Joseph Wells

Cross-species neural transplants of embryonic septal nuclei to the hippocampal formation of adult rats

SummaryIn the absence of immunosuppressive treatment, suspensions of cells from the developing septal region of mouse embryos were transplanted successfully into the denervated hippocampal formations of adult rat hosts. The longitudinal recovery of acetylcholinesterase (AChE)-containing fibers in the host was the index of transplant success. In transplant recipients, the fornix-fimbrial interconnection between the septum and hippocampal formation was severed unilaterally, and two 5 μl aliquots of cell suspension were injected into the hippocampal formations of host rats. Five sets of controls included one in which animals received no surgical intervention (Normal Controls), and another which was subjected to a sham operation (Sham Controls). The fornix-fimbria pathway was transected unilaterally in Lesion Control animals, while Hippocampal Controls received the same lesion plus two injections of non-cholinergic cells from the hippocampal formations of mouse embryos. Injection Controls were subjected to a fornix-fimbria transection and given two injections of debris and dead cells in saline. The cross-species transplants induced the return of a normal AChE laminar pattern in the recipient rats. The density of the laminar pattern, quantified with laser densitometry, was greatest in transplants that had survived for one week, but only in sections adjacent to the injection sites. Although the density decreased from the first through third weeks of survival, overall density of AChE staining stabilized from the fourth through 17th weeks of survival. Because the success rates of these cross-species transplants were similar to those reported for homogenic tissue, it was concluded that the rat brain is a suitable host for xenogenic transplants of septal. neurons from embryonic mice.

Transneuronal changes of the inhibitory circuitry in the macaque somatosensory thalamus following lesions of the dorsal column nuclei.

The inhibitory circuitry of the ventroposterolateral nucleus (VPL) of the macaque somatosensory thalamus was analyzed in normal animals and in those surviving for a few days or several weeks following a unilateral lesion of the cuneate nucleus, the source of medial lemniscal (ML) axons carrying information from the contralateral upper extremity. Inhibitory synaptic terminals in the VPL were defined as those that contain flattened or pleomorphic synaptic vesicles and that can be shown to be immunoreactive for γ‐aminobutyric acid (GABA). There are two types of these profiles: F axon terminals that arise from neurons of the thalamic reticular nucleus, and perhaps from VPL local circuit neurons (LCNs); and the dendritic appendages of LCNs that form presynaptic dendrites (PSDs). ML terminals normally have extensive synaptic interactions with PSDs but not with F axon terminals. Electron microscopic analyses revealed that cuneatus lesions resulted in a rapid loss of ML terminals and a statistically significant reduction in both F and PSD synaptic profiles. Confocal scanning microscopy also demonstrated a profound loss of GABA immunoreactivity in the deafferented VPL. These changes persisted for more than 20 weeks, without any evidence of reactive synaptogenesis of surviving sensory afferents or of inhibitory synapses. The changes in GABA circuitry are transneuronal, and the possible mechanisms that may underlie them are discussed. It is suggested that the altered GABAergic circuitry of the VPL in the monkey may serve as a model for understanding changes in somatic sensation in the human following peripheral or central deafferentation.

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.

Cross-species septal transplants: recovery of choline acetyltransferase activity ☆

Following interruption of the fornix-fimbria pathway, hippocampal choline acetyltransferase (ChAT) activity was restored gradually by cross-species cell suspension transplants of embryonic septum. The hippocampal segment closest to the implant reached 35% of normal 17 weeks after transplantation. The overall restoration of ChAT by xenogenic cell suspension had many similarities to that reported for homogenic solid and cell suspension septal grafts. The time course of the recovery of ChAT activity was different from the time course of the ingrowth of acetylcholinesterase stained fibers reported previously.

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.

Extensive Dual Innervation and Mutual Inhibition by Forelimb and Hindlimb Inputs to Ventroposterolateral Nucleus Projection Neurons in the Rat

The stimulation of brachial plexus and sciatic nerve resulted in a precisely timed, synchronous volley of inputs to ventroposterolateral (VPL) neurons from either forelimb or hindlimb. Such stimulation activated sensory fibers of all modalities and was therefore modality-nonspecific. Extracellular recordings of modality-nonspecific single-unit evoked responses from VPL showed that 13% of VPL projection neurons responded to both forelimb and hindlimb inputs. We also demonstrated mutually inhibitory interactions between inputs from forelimb and hindlimb in 45% of VPL units. Unlike the somatotopic map produced by others using modality-specific inputs, the modality-nonspecific evoked response map of VPL had a broadly overlapping distribution of evoked responses. This was especially true for the more caudal aspects of VPL. When the delivery of stimuli was appropriately timed, forelimb inputs caused the inhibition of responses to forelimb stimulation; similarly, hindlimb inputs inhibited responses to forelimb stimulation. The inhibition had a variable duration that may reflect a combination of processes, including recurrent inhibitory collateral input from the thalamic reticular nucleus (TRN) or an intrinsic hyperpolarizing inhibitory afterpotential of the VPL neuron. The presence of an extensive converging input on VPL neurons and an inhibitory correlate to this overlapping of inputs may explain the shifting of VPL maps following lesions of peripheral nerve, spinal cord, or dorsal column nuclei (DCN).

Synaptic rearrangement in the ventrobasal complex of the mouse following partial cortical deafferentation.

Morphological rearrangement has been shown in certain areas of the adult mammalian central nervous system in response to afferent denervation. Lynch et al. 6 and Steward et al. 1°,11 have demonstrated newly formed entorhinal fibers in the hippocampus of the adult rat in response to ipsilateral deafferentation. Raisman v has found synaptic reorganization in the lateral septal nucleus after removal of inputs from either the hippocampus or the medial forebrain bundle. Comparable changes also have been demonstrated in other areas of the CNS including the spinal cord 5 and parts of the visual system 3. The present study indicates that synapses can reform in ventrobasal thalamus (VB) after partial loss of the principal somatic sensory (Sm I) cortical input. To identify the pattern of cortical termination in VB, lesions were placed in Sm I of the right cortical hemisphere of C57B1/6J mice, and the subsequent degeneration was traced to VB using the Fink-Heimer stain for degenerating axon terminals. The somatotopic distribution of the cortical input was similar to that demonstrated by Cowan et al. 1. A small area in the lateral part of VB was chosen as a sample area for ultrastructural study of the effect of deafferentation. This area was chosen for study since the rather distinct lateral border of VB could be used as a consistent landmark for uniform sampling. For deafferentation of the sample area, a small (1 m m × 1 m m × depth of cortex) lesion was placed stereotaxically in the medial part of Sm I cortex. The results of the degeneration study (above) confirmed that these lesions produced a well-defined patch of degenerating terminals in the lateral part of VB. The sample area was within the borders of this degeneration field. From electron micrographs of the sample area in 4 normal animals, 4 categories of vesicle-containing profiles were established on the basis of consistent differences in their morphology, and the number of synaptic contacts made by each of these types as well as the total were computed per 100 sq. # m of neuropil (excluding blood vessels and cell bodies). For quantitation a synapse was defined as any profile containing three or more vesicles which were adjacent to an

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.