neocortex was not revealed in the transplants. Furthermore, a 1.4-fold decrease in numerical density of the entire neuron population was found compared to the control. The proportion of GABAergic nerve cells in the transplanted tissue was reduced even more dramatically­ by 13.1 times. The dimensions of all types of neurons, especially GABAergic cells, were greater in the transplants in oculo compared to neocortex in situ. The increase in size occurred mostly due to the cytoplasm. Thus, the nuclei of GABA-positive neurons in the transplants were larger by 1.2 times compared to the control and their perikarya were larger by 1.5 times. The obtained results showed that the conditions in the anterior eye chamber the most dramatically affect the differentiation of GABAergic neurons, and cell hypertrophy, probably, is the functional compensation of the decrease in their number. Considering the literature data on the increased excitability and synchronized neuronal activity in the intraocular transplants, it can be assumed that these transplants can be used as a model for studying the cellular mechanisms of nervous tissue epileptization under disinhibition conditions.

[Development of dissociated cells of various rat cns primordia after transplantation into the damaged nerve].

The purpose of this paper was to examine the possibilities of engraftment, and to study the differentiation of the dissociated cells from the embryonic primordia of the spinal cord and the neocortex of Wistar rats, after their transplantation into the sciatic nerve of adult animals. The cell suspension obtained as a result of a dissociation of fragments of the cervical spinal cord and the anterior cerebral vesicle from rat fetuses at day 15 of development, was injected into the proximal segment of a previously damaged sciatic nerve. Using the immunocytochemichal marker of neural stem/progenitor cells (Msi-1) the transplanted cells were identified in the nerve trunks after 1 day after the operation. After 21 day some of these cells underwent differentiation into NeuN-immunopositive neurons, however their number was small. Thus, dissociated precursor cells from embryonic rat spinal cord and neocortex survive for three weeks under conditions of transplantation into the damaged nerve and retain the ability to differentiate into neurons, but the number is small. Most of the cells in the neocortex transplants, unlike those from spinal cord transplants, within 21 days after the operation were represented by the ependymocytes.

Astrogliogenesis in heterotopic allotransplants of rat embryonic neocortex.

We studied gliogenesis in transplants of rat embryonic neocortex (E14-15) in 3, 7, 15, and 30 days and 12-13 months after transplantation into the sciatic nerve of adult animals. Immunogistochemical reactions to intermediate filament proteins nestin, vimentin, glial fibrillary acidic protein were used. In transplants, vimentin- and nestin-positive precursor cells differentiate into astrocytes earlier that in the developing rat neocortex (in situ). One year after transplantation, some astrocytes start to express nestin and vimentin, which attests to the development of reactive gliosis in the transplants.

Spatial and temporal bias in the mitotic origins of somatostatin- and parvalbumin-expressing interneuron subgroups and the chandelier subtype in the medial ganglionic eminence.

GABAergic interneurons modulate cortical activity through the actions of distinct subgroups. Recent studies using interneuron transplants have shown tremendous promise as cell-based therapies for seizure disorders, Parkinson's disease, and in the study of neocortical plasticity. Previous reports identified a spatial bias for the origins of parvalbumin (PV)- and somatostatin (SST)-expressing interneuron subgroups within the medial ganglionic eminence (MGE). In the current study, the mitotic origins of these interneurons are examined by harvesting MGE cells at 2 time points and evaluating their neurochemical profiles after transplantation into neonatal mouse cortex. Although the dorsal MGE (dMGE)-SST and ventral MGE (vMGE)-PV bias were confirmed, both subgroups originate from progenitors located throughout the MGE. The dMGE bias was also found for SST subgroups that coexpress calretinin or reelin. In contrast, another major subgroup of SST interneuron, neuropeptide Y-expressing, does not appear to originate within the MGE. Finally, novel evidence is provided that a clinically important subtype of PV-expressing interneuron, the chandelier (axo-axonic) cell, is greatly enriched in transplants from the vMGE at embryonic day 15. These findings have important implications both for the study of interneuron fate determination and for studies that use interneuron precursor transplantation to alter cortical activity.

Serotonin is involved in the regulation of histogenetic processes in rat embryonic neocortex.

We compared the dynamics of the development of ectopic transplants of embryonic (day 14) primordial neocortex from rats injected with serotonin inhibitor (para-chlorophenylalanine; 400 mg/kg) on day 11 of pregnancy and transplants of similar primordial neocortex incubated before transplantation in a medium with serotonin (3 microg/ml). The study of mitotic activity and differentiation of transplanted cells showed that serotonin promoted survival of the transplanted neuroepithelial cells and their differentiation into nerve cells, and is involved in the regulation of their proliferation. We hypothesized that serotonin accelerated the cell cycle of transplanted cells, thus accelerating the neuron differentiation.

