Neurogenesis: A process ontogenically linked to brain cavities and their content, CSF.

Neurogenesis is the process underlying the development of the highly evolved central nervous system (CNS) in vertebrates. Neurogenesis takes place by differentiation of specific Neural Precursor Cells in the neurogenic niche. The main objective of this review is to highlight the specific relationship between the brain cavities, and neurogenesis from neural precursors. Brain cavities and their content, Cerebrospinal Fluid (CSF), establish a key relation with the neurogenic "niche" because of the presence in this fluid of neurogenic signals able to control neural precursor cell behaviour, inducing precursor proliferation and neuronal differentiation. This influence seems to be ontogenically preserved, despite the temporal and spatial variations that occur throughout life. In order to better understand this concept, we consider three main life periods in the CSF-Neurogenesis interaction: The "Embryonic" period, which take place at the Neural Tube stage and extends from the isolation of the neural tube at the end of "neurulation" to the beginning of Choroid Plexus activity; the "Fetal" period, which includes the remaining developmental and the early postnatal stages; and the "Adult" period, which continues for the rest of adult life. Each period has specific characteristics in respect of CSF synthesis and composition, and the location, extension and neurogenic activity of the neurogenic niche. However, CSF interaction with the neurogenic niche is a common factor, which should be taken into account to better understand the ontogeny of neuron formation and replacement, as well as its potential role in the success or failure of therapies for the ageing, injured or diseased brain.

The use of flow cytometry to examine calcium signalling by TRPV1 in mixed cell populations.

Flow cytometric analysis of calcium mobilisation has been in use for many years in the study of specific receptor engagement or isolated cell:cell communication. However, calcium mobilisation/signaling is key to many cell functions including apoptosis, mobility and immune responses. Here we combine multiplex surface staining of whole spleen with Indo-1 AM to visualise calcium mobilisation and examine calcium signaling in a mixed immune cell culture over time. We demonstrate responses to a TRPV1 agonist in distinct cell subtypes without the need for cell separation. Multi parameter staining alongside Indo-1 AM to demonstrate calcium mobilization allows the study of real time calcium signaling in a complex environment.

Bcr-Abl protein tyrosine kinase activity induces a loss of p53 protein that mediates a delay in myeloid differentiation.

Chronic myeloid leukaemia is a haemopoietic stem cell disorder, the hallmark of which is the expression of the Bcr-Abl Protein Tyrosine Kinase (PTK). We have previously reported that activation of a temperature sensitive Bcr-Abl PTK in the multipotent haemopoietic cell line FDCP-Mix for short periods resulted in subtle changes including, a transient suppression of apoptosis and no inhibition of differentiation. In contrast, activation of the Bcr-Abl PTK for 12 weeks results in cells that display a delay in differentiation at the early granulocyte stage. Flow cytometric analysis also indicates that the expression of cell surface differentiation markers and nuclear morphology are uncoupled. Furthermore, a significant number of the mature neutrophils display abnormal morphological features. Prolonged exposure to Bcr-Abl PTK results in interleukin-3 independent growth and decreased p53 protein levels. FDCP-Mix cells expressing a dominant negative p53 and p53null FDCP-Mix cells demonstrate that the reduction in p53 is causally related to the delay in development. Returning the cells to the restrictive temperature restores the p53 protein levels, the growth factor dependence and largely relieves the effects on development. We conclude that prolonged Bcr-Abl PTK activity within multipotent cells results in a reduction of p53 that drives a delayed and abnormal differentiation.

Flt3 ligand can promote survival and macrophage development without proliferation in myeloid progenitor cells.

Flt3 ligand elicits a variety of effects on early hemopoietic progenitors by occupying its cognate receptor, Flt3, a member of the type III tyrosine kinase receptor family. The cytokines macrophage colony-stimulating factor (M-CSF) and stem cell factor (SCF) bind to related members of this tyrosine kinase receptors family, c-fms and c-kit, respectively. The relative effects of the cytokines M-CSF, SCF, and Flt3L on the proliferation and development of the late myeloid progenitors granulocyte-macrophage colony-forming cells (GM-CFC) were investigated. Distinct biologic responses were stimulated by ligand binding to these different tyrosine kinase receptors in enriched GM-CFC. M-CSF stimulated GM-CFC to proliferate and develop into macrophages. SCF, on the other hand, stimulated GM-CFC to develop into neutrophils. Flt3 ligand had a relatively small proliferative effect on enriched GM-CFC compared to SCF and M-CSF and had no ability to either stimulate colony formation or synergize with these two cytokines in promoting DNA synthesis, colony formation, or expansion in liquid culture. Flt3 ligand, however, was capable of maintaining the clonogenic potential of GM-CFC and acted as an anti-apoptotic agent as assessed using the Annexin-V apoptosis assay. GM-CFC cultured in Flt3 ligand eventually formed macrophages and neutrophils in liquid culture. Labeling with the membrane-associated cell tracker dye PKH26 indicated that the majority of the enriched GM-CFC responded to Flt3 ligand by undergoing limited proliferation and macrophage development, whereas other cells survived but did not proliferate and differentiate into macrophages. Thus, Flt3 ligand promoted survival and stimulated development without proliferation in primary-enriched myeloid progenitor cells.

