Human pontine glioma cells can induce murine tumors.

Diffuse intrinsic pontine glioma (DIPG), with a median survival of only 9 months, is the leading cause of pediatric brain cancer mortality. Dearth of tumor tissue for research has limited progress in this disease until recently. New experimental models for DIPG research are now emerging. To develop preclinical models of DIPG, two different methods were adopted: cells obtained at autopsy (1) were directly xenografted orthotopically into the pons of immunodeficient mice without an intervening cell culture step or (2) were first cultured in vitro and, upon successful expansion, injected in vivo. Both strategies resulted in pontine tumors histopathologically similar to the original human DIPG tumors. However, following the direct transplantation method all tumors proved to be composed of murine and not of human cells. This is in contrast to the indirect method that included initial in vitro culture and resulted in xenografts comprising human cells. Of note, direct injection of cells obtained postmortem from the pons and frontal lobe of human brains not affected by cancer did not give rise to neoplasms. The murine pontine tumors exhibited an immunophenotype similar to human DIPG, but were also positive for microglia/macrophage markers, such as CD45, CD68 and CD11b. Serial orthotopic injection of these murine cells results in lethal tumors in recipient mice. Direct injection of human DIPG cells in vivo can give rise to malignant murine tumors. This represents an important caveat for xenotransplantation models of DIPG. In contrast, an initial in vitro culture step can allow establishment of human orthotopic xenografts. The mechanism underlying this phenomenon observed with direct xenotransplantation remains an open question.

Effect of neural transplantation on depressive behavior in rats with lesioned nucleus basalis magnocellularis.

Recent data of our group have shown that bilateral electrolytic lesions of the nucleus basalis magnocellularis (NBM) in rats reduced the escape behavior deficit that occurs in the learned helplessness test. The present study was done to establish the effect of intracerebral neural transplantation on the change in escape behavior of NBM-lesioned adult male Wistar rats in the learned helplessness test. At 2 days (NBM-ET) or 10 days (NBM-DT) after bilateral electrolytic NBM-lesions, small fragments of fetal frontal cortex (18th day of gestation) were allotransplanted into the lesioned NBM. Ten days after neural transplantation, the learned helplessness test was performed. The number of shocks that animals received before making an escape response was significantly reduced in NBM-lesioned rats (p < .001, compared to intact control and sham-operated rats). In comparison to NBM-lesioned and sham-ET rats, the NBM-ET rats showed a marked (p < .001) increase in the number of shocks delivered before the animal made such an escape response. On the other hand, NBM-DT rats did not show this increase. These results indicate that neural transplantation performed at an early time after lesioning of NBM reversed the effect of this lesion in rats exposed to learned helplessness test.

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.

Functional recovery of skilled forelimb use in rats obliged to use the impaired limb after grafting of the frontal cortex lesion with homotopic fetal cortex.

The long-term effect of transplanting embryonic frontal cortex into a unilateral frontal cortex lesion has been studied in adult rats. Before surgery, activity in an open field, muscular strength of both forelimbs, and performance in a paw-reaching-for-food task were scored in 26 rats. In 21 animals a unilateral cortex lesion was then made in the forelimb motor area of the hemisphere contralateral to the preferred paw in the paw-reaching-for-food task, while the other 5 animals were sham-operated. On retesting, the lesion animals changed the preferred paw. A solid homotopic transplant of embryonic tissue (embryonic day 17) was then placed in the lesion cavity in 11 of the lesion rats. Three months later neither lesion alone nor lesion plus transplantation affected open field behavior and muscular strength, but the lesion permanently affected performance in the paw-reaching-for-food task, as shown by a change of preferred paw and a functional deficit in the paw contralateral to the lesion. Transplantation ameliorated the deficits caused by the lesion, but this was only evident when animals were forced to reach with the paw contralateral to the lesion plus transplant. The behavioral results were independent of the size of the lesion and graft. Connections between graft and host tissue were studied by means of the fluorescent tracer 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI). A dense array of labeled fibers was found in the host cortex adjacent to the transplant. The results suggest that functional recovery depends on grafting but is only evident when the animal is obliged to use the affected limb.

Stage of specification of the spinal cord and tectal projections from cortical grafts.