[Study of effect of serotonin on histogenesis of rat embryonal neocortex at model of ectopic neurotransplantation].

A comparative study has been performed of dynamics of development of ectopic transplants of embryonal (E14) neocortex anlages obtained from intact rats and from the rats administered with serotonin inhibitor para-chlorophenylalanine (PCPA) at the 11th day of pregnancy as well as after incubation of such anlages in the serotonin-containing medium. The goal of this work was to elucidate effect of serotonin on division and differentiation of embryonal neocortex cells. Study of degeneration, mitotic activity, and differentiation (by using immunohistochemical detection of nerve cell nuclear protein--NeuN) of transplanted cells has allowed establishing that serotonin promotes survival and differentiation of transplant neuroepithelial cells as well as participates in regulation of their proliferation. It is suggested that serotonin produces stimulation effect on the cell cycle rate of transplantated cells to thereby accelerate neuronal differentiation.

Engraftment and differentiation of neocortical progenitor cells transplanted to the embryonic brain in utero.

Transplantation of neural progenitors or stem cells is a most useful tool to investigate the relative contribution of cell-autonomous mechanisms and environmental cues in the regulation of cell specification and differentiation during CNS development. To assess the capability of neocortical progenitor cells to integrate into foreign brain regions, here we examined the fate of precursor cells isolated from the dorsal telencephalon of E12 ss-actin-EGFP transgenic mouse embryos after heterotopic/heterochronic transplantation to the E16 rat brain in utero. Our observations show that donor cells were able to penetrate, survive and produce mature cell types into wide regions of the host CNS. Namely, EGFP-positive cells acquired site-specific neuronal identities in many telencephalic regions, including neocortex, hippocampus, olfactory bulb and corpus striatum. In contrast, incorporation into more caudal sites was much less efficient. A fraction of donor cells formed large aggregates that remained segregated from the host milieu. Such aggregates contained mature neurons and glia, including some EGFP-negative elements of host origin, and developed the complex organization of the mature nervous tissue. On the other hand, transplanted cells that engrafted in the parenchyma of extratelencephalic regions predominantly generated glial types. The few neurons failed to acquire obvious site-specific phenotypic traits and did not integrate into the local host architecture. Altogether, our observations indicate that E12 neocortical progenitors are already committed towards regional identities and are unable to modify their phenotypic choices when exposed to heterotopic environmental conditions along different rostro-caudal domains of the embryonic CNS.

Transplantation of embryonic porcine neocortical tissue into newborn rats.

Several previous studies, suggesting the potential use of embryonic xenografts in the treatment of neurological disorders, indicate that neural growth and axonal guidance factors may function across species. In this light, blocks of fetal porcine neocortex were grafted into small cortical lesion cavities made in newborn rats. Sacrifice at 3-12.5 weeks posttransplantation revealed healthy looking grafts in several animals. Apparent graft rejection evidenced by areas of necrosis and OX1 reactivity was observed in some of the older transplants. Treatment of nursing mothers or of postweaning newborns with cyclosporin A did not appear to promote graft survival. Some transplants grew to extremely large proportions and were characterized by bands of cells and bundles of axons as observed using immunohistochemical staining for pig neurofilament. Neurofilament-positive axons projected from several of the grafts to course through the corpus callosum to the contralateral cortex or to course ipsilaterally within the subcortical white matter, where labeled fibers could be traced to the midbrain crus cerebri in older transplants. Bundles of axons were also observed coursing within the ipsilateral caudate putamen where terminal branching was apparent. The normal course of transplant efferents within the host brain indicates that growing pig axons can respond to rodent axonal guidance factors.

Suspensional reaggregates of human foetal neocortex and tegmentum as objects of neurotransplantation.

