Brad P. Vietje

Selective lesions of granule cells by fluid injections into the dentate gyrus

Granule cells were selectively lesioned by injections of fluid into the infragranular cleavage plane in the dentate gyrus. The granule cells were axotomized by the cavity created by the fluid and 6 days after the injection there were no granule cells at the injection site. The size of the granule cell loss could be altered by varying the volume and rate of the injection. The loss of granule cells led to a shrinkage of the molecular layer and to a reactive gliosis. The lesion also caused an increase in the density of AChE and Timm staining in the molecular layer above the lesion. Although the increased density of AChE and Timm staining may have been due in part to the shrinkage of the molecular layer, part was due to the growth of inputs in response to the loss of granule cells and/or to the axotomy of the input terminals. The changes seen in the molecular layer above the lesion site ended abruptly at the margins of the lesion and the adjacent molecular and granule cell layers appeared normal.

Increase in acetylcholinesterase in the molecular layer of the dentate gyrus in the absence of septal inputs following selective granule cell lesions

Granule cell lesions cause an increase in acetylcholinesterase (AChE) staining in the molecular layer of the dentate gyrus. The source of this response was examined by combining granule cell lesions with lesions of the fornix-fimbria, thereby removing the cholinergic input from the septum to the hippocampus. The increased AChE staining was present in animals with granule cell lesions regardless of whether the fornix was lesioned or intact. The increase in AChE staining occurred without a corresponding increase in choline acetyltransferase staining. These findings suggest that an AChE-positive, but non-cholinergic, sprouting response occurred within the dentate gyrus following selective lesions of the granule cells. The source of this sprouting may be from AChE-positive hilar interneurons.

Chapter 13 Neural transplantation of horseradish peroxidase-labeled hippocampal cell suspensions in an experimental model of cerebral ischemia

Publisher Summary One of the major obstacles in assessing the use of neuronal transplants to replace cells that are lost because of ischemia is the inability to identify and distinguish homotypically-transplanted cells from similar cells in the host brain. This chapter discusses the use of horseradish peroxidase (HRP) to label cell suspensions of fetal rodent hippocampus that were injected into the hippocampi of post-ischemic rats to determine whether homotypically transplanted cells could be distinguished from those of the host. When homotypic cells are to be transplanted and are labeled with HRP, a label is required, which might help in distinguishing cells of transplant origin from those of the host. One factor influencing the transplant survival appears to be the placement of the cells within the hippocampus. Whether the minor differences in histocompatibility between the donor tissue and host in combination with the cytotoxicity factors influence cellular survival is an issue that remains to be resolved. The chapter suggests that neural transplantation may be feasible for treatment for focal lesions produced during ischemia. Sufficient cell growth and development that occurred in the four vessel occlusion (VO) animals, justify further transplantation experiments in this model.

Xenografts of brain cells labeled in cell suspensions show growth and differentiation in septo-hippocampal transplants.

Embryonic mouse brain cells from the basal forebrain region were labeled in cell suspensions and transplanted into the denervated hippocampal formation of adult rats. Many labeled cells had the appearance of typical pyramidal neurons with dendrites that had both growth cones and neurites. Labeled neurons and glia were seen at several sites in the hippocampal formation. The neurons were located predominantly along the dentate granule cell layer and the pyramidal neurons had a preferred orientation of their apical dendrites toward the molecular layer. Since it was rare to see a surviving labeled neuron within the injection site, migration away from the injection site seemed important for survival of the cells. The methods used in these experiments should become an important adjunct to the methods for studying the migration, differentiation and growth of neurons and glia.

Interactions between donor and host tissue following cross-species septohippocampal transplants

Interactions between donor and host tissues following xenogeneic transplantation were studied using the neural cell surface antigen, Thy 1.2, as a marker for the donor tissue. Dissociated septal cells from Thy 1.2-positive fetal mice were transplanted to the dentate gyrus of Thy 1.2-negative adult rats. At post-transplantation survival times between 1 and 5 months, an antibody to Thy 1.2 was used to identify donor tissue. The results demonstrate that the donor tissue was capable of migrating and developing within the host following transplantation. Thy 1.2-positive cells and processes were consistently found within the supragranular, infragranular, and molecular layers of the dentate gyrus, and occasionally within the hilus, suggesting that mechanisms existed within the host which influenced the development of the transplanted tissue. Additionally, the survival and growth of the Thy 1.2-positive neurons differed from previous reports describing the growth of acetylcholinesterase (AChE)-positive fibers from xenogeneic transplants. This finding suggested that in addition to growing within the host, xenogeneic transplants may also stimulate a compensatory sprouting response from the host.