In order to determine the embryonic age at which the hodological phenotype developed by neocortical cells is specified, we have examined the spinal or tectal projections developed by embryonic (E) grafts of presumptive frontal or occipital neocortex placed into the frontal or occipital neocortex of newborn host rats. Grafts of E13, E14 and E16 cells of the frontal cortex transplanted into the occipital cortex of newborns are capable of developing and maintaining in adulthood a spinal cord axon. Grafts of E12 cells do not project to the spinal cord but send fibres to the superficial layers of the tectum. In addition, following transplantation into the frontal cortex, early embryonic (E12) cells from the presumptive occipital cortex are capable of differentiating into neurons with spinal cord projection but are practically incapable of developing a tectal projection. When grafted at E14 into the frontal cortex, occipital cells lose the capacity to project to the spinal cord but become able to send fibres to the tectum. Taken together, these findings indicate that young (E12) embryonic frontal and occipital cortical cells are competent to subsequently differentiate into neurons projecting to the spinal cord or tectum according to instructive signals available in the cortical territory where they complete their development. By E13/E14, some cortical cells are specified and their capacity to contact targets that are not appropriate to their embryonic origin is much reduced. These findings are consistent with the notion that cortical specification involves progressive restriction in cell multipotentiality and fate specification toward region-specific phenotypes.

Neonatal lesion of the rat's frontal cortex and subsequent transplantation of embryonic frontal cortex: evidence of appropriate synaptic integration of the graft neurons within the host thalamo-fronto-striate circuit.

Previous observations in intact rats have indicated that axons from the ventrolateral thalamic nucleus (VL) establish direct axo-somatic or axo-dendritic contacts onto frontal cortical neurons projecting to the striatum. The embryonic frontal cortex was grafted into the damaged frontal cortex of newborn rats to study the capacity of homotopic transplants to restore the thalamo-fronto-striate pathway. Several months later, grafted neurons projecting to the striatum were identified by injecting a retrograde neurotracer (subunit b of the cholera toxin) into the ipsilateral caudate putamen. In the same animal, axons and terminations from the VL were labeled within the transplant with an anterograde neurotracer (Phaseolus vulgaris leuco-agglutinin) injected into the ipsilateral VL. The findings show that VL axons establish direct synaptic contacts onto grafted neurons projecting to the striatum. Although the synaptic contacts were scarce in the transplants, their organization was similar to that observed in intact rats. The contacts were axo-somatic or axo-dendritic. Our observations for the first time indicate that synaptic contacts are formed in cortical grafts and that fetal frontal cortex is susceptible to develop appropriate synaptic integration within the host thalamo-fronto-striate system.

Learning and memory in nucleus basalis magnocellularis-lesioned rats after transplantation of fetal frontal cortex.

The effect of fetal frontal cortex transplantation on behaviour performance was examined in adult male Wistar rats with lesions of the nucleus basalis magnocellularis (NBM). Compared to intact and sham-operated controls, the rats tested ten or twenty days after bilateral electrolytic lesions of NBM exhibited the significant learning and memory impairments (acquisition and performance of two-way active avoidance) whereas spontaneous motor activity was not significantly altered. The animals which received allotransplants of fetal frontal cortex (from 18-day gestational rat fetuses) into NBM, two ("early" transplantation-NBM-ET) or ten ("delayed" transplantation-NBM-DT) days after lesioning, respectively, manifested the complete amelioration of noticed impairments when tested ten days after transplantation procedure. Corresponding sham-transplants groups (NBM-SET and NBM-SDT) showed only slightly improvement of acquisition but not performance of two-way active avoidance. The ability of the transplants to restore learning and memory in the NBM lesioned rats suggests that graft of fetal frontal cortex can functionally influence neuronal activity of the lesioned host brain.

Development of projections from transplants of embryonic medial or lateral frontal cortex placed in the lateral frontal cortex of newborn hosts.

Several recent experiments using neocortical transplantation paradigms indicated that embryonic neurons grafted in a heterotopic locus retain development characteristics corresponding to their site of origin. In the present study, limited portions of lateral (lateral-to-lateral) or medial (medial-to-lateral) sectors of embryonic (E16) frontal cortex were grafted into the lateral frontal cortex of newborn rats. A retrograde tracer was injected 3-4 months later into the dorsomedial or ventrolateral sectors of the host caudate-putamen (CPU). The results indicate that the mediolateral arrangement of striatal projection developed by lateral-to-lateral transplants is virtually identical to that found in intact rats. A very weak proportion of the transplanted cells distribute fibers to the dorsomedial sector of the CPU. In marked contrast, the proportion of efferents from medial-to-lateral transplants projecting to the dorsomedial CPU is by far larger than the one directed to the ventrolateral CPU. Our findings provide evidence that even within one single neocortical area (the frontal neocortex) some degree of prespecification (medial versus lateral patterns of efferent projections) is already present at E16.