Suspensional reaggregates were obtained from human neocortical and tegmental anlagen (7 weeks of gestation), using 0.1% tripsin solution, and cultivated in Medium 199. Suspensional reaggregates, formed after 2 days in vitro, were grafted into the Wistar rat striatum. Incipient stages of histogenesis in the reaggregates and their interaction with host brain were investigated using light and electron microscopy, with antibodies against vimentin, glial fibrillary acidic protein (GFAP), proliferating cell nuclear antigen (PCNA), ferritin, as well as lectin ricinus communis agglutinin (RCA). The reaggregates showed a low level of tissue organisation. An intermediate condition between suspension and the true tissue could be observed in them. These reaggregates had two evident features: a rather irregular cell arrangement (without parallel bundles of radial glia), and the presence of special intercellular junctions. Some cells made up fragments of neuroepithelial sheet in the form of true rosettes. The one-week-old grafts were integrated with the host brain as well as dissociated and contained host astrocytes. Degenerated cells and detritus appeared rarely. The data of this work let us conclude that the suspensional aggregate grafting combines some advantages of suspensional and solid grafting methods.

Long-term injured purkinje cells are competent for terminal arbor growth, but remain unable to sustain stem axon regeneration.

Long-distance axon regeneration requires the activation of a specific set of neuronal growth-associated genes. Adult Purkinje cells fail to upregulate these molecules in response to axotomy and show extremely weak regenerative properties. Nevertheless, starting from several months after injury, transected Purkinje axons undergo spontaneous sprouting. Here, we asked whether long-term injured Purkinje cells acquire novel intrinsic growth properties that enable them to upregulate growth-associated genes and sustain axon regeneration. To test this hypothesis, we examined axon growth and cell body changes in adult rat Purkinje neurons following axotomy and implantation of embryonic neocortical tissue or Schwann cells into the injury track. Purkinje cells that survived over 6 months after injury/transplantation displayed profuse sprouting in the injured cerebellum and developed extensive networks of terminal branches into embryonic neocortical grafts. In addition, severed Purkinje axons exposed to these transplants 6 months after injury grew faster than their counterparts confronted with the same environment immediately after axotomy. Nevertheless, long-term injured Purkinje cells failed to regenerate stem neurites into Schwann cell grafts, and, under all experimental conditions, they did not upregulate growth-associated molecules, including c-Jun, GAP-43, SNAP-25, and NADPH-diaphorase. These results indicate that the long-term injured Purkinje cells remain unable to activate the gene program required to sustain axon regeneration and their plasticity is restricted to terminal arbor remodeling. We propose that the delayed growth of injured Purkinje cells reflects an adaptive phenomenon by which the severed axon stump develops a new terminal arbor searching for alternative connections with local partners.

Late-stage immature neocortical neurons reconstruct interhemispheric connections and form synaptic contacts with increased efficiency in adult mouse cortex undergoing targeted neurodegeneration.

In the neocortex, the effectiveness of potential cellular repopulation therapies for diseases involving neuronal loss may depend critically on whether newly incorporated cells can differentiate appropriately into precisely the right kind of neuron, re-establish precise long-distance connections, and reconstruct complex functional circuitry. Here, we test the hypothesis that increased efficiency of connectivity could be achieved if precursors could be more fully differentiated toward desired phenotypes. We compared embryonic neuroblasts and immature murine neurons subregionally dissected from either embryonic day 17 (E17) (Shin et al., 2000) or E19 primary somatosensory (S1) cortex and postnatal day 3 (P3) purified callosal projection neurons (CPNs) with regard to neurotransmitter and receptor phenotype and afferent synapse formation after transplantation into adult mouse S1 cortex undergoing targeted apoptotic degeneration of layer II/III and V CPNs. Two weeks after transplantation, neurons from all developmental stages were found dispersed within layers II/III and V, many with morphological features typical of large pyramidal neurons. Retrograde labeling with FluoroGold revealed that 42 +/- 2% of transplanted E19 immature S1 neurons formed connections with the contralateral S1 cortex by 12 weeks after transplantation, compared with 23 +/- 7% of E17 neurons. A greater percentage of E19-derived neurons received synapses (77 +/- 1%) compared with E17-derived neurons (67 +/- 2%). Similar percentages of both E17 and E19 donor-derived neurons expressed neurotransmitters and receptors [glutamate, aspartate, GABA, GABA receptor (GABA-R), NMDA-R, AMPA-R, and kainate-R] appropriate for endogenous adult CPNs progressively over a period of 2-12 weeks after transplantation. Although P3 fluorescence-activated cell sorting-purified neurons also expressed these mature phenotypic markers after transplantation, their survival in vivo was poor. We conclude that later-stage and region-specific immature neurons develop a mature CPN phenotype and make appropriate connections with recipient circuitry with increased efficiency. However, at postnatal stages of development, limitations in survival outweigh this increased efficiency. These results suggest that efforts to direct the differentiation of earlier precursors precisely along specific desired neuronal lineages could potentially make possible the highly efficient reconstruction of complex neocortical and other CNS circuitry.

