Corticofugal projections from medial primary somatosensory cortex avoid EphA7-expressing neurons in striatum and thalamus.

Within the first two postnatal weeks, corticostriatal axons from the primary somatosensory cortex (S1) form topographic projections that organize into characteristic bands of axon terminals in the dorsolateral striatum. Molecules regulating the development of these topographically organized projections are currently unknown. Thus, the present study investigated whether EphA receptor tyrosine kinases, which regulate axonal guidance in the visual system via axon repulsion, could participate in the formation of corticostriatal connections during development. Prior studies indicate that EphA7-expressing striatal neurons are organized into banded compartments resembling the matrisome innervation pattern formed by cortical afferents from the S1 cortex and that ephrin-A5, a known EphA7 ligand, is expressed in a medial (high) to lateral (low) gradient in S1. Thus, we hypothesized that the organization of EphA7-expressing striatal neurons in banded domains provides a repulsive barrier preventing corticostriatal axons containing EphA7-ligands from innervating inappropriate regions of the striatum. To evaluate this, we injected the anterograde tracer, biotinylated dextran amine (BDA), into two locations in medial areas of S1 (the anterior and posterior whisker fields), which are reported to express high levels of ephrin-A5 during development. Injections were made in mouse pups on postnatal day 9 (P9) and the animals were processed for immunohistochemistry on P12. Our data demonstrate that projections from both the forelimb/anterior whisker field and the posterior whisker field avoid EphA7-expressing neurons and terminate in a banded pattern in regions with very low EphA7-expression. We also determined that corticothalamic projections from medial S1 also exhibit a restricted distribution in the thalamus and avoid neurons expressing EphA7. Thus, our results support the hypothesis that the anatomical organization of striatal and thalamic neurons expressing EphA7 receptors restricts the topographic distribution of cortical afferents from medial regions of S1 which express high levels of ephrin-A5.

Ephrin-A binding and EphA receptor expression delineate the matrix compartment of the striatum.

The striatum integrates limbic and neocortical inputs to regulate sensorimotor and psychomotor behaviors. This function is dependent on the segregation of striatal projection neurons into anatomical and functional components, such as the striosome and matrix compartments. In the present study the association of ephrin-A cell surface ligands and EphA receptor tyrosine kinases (RTKs) with the organization of these compartments was determined in postnatal rats. Ephrin-A1 and ephrin-A4 selectively bind to EphA receptors on neurons restricted to the matrix compartment. Binding is absent from the striosomes, which were identified by mu-opioid receptor immunostaining. In contrast, ephrin-A2, ephrin-A3, and ephrin-A5 exhibit a different mosaic binding pattern that appears to define a subset of matrix neurons. In situ hybridization for EphA RTKs reveals that the two different ligand binding patterns strictly match the mRNA expression patterns of EphA4 and EphA7. Ligand-receptor binding assays indicate that ephrin-A1 and ephrin-A4 selectively bind EphA4 but not EphA7 in the lysates of striatal tissue. Conversely, ephrin-A2, ephrin-A3, and ephrin-A5 bind EphA7 but not EphA4. These observations implicate selective interactions between ephrin-A molecules and EphA RTKs as potential mechanisms for regulating the compartmental organization of the striatum.

Truncated trkB receptors on nonneuronal cells inhibit BDNF-induced neurite outgrowth in vitro.