Development of spinal cord projections from neocortical transplants heterotopically placed in the neocortex of newborn hosts is highly dependent on the embryonic locus of origin of the graft.

Previous experiments based on heterotopic transplantation paradigms have indicated that the distribution of efferents developed by layer V pyramidal cells seems to be related to where in the neocortex the cells develop and not to where they were generated. The present study was undertaken in an attempt to obtain a quantitative estimation of the weight of extrinsic factors in the development of neocortical efferents. Fragments of embryonic (E15-E19) frontal or occipital cortex were grafted homotopically or heterotopically into the frontal or occipital cortex of newborn rats. As adults, the hosts received an injection of a retrograde tracer into the pyramidal tract decussation, and the distribution of the subsequent cell labeling was examined in each category of transplant. The mean numbers of labeled cells were 725 in frontal-to-frontal transplants and 250 in frontal-to-occipital transplants. In occipital-to-frontal transplants, the numbers of labeled cells were extremely low, ranging from 0 to 14. Finally, as expected, practically no cell labeling was found in occipital-to-occipital transplants. Thus, transplants of presumptive frontal origin systematically develop and maintain in adulthood a spinal cord projection even though they are placed in the host occipital cortex. Conversely, transplants of presumptive occipital origin are practically incapable of maintaining a spinal cord projection in adulthood even though they are placed in the host frontal cortex. It seems, therefore, that the generation of regional differences in efferent connectivity found in the mature cortex depends on early regional specification within the neocortical neuroepithelium.

The topographic distribution of the efferents from neocortical neurons is not only dependent upon where in the neocortex the cells develop. A transplantation study within one single neocortical region.

It has been proposed that the distribution of efferents developed by neocortical neurons depends upon where in the neocortex the cells develop, not where they were generated. However, the capacity of diverse isocortical areas to differentiate connectional characteristics belonging to other isocortical areas has recently been questioned in several experiments using heterotopic transplantation paradigms. The present study was designed to determine whether the principle of multipotentiality is still valid within one single isocortical region. Mediolateral bands of embryonic (E16) frontal neocortex were dissected out and grafted into the left frontal cortex of neonate hosts according to either correct or inverted mediolateral orientation. Five to six months after grafting, a retrograde tracer was injected into the dorsomedial or ventrolateral left neostriatum of the host. The mediolateral distribution of the cell labeling within the transplant was then compared to that of an equivalent frontal cortical area (ECA) in control cases. The results indicate that strips of embryonic frontal neocortex transplanted according to a correct mediolateral orientation are able to develop a projection towards the host striatum whose mediolateral topographical distribution is not significantly different from that arising from the frontal neocortex of control animals. The percentages of transplant cells labeled in the medial or lateral division of the grafts were not significantly different from those found medially or laterally in the ECA in control cases. Following inversion of the mediolateral orientation of the grafts at the time of transplantation, the percentages of cells labeled in the medial or lateral division of the grafts were nearly equal whatever the site of tracer deposit within the host neostriatum. These results indicate that even within one single neocortical region the principle of areal interchangeability is not entirely validated and that the development of neocortical efferents is not only guided by extrinsic factors.

Behavioural consequences of frontal cortex grafts and enriched environments after sensorimotor cortex lesions.

Past studies have experienced difficulty in achieving graft survival and behavioural recovery after sensorimotor cortex lesions. In the present work, adult female rats trained preoperatively to cross a narrow beam for food reward were maintained in standard group cages or an enriched environment, commencing one week after a unilateral lesion. One month post-lesion, half of these rats received multiple suspension grafts of (E20) fetal frontal cortex, placed adjacent to the lesion cavity, and 8 days later recovery of beam-walking skills was examined for a six-week period. The grafts survived in all cases with an appropriate lesion, a notable result given the one month lesion-graft delay, but graft volume was not influenced by postoperative environment. The substantial lesion-induced deficits evident just prior to differential housing showed a marked reduction by the start of post-graft testing, but relative to intact controls a persistent deficit in foot slip errors occurred in all lesion groups. Irrespective of graft status, postoperative enrichment prevented the occurrence of severe foot slips, especially early in retraining. The frontal grafts, however, enhanced beam-walking recovery by reducing the overall frequency of foot slips on early post-grafting sessions, an effect we suggest is related to graft-derived trophic influences, but this measure was not significantly improved by postoperative enrichment.

