LacZ-expressing olfactory ensheathing cells do not associate with myelinated axons after implantation into the compressed spinal cord.

Studies have shown that implanting olfactory ensheathing cells (OECs) may be a promising therapeutic strategy to promote functional recovery after spinal cord injury. Several fundamental questions remain, however, regarding their in vivo interactions in the damaged spinal cord. We have induced a clip compression injury at the T10 level of the spinal cord in adult rats. After a delay of 1 week, OECs isolated from embryonic day 18 rats were implanted into the cystic cavity that had formed at the site of injury. Before implantation, OECs were infected with a LacZ-expressing retrovirus. At 3 weeks after implantation, LacZ-expressing OECs survived the implantation procedure and remained localized to the cystic cavity. At the electron microscopic level, the cystic cavity had clusters of LacZ-expressing OECs and numerous Schwann cells lacking LacZ expression. Although labeled OECs made no direct contact with axons, unlabeled Schwann cells were associated with either a single myelinated axon or multiple unmyelinated axons. Positively labeled OEC processes often enveloped multiple Schwann cell-axon units. These observations suggest that the role of OECs as the primary mediators of the beneficial effects on axon growth, myelination, and functional recovery after spinal cord injury may require re-evaluation.

TrkA-expressing trigeminal sensory neurons display both neurochemical and structural plasticity despite a loss of p75NTR function: responses to normal and elevated levels of nerve growth factor.

In neural crest-derived sensory ganglia, approximately half of the neuronal population expresses the transmembrane trkA receptor that is required for neuronal binding of target-derived nerve growth factor (NGF). These same neurons also express the p75 neurotrophin receptor (NTR) that increases the affinity of trkA for NGF. Depleting p75NTR expression reduces both the survival of trkA-positive sensory neurons and their afferent innervation of peripheral targets. In this investigation, we assessed the neurochemical and structural plasticity of trigeminal sensory neurons in p75NTR-deficient mice in response to either normal or elevated levels of NGF during postnatal development and into adulthood. Although p75NTR-deficient mice have 30% fewer trigeminal neurons, levels of trkA mRNA expression are modestly elevated in these mutant mice as compared to control mice. The density of central afferent axons and local levels of NGF are, however, comparable between mutant and control animals. Thus, despite the survival of fewer trigeminal neurons, neither ganglionic levels of trkA mRNA expression nor the density of central afferent projections are depleted in p75NTR-deficient mice. In response to elevated levels of NGF protein, transgenic mice with and without p75NTR expression display both increased levels of trkA mRNA expression and a greater density of trigeminal central afferent axons as compared to control mice. These data further reveal that an absence of p75NTR function in trigeminal sensory neurons does not diminish their capacity for NGF-dependent plasticity, namely trkA mRNA expression and collateral growth of central afferent axons.

Absence of p75(NTR) expression reduces nerve growth factor immunolocalization in cholinergic septal neurons.

