Axonal regrowth of layer II-III visual-projecting cortical neurons in rats fails beyond eye opening.

Fetal neurons (embryonic age E16) of occipital origin grafted in the visual cortex of albino rats at increasing postnatal stages (P0, P7, P15, P30, P60, P120) can be activated by photic stimulation. Inputs originate from five major areas of the brain ipsilateral to the graft, namely, the claustrum, the periallocortex/proisocortex, the isocortex, the visual thalamus, and some unspecific subthalamic and hypothalamic nuclei. All inputs decrease in number with the age at which grafting was performed. Isocortical afferents exhibit furthermore a progressive laminar shaping. In neonates, layer II-III and layer V-VI neurons contribute equally to the graft input. In adults, grafts receive prominent input (approximately 70-80%) from layer VI neurons whereas layer II-III neurons account for less than 10%. Proportions of layer IV (approximately 2-4%) and layer V (approximately 15-20%) neurons innervating the graft remain stable, irrespective of the age of the recipient. The adult pattern of connectivity between the host brain and the graft establishes in frontal and temporal areas 1 week earlier than in occipital areas. It is nearly completed in postnatal day 15 (P15) grafted recipients. Supragranular neurons would be thus unable to innervate and to make stable synapses at the graft level beyond P15, i.e., when eyes open. Some infragranular neurons (supposedly remnants of the earliest generated cortical cell population) still have this capacity in adults.

Early (E12) cortical progenitors can change their fate upon heterotopic transplantation.

To help understand how the cortical map is set up during the early stages of corticogenesis, we have examined the developmental fate of embryonic day (E) 12 cortical progenitors in the rat. We have analysed the pattern of thalamic connections and cytoarchitectonic organization developed by progenitor cells removed at E12 from the presumptive parietal or occipital cortex and grafted into the parietal cortex of newborn hosts. Occipital progenitors grafted into the parietal cortex differentiated into neurons that developed reciprocal connections with the ventrobasal complex of the host thalamus. They could also form barrel-like structures, within which axons of the ventrobasal complex were distributed in dense patches. Some of these barrel-like structures were arranged in rows. Moreover, these progenitors failed to develop characteristic traits of occipital cortex cells as they did not establish connections with the dorsal lateral geniculate nucleus. We propose that cortical progenitors are not committed at E12 and, upon heterotopic transplantation, have the capacity to respond to local cues and to subsequently differentiate and maintain major phenotypic characteristics of neurons in their new environment. Only early progenitors are multipotent. By E13/E14, indeed, most cortical cells become irreversibly committed and upon heterotopic transplantation differentiate neurons with phenotypic characteristics of their cortical site of origin (Pinaudeau et al., 2000, Eur. J. Neurosci., 12, 2486-2496).

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.

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.

Specification of somatosensory area identity in cortical explants.

The H-2Z1 transgene is restricted to a subset of layer IV neurons in the postnatal mouse cortex and delineates exactly the somatosensory area. Expression of the H-2Z1 transgene was used as an areal marker to determine when the parietal cortex becomes committed to a somatosensory identity. We have shown previously that grafts dissected from embryonic day 13.5 (E13.5) H-2Z1 cortex and transplanted into the cortex of nontransgenic newborns express H-2Z1 according to their site of origin. Expression was not modified on heterotopic transplantation (). In the present study, whole cortical explants were isolated at E12.5 from noncortical tissues. The explants developed a regionalized expression of H-2Z1, indicating that regionalization takes place and is maintained in vitro. We used this property and confronted embryonic H-2Z1 cortex with presumptive embryonic sources of regionalizing signals in an in vitro grafting procedure. A great majority of E11.5-E13.5 grafts maintained their presumptive expression of H-2Z1 when grafted heterotopically on nontransgenic E13.5-E15.5 explants. However, a significantly lower proportion of E11.5 parietal grafts expressed H-2Z1 in occipital compared with parietal cortex, indicating that somatosensory identity may be partially plastic at E11.5. Earlier stages could not be tested because the E10.5 grafts failed to develop in vitro. The data suggest that commitment to the expression of a somatosensory area-specific marker coincides with the onset of neurogenesis and occurs well before the birth of the non-GABAergic neurons that express H-2Z1 in vivo.

Abnormalities in the development of the tectal projection from transplants of embryonic occipital cortex placed in the damaged occipital cortex of newborn rats.