Efferents of frontal or occipital cortex grafted into adult rat's motor cortex.

Phaseolus vulgaris leucoagglutinin (PHA-L) was used to examine the efferent connectivity of embryonic (E16) frontal (homotopic) or occipital (heterotopic) neocortical transplants placed into--or in the vicinity of--lesion cavities made in the frontal cortex of adult recipients. Homotopic transplants projected towards the host sensorimotor cortex and, in most cases, into the lateral caudate-putamen (CPu). Heterotopic transplants projected into the anterior cingulate cortex and, in most cases, distributed terminals into the medial CPu. It is suggested that embryonic neocortical tissue placed into a damaged cortical site of an adult recipient develops a pattern of efferents corresponding to its cortical origin.

The ontogeny of estrogen receptors in heterochronic hippocampal and neocortical transplants demonstrates an intrinsic developmental program.

We investigated the intrinsic vs. environmental regulation of estrogen receptor (ER) ontogeny in the neocortex, hippocampus and hypothalamus by employing a heterochronic transplantation paradigm. These studies were based on previous reports demonstrating that neural ER develop asynchronously with quantitatively distinct ontogenetic profiles in various brain regions. Fetal (E14-15) hippocampal, frontal cortical or hypothalamic preoptic area (HPOA) primordial tissue was grafted into frontal cortical lesion cavities made in newborn (PND-0) rats. Thus, the grafted tissue was 1 week younger than the host. Two and 4 weeks following transplantation surgery, which corresponds to a theoretical donor age of PND-7 and PND-21, the grafts, a region of the host neocortex surrounding the transplant, and the host hippocampus, frontal cortex or HPOA (depending on graft type) were assayed for ER content using in vitro binding assays. ER concentration in hippocampal grafts at theoretical age PND-7 were significantly higher than those found in the host (PND-14) hippocampus and in the host neocortex adjacent to the transplant. By theoretical graft age PND-21, ER concentration in hippocampal transplants had decreased to levels comparable to those found in the host. This developmental pattern is analogous to that previously reported for the in situ hippocampus. A similar profile of ER concentration corresponding to the donor age developmental timetable was observed in neocortical grafts. ER levels in HPOA grafts did not change from theoretical donor age PND-7 to PND-21, which also corresponds to the normal ontogenetic profile. These data suggest that region-specific developmental patterns of ER expression in the rat brain are specified by embryonic day 14.

Motor deficits are produced by removing some cortical transplants grafted into injured sensorimotor cortex of neonatal rats.

Fetal frontal cortex was transplanted into cavities formed in the right motor cortex of neonatal rats. As adults, the animals were trained to press two levers in rapid succession with their left forelimb to receive food rewards. Once they had reached an optimal level of performance, the effect of removing their transplants was assessed. Surgical removal of transplants significantly impaired the performance of 2 of 4 subjects. Placing a cross-strain skin graft to induce the immunological rejection of the transplants produced a behavioral deficit in 1 of 2 subjects with complete transplant removal. Skin grafts produced no behavioral effects in four subjects that had surviving transplants. Since the motor deficits produced by transplant removal resembled those observed following the removal of normal motor cortex, we propose that these three transplants functioned within the host brain. Histology showed that the procedures used to remove cortical grafts did not injure any host brains. Therefore, host brain damage is unlikely to account for the behavioral deterioration that followed transplant removals.

Fetal cortical transplants reduce motor deficits resulting from neonatal damage to the rat's frontal cortex.

Motor deficits in the execution of grasping movements of the right forelimb were compared in normal female Wistar rats, in animals which sustained a neonatal lesion of the left frontal cortex and in animals which received immediately after the lesion a transplant obtained from the frontal cortex of E16 embryos. Behavioral testing was carried out from postnatal day 48 (D48) to D108. The animals were placed at the center of a circular wire grid which was turned upside down so that they hung by their 4 paws at a distance of 40 cm above the floor. The precision of grasping movements of the right limb and the number of falls were recorded during a 1 min session of active moving across the grid. The lesioned subjects were most impaired on both motor indices whereas the grafted animals although performing slightly poorer than the controls were, however, less impaired than the lesioned animals. Fetal cortical transplants, therefore, seem to promote functional recovery from neonatal cortical damage.