Neuropeptide control of bone marrow neutrophil production. A key axis for neuroimmunomodulation.

Nerve fibers project into the bone marrow and terminate in association with stromal cells. Nerve terminals are also associated with antigen-processing and -presenting cells throughout the body and have been shown to be important in leukocyte trafficking and wound healing as well as hemopoiesis. Here we show that neuropeptide input to the bone marrow is vital to normal granulopoiesis and that deletion of the neuropeptides, substance P, and calcitonin gene-related peptide (CGRP), with the neurotoxin, capsaicin, abrogates normal blood cell production. Norepinephrine, neurokinins a and 2, and vasoactive intestinal peptide all have inhibitory effects on in vitro CFU-GM colony formation. Substance P, neurokinin 1, nerve growth factor, and CGRP have stimulatory effects on CFU-GM. Furthermore, in vitro experiments show that, apart from CGRP, all the neuroactive substances we tested operate through effects on accessory cells, stimulating the release of regulatory molecules that have a direct effect on purified CFU-GM.

Neural control in the immunocytotoxic destruction of muscles in Diptera.

Following successful escape from the puparium (eclosion), sets of muscles in all three segments of dipteran flies degenerate. Whereas the head and abdominal muscles degenerate in response to hormonal triggers released before, and immediately after eclosion, the thoracic muscles require a specific neural trigger encountered following eclosion. Evidence is presented for the role of neural activity in the activation of immunocytes that destroy the thoracic muscles. Removal of the neural input by severing the nerve to any particular muscle results in survival of the muscle and inactivation of the immunocyte. The destruction process can be stopped at any time by severing the nerve and the muscle fibres that remain continue to show normal physiology and response to stimulation. Elcctrophysiological recordings of the response to lethal attack are presented together with ultrastructural evidence demonstrating the invasion of muscle fibres by processes of the immunocyte.

Induction of ACTH- and TNF-alpha-like molecules in the hemocytes of Calliphora vomitoria (Insecta, Diptera).

Three basic cell types are described in the hemolymph of newly enclosed adult Calliphora vomitoria: prohemocytes, plasmatocytes, and granular cells. In addition, anucleate cellular fragments with some inclusions are observed. Cell division is found only in the prohemocytes. Plasmatocytes and granular cells are capable of in vitro bacterial phagocytosis and take part in capsule formation. Adrenocorticotropin hormone (ACTH)-like molecules are found in both cell types. The staining of the plasmatocytes is related to the functional activation of the cells. Indeed, positive staining is only observed during capsule formation, suggesting an induction of ACTH expression. The behaviour of tumor necrosis factor (TNF)-alpha-like molecules is similar. In conclusion, these data provide further support for the close, evolutionary relationship between the immune and the neuroendocrine systems.

Coordinated host defense through an integration of the neural, immune and haemopoietic systems.

Interactions between the neural and immune systems exist through humoral factors operating via the hypothalamic-pituitary-adrenal axis and cytokines acting over a relatively long distance. Anatomical evidence also suggests direct, hard-wired pathways of interaction and control through innervation of lymphoid organs and peripheral sites involved in host defense, including the thymus, spleen, lymph nodes, and skin. Recent evidence has demonstrated: 1) neural control of the bone marrow haemopoietic system, 2) interactions between peripheral nerve endings in the skin and epidermal Langerhans cells, and 3) peripheralization of leukocytes in the initial stages of stress. This leads us to propose that the nervous system is involved in host monitoring and coordination of host defense systems. If the brain is to have appropriate control of host defense mechanisms it must have: (a) afferent inputs monitoring host defense status, (b) efferent control pathways that modulate primary reactions to infection and damage, (c) efferent activation pathways to the myeloid defense system while the specific, lymphoid immune system is activated, and (d) inhibition of the proliferative lymphocytic response if the infection has been dealt with. We are investigating whether such a model, which allows for control and coordination of both the initial myeloid defense system and of the acquired immune response, is observed in mammals.

The functional outcome of shunting H-Tx rat pups at different ages.

"Normal", untreated hydrocephalic and CSF shunted hydrocephalic Texas (H-Tx) rat pups at different ages and Sprague-Dawley controls were tested for spatial learning and memory acquisition in an 8-arm radial maze. Shunting 5 day old hydrocephalic pups improved their performance to the level of that of "normal" litter-mates, whereas shunting at 10 days produced no improvement, and the performance of these animals was comparable to that of the untreated hydrocephalics. None of the H-Tx rats, whether treated, untreated or "normal", performed as well as the Sprague-Dawley controls. These findings suggest that shunting prior to skull plate fusion, which occurs at approximately the tenth post-natal day, is capable of protecting brain function involved in memory and learning. The poor functional scores achieved by the "normal" as well as the treated and untreated H-Tx animals compared to the control Sprague-Dawleys, raises the possibility that the H-Tx rat has brain deficits in addition to those caused by hydrocephalus.