The differentiation of dentate granule cells following transplantation.

Immature cells transplanted into an adult host must adapt to their new environment. In the present study we have shown the dendritic development of dentate granule cells following transplantation. The adult host granule cells were lesioned by a fluid injection into the infragranular cleavage plane of the dentate gyrus. Few, if any, granule cells survived the lesion and the molecular layer (ML) shrank. When allogeneic neonatal granule cells were included in the fluid, the host granule cells were simultaneously killed and replaced. In order to visualize the dendrites, the granule cells were filled with Lucifer yellow (LY) in fixed sections and subsequently immunoreacted with an antibody to LY. The granule cell dendrites in the transplant were shorter in length, had a greater cross-sectional area, had more spines, and were more coiled and bent than control granule cell dendrites. The dendrites in the transplant formed functional synapses as indicated by cytochrome oxidase histochemistry and the transplant prevented xc03some of the ML shrinkage. Acetylcholinesterase (ACHE) xkreaction product increased both in lesioned and in transplant groups. The laminar pattern of ACHE in the control ML was not seen after the lesion and did not return in animals with successful transplants. We conclude that (i) the dendrites of neurons in the transplant adapted to the adult host environment and a shrinking ML with remarkable structural plasticity; (ii) the transplant prevented some of the shrinkage of the ML; (iii) the transplant could not reverse some of the lesion-induced changes in host organization, such as the organization of ACHE inputs to the ML; and (iv) a phenotypically specific population of transplanted neurons can replace traumatically lesioned neurons of the same type even if the host conditions continue to change.

Migration and Differentiation of Xenogenic and Homogenic Brain Cells Transplanted into the Adult Rat Hippocampusa

Brain cells from the septal-basal forebrain region of embryonic mice and rats were prepared as cell suspensions and labeled by horseradish peroxidase (HRP). The labeled cell suspensions were injected into the hippocampal formation of adult rats that were partially denervated by fornix lesions, and cross-species transplants were compared to transplants between the same species. One to 2 weeks after the transplants were injected, the rat hosts were perfused and the brain processed histochemically to show HRP. In the transplants between different species, four significant observations were made. First, the mouse cells, labeled by HRP, migrated away from the injection site (FIG. 1 j. The route of the migration was frequently along the dentate granule cell layer and occurred over considerable distances-up to 1.5 mm. Second, the injection site rarely contained labeled cells that looked like viable neurons. The injection site itself contained cells, but the only HRP reaction product was seen as debris or as particulate matter in phagocytic cells. Third, two types of neurons survived the xenogenic transplantspyramidal neurons and multipolar neurons. Fourth, some pyramidal neurons had highly differentiated dendritic arbors whereas others had only rudimentary branches. The thick apical dendrites of the pyramidal neurons were preferentially oriented toward the molecular layer of the dentate gyrus, much like the dendrites of the host granule cells. Thus, the cues for migration and orientation must still be present in the adult rat dentate gyrus and available to cells from a different species. The multipolar neurons showed no preferred orientation of their dendrites. In contrast to the xenogenic transplants, when the transplants were made between animals of the same species, many more labeled cells survived. The labeled cells were largely within the injection site with just a few cells having migrated out of the transplant site (FIG. 2). Those labeled cells that did leave the injection site were still close to the injection site between 1 and 2 weeks after transplantation. The most commonly labeled cell type seen in the homogenic transplants was a fusiform neuron with long dendrites that had few branches. This cell type was not seen in the xenogenic transplants. The homogenic transplants had begun to organize themselves within the injection site itself. Some labeled cells appeared to have started to form a row. The

Cell-sized microspheres in the hippocampus show cleavage planes and passive displacement.