Septal axons provide a cholinergic innervation to the nerve growth factor (NGF)-producing neurons of the mammalian hippocampus. These cholinergic septal afferents are capable of responding to target-derived NGF because they possess trkA and p75(NTR), the two transmembrane receptors that bind NGF and activate ligand-mediated intracellular signaling. To assess the relative importance of p75(NTR) expression for the responsiveness of cholinergic septal neurons to hippocampally derived NGF, we used three lines of mutant and/or transgenic mice: p75(-/-) mice (having two mutated alleles of the p75(NTR) gene), NGF/p75(+/+) mice (transgenic animals overexpressing NGF within central glial cells and having two normal alleles of the p75(NTR) gene), and NGF/p75(-/-) mice (NGF transgenic animals having two mutated alleles of the p75(NTR) gene). BALB/c and C57B1/6 mice (background strains for the mutant and transgenic lines of mice) were used as controls. Both lines of NGF transgenic mice possess elevated levels of NGF protein in the hippocampus and septal region, irrespective of p75(NTR) expression. BALB/c and C57Bl/6 mice display comparably lower levels of NGF protein in both tissues. Despite differing levels of NGF protein, the ratios of hippocampal to septal NGF levels are similar among BALB/c, C57B1/6, and NGF/p75(+/+) mice. Both p75(-/-) and NGF/p75(-/-) mice, on the other hand, have markedly elevated ratios of NGF protein between these two tissues. The lack of p75(NTR) expression also results in a pronounced absence of NGF immunoreactivity in cholinergic septal neurons of p75(-/-) and NGF/p75(-/-) mice. BALB/c, C57B1/6, and NGF/p75(+/+) mice, on the other hand, display NGF immunoreactivity that appears as discrete granules scattered through the cytoplasm of cholinergic septal neurons. Elevated levels of NGF in the hippocampus and septal region coincide with hypertrophy of cholinergic septal neurons of NGF/p75(+/+) mice but not of NGF/p75(-/-) mice. Levels of choline acetyltransferase (ChAT) enzyme activity are, however, elevated in the septal region and hippocampus of both NGF/p75(+/+) and NGF/p75(-/-) mice, compared with control mice. These data indicate that an absence of functional p75(NTR) expression disrupts the normal cellular immunolocalization of NGF by cholinergic septal neurons but does not affect the ability of these neurons to respond to elevated levels of NGF, as determined by ChAT activity.

Enhanced neurotrophin-induced axon growth in myelinated portions of the CNS in mice lacking the p75 neurotrophin receptor.

Axonal growth in the adult mammalian CNS is limited because of inhibitory influences of the glial environment and/or a lack of growth-promoting molecules. Here, we investigate whether supplementation of nerve growth factor (NGF) to the CNS during postnatal development and into adulthood can support the growth of sympathetic axons within myelinated portions of the maturing brain. We have also asked whether p75(NTR) plays a role in this NGF-induced axon growth. To address these questions we used two lines of transgenic mice overexpressing NGF centrally, with or without functional expression of p75(NTR) (NGF/p75(+/+) and NGF/p75(-/-) mice, respectively). Sympathetic axons invade the myelinated portions of the cerebellum, beginning shortly before the second week of postnatal life, in both lines of NGF transgenic mice. Despite the presence of central myelin, these sympathetic axons continue to sprout and increase in density between postnatal days 14 and 100, resulting in a dense plexus of sympathetic fibers within this myelinated environment. Surprisingly, the growth response of sympathetic fibers into the cerebellar white matter of NGF/p75(-/-) mice is enhanced, such that both the density and extent of axon ingrowth are increased, compared with age-matched NGF/p75(+/+) mice. These dissimilar growth responses cannot be attributed to differences in cerebellar levels of NGF protein or sympathetic neuron numbers between NGF/p75(+/+) and NGF/p75(-/-) mice. Our data provide evidence demonstrating that growth factors are capable of overcoming the inhibitory influences of central myelin in the adult CNS and that neutralization of the p75(NTR) may further enhance this growth response.

Nerve growth factor-induced growth of sympathetic axons into the optic tract of mature mice is enhanced by an absence of p75NTR expression.

Postganglionic sympathetic axons display a remarkable ability for new collateral growth in response to local increases in nerve growth factor (NGF). Elevating NGF levels within the brain also induces the directional growth of sympathetic axons, but not within myelinated pathways of adult mammals. In this investigation, we provide in vivo evidence that sympathetic axons are capable of NGF-induced collateral growth through the microenvironment of mature myelinated pathways, especially in the absence of the p75 neurotrophin receptor (NTR). In transgenic mice overexpressing NGF centrally and expressing p75NTR, only a few varicose sympathetic axons invade the optic tract after the first month of postnatal life. In other transgenic mice overexpressing NGF centrally but lacking p75NTR expression, the incidence of sympathetic axons within this myelinated tract substantially increases. Moreover, numerous unmyelinated sympathetic axons cluster together to form large processes extending through the optic tract; such structures are first seen 8 weeks after birth. Only these large axon bundles display prominent immunostaining for GAP-43, which is preferentially localized to the sympathetic fibers, since nonmyelinating Schwann cells are not associated with these axon bundles. These data provide the first direct evidence that sympathetic axons are indeed capable of NGF-induced collateral growth into myelinated tracts of mature mammals, and that their continued growth through this microenvironment is markedly enhanced by the absence of p75NTR expression. We propose that p75NTR among sympathetic axons may either directly or indirectly limit collateral branching of these fibers in response to increased levels of NGF.