The function of truncated trkB receptors during nervous system plasticity and regeneration is currently unknown. The extensive nonneuronal localization of truncated trkB-T1 receptors, coupled with their up-regulation by CNS glial cells in response to injury, has led to the speculation that these receptors may sequester BDNF and NT-4/5 to reduce their local availability and, thus, limit axonal sprouting. Conversely, trkB-T1 receptors could bind and present neurotrophins to injured axons and facilitate their regeneration in a manor analogous to that proposed for p75(NTR) receptors on Schwann cells. To address this issue, we used an in vitro coculture paradigm in which wild-type 3T3 NIH fibroblasts or two different 3T3 cell clones stably expressing trkB-T1 receptors served as monolayer substrates upon which to evaluate the effect of trkB-T1 receptors on nonneuronal cells to influence neurotrophin (NGF, BDNF, NT-3, and NT-4/5)-induced neurite outgrowth from retinoic acid (RA)-treated SY5Y neuroblastoma cells. In these experiments, BDNF and NT-4/5 produce a strong phosphorylation of trk receptors on the RA-SY5Y cells and induce differentiation of the SY5Y cells (as measured by the development of neurofilament-positive neuritic processes). This ability of the trkB ligands to stimulate neurite outgrowth is dose dependent since increasing concentrations of BDNF (5, 25, and 100 ng/ml) result in an increased percentage of SY5Y cells developing neurites and in progressively longer neurites from SY5Y cells on the control 3T3 monolayers. In these experiments, BDNF and NT-4/5 induce the strongest neurite outgrowth, followed by NT-3 and then NGF. When trkB-T1 receptors are present on the 3T3 cell substratum both BDNF- and NT-4/5-induced neurite extension from the SY5Y cells are strongly inhibited. In contrast, NGF-induced neurite growth is unaffected and NT-3-associated growth is somewhat reduced. These results suggest that the inhibitory effect of the trkB-T1 receptors on the nonneuronal cell substrates is selective for neurite outgrowth that is mediated via the trkB-kinase receptors on the neuroblastoma cells. This ability of trkB-T1 receptors on the nonneuronal substratum to inhibit BDNF-induced neurite outgrowth can be overcome by the addition of high concentrations of BDNF (1 microg/ml). Binding assays using 125I-BDNF suggest that this inhibitory effect could be mediated via binding and internalization of BDNF by the trkB-T1 receptors on the 3T3 cells. These results provide strong support for the hypothesis that the up-regulation of trkB-T1 receptors on astrocytes following CNS lesions enhances the sequestration of the trkB ligands, BDNF and NT- 4/5, at the site of reactive gliosis and, thus, contributes to the inhibition of CNS axonal regeneration from neurons expressing trkB-kinase receptors by removing their ligands from the extracellular environment.

Developmental and mature expression of full-length and truncated TrkB receptors in the rat forebrain.

The neurotrophins brain-derived neurotrophic factor (BDNF) and NT-4/5 exert their trophic effects on the nervous system via signaling through trkB receptors. These receptors occur as splice variants of the trkB gene that encodes a full-length receptor containing the signal transducing tyrosine kinase domain as well as truncated forms lacking this domain. Because the importance of the trkB isoforms for development and maturation of the nervous system is unknown, we have examined the expression of trkB receptor isoforms during development of the rat forebrain using 1) a sensitive ribonuclease protection assay to distinguish full-length and truncated trkB transcripts, 2) western blot analysis to characterize developmental changes in trkB proteins, and 3) immunohistochemistry to determine the cellular localization of trkB receptors. In the rat forebrain, adult mRNA levels for full-length trkB are reached by birth, whereas truncated trkB message does not peak until postnatal days 10-15. Western blot analysis indicates that full-length trkB protein is the major form during early development, whereas truncated trkB protein predominates in all forebrain regions of late postnatal and adult rats. These data also suggest that the glycosylation state of these receptors changes during postnatal maturation. TrkB immunoreactivity is present predominately within neurons, where it is localized to axons, cell soma, and dendrites. Strong dendritic immunostaining is particularly evident in certain neuronal populations, such as pyramidal neurons in the hippocampus and in layer V of the neocortex. The dendritic localization of trkB receptors supports the hypothesis that dendrites, as well as axons, are important sites for neurotrophin actions in the central nervous system.

The basal forebrain cholinergic system and object memory in the rat.

Rats with near complete destruction of basal forebrain cholinergic neurons from intracerebroventricular injections of 192 IgG-saporin were trained on object discrimination problems and then retrained two weeks later to measure retention. Despite dramatic reductions of acetylcholinesterase-positive fibers in hippocampus and neocortex, these animals did not differ from controls on an analysis of savings scores. Thus, the basal forebrain cholinergic system may serve functions that support non-spatial memory but are not specifically mnemonic in nature.

Regulation of TrkA and ChAT expression in developing rat basal forebrain: evidence that both exogenous and endogenous NGF regulate differentiation of cholinergic neurons.