Study of the motor corticospinal system in the developing rat fetus: comparison of Wistar and normal and hydrocephalic HTx rats.

The motor corticospinal system can be identified from day E14 in Wistar and HTx fetuses. There are no significant anatomical differences between the two species of rats. In addition, in day E17 Wistar and HTx fetuses cell counts in the cortical mantle (cortical plate, intermediate zone and germinal matrix) are similar. However, in day E20 fetuses there are significant differences in the number of cells in the cortical mantle of the hydrocephalic HTx fetuses compared to that in the Wistar and normal HTx fetuses, their total number of cells being reduced compared to that of the normal HTx and Wistars. Breakdown of the numbers of cells in the different layers shows that in the hydrocephalics there is a significant reduction in the number of cells in the germinal matrix and intermediate zone but, although the number of cells is also reduced in the cortical plate, the reduction is not significant. Measurements of the anterior/posterior width of the pyramid show that its growth is almost complete by day E17 and that on day E20 the measurements are similar in Wistar and normal and hydrocephalic HTx fetuses. These findings suggest that it is only cells generated after day E17 that are missing from the cortex of day E20 hydrocephalic rats. It is known that the motor corticospinal tract axons arise from pyramidal cells in layers 6, 5 and 4 of the cortical plate. These layers are generated earlier than layers 3 and 2 and are almost certainly in place by day E17 and account for why motor corticospinal tract function is spared in younger animals with established hydrocephalus.

Changes in the CSF fluid pathways in the developing rat fetus with early onset hydrocephalus.

Interest in the factors involved in the abnormal cortical development of the HTx rat fetus have led us to re-examine the structural and morphological changes in the CSF pathways preceding constriction and blockage of the cerebral aqueduct. Histological analysis was carried out on coronal and sagittal sections from HTx and Wistar fetuses. The aqueduct is found to be a broad channel extending from the posterior end of the third ventricle that ends in a blind pouch above the developing cerebellum. The aqueduct drains into the fourth ventricle via a vertically orientated, narrow channel lying between the posterior aspect of the pontine flexure and the anterior surface of the cerebellum. On Day E18 the connecting channel between the aqueduct and the fourth ventricle is blocked by apposition of its walls. 24 hours later the lateral ventricles begin to dilate and the anterior end of the aqueduct is blocked and the connecting channel between the aqueduct and the fourth ventricle reopens. The cause of these sequential changes in the CSF fluid pathways remains speculative.

Effects of hydrocephalus on nitric oxide neurones in the brain of the HTx rat.

The effect of hydrocephalus on the nitric oxide system in the brain of the HTx rat brain was investigated. We used NADPH-diaphorase histochemistry to stain neurones containing the enzyme, nitric oxide synthase. Comparison of normal and hydrocephalic HTx rat brains showed a reduction in stained neurones and a greatly depressed staining intensity in those that were labelled in the hydrocephalic brain. Also, there was an apparent reduction in number and length of radiating fibres from stained neurones. In addition to these changes we also observed a clear association between stained neurones and the microvasculature of the brain. This suggests that changes in the activity of these neurones may have a significant impact on the normal perfusion of the brain.

Cell death in the brain of the HTx rat.

The structural changes in the hydrocephalic brain suggest that cell death must be a significant factor in the loss of cortical mantle thickness and cell numbers. In the developing hydrocephalic brain a similar conclusion might also be reached. We present data suggesting that in the developing hydrocephalic HTx rat brain, there are at least two processes that underlie the structural deficit in the cortex. The first is abnormal development of the cortex probably resulting from a combination of the genetic defect and blockage of CSF circulation in the fetus. The second is a direct consequence of raised intracranial pressure and is manifested in a lack of development of glial cells in the neonatal brain and in the loss of myelination and neurones as pressure rises after birth, particularly after skull plate fusion. We find little evidence of increased cell death by apoptosis, recording instead a decrease in apoptotic index. There is evidence that necrosis must have occurred.

Cross-talk between neural and immune receptors provides a potential mechanism of homeostatic regulation in the gut mucosa.

The relationship between elements of the immune system and the nervous system in the presence of bacteria has been addressed recently. In particular, the sensory vanilloid receptor 1 (transient receptor potential cation channel subfamily V member 1 (TRPV1)) and the neuropeptide calcitonin gene-related peptide (CGRP) have been found to modulate cytokine response to lipopolysaccharide (LPS) independently of adaptive immunity. In this review we discuss mucosal homeostasis in the gastrointestinal tract where bacterial concentration is high. We propose that the Gram-negative bacterial receptor Toll-like receptor 4 (TLR4) can activate TRPV1 via intracellular signaling, and thereby induce the subsequent release of anti-inflammatory CGRP to maintain mucosal homeostasis.