Prolonged exposure to elevated levels of endogenous nerve growth factor affects the morphological and neurochemical features of sympathetic neurons of postnatal and adult mice.

It is well documented that acute increases of target-derived nerve growth factor affect the morphological and neurochemical features of post-ganglionic sympathetic neurons. It has yet to be determined, however, whether similar changes are still evident after prolonged exposure to increased levels of endogenous nerve growth factor. Using a transgenic line of mice which overexpresses nerve growth factor in the brain commencing after the first week of postnatal life and continuing into adulthood, we have shown previously that sympathetic axons sprout into the nerve growth factor-rich cerebellum of these animals; no such axons are seen in the cerebellum of age-matched wild type animals. The aim of this study was to examine and characterize the effects of chronically elevated levels of endogenous nerve growth factor on sympathetic neurons of the superior cervical ganglion. In comparison to adult wild type mice, adult transgenic animals possessed hypertrophied ganglia which displayed both an increase in sympathetic somal size and a decrease in their density. At the electron microscope level, sympathetic somata of the adult transgenic animals had numerous electron-dense lysosome-like structures in the cytoplasm, as compared to that seen in the sympathetic somata of adult wild type animals. Immunodetection of nerve growth factor in the sympathetic somata revealed that the staining intensity in postnatal (day 28) transgenic mice was greater than that in age-matched wild type mice. By adulthood, however, such differences in the intensities of nerve growth factor immunostaining were no longer evident. In situ hybridization analyses of trkA receptor messenger RNA revealed that levels of expression among somata of similar sizes were comparable between the transgenic and wild type neuronal populations of both postnatal day 28 and adult animals. A small subpopulation of sympathetic somata in postnatal transgenic mice displayed a marked increase in p75NTR messenger RNA expression in comparison to somata of a similar size in age-matched wild type animals. By adulthood, the proportion of sympathetic somata in the transgenic animals possessing elevated levels of p75NTR messenger RNA expression had increased. These results reveal that chronically elevated levels of endogenous nerve growth factor in the postnatal and adult mouse brain can induce both structural and neurochemical remodelling of sympathetic neurons. The preferential increase in p75NTR messenger RNA expression among sympathetic somata of transgenic mice may be required for their growth of collateral axons into the nerve growth factor-rich cerebellum during postnatal development and may facilitate the increased immunodetection of nerve growth factor on these aberrant sympathetic axons in adult transgenic animals.

Successful survival of grafted transgenic neural plate cells in adult central nervous system environment.

1. Accumulating evidence indicates that damaged brain functions can be ameliorated in a variety of animal models by the grafting of fetal neuronal cell or tissue into damaged brain. Clinical trials are under way to determine whether human fetal mesencephalic tissue can ameliorate motor functions in patients with Parkinson's disease. 2. Autopsy findings of parkinsonian patient implanted with human fetal mesencephalic tissue clearly revealed that the fetal neuronal graft can survive for an extended period of time in the human brain and densely reinnervate the surrounding host striatal tissue. 3. It is, however, still important to obtain more practical, effective, and ethically justifiable donor material for the future clinical application of the procedures. Desirable properties for the donor cells include long-term survival in the brain, neuronal cell type for the reconstruction of damaged neural circuits, and susceptibility to genetic manipulation for the practical use. 4. With the development of molecular biology techniques, genetic modification and transplantation of the donor neuronal cells might be a feasible way to cure many kinds of central nervous system diseases toward a "graft-gene therapy."

Absence of the p75 neurotrophin receptor alters the pattern of sympathosensory sprouting in the trigeminal ganglia of mice overexpressing nerve growth factor.