TrkA is a receptor tyrosine kinase whose activation transduces NGF signaling. TrkA expression has been demonstrated in NGF-responsive adult basal forebrain cholinergic neurons (BFCNs). Several lines of evidence have suggested that endogenous NGF plays a role in the development and differentiation of these neurons. We examined TrkA expression during development. TrkA mRNA and protein were present in basal forebrain neurons during the entire postnatal period; the distribution of neurons bearing these markers was identical to that for those containing choline acetyltransferase (ChAT) mRNA, suggesting that, as in the adult, TrkA gene expression is localized to BFCNs. The expression of TrkA and ChAT followed a very similar temporal pattern, suggesting regulation by the same factor(s). We discovered that NGF administration in vivo activated TrkA receptors, and increased both TrkA and ChAT mRNA; conversely, anti-NGF infusions suppressed expression of both genes. These results suggest that endogenous NGF regulates expression of TrkA and ChAT. Finally, while NGF infusion increased the size of developing BFCNs, NGF antibodies inhibited the normal developmental increase. The results are evidence that endogenous NGF acts on developing BFCNs to enhance gene expression and cellular differentiation.

Entorhinal-hippocampal connections and object memory in the rat: acquisition versus retention.

Investigations of the neurobiology of memory using experimental animals have modeled many of the characteristic features of amnesia seen in human clinical populations. To examine long-term memory, however, animal models of amnesia often employ extended measures of acquisition, which stand in contrast to the retention measures used with humans. To determine the role of entorhinal-hippocampal circuitry on both information acquisition and long-term retention, rats with bilateral transections of the angular bundle were trained on three object discrimination problems and then retrained two weeks later to measure retention. Animals with discrete lesions of the angular bundle, which disrupted perforant path connections from the entorhinal cortex to the hippocampus and efferent hippocampal-cortical projections, acquired the object discrimination problems normally but showed a marked deficit in retention. These findings are important because they indicate that the role of entorhinal-hippocampal connections may be limited to maintaining some types of information (e.g., single object discriminations) for retention. This dissociation, moreover, suggests that behavioral paradigms that include a measure of retention may be particularly important for characterizing the mnemonic functions of the hippocampal/parahippocampal region.

Cholinergic deafferentation of dorsal hippocampus by fimbria-fornix lesioning differentially regulates subtypes (m1-m5) of muscarinic receptors.

Unilateral aspiration lesions of the rostral supracallosal stria/cingulum bundle and fimbria-fornix were performed on adult female rats. Ten and 24 days post lesioning, an elevation (17%; p < 0.01) of total muscarinic receptors was observed in lesioned versus control hippocampi. By using antisera selective for each of the five molecularly defined subtypes (m1-m5) of muscarinic receptors, significant changes were observed in the levels of expression for at least four receptor proteins. Three receptor subtypes increased in density: m1 by 14% (from 943 to 1,078 fmol/mg); m3 by 77% (from 150 to 268 fmol/mg); and m4 by 29% (from 220 to 285 fmol/mg). In contrast, a 22% decrease in the density of m2 receptors was found (from 220 to 173 fmol/mg). Detectable levels of m5 receptors were low in the hippocampus (approximately 1% of total receptors), and reliable measurements were not obtained. The directions of these changes are likely to be related to the pre- or postsynaptic localization of these receptor subtypes.

Isolation and characterization of Bsk, a growth factor receptor-like tyrosine kinase associated with the limbic system.

Neuronal degeneration has been shown to be involved in various neurological disorders. Growth/trophic factors and their receptors are known to be important for the regeneration and survival of neurons. We report here the molecular cloning of a receptor-like protein tyrosine kinase, bsk, (for brain specific kinase). Bsk is highly related to the eph/elk receptor-like kinase family members. Northern blot analysis shows that it is expressed specifically in the brain, with no expression detected in adult heart, spleen, lung, liver, skeletal muscle, and kidney. In situ hybridization analysis of adult mouse brain sections indicates that bsk is expressed at high levels in the hippocampus, tenia tecta, indusium griseum, and the piriform cortex, major components of the limbic system that are important for learning and memory. In addition, elevated levels of expression are found in other areas of the limbic system such as the amygdala, medial septum, and nucleus of the diagonal band, and in the olfactory bulb, which has close connections to the limbic system. The highest level of expression is found in the CA3 region of the hippocampus and the pyramidal cell layer of the piriform cortex. In 16.5 day mouse embryos, bsk is expressed predominantly in the primordial cortex of the telencephalon. An antibody against a C-terminal peptide of bsk recognized a 105 kD protein in the 16.5 day embryonic head extract. Our analysis shows that bsk is a growth factor receptor-like protein tyrosine kinase and that its greatest expression in the adult brain is associated with components of the limbic system.

