Platelet-derived growth factor promotes survival of rat and human mesencephalic dopaminergic neurons in culture

The effect of two isoforms of platelet-derived growth factor (PDGF), PDGF-AA and PDGF-BB, was tested on dissociated cell cultures of ventral mesencephalon from rat and human embryos. PDGF-BB but not PDGF-AA reduced the progressive loss of tyrosine hydroxylase- (TH)-positive neurons in rat and human cell cultures. The mean number of TH-positive cells in the PDGF-BB-treated rat culture was 64% and 106% higher than in the control cultures after 7 and 10 days in vitro, respectively. Corresponding figures for human TH-positive neurons were 90% and 145%. The influence of PDGF-BB was specific for TH-positive neurons and not a general trophic effect, since no change of either total cell number or metabolic activity was found. In PDGF-BB-treated cultures of human but not rat tissue the TH-positive neurons had longer neurites than observed in control or PDGF-AA-treated cultures. These data indicate that PDGF-BB may act as a trophic factor for mesencephalic dopaminergic neurons and suggest that administration of PDGF-BB could ameliorate degeneration and possibly promote axonal sprouting of these neurons in vivo.     

Astrocyte-dependent and -independent phases of the development and survival of rat embryonic day 14 mesencephalic, dopaminergic neurons in culture

A primary neuronal culture was prepared from the ventral mesencephalon, centered on the A8, A9 and A10 dopaminergic nuclei of the embryonic day 14 rat, and studied from 12 h to 28 days. At 12 h after plating, and before cell death ensued, 95% of the cells stained positive for neuron specific enolase; 20% for tyrosine hydroxylase; 5% for vimentin and < 0.1% for glial fibrillary acidic protein. In the presence of the mitotic inhibitor cytosine arabinoside (2.0 microM), neuronal growth and survival were surprisingly normal up to the ninth day in culture, but deteriorated rapidly thereafter. In the absence of a mitotic inhibitor, and in the presence of proliferating but non-confluent glia, the tyrosine hydroxylase positive neurons that survived to the 10th day, had retracted neurites and a rounded soma, suggesting an inhibition of cell development. Those tyrosine hydroxylase positive neurons that survived this adverse phase of development tended to produce elaborate neuritic profiles after the 11th day, coincident with confluence of the astrocyte monolayer at the 12th day. By the 21st day in culture, and persisting up to the 28th day, 60% (61 +/- 10, n = 20) of the surviving neurons stained positive for tyrosine hydroxylase. When plated on an established, ventral mesencephalic monolayer of astrocytes, at the seventh day in culture, neuritic growth and branching of the tyrosine hydroxylase positive neurons were greater, compared with similar neurons grown on poly-D-lysine, and the signs of arrested development (retraction of neurites and rounded soma) seen at the 10th day after plating on poly-D-lysine, were not observed. We conclude that in the primary culture studied, and under the experimental conditions used, the survival of dopaminergic neurons was independent of glia during the first nine days, and critically dependent on glia thereafter. The resurgence of growth of dopaminergic neurons after 10 days in vitro, and their subsequent selective survival in culture, suggest that confluent type-1 astrocytes produce factors that act selectively on the dopaminergic neuronal phenotype. The successful identification of these dopaminergic-specific, neurotrophic factors could lead to an increased understanding of the etiology of Parkinson's disease, and suggest new directions for therapeutic intervention.     

Involvement of protease-activated receptor-1 in the in vitro development of mesencephalic dopaminergic neurons

In situ hybridization studies have revealed high levels of protease (thrombin)-activated receptor-1 messenger RNA in the mesencephalon of rats, suggesting that dopaminergic neurons are a target for thrombin's actions. We have evaluated the effect of thrombin receptor activation, either by thrombin or by thrombin receptor agonist peptide, a 14 amino acid agonist of protease-activated receptor-1, on tyrosine hydroxylase-positive neurons. Pure cultures of rat mesencephalic neurons or co-cultures of mesencephalic neurons and glial cells were treated with either thrombin or thrombin receptor agonist peptide the day after plating. Tyrosine hydroxylase-positive cell counting, [3H]dopamine uptake and morphometric analysis were performed on day 5. Thrombin and thrombin receptor agonist peptide influenced neurite elongation, branching and the number of primary, secondary and tertiary neurites of tyrosine hydroxylase-positive neurons. In pure cultures, the most significant effects of thrombin and thrombin receptor agonist peptide were to delay branching and to increase the centrifugal growth of neurites without affecting the total neuritic length. Thrombin (up to 10 nM) and thrombin receptor agonist peptide did not affect the number of tyrosine hydroxylase-positive neurons or [3H]dopamine uptake. Neurotrophin-4 also influenced the morphology of tyrosine hydroxylase-positive neurons. The increase of neuritic length initiated by this neurotrophin is complementary to the radial elongation induced by protease-activated receptor-1 activation. When neurons were cultured in the presence of glial cells, the effects of thrombin and thrombin receptor agonist peptide on most of these parameters were larger than those observed with pure cultures. Thus, thrombin is able to initiate a complex remodelling of the architecture of tyrosine hydroxylase-positive neurons through the activation of protease-activated receptor-1. These results provide further support for the involvement of protease-activated receptor-1 activation in the development and differentiation of the central nervous system.     