Sympathetic axons invade the trigeminal ganglia of mice overexpressing nerve growth factor (NGF) (NGF/p75(+/+) mice) and surround sensory neurons having intense NGF immunolabeling; the growth of these axons appears to be directional and specific (). In this investigation, we provide new insight into the neurochemical features and receptor requirements of this sympathosensory sprouting. Using double-antigen immunohistochemistry, we demonstrate that virtually all (98%) trigeminal neurons that exhibit a sympathetic plexus are trk tyrosine kinase receptor (trkA)-positive. In addition, the majority (86%) of those neurons enveloped by sympathetic fibers is also calcitonin gene-related peptide (CGRP)-positive; a smaller number of plexuses (14%) surrounded other somata lacking this neuropeptide. Our results show that sympathosensory interactions form primarily between noradrenergic sympathetic efferents and the trkA/CGRP-expressing sensory somata. To assess the contribution of the p75 neurotrophin receptor (p75(NTR)) in sympathosensory sprouting, a hybrid strain of mice was used that overexpresses NGF but lacks p75(NTR) expression (NGF/p75(-/-) mice). The trigeminal ganglia of NGF/p75(-/-) mice, like those of NGF/p75(+/+) mice, have increased levels of NGF protein and display a concomitant ingrowth of sympathetic axons. In contrast to the precise pattern of sprouting seen in the ganglia of NGF/p75(+/+) mice, sympathetic axons course randomly throughout the ganglionic neuropil of NGF/p75(-/-) mice, forming few perineuronal plexuses. Our results indicate that p75(NTR) is not required to initiate or sustain the growth of sympathetic axons into the NGF-rich trigeminal ganglia but rather plays a role in regulating the directional patterns of axon growth.

Effects of elevated levels of nerve growth factor on the septohippocampal system in transgenic mice.

Elevating target-derived levels of nerve growth factor (NGF) in peripheral organs of postnatal mammals is known to enhance the survival of postganglionic sympathetic neurons and to promote the terminal arborization of sympathetic axons within such NGF-rich target tissues. Although increasing levels of NGF in the central nervous system can ameliorate cholinergic function of damaged and aged neurons of the medial septum, it remains undetermined whether the postnatal development of this neuronal population and their projections that innervate the hippocampus are likewise affected by elevated levels of target-derived NGF. To address this question, the cholinergic septohippocampal pathway was examined in adult transgenic mice which display elevated levels of NGF protein production in the dorsal hippocampus during postnatal development. Adult transgenic mice possessed a cholinergic population of septal neurons approximately 15% larger than that seen in age-matched control animals. Despite increased numbers of cholinergic septal neurons, as well as elevated levels of hippocampal NGF, the density of cholinergic septal axons in the outer molecular layer of the hippocampal dentate gyrus of adult transgenic animals was comparable with that found in wild-type controls. These results reveal that elevating levels of target-derived NGF during postnatal development can increase the population size of the cholinergic septal neurons but does not alter their pattern of afferent innervation in the hippocampus of adult mice.

Glial overexpression of NGF enhances neuropathic pain and adrenergic sprouting into DRG following chronic sciatic constriction in mice.

Adrenergic sprouts within axotomized dorsal root ganglia (DRG) may contribute to neuropathic pain, and may arise under the influence of nerve growth factor (NGF). We investigated effects of chronic constriction injury (CCI) on behavior and sprouting in mice in which NGF overexpression is driven by a glial protein (GFAP) promotor. GFAP-NGF mice were naturally hyperresponsive to radiant heat, and had enhanced ipsilateral responses to thermal and mechanical stimulation following CCI compared to wild-type mice. Sympathetic axons were already present in intact DRG of GFAP-NGF mice. Following CCI, sprouting in ipsilateral and to a lesser extent contralateral DRG occurred in both genotypes, but the sprout density 2 weeks post-lesion was much greater in GFAP-NGF mice. These results demonstrate a connection between the endogenous ectopic overexpression of NGF and (1) neuropathic pain behaviour and (2) sympathetic sprouting in the DRG.

p75-deficient sensory axons are immunoreactive for the glycoprotein L1 in mice overexpressing nerve growth factor.