Changes in astroglial GLT-1 expression after neural transplantation or stab wounds.

Uncontrolled release of glutamate from damaged brain initiates events that result in excitotoxic neuronal death. Glutamate uptake by specialized astroglial transporters is essential for control of extracellular glutamate levels. Many studies have demonstrated a reduction in astrocytic GLT-1 expression after different forms of injury. Because extensive neuronal death does not occur after direct cortical stab wounds and viable developing neurons populate fetal CNS grafts, we hypothesized that reactive astroglia associated with these procedures might maintain or up-regulate GLT-1. We examined the temporal and spatial distribution of GLT-1, GFAP and nestin proteins by confocal double-label immunohistochemistry combined with a new methodology in which precise brain areas are microdissected and analyzed for protein content by immunoaffinity chromatography. In stab wounds, GLT-1 protein content did not change compared to normal cortex, as determined by direct protein measurements; GLT-1 colocalized with nestin- and GFAP(+) astroglia adjacent to the lesion. In contrast, host reactive astroglia adjacent to grafts significantly upregulated GLT-1 by 3 days postoperative. The GFAP protein analysis suggests that increased GLT-1 is not the result of greater numbers of activated astroglia around grafts, but that developing graft tissue influences adjacent host astroglia to upregulate GLT-1. GLT-1 protein within grafts was rapidly accelerated to mature levels by just three days, and was expressed by the nestin(+) cell population. These data, which demonstrate immunoexpression of GLT-1 protein combined with a new method for protein measurement in situ indicate that, in contrast to other injury models, astroglial GLT-1 is upregulated or maintained following invasive CNS procedures. (c)2002 Elsevier Science (USA).

Attraction exerted in vivo by grafts of embryonic neocortex on developing thalamic axons.

In a previous study we provided evidence that embryonic (E) day 16 frontal cortical cells grafted into the occipital cortex of newborn rats receive inputs from the ventrolateral (VL) and ventromedial (VM) thalamic nuclei which, normally, project to the frontal cortex (25). The present study was designed to examine further the conditions of development of the thalamic innervation of heterotopic neocortical grafts. We demonstrate that VL/VM axons do not provide transitory aberrant input to the occipital cortex either in intact newborn animals or in rats having received neonatal occipital lesion and subsequent graft of E16 occipital cells. These findings indicate, therefore, that the VL/VM projection to the graft does not result from the stabilization of an initial widespread cortical projection from these thalamic nuclei occurring either spontaneously or in response to the lesion and homotopic transplantation procedures. We also show that the VL/VM projection to frontal-to-occipital grafts develops within a few days posttransplantation and is maintained in adulthood. Finally, this study establishes that most VL/VM axons which enter the grafts are not collaterals of thalamofrontal axons. After having reached the cortex, they proceed caudally primarily within the infragranular layers. The findings of this and previous (25) in vivo studies for the first time provide evidence that developing thalamic axons have the capacity to respond to signals from grafts of E16 cortical cells and are capable of deviating their trajectory to establish contact with the grafts. Only those axons arising from thalamic nuclei appropriate for the cortical locus of origin of the grafted cells respond to the guidance signals. The mechanisms by which the thalamic axons find their way to the graft probably rely on cell-contact signaling and/or long-range attraction exerted by diffusible molecules.

[Effect of the foreign gene GDNF on development of homo- and xenografts in the rat brain]. / Vliianie chuzherodnogo gena GDNF na razvitie transplantatov i ksenotransplantatov v mozge krys.

A transgenic line of Drosophila melanogaster was selected which carried the following genes: Delta, lacZ (for bacterial galactosidase), and human GDNF (for glial cell line-derived neurotrophic factor). Drosophila neuroectodermal embryonic cells were transplanted with the embryonic neurohomografts into the occipital brain region of an adult rat. Xenografts were found to block scar formation at the graft-host tissue boundary, stimulated homograft development (so that it was twice as large as the control homograft transplanted alone with no xenograft added), and noticeably improved vascularization of the homograft area.

[Features of the development of homo- and heterotopic allotransplants of rat embryonal neocortex]. / Osobennosti razvitiia gomo- i geterotopicheskikh allotransplantatov émbrional'nogo neokorteksa krys.