Role of the parahippocampal region in spatial and non-spatial memory: effects of parahippocampal lesions on rewarded alternation and concurrent object discrimination learning in the rat.

Rats with aspiration or excitotoxic (NMDA) lesions of the parahippocampal region were trained on a series of behavioral tasks which consisted of: (1) a test of spatial memory (discrete trial rewarded alternation), (2) a black-white discrimination, and (3) a test of non-spatial memory commonly used in primate models of amnesia (visual concurrent object discrimination). Rats in both lesion groups were severely impaired on the concurrent discrimination, even though they were able to learn the black-white discrimination normally. Animals with aspiration lesions were also impaired on the spatial memory task, whereas those with NMDA lesions did not differ from controls. The results indicate that concurrent object discrimination is a particularly sensitive measure of hippocampal/parahippocampal functions and suggest that these structures in the rat may serve mnemonic functions which are qualitatively similar to those of human and non-human primates.

Effects of delayed transplantation of cultured Schwann cells on axonal regeneration from central nervous system cholinergic neurons.

The introduction of transplants consisting of cultured Schwann cells and their associated extracellular matrix (Sc/ECM) into a central nervous system (CNS) lesion cavity facilitates axonal regeneration from injured, adult mammalian neurons with subsequent reinnervation of their appropriate target (Kromer and Cornbrooks: Proceedings of the National Academy of Sciences of the United States of America 82:6330-6334, 1985). In the present study, the effects of a delayed transplantation procedure on the time course of this regenerative response were evaluated. For these experiments, bilateral CNS lesions were created between the septum and hippocampus by removing the fimbria-fornix pathway. Lesion cavities received either no transplants, transplants of collagen, or Sc/ECM transplants at the time the lesion was created or 6 days later. When no transplants or transplants of collagen were used, axonal sprouts extended for very short distances into the lesion cavity. These axons were not preferentially associated with the collagen transplants nor maintained at long post-lesion survival times. In animals that received Sc/ECM transplants, the number of sprouting axons and the progression of axonal growth along the transplants was much more extensive than for the collagen transplants. Although more axons were detected in cavities that received transplants immediately after the fimbria-fornix lesion, axonal regeneration along the transplants was similar regardless of whether there was a delay in transplanting the Schwann cells. By using histochemical techniques to identify acetylcholinesterase (AChE), regenerating AChE-positive axons were first detected in the cavity at 3 days post-transplantation, were associated with the Sc/ECM transplants by 5 days, and crossed the cavity within 8 days post-transplantation. Regenerating, neurofilament-positive axons crossed the CNS-Sc/ECM transplant interfaces in association with laminin-positive, glial fibrillary acidic protein-positive cellular pathways. Upon reaching the caudal end of the Sc/ECM transplant, the cholinergic axons abandoned the transplant and oriented directly toward the adjacent hippocampus. Both the simultaneous and delayed transplantation paradigms demonstrated a similar reinnervation pattern of AChE-positive fibers in the hippocampus, but there was a more rapid penetration and more extensive arborization of fibers in animals receiving the delayed transplants. Cholinergic fibers initially invaded the dentate gyrus molecular layer and hilus between 8 and 14 days post-transplantation. By 45 days post-transplantation, AChE-positive axons were detected throughout the dentate gyrus and regio inferior, but few fibers were present in regio superior of the hippocampus.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of sciatic nerve transplants after fimbria-fornix lesion: examination of the role of nerve growth factor.

At two weeks post-transplantation, sciatic nerves inserted into the lesioned septo-hippocampal pathway contain NGF levels more than twice that of normal nerves. These transplanted nerves also contain regenerating cholinergic axons. Moreover, transplanted animals exhibit septal NGF levels that are significantly greater than in animals with lesions only. These results suggest a role for NGF in the ingrowth of axons into the transplants and in the increase in ChAT(+) septal neurons previously observed at this post-transplant time.