Axotomized, adult basal forebrain neurons can innervate fetal frontal cortex grafts: a double fluorescent tracer study in the rat.

The ability of axonal regeneration of identified adult basal forebrain (BFB) neurons was examined after homotopic grafting of fetal neocortical tissue to a lesion cavity in the frontal neocortex. Using a four step experimental procedure, adult rats first received an injection of the fluorescent dye Fluoro-Gold (FG) into the sensorimotor cortex in order to label those neurons with projections to the area by retrograde axonal transport. After one week the injection area was removed by aspiration, leaving a cavity in the neocortex. One week later a block of fetal (E14) frontal cortical tissue was placed in the cavity. The animals were then allowed to survive for 6 weeks before a second fluorescent tracer, Nuclear Yellow (NY), was injected into the transplant. The animals were sacrificed 24 h later and analyzed by fluorescence microscopy. Both single labeled, FG and NY containing neurons and double labeled neurons containing both tracers were found in the BFB. The results demonstrate that adult BFB neurons can reestablish cortical projections into fetal cortical grafts (double labeled neurons), and they suggest that other BFB neurons, not initially innervating the lesioned cortical area, have sprouted into the transplant (NY labeled neurons).

Transplantation of fetal frontal cortex onto degenerating thalamus of cats and kittens.

Tissue from fetal frontal cortex survived after transplantation onto the surface of the left thalamus in 2 kittens and 2 adult cats which 7 days previously had sustained a left cerebral hemispherectomy. There were nerve fiber connections with host tissue (WGA-HRP, Loyez myelin stain) only in the neonatal animals. The grafts contained surviving neurons in all but in one adult cat which survived 301 days. The grafts had little effects on the retrograde ventral thalamic degeneration typically seen following hemispherectomy. However, the dorsal lateral geniculate nucleus adjacent to the transplants showed reduced neuronal loss and gliosis compared to controls. Magnetic resonance imaging was successfully used to visualize the grafts in vivo and suggested a decrease in size as well as changes in composition for a graft systematically followed for 120 days posttransplantation. Cytochrome oxidase histochemistry indicated sustained metabolic activity in transplants containing surviving neurons. This study introduces the cat as a useful model for brain tissue transplantation in a classical, myelinated sensorimotor system.

Fetal cortical transplants reduce the thalamic atrophy induced by frontal cortical lesions in newborn rats.

Fetal neocortical tissue was grafted into frontal cortex lesion cavities made in newborn rats. After survival periods extending up to 14 months, volumetric measurements of the total thalamus and of the lateral, medial and anterior thalamic compartments showed an amelioration of the thalamic atrophy that normally is found after cortical lesions. These results correspond to previous findings demonstrating interconnections between fetal cortical transplants and the host thalamus.

Intrahippocampal grafts of fetal basal forebrain tissue alter place fields in the hippocampus of rats with fimbria-fornix lesions.

Intrahippocampal grafts of fetal basal forebrain tissue have been shown to restore several aspects of neural function, including some degree of behavioral recovery in spatial memory tasks, in rats with fimbria-fornix lesions. Place fields, the behavioral correlates of complex-spike unit activity recorded in the hippocampus of rats, are altered by fimbria-fornix lesions, and provide an important measure of the functioning circuitry of the hippocampus after grafts. To investigate the effects of grafts on hippocampal circuitry, complex-spike units were recorded while the rats traversed a radial maze. Quantitative analyses of spatial activity showed that units in normal rats had spatially clustered, reliable place fields that were stable despite alterations of the maze. In contrast, units in rats with fimbria-fornix lesions had more dispersed, less reliable place fields that were disrupted when the maze was covered or rotated. Compared to rats with fimbria-fornix lesions, rats with grafts and units with more tightly clustered, more reliable, and more stable place fields when the maze was altered. The results suggest that: (1) fimbria-fornix lesions disrupt some aspects of complex-spike place field activity; (2) the functioning of hippocampal circuitry is influenced by fetal basal forebrain grafts; and (3) the grafts may ameliorate the effects of lesions on spatial behaviors by influencing critical aspects of place field activity in the hippocampus.