Nerve growth factor (NGF) regulates the expression of the glycoprotein L1 among neural cell populations. The purpose of this investigation was to determine whether NGF equally affects the immunolocalization of L1 on both sympathetic and sensory axons, and whether the functional expression of the p75 neurotrophin receptor (p75NTR) is required for the immunodetection of this glycoprotein on peripheral axons. Two lines of transgenic mice overexpressing NGF among glial cells were used in this study: (1) one line of mice possessing two normal alleles for p75NTR, and (2) another line of mice possessing two mutated alleles for p75NTR. In both types of animals, sensory axons stained immunohistochemically for calcitonin gene-related peptide and sympathetic axons stained immunohistochemically for tyrosine hydroxylase invaded the deep white matter portions of the cerebellum (a central structure containing high levels of transgene expression and synthesis); the cerebella of wild type (C57Bl/6) and p75NTR-deficient mice lacked these sensory and sympathetic fibers. Both lines of transgenic animals also possessed a dense plexus of L1-immunoreactive axons in their cerebella; the spatial distribution of these L1-immunostained axons paralleled that seen for the sensory and sympathetic axons. A unilateral removal of the superior cervical ganglion in both lines of transgenic animals caused a complete reduction of tyrosine hydroxylase-immunopositive axons in the cerebellum but did not affect the density of L1-immunopositive axons. From these in vivo data, we conclude that collateral branches of sensory axons which invade a NGF-rich target area display L1 immunoreactivity, and that such immunodetection does not require the functional expression of p75NTR.

Sympathetic and sensory innervation of the extracerebral vasculature: roles for p75NTR neuronal expression and nerve growth factor.

The extracerebral vasculature receives a postnatal innervation of noradrenergic sympathetic axons and nociceptive sensory axons. These axons are responsive to the neurotrophin nerve growth factor (NGF), in that they possess the transmembrane receptors p140proto-trkA and p75neurotrophin receptor (NTR) which bind NGF. p75NTR-deficient mice display reduced patterns of sympathetic innervation of the pineal gland and sensory innervation of the skin (Lee et al., 1992, 1994a). The goal of this investigation was to determine whether an absence of p75 expression likewise perturbs the sympathetic and sensory innervation of the extracerebral vessels of adult mice, and if so, whether increasing levels of NGF within the target field is capable of enhancing this perturbed axon growth. Four lines of mice were used: wild-type C57Bl/6 mice, transgenic mice overexpressing NGF in the brain, p75NTR-deficient mice, and hybrid mice which overexpress NGF in the brain but lack p75NTR expression. Sympathetic and sensory innervation of the meningeal arteries were severely perturbed in p75NTR-deficient mice. Wild-type and hybrid mice displayed comparable patterns of sympathetic and sensory axons along the dural arteries. Transgenic mice, however, possessed the greatest degree of arterial innervation. These data reveal that while p75NTR expression may be a critical factor for initiating axon growth along the extracerebral vasculature during postnatal development, the sympathetic and sensory nervous systems display a remarkable degree of NGF-induced axonal plasticity, such that increased levels of NGF can ameliorate perturbed patterns of arterial innervation in p75-deficient mice.

Sympathetic axons surround nerve growth factor-immunoreactive trigeminal neurons: observations in mice overexpressing nerve growth factor.