Mechanisms of regulation of cell division in the developing neocortex are largely unknown. The aim of the present study was to investigate the influence of a microenvironment on the fetal neocortex histogenesis. The fetal neocortex from 15-day old Wistar rat embryo was grafted into the neocortex, crushed sciatic nerve and anterior chamber of eye of adult rats. A comparative study of graft development was carried out on 1, 3, 7, 10, 30 days using histological (Nissl stain, hematoxylin-eosin) and immunohistochemical (monoclonal antibody to proliferating cell nuclear antigen, and to glial fibrillary acidic protein) methods. Grafted neuroepithelial cells proliferated in grafts that developed in the neocortex and the anterior chamber of eye for 7 days, and in the sciatic nerve for 10 days. In all grafts differentiating neuroblasts, young neurons and mature neurons were observed 7, 10 and 30 days later, respectively. In 10 days, transplants in the nerve have a glial capsule, in contrast to other sites of grafting. The capsule consists of ependymocytes with microvilli and cilia 30 days later. These cells are GFAP-positive. Our results indicate epigenetic influence on the development of neuroepithelial precursors. The microenvironment of the peripheral nerve is suggested to promote glyogenesis in developing grafts. Afferent inputs do not influence the proliferative potency of brain cell precursors.

Expression of zinc-positive cells and terminals in fetal neocortical homografts to adult rat depends on lesion type and rearing conditions.

Zinc-positive neurons and terminals, known to be associated with the glutamatergic projections in the brain, can be demonstrated by the histochemical Timm method and later modifications thereof. The adult rat neocortex contain a uniform lamination of zinc-positive cells with specific projections to, e.g., the striatum. We have previously reported that fetal neocortical grafts implanted in the adult rat neocortex combined with rearing in an enriched environment can improve behavioral functions and reduce the secondary atrophy of thalamus after cortex infarction in adult rats. In order to examine whether the expression of zinc positivity is ontogenetically inherent to neocortical neurons we grafted fetal neocortical tissue to aspiration or ischemic lesions of the frontoparietal neocortex of adult rats, followed by histochemical visualization of the vesicular zinc pool by selenite or sulfide. One further aim of the study was to elucidate to what extent the distribution of zinc-containing neurons and terminals in the grafts depended on rearing under different environmental conditions. The foremost finding of the present study was that the overall density of zinc-containing terminals in fetal cortical transplants placed in brain infarcts of adult spontaneously hypertensive rats is higher when the rats are reared in an enriched environment. Moreover, the presence and expression of zinc-positive neurons and terminals do not seem to be ontogenetically inherent to the cortical neurons as the fetal neocortical grafts placed in aspiration lesions contained no zinc-selenide-positive neurons and few or no zinc-selenide-positive terminals. The presence or expression of zinc-positive cells may thus be induced by ingrowth of fibers and terminals from the host brain as transplants placed in the ischemic lesions expressed both zinc-positive neurons and terminals.

Protein synthesis inhibition in neocortical grafts evaluated by systemic amino acid uptake autoradiography.

The temporal pattern of protein synthesis inhibition was examined in grafted neocortical neurons using [(3)H]valine in vivo autoradiography. Neuronal uptake levels of systemically administered (3)H-labeled amino acids which cross the blood-brain barrier (BBB) via endothelial cell neutral carriers have long been a hallmark in studies of experimental ischemic pathology; there is likely a strong correlation between persistent protein synthesis inhibition and the progression of cell damage. Because the grafting procedure involves the loss of blood flow and the subsequent reperfusion of the donor tissue there are, mechanistically, important similarities to reversible ischemia models. The effects of ischemic injury on grafted CNS neurons are not fully understood. Quantitative analysis of grain distribution in individual graft or control (adjacent host cortex) neurons indicated an initial breakdown of the amino acid barrier system, subsequent recovery, and progressive reduction of amino acid uptake by 1 year. Up to 3 weeks after surgery grafts were flooded with the [(3)H]valine tracer but individual neurons contained relatively few silver grains. After this time, the tracer was normally distributed within graft neurons but at significantly lower levels than in controls. Grain density gradually decreased over time such that 12-month grafted neurons had approximately half that compared to control and only 58% of that in 2-month grafts; the 12-month levels were comparable to those observed at early (10 days) postoperative times. Autoradiography of immunostained sections for MAP-2, SMI 311 (neurofilament marker), and neuron-specific enolase showed reduced expression of these proteins in neurons coupled with weak amino acid tracer uptake. The results further suggest that grafted neurons bear intriguing similarities to neurons placed at ischemic risk, particularly "penumbral" neurons, which are affected by reduced blood flow and are metabolically weakened. The loss of BBB properties in early grafts may also extend to the endothelial cell amino acid carrier system, and the delayed revascularization process could affect neuronal uptake mechanisms.