Object recognition memory in the rat: the role of the hippocampus.

Object recognition memory of rats with fimbria-fornix or ventral temporal lesions was evaluated with a behavioral protocol (delayed non-matching-to-sample task with trial-unique stimuli) similar to that used to test recognition functions in primates. Animals with damage to the hippocampal system showed no evidence of lasting impairment on the object recognition task with retention intervals up to 30 s. In contrast, rats with fimbria-fornix lesions displayed severe and enduring deficits on a test of spatial memory, i.e. rewarded alternation, with but 5 s delays. These results provide further evidence that a dissociation exists between the types of memory that are and are not lost following damage to the hippocampus. Whereas the hippocampus is necessary for some types of mnemonic processes, other types of recognition functions (e.g. perceptual recognition) may be fully mediated in regions of sensory and/or association neocortex without the involvement of the hippocampus.

Transplants of cholinergic septal explants reinnervate adult rodent hippocampus.

Embryonic septal-basal forebrain tissue was grown in explant culture for 1, 4 or 5 days prior to transplantation to the hippocampus of adult rats denervated of its septal input by a fornix/fimbria transection. The explant transplants were compared with transplants of septal cell suspensions. Analysis of fiber ingrowth by acetylcholinesterase (AChE) histochemistry at various timepoints post-transplantation shows little difference in the developmental time course or extent of cholinergic axon ingrowth into the host hippocampus between the suspension transplants and the explant transplants. Septal explants in culture for 5 days were as effective as those in culture for one day prior to transplantation in providing cholinergic reinnervation to the host hippocampus. Studies using antibodies to choline acetyltransferase (ChAT) and the fluorescent retrograde tracer, fast blue, confirmed that the AChE-positive cells in the transplants were cholinergic and that the innervation to the host hippocampus came from the transplanted cholinergic neurons. Thus the results demonstrate that embryonic cholinergic septal neurons can be maintained in short-term culture prior to transplantation without adversely affecting their ability to innervate an appropriate CNS target in vivo.

Nerve growth factor treatment after brain injury prevents neuronal death.

Cholinergic neuronal degeneration after axotomy has been proposed to be due to the loss of a retrogradely transported neurotrophic factor, possibly nerve growth factor (NGF). To test this hypothesis, NGF was continuously infused into the lateral ventricles of adult rats that had received bilateral lesions of all cholinergic axons projecting from the medial septum to the dorsal hippocampus. After 2 weeks of NGF treatment, identification of cholinergic neurons by the presence of the biosynthetic enzyme choline acetyltransferase revealed a dramatic increase (350%) in the survival of the axotomized septal cholinergic neurons. Thus, NGF or an NGF-like molecule can act as a neurotrophic factor for these neurons.

Identification of trophic factors and transplanted cellular environments that promote CNS axonal regeneration.

As indicated in this review, we have begun to elucidate cellular environments and trophic factors that promote the regeneration of adult mammalian CNS neurons. In the present paradigm, bilateral aspiration lesions of the fornix-fimbria are used to axotomize septal neurons and transect the septal cholinergic projection to the dorsal hippocampus in order to evaluate the influence of trophic factors, such as NGF, on neuronal survival and the ability of cellular transplants of PNS tissue to promote axonal regeneration in vivo. Initial results demonstrate that NGF is a potent trophic molecule that prevents retrograde degeneration of septal cholinergic neurons. Observations from transplantation studies demonstrate that viable Schwann cells obtained from PNS nerve grafts or Schwann cell-ECM cultures provide a favorable cellular milieu for CNS regeneration. These cellular transplants induce a remarkable sprouting response from septal cholinergic neurons and promote the rapid elongation of septal axons that reinnervate the denervated hippocampus. In stark contrast to the Schwann cell-laden transplants, transplants including only ECM channels synthesized by cultured Schwann cells do not promote axonal regeneration within the time periods that we have examined. Therefore, we hypothesize that viable Schwann cells are crucial for the process of regeneration because they contribute both trophic and tropic factors to the injured CNS neurons. The significant early sprouting phenomenon associated with transplants containing Schwann cells strongly suggests that soluble Schwann cell-synthesized factors induce axon elongation and possibly enhance the survival of injured septal neurons. The trophic factors probably function in a manner similar, if not identical, to the action of NGF on axotomized septal neurons. Moreover, Schwann cells appear to provide tropic signals, such as LAM or a LAM-NGF complex, that can act, when in the proper stereoconfiguration, to promote the elongation and orientation of regenerating axons. Thus, our current data indicate that in order to promote optimal axonal regeneration from injured CNS neurons, both trophic and tropic factors must be supplied from exogenous sources.