Fluorescent microspheres (6 or 10 micron in average diameter) dispersed in fluid were injected into the hippocampus, neocortex or striatum. In the hippocampus the microspheres were located in one of three cleavage planes. Cleavage planes were found above the alveus, in the obliterated hippocampal fissure and on the hilar side of the dentate granule cells. When the injections were made into the infragranular cleavage plane, the adjacent granule cells degenerated, presumably because the cavity separated the axons from their cell bodies. Some microspheres were passively displaced beyond the boundary of the injection site. If the microspheres gained access to the subarachnoid space, some of the displaced microspheres were found at considerable distances from the injection site. There were no cleavage planes in neocortex or striatum but there was passive displacement of microspheres into the host parenchyma. In cell suspension transplants, the passive displacement of cells should be distinguished from migration and the possibility of a widespread distribution of transplanted cells needs to be considered.

The role of somatosensory information in a constrained locomotor task

The purpose of this project was to study the role of somatosensory information in the performance of a constrained locomotor task by rats and to further examine the influence of structural recovery in the somatosensory thalamus, specifically the ventral posterolateral nucleus (VPL). Groups of rats were trained to traverse an elevated, one inch bar for a reward. The time taken to run across the bar (run time) was used as a measure of the success of the goal-directed behavior. The movement pattern of the hindlimb during the swing phase of the locomotor task was quantified from videotape on Preoperative (PRE) Day 15 and during the 46-day postoperative period. The movement pattern was characterized using six different parameters: the area, the X and Y values of the centroid under the normalized curve of the hindlimb trajectory, the vertical displacement of the hindlimb in the flexion and extension phases of the swing cycle, the maximum instantaneous hindlimb velocity, and the proportion of time spent in the acceleration versus deceleration phases of the swing cycle. In order to disrupt the central pathways for somatosensory information, lesions were made in (i) the right gracile nucleus (GN) (n = 18), (ii) bilateral GN (n = 7), (iii) the right GN and the left VPL (n = 6), and (iv) bilateral VPL (n = 8), and (v) sham-operated animals (n = 5). The run time and the pattern of the hindlimb swing cycle were used as measures of loss and recovery of function. Only the bilateral VPL group showed an impairment in run time and they recovered by Postoperative (POST) Week 4. All groups demonstrated an impairment in initial flexion of the hindlimb during the swing cycle that recovered in the right GN group only. On POST Day 49, the right GN, bilateral GN, and the sham groups received injections of 5% WGA-HRP into both CN to determine the location of these projections in VPL. The CN projections were not redistributed into the gracile area of VPL after GN lesions. Since our previous study (24) had shown the number of synapses in VPL returned to normal after dorsal column nuclei (DCN) lesions by POST Day 50, the recovery of the number of synapses alone was not sufficient to restore the normal gait pattern, while the recovery of the run time preceded the complete recovery of the number of synapses.(ABSTRACT TRUNCATED AT 400 WORDS)

Dendritic outgrowth from neural cells transplanted to the hippocampal fissure.

Transplants of cell suspensions that were either selective for granule cells or contained all hippocampal cell types were placed in the hippocampal fissure or in the infragranular cleavage plane (IGCP) of the dentate gyrus. Several transplants were found in both areas in the same dentate gyrus. After a variety of post-transplant survival times, neurons of both the donor and the host were filled with lucifer yellow in fixed sections. Sections were also immunoreacted with antibodies to glial fibrillary acidic protein (GFAP), vimentin, neural cell adhesion molecule (NCAM and HNK-1/NCAM) and were histochemically reacted for ACHE. Dendrites of neurons from transplants of cells of the whole hippocampus usually stayed within the transplant. If a dendrite from such transplants did grow out of the transplant, it grew into the molecular layer (ML) of the host dentate gyrus and not into the hilus of the host. Dendrites from granule cell selective transplants grew into the ML of the host and those that grew from fissure transplants were inverted from the normal orientation of host granule cell dendrites. Dendrites also grew out of the transplant in the absence of reactive gliosis. Transplants of cells from the whole hippocampus placed in the IGCP showed the greatest ingrowth of acetylcholinesterase (ACHE) fibers. In granule cell transplants made concurrently into the fissure and the IGCP, donor granule cell dendrites grew into the host ML from both sites, demonstrating that a gradient of tropic factors across the ML could not account for the direction and orientation of the dendritic outgrowth, since a gradient that directed the growth of one set of dendrites would work against the dendrites growing in the opposite direction.