It has been postulated that the aberrant projection of sympathetic axons to individual primary sensory neurons may provide the morphological basis for pain-related behaviors in rat models of chronic pain syndrome. Since nerve growth factor (NGF) can elicit the collateral sprouting of noradrenergic sympathetic terminals, it might be predicted that NGF plays a role in mediating the sprouting of sympathetic axons into sensory ganglia. Using a line of transgenic mice overexpressing NGF among glial cells, it was first found that trigeminal ganglia from adult transgenic mice possessed significantly higher levels of NGF protein in comparison to age-matched wild-type mice; as well, detectable levels of NGF mRNA transgene expression were present in both the ganglia and brain stem. Within the trigeminal ganglia, a small proportion of the sensory neuronal population stained immunohistochemically for NGF; a higher percentage of NGF-positive neurons was evident in transgenic mice. New sympathetic axons extended into the trigeminal ganglia of transgenic mice only and formed perineuronal plexuses surrounding only those neurons immunostained for NGF. In addition, such plexuses were accompanied by glial processes from nonmyelinating Schwann cells. From these data, we propose that accumulation of glial-derived NGF by adult sensory neurons and its putative release into the ganglionic environment induce the directional growth of sympathetic axons to the source of NGF, namely, the cell bodies of primary sensory neurons.

Sympathetic and sensory axons invade the brains of nerve growth factor transgenic mice in the absence of p75NTR expression.

Collateral sprouting, a nerve growth factor (NGF)-mediated growth response of undamaged peripheral axons, can be divided into reparative and aberrant axonal growth. We have previously shown that aberrant growth occurs in transgenic mice overexpressing NGF centrally under the control of the glial fibrillary acidic protein promoter. Both sympathetic and sensory fibers, stained immunohistochemically for tyrosine hydroxylase and calcitonin gene-related peptide, respectively, invade the cerebellum of postnatal transgenic mice, whereas no such axons are seen in age-matched wild-type cerebellum. Recent examination of mice possessing a null mutation for p75NTR has suggested that axon growth may be influenced by the functional expression of this receptor. To address the potential role of p75NTR in axon growth, we have generated a new line of hybrid mice overexpressing NGF but lacking functional p75NTR expression. Postnatal (day 14) hybrid cerebellum possessed fewer aberrant sensory and sympathetic fibers compared to their age-matched transgenic counterparts. By adulthood, however, hybrid cerebellum displayed a robust plexus of axons stained immunohistochemically for calcitonin gene-related peptide and tyrosine hydroxylase. No neuronal or nonneuronal localization of p75NTR-immunoreactive elements was observed in postnatal and adult hybrid cerebellum. Interestingly, sympathetic axons within the hybrid cerebellum displayed a markedly reduced axon density and staining intensity for NGF, suggesting a possible alteration in axonal sequestration of NGF. These results show that p75NTR is not vital for new growth of NGF-sensitive sympathetic and sensory axons and that immunohistochemical detection of NGF at sympathetic axons requires the functional expression of p75NTR.

Sympathetic axons invade the brains of mice overexpressing nerve growth factor.

Transgenic mice that overexpress nerve growth factor (NGF) in cells producing glial fibrillary acidic protein were used to determine whether sympathetic axons will invade the undamaged, postnatal mammalian brain. By using reverse transcriptase-polymerase chain reaction, NGF mRNA transgene expression was detectable in the hippocampi and cerebella of transgenic mice but not in age-matched, wild type mice. Elevated levels of NGF protein were detected in the hippocampi and cerebella of postnatal and adult transgenic animals as well as in conditioned media from transgenic cerebellar astrocytes in culture. The brains of these transgenic mice were found to contain postganglionic sympathetic fibers, as identified by their immunohistochemical staining for tyrosine hydroxylase and by their disappearance following superior cervical ganglionectomy. In the cerebellum, a robust plexus of sympathetic fibers was evident in the deep white matter and in the inferior cerebellar peduncles. These axons within the cerebellum were observed as early as 14 days after birth and dramatically increased in number with age. Sympathetic axons were also associated with the large blood vessels of the hippocampal fissure and were present within the hilar region of the dentate gyrus. NGF immunoreactivity was present within the sympathetic axons as well as within glial cells in the transgenic cerebellum and hippocampus. Wild type mice, however, lacked similar patterns of immunostaining. These results demonstrate that elevated expression of NGF in the intact mammalian brain results in the growth of sympathetic axons into the central nervous system in the absence of injury.