Development and neuronal organization of dissociated and reaggregated embryonic cerebellum after intracephalic transplantation to adult rodent recipients.

Embryonic (E13) rat cerebellar primordia were dissociated and aggregated into tissue pellets by centrifugation. After transplantation into premade intracephalic cavities in adult rats, transplant development was evaluated at 2, 4 and 6 weeks survival. Within the cerebellar pellets there is an initial sorting of large neurons (Purkinje cells from deep nuclear cells) followed by the segregation of developing cortical cells into a trilaminar organization. These results suggest that this preparation should be useful for analyzing cellular interactions that determine cerebellar cytoarchitectural organization.

Transplants of Schwann cell cultures promote axonal regeneration in the adult mammalian brain.

Transplantation of embryonic brain tissue or mature peripheral nerves into the adult mammalian central nervous system promotes axonal regrowth from axotomized central nervous system neurons; however, the cellular origin and molecular nature of the factors promoting axonal growth in vivo are unknown. To further characterize cellular environments that facilitate regeneration of central nervous system axons, we developed a methodology whereby cultured cell preparations can be transplanted into the brain of mature mammals. For this procedure, lesions are produced in the septal-hippocampal system of adult rats, and selected regions from collagen-supported Schwann cell/neuron cultures (consisting of Schwann cells, extracellular matrix, and degenerating neuronal processes and myelin but devoid of neuronal perikarya and fibroblasts) are positioned within the intracephalic cavity so that they bridge the lesion gap (approximately 3 mm) separating the septum and hippocampus. At various time up to 3 weeks after transplantation, specimens were prepared for acetylcholinesterase histochemistry and the immunocytochemical localization of laminin (an extracellular matrix protein) and C-4 (a Schwann cell membrane antigen). All specimens (from uninjured controls and from animals with either acellular collagen or mature Schwann cell/extracellular matrix transplants) contained laminin immunoreactivity associated with the meninges, choroid plexus, ependyma, and cerebral blood vessels. All animals with transplants showed prominent laminin staining on astrocytic processes along the intracephalic cavity, but only the Schwann cell/extracellular matrix transplants exhibited dense laminin and C-4 immunoreactivity within the cellular portion of the transplants. Regeneration of acetylcholinesterase-positive septal fibers occurred only in animals containing Schwann cell/extracellular matrix transplants. By 6 days after transplantation, acetylcholinesterase-positive fibers were observed both on laminin-positive cellular tissue strands connecting the septum and the Schwann cell/extracellular matrix transplants and on the initial portions of the transplants. By day 14, acetylcholinesterase-positive fibers traversed the entire lesion cavity in intimate association with the laminin- and C-4-positive cellular layer of the transplants and reinnervated the host hippocampus. However, cholinergic fibers were not associated with all laminin-containing processes along the lesion cavity nor did they grow along acellular collagen transplants. These results indicate the presence of factors in transplants of cultured Schwann cells and their associated extracellular matrix that promote rapid regeneration of central nervous system cholinergic axons in vivo.

Outbred Sprague-Dawley rats from two breeders exhibit different incidences of neuroanatomical abnormalities affecting the primary cerebellar fissure.

A significant variation in the incidence of alterations in the cellular organization along the primary fissure of the cerebellum is observed in outbred Sprague-Dawley rats obtained from Hilltop Animal Labs (89.9%) and Charles River Canada (34.1%). In this abnormality, there are areas in the depths of the primary fissure which lack pial cells, exhibit fusion of the apposing molecular layers, and contain ectopic granule cells, often with an associated disorganization of the cortical laminae. The extent and incidence of the abnormality can vary between individuals and litters, but the general incidence rate for a given breeder appears to be consistent over extended breeding cycles. This finding has serious implications for the use of outbred animals from different breeders when collecting data during the course of an experiment and when comparing and replicating data from different laboratories.