We have examined the degree of precision in the topographic arrangement of the tectal projection developed by homotopic transplants of embryonic occipital cortex and tried to determine whether the development of the corticotectal projection is exclusively dependent on environmental cues or is also controlled by intrinsic factors. Transplants of embryonic (E16) occipital cortex were grafted into various areas of the occipital cortex (Oc1 or Oc2) of newborn rats and the organization of the tectal projection arising from the transplants was subsequently examined by injecting different neurotracers into the transplants. Our results indicate that in most cases the laminar and tangential distributions of the tectal projections from the transplants were abnormal. Indeed, whatever the location of the transplant in the host occipital cortex and whatever the placement of the injection into the transplant, a hybrid distribution of the tectal labeling was found, reminiscent of the pattern observed following tracer deposits in both Oc1 and Oc2 in intact animals. Since the grafts were composed of cells of both Oc1 and Oc2 embryonic origin, it is likely that the hybrid pattern of efferents reflects the heterogeneity of the embryonic origin of the cells composing the graft. These findings provide evidence that the development of the topographic distribution of neocortical efferents is not only dependent on factors extrinsic to the cortex and further indicate that even within one single cortical region, the occipital cortex, different areas (Oc1 vs Oc2) are not totally interchangeable. These findings might have important implications in transplantation experiments aiming at the reconstruction of damaged neocortical circuitry where a precise "point-to-point" reconstruction of the circuitry is expected.

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.

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.

Behavioral and morphological studies of fetal neural transplants into SCN-lesioned rats.

We have studied the effects of fetal neuronal grafts on the temporal pattern of drinking behavior of suprachiasmatic nuclei (SCN)-lesioned adult rats. Additionally, in an independent set of animals, the immunohistochemical staining for vasopressin, vasoactive intestinal polypeptide, and neuropeptide Y and the retinal connections to the hypothalamus were studied. The behavioral experiments indicate that anterior hypothalamic transplants induced reorganization of the temporal pattern of drinking behavior when placed in the third ventricle of adult hosts bearing complete SCN lesions, but not when placed in a cavity in the occipital cortex. Such rhythmicity persists only when the animals were recorded under constant darkness but not under constant light, indicating that the restored rhythmicity was generated endogenously but that the oscillator was extremely sensitive to light. Fetal occipital cortex induced reorganization of the temporal pattern of previously arrhythmic hosts, but it disappeared when the animals were recorded under constant light or constant darkness. It is clear that this rhythmicity was exogenous. In contrast to the cortical transplants, the hypothalamic transplants showed a morphological organization similar to that found in the normal hypothalamus regardless of their placement in the host brain. From these observations it is concluded that development of neocortex is more affected by environmental factors than that of the hypothalamus. Both hypothalamic and cortical transplants induced sprouting of retinal fibers into the anterior hypothalamus and the grafted tissue. It is possible that such fibers could be the neuroanatomical substrate by which rhythmicity is induced by cortical tissue.

Selective elimination of axons extended by developing cortical neurons is dependent on regional locale: experiments utilizing fetal cortical transplants.

In adult rats, cortical neurons that extend an exon through the pyramidal tract (a major subcortical efferent projection of the neocortex) are limited to layer V of about the rostral two-thirds of the neocortex. In neonates, however, pyramidal tract neurons are distributed throughout the neocortex, but all of those found in certain areas, such as the posterior occipital region (including primary visual cortex) selectively lose their pyramidal tract axon (Stanfield et al., 1982) yet maintain axon collaterals to other subcortical targets (O'Leary and Stanfield, 1985). To determine if the regional location of a developing pyramidal tract neuron critically influences the maintenance or elimination of the axon collaterals it initially extends, pieces of cortex from embryonic day 17 (E17) rat fetuses (exposed to 3H-thymidine on E15) were transplanted heterotopically into the cortex of newborn (PO) rats; rostral cortex was placed into the posterior occipital region (R----O), or posterior occipital cortex into a rostral cortical locale (O----R). The retrograde tracers Fast blue (FB) and Diamidino yellow (DY) were used to assay for the presence of specific populations of cortical projection neurons within the autoradiographically identified transplants. In terms of the extension and maintenance of pyramidal tract axons, the transplanted neurons behave like the host neurons of the recipient cortical region rather than like those of their site of origin. At P40, following FB injections into the pyramidal decussation on P34, pyramidal tract neurons are labeled within the O----R transplants, but none can be labeled within R----O transplants, although in the same R----O cases transplanted neurons are labeled by an injection of DY in the superior colliculus. However, at P13 pyramidal tract neurons can be identified within the R----O transplants, as well as in the host occipital cortex, following injections made on P9, a period when the distribution of pyramidal tract neurons in normal rats is widespread (Stanfield and O'Leary, 1985b). In a second series of host rats, on P34 FB was injected in the pyramidal decussation of the O----R cases, or in the superior colliculus of the R----O cases, and in both groups DY was injected into the region of contralateral cortex homotopic for the new location of the transplant. On P40, in both the O----R and R----O transplants, many neurons singly labeled with FB or DY are found, but no double dye-labeled cells are seen.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurotrophic and behavioral effects of occipital cortex transplants in newborn rats.