Sensory nociceptive axons invade the cerebellum of transgenic mice overexpressing nerve growth factor.

Transgenic mice possessing elevated levels of mRNA expression and synthesis for the neurotrophin nerve growth factor among astrocytes display a robust ingrowth of new sympathetic fibers to the cerebellum. In this investigation, we report that the cerebellum of these mice also possesses a dense plexus of aberrant axons of sensory origin. Axons stained immunohistochemically for calcitonin gene-related peptide were seen in the transgenic cerebellum as early as one week after birth. The density of these axons dramatically increased with age. Immunopositive axons were confined predominantly to the deep white matter of the cerebellum in the adult transgenic mice, with a smaller number of axons seen coursing along blood vessels in the gray matter. Axons stained immunohistochemically for the neurotrophin receptor, p75NTR, displayed a similar pattern of distribution and density as those immunostained for calcitonin gene-related peptide. Wild-type post-natal and adult animals lacked such calcitonin gene-related peptide- and p75NTR-immunoreactive axons in the cerebellum. Retrograde labelling revealed that these axons within the transgenic cerebellum originated from neurons in the sensory trigeminal and dorsal root ganglia (upper cervical levels). This investigation demonstrates that overexpression of nerve growth factor is capable of inducing the directional growth of collateral axons of sensory neurons into the undamaged mammalian central nervous system.

Distribution of calretinin-immunoreactive septal axons in the normal and deafferented medial habenula of adult rats.

To characterize the neural circuitry and plasticity of the septohabenular pathway, the present study analyzes the distribution of calretinin-immunoreactive fibers within the normal and deafferented medial habenula (MHb) at the light and ultrastructural levels. In the adult rat, a dense plexus of calretinin-positive fibers was found throughout the entire MHb neuropil; these immunoreactive terminals formed asymmetric synaptic contacts with unstained dendritic profiles. Calretinin-positive axons that innervate the MHb originated from neurons of the ipsilateral posterior septum, specifically those of the nucleus septofimbrialis and the nucleus triangularis. Unilateral deafferentation of the MHb resulted in the complete loss of calretinin-immunostained fibers within the ipsilateral MHb after 7 days; no reduction was apparent on the contralateral side. Four weeks after unilateral MHb deafferentation, new calretinin-immunoreactive fibers were found confined to the caudal regions of the MHb, these axons again formed asymmetrical contacts with unstained dendritic profiles. No calretinin-positive axons, however, were found within the MHb at 4 weeks following bilateral deafferentation, thus suggesting that the source of these new fibers within the long-term deafferented MHb arises from the contralateral septal neurons. Supporting this idea, injections of biotinylated dextran amine into the 4-week deafferented MHb resulted in retrogradely labeled somata observed in the contralateral posterior septum. These data reveal that septal projections to the MHb, which are normally ipsilateral, respond to a unilateral deafferentation by extending contralateral fibers that cross the midline at the habenular commissure and reinnervate the caudal regions of the nucleus.

Implantation of olfactory ensheathing cells in the adult rat brain following fimbria-fornix transection.

Ensheathing cells from fetal rat olfactory bulb were implanted into th e damaged adult rat brain to assess whether these cultured cells would survive in nonolfactory CNS areas and support the regrowth of nonolfactory axons. Cultures of primary ensheathing cells prelabeled with WGA-Au were embedded in a collagen matrix and implanted into a lesion cavity immediately following ablation of the fimbria-fornix in adult rats; the animals were sacrificed 4 weeks after surgery. Labeled ensheathing cells were observed only within the graft and not in the adjacent neural tissue. Immunostaining for p75 neurotrophin receptor revealed two characteristic morphologies of ensheathing cells within the graft; slender, spindle-shaped cells and larger, flattened cells. Although GFAP immunostaining revealed an intense glial reaction around the margin of the wound with host astrocytes sending cytoplasmic processes into the collagen matrices, no immunoreactive cells were found within the grafts. Histochemical detection of AChE revealed numerous reactive fibers confined to the regions of the grafts possessing ensheathing cells. Axons within the grafts were also immunoreactive for GAP-43. These initial experiments provide encouraging data concerning the survival of ensheathing cells for a minimum of 4 weeks following implantation into the adult rat brain and their ability to support the growth of new axons.