Cell suspensions of embryonic occipital cortex were transplanted into newborn rats with large unilateral visual cortex lesions. When the animals were adults, they were tested on a difficult visual discrimination, and subsequently their brains were analyzed for possible neurotrophic effects of the transplants on nonvisual cortical areas which normally form connections with the occipital cortex. Behaviorally, animals with lesions and transplants learn to discriminate between columns and rows of squares at a rate which is identical to normal rats while animals with lesions and no transplants are impaired. Volume and cell-density measures show that the transplants also rescue neurons in cortical area 8 that would normally degenerate following the cortical lesion. No such neurotrophic effect of the transplants is found in cortical area 24 or area 17 contralateral to the lesion. In rats with lesions and no transplants, there is a significant correlation between the amount of area 8 remaining after the lesion and trials to criterion on the columns-rows discrimination, a relationship that does not exist in transplant animals because of their normal learning curve and the consistent sparing of area 8. Injections of HRP into the visual cortex contralateral to the lesion result in variable numbers of labeled cells within the transplant. However, there is no consistent relationship between the number of transplant cells which project to the opposite hemisphere and learning of the discrimination. It is suggested that the learning deficit following the lesion is largely attentional and that the sparing of cortical area 8 (which in rats may include the analog of the frontal eye fields present in the primate cortex) contributes to the sparing of function.

Cotransplantation of embryonic mouse retina with tectum, diencephalon, or cortex to neonatal rat cortex.

Retinae from embryonic mice were transplanted to the occipital cortex of neonatal rats together with their normal target regions, tectum or diencephalon, from embryonic mice or rats. In control experiments, retinae were cotransplanted with embryonic rat occipital cortex. In over 80% of the experimental animals, both transplants differentiated and grew. Ganglion cells in the retinae cotransplanted close to tectum or diencephalon survived for at least 15 weeks. Their survival was associated with the development of a distinct optic fiber layer and outgrowth of axons from the transplanted mouse retina. Specific innervation of distinct patches within the cotransplanted rat tectum or diencephalon was demonstrated by the use of an anti-mouse antibody. The innervated regions, which could be as far away as 1.3 mm from the retinae, were correlated with cytological features of the cotransplanted tectum or diencephalon. By contrast, the host cortex was never innervated by the transplanted retinae. In the control animals in which the retinae were cotransplanted with occipital cortex and in four animals in which the cotransplants lay more than 2.7 mm apart, no ganglion cells were identified and there was no evidence of an optic fiber layer, outgrowth of axons, or innervation. These results support the idea that in order to survive, retinal ganglion cells need to innervate an appropriate target region. Further, the specific innervation of regions within the cotransplanted tectum or diencephalon suggests that these target regions are able to exert a tropic influence on the axons of retinal ganglion cells, even in the absence of many of the normal structure cues.

The physiological identification of pyramidal tract neurons within transplants in the rostral cortex taken from the occipital cortex during development.

Axons from neurons in the occipital cortex transiently extend to the pyramidal tract (PT) during the early postnatal development of rats. Normally, these axons are eliminated by the end of the third postnatal week. However, if a portion of fetal occipital cortex is transplanted to the parietofrontal region in newborn hosts then some neurons in the transplant will extend pyramidal tract axons and maintain them. Intracortical microstimulation and electrophysiological recording techniques were used to identify the physiological characteristics of the transplanted pyramidal tract cells and to determine if motor effects could be elicited from the occipital transplant. Microstimulation of the transplant did not reliably evoke movement but the low density and disarray of PT cells within the transplant might account for this. Recording from within the transplant revealed that the overall cell activity was depressed. We were able to identify neurons within the transplant which responded antidromically to stimulation of the pyramidal tract, indicating that their axons have the capacity to conduct impulses and are therefore likely to have developed some viable connections. The functional significance of such projections remains uncertain.

Transplantation of the fetal occipital cortex to the third ventricle of SCN-lesioned rats induces a diurnal rhythm in drinking behavior.

Fetal hypothalamic transplants which include the suprachiasmatic nuclei (SCN), were shown previously to be capable of restoring circadian rhythmicity as manifested by both diurnal and free-running rhythms in drinking behavior in rats rendered arrhythmic by SCN lesions. The question arises as to whether the transplant must be homologous tissue or whether any other fetal brain tissue can produce similar effects. In this study rats with a lesion of the SCN and with clear loss of drinking rhythms received grafts of fetal occipital cortex placed into the third ventricle. Following the graft, animals were placed in LD conditions for 8 weeks and then in DD for another 8 weeks. The results indicate that the cortical graft induced recovery of a drinking rhythm under LD lighting conditions but that under DD the rhythm was lost again. These results suggest that non-hypothalamic tissue can mediate recovery of a diurnal rhythm but that hypothalamic tissue including the SCN is required to restore circadian function with maintenance of free-running rhythms.