Transgenic neural plate contributes neuronal cells that survive greater than one year when transplanted into the adult mouse central nervous system.

Neural plate cells from the early embryo may have a number of important advantages as donor material for the delivery of foreign genes into the diseased adult central nervous system (CNS). Mesencephalic neural plate from transgenic GT4-2 mice was used as a source of marked donor cells to determine whether transgene-expressing embryonic CNS progenitor cells can be used as donor material for implantation into the adult mouse brain. Transgenic mouse embryos from this line express the Escherichia coli beta-galactosidase (beta-gal) gene throughout early CNS development. At the early somite stage (Embryonic Day 8.5), mesencephalic neural plate tissue from heterozygous embryos was dissected out and either transferred into culture for characterization or immediately implanted into the striatum or lateral ventricle of adult wild-type CD-1 mice. Explants of neural plate tissue possessed intense beta-gal activity and produced extensive outgrowth of neurofilament-positive processes after 6 days in vitro. Many beta-gal-positive cells migrated away from the explanted tissue mass. Grafts of transgenic neural plate tissue in the normal adult mouse striatum, sampled 2 weeks to 1 year after implantation, possessed healthy beta-gal-positive cells. More detailed analysis of grafts 3 months after implantation indicated that most beta-gal-positive cells were also immunoreactive for neurofilament and microtubule-associated proteins, two neuron-specific markers. In addition, extensive neurofilament-positive axonal tangles were evident within the grafts among the beta-gal-positive cells. Electron microscopic (EM) findings of implanted tissue stained with Bluo-Gal revealed many beta-gal-positive neurons received synaptic contacts from other cells. A few donor-derived astrocytes were also found in the grafts by EM analysis. No obvious signs of immunological rejection, or of significant decrease in graft volume, were observed at any age. Some beta-gal-positive cells were observed to lie up to 230 microns away from the main graft mass in both striatal and intraventricular implantations. These data suggest that the neural plate can contribute a long-surviving population of neuronal and astrocytic cells when transplanted into the adult CNS.

Somatic gene transfer of nerve growth factor promotes the survival of axotomized septal neurons and the regeneration of their axons in adult rats.

Intracerebral grafts consisting of primary fibroblasts genetically engineered to express NGF were used to assess the regenerative capacity of cholinergic neurons of the adult rat septum. Our data reveal that NGF-producing grafts sustain a significantly higher proportion of NGF receptor-immunoreactive septal neurons following axotomy (approximately 65-75%) than do grafts of noninfected fibroblasts. In addition, NGF promotes the regeneration of septal axons. Following the ablation of cholinergic septal projections to the hippocampus, NGF-producing grafts placed within the lesion cavity contain large numbers of AChE-positive axons; control grafts, on the other hand, lack such cholinergic axons. Ultrastructural examination reveals that unmyelinated axons within NGF-producing grafts use many different substrates for growth, including astrocytes and components of the extracellular matrix. Grafts of control fibroblasts possess the same cellular and matrix substrates but contain only a small population of axons, probably of peripheral origin. AChE-positive axons growing through NGF-producing grafts provide a new topographically organized input to the deafferented hippocampal dentate gyrus. Furthermore, regenerating septal axons terminate predominantly on the dendritic processes of granular neurons. The dentate gyrus ipsilateral to grafts of noninfected fibroblasts, on the other hand, remains devoid of AChE-positive fibers. From these results, we conclude that the availability of NGF is a necessary requirement to sustain axotomized cholinergic septal neurons and to promote axon regeneration and cholinergic reinnervation of dentate granular neurons by these lesioned neurons. The presence of many permissive substrates (e.g., astrocytes, basal lamina, and collagen) alone, however, is not sufficient to induce axon regrowth from adult septal neurons.