Transplantation of embryonic occipital cortex to the brain of newborn rats: a Golgi study of mature and developing transplants.

Segments of the occipital cortex were taken from rat embryos (E16-E19) and transplanted to the cerebral cortex or the tectal region of a newborn rat host. With the aid of Golgi impregnation techniques, neuron morphology was studied in cortical transplants which had survived for 1 week or more in the host brain. In mature transplants (greater than 4 weeks) three main groups of neurons, termed groups I-III, were identified. Group I neurons resembled pyramidal neurons of the intact cerebral cortex. No preferential orientation of either soma or dendrites of group I neurons was observed in the transplants, and some group I neurons had curved apical dendrites. Group II neurons had predominantly stellate form and their dendrites were densely covered with spines. Paucity or absence of dendritic spines characterized group III neurons which exhibited various dendritic topologies. Different neuron types were also recognized in immature transplants growing for 1 and 2 weeks in the host brain. The sequence of dendritic maturation of transplanted cortical neurons is similar to that seen in intact cortex, although the stage reached related more to the actual age of the transplant than to that of the host. Thus, group I neurons in the 1-week-old transplants taken from E16 embryos had not attained the same complexity of branching as pyramidal neurons in the surrounding host cortex, but rather resembled slightly younger cells more like those found in the cerebral cortex of the newborn rat. These results show, therefore, that at least the basic cell classes identified in intact visual cortex can also be recognized in the cortical transplants. This will provide a foundation for studies defining which cells project to the host brain and which are involved in particular intrinsic connections.

Transplantation of embryonic occipital cortex to the tectal region of newborn rats: a light microscopic study of organization and connectivity of the transplants.

Occipital cortex was taken from fetal rats and transplanted to the tectal region of newborn rats, where it developed a specific structural identity reflecting in part its cortical origin. The implants showed locally distributing intratransplant connections, and the majority formed connections with defined regions of te host cerebral cortex and the brainstem. A sparse afferent projection from the host had its origin in visual, somatosensory, and cingulate areas of te cortex, pretectum, superior colliculus, central gray, hypothalamus, pontine reticular formation, raphe nuclei, and the locus coeruleus. No input was identified from either the retina or the dorsal thalamus. Efferent fibers were observed in normal fiber preparations as compact bundles running through the host brainstem along two main routes, one group of bundles in a dorsal position and a second group more ventral. Efferent fibers traveling rostrally along the first pathway distributed in the lower part of the stratum griseum superficiale and in the intermediate laminae of the superior colliculus, and in some cases they reached the pretectum and the lateral posterior thalamic nucleus. Deep efferent fibers ran rostrally and caudally in the central gray, and in some cases laterally directed fibers were seen to distribute in the midbrain tegmentum and reticular formation, in one case reaching the pontine gray. The finding that most afferent and many efferent connections of cortical transplants are uncharacteristic of normal cortex stands in marked contrast to retina and tectum, which, when transplanted to the same region, make relatively normal connections.

Transplantation of embryonic occipital cortex to the brain of newborn rats. An autoradiographic study of transplant histogenesis.

The neurogenesis of immature cerebral cortex transplants was investigated using tritiated thymidine (3HT) autoradiography. Cortical tissue taken from rat fetuses during their last week of gestation (E15-E21) was transplanted to the tectum or cerebral cortex of newborn rat hosts. At different times after transplantation, a single injection of 3HT was given to the host. Most of the experimental animals were killed after the transplants had grown to maturity (5-12 weeks), and some were studied shortly after the tracer had been given. In other experiments, donor tissue was used that was labeled in utero up to 1 day before being transplanted on E16, E17, E18, or E19. It was found that neurons labeled before transplantation survived and differentiated in the graft. Removal of the graft from its natural context did not prevent 3HT incorporation into surviving precursor neurons, indicating continuation of neurogenesis in the cortical transplants. In transplants from E16 donors neurons continued to be generated for 5-6 days after transplantation. Termination of neurogenesis occurred at successively earlier times in transplants taken from correspondingly older embryos. Independent of size and position of the transplant, application of 3HT after "projected" transplant ages of E23 and older labeled only nonneuronal cells. This suggests a time schedule of neuron generation in the cortical transplants similar to that observed during normal development of the cerebral cortex, which is not disturbed by the transplanting procedure.