Calcitonin gene-related peptide reduces brain injury in a rat model of focal cerebral ischemia

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide is an endogenous vasodilating neuropeptide with a dense concentration in the trigeminocerebrovascular system. It is hypothesized that depletion of this peptide contributes to delayed cerebral ischemia after subarachnoid hemorrhage and that an exogenous supply of calcitonin gene-related peptide will augment ischemic cerebral blood flow and reduce neuronal injury. METHODS: In this study we have investigated the effect of an intravenous infusion of calcitonin gene-related peptide (100 ng/kg per minute), started 1 hour before and continued throughout 4 hours of focal cerebral ischemia, on cerebral blood flow and the volume of brain injury in a rat model of middle cerebral artery occlusion. RESULTS: Calcitonin gene-related peptide produces a significant improvement in ischemic cerebral blood flow (32 +/- 2 compared with 13 +/- 2 mL/100 g per minute in the controls; t = 6.92, P < .0001) with a concomitant reduction in the volume of ischemic brain injury (102 +/- 22 compared with 234 +/- 19 mm3; t = 4.47, P < .001). CONCLUSIONS: These findings lend support for the potential use of this peptide in the prophylactic treatment of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage.     

Calcitonin gene-related peptide and spinal afferents partly mediate postoperative colonic ileus in the rat

A phylogenetic tree of a gene family of nicotinic acetylcholine receptor subunits was constructed using 84 nucleotide sequences of receptor subunits from 18 different species in order to elucidate the evolutionary origin of receptor subunits. The tree constructed showed that the common ancestor of all subunits may have appeared first in the nervous system. Moreover, we suggest that the alpha 1 subunits in the muscle system originated from the common ancestor of alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, and beta 3 in the nervous system, whereas the beta 1, gamma, delta, and epsilon subunits in the muscle system shared a common ancestor with the beta 2 and beta 4 subunits in the nervous system. Using the ratio (f) of the number of nonsynonymous substitutions to that of synonymous substitutions, we predicted the functional importance of subunits. We found that the alpha 1 and alpha 7 subunits had the lowest f values in the muscle and nervous systems, respectively, indicating that very strong functional constraints work on these subunits. This is consistent with the fact that the alpha 1 subunit has sites binding to the ligand, and the alpha 7-containing receptor regulates the release of the transmitter. Moreover, the window analysis of the f values showed that strong functional constraints work on the so-called M2 region in all five types of muscle subunits. Thus, the window analysis of the f values is useful for evaluating the degree of functional constraints in not only the entire gene region, but also the within-gene subregion.     

Differentiation of mesencephalic progenitor cells into dopaminergic neurons by cytokines

1. This study examines the effects of exercise training and chronic ethanol consumption on cholinesterase activity and its relationship to lipid peroxidation in blood and brain regions of rat. 2. Exercise training (6.5 weeks) decreased acetylcholinesterase (AChE) activity significantly (64% of control) in hypothalamus and increased AChE activity in cerebral cortex (149% of control), whereas, malondialdehyde (MDA) levels increased in hypothalamus (129% of control) and decreased in cortex, striatum, and cerebellum (50%, 69% and 75% of control), respectively. 3. Chronic ethanol ingestion (2.0 gm/kg, p.o. for 6.5 weeks) significantly increased butyrylcholinesterase (BuChE) activity in plasma (136% of control) and decreased AChE activity in hypothalamus (63% of control), whereas, MDA levels increased in hypothalamus, cortex, and plasma (140%, 130% and 220% of control), respectively. 4. The combination significantly increased BuChE activity (173% of control) in plasma and decreased AChE activity (71% of control) in hypothalamus and (57% of control) in cerebellum, whereas, MDA levels increased in hypothalamus, cerebellum, medulla and plasma (134%, 128%, 140% and 309% of control), respectively. 5. Exercise training, chronic ethanol ingestion, and combination selectively inhibited hypothalamic AChE and the inhibition was correlated with increased lipid peroxidation (r = 0.11, 0.41 and 0.45) which may perturb hypothalamic function. The combination enhanced the peripheral stress response by increasing plasma BuChE activity and lipid peroxidation.     

IL-7 promotes the survival and maturation but not differentiation of human post-thymic CD4+ T cells

This study examines the influence of IL-7 on post-thymic CD4+ T cells using cord blood as a model system. Survival of naive cord blood T cells in the presence of IL-7 alone was significantly prolonged by up-regulating bcl-2, thereby preventing apoptosis while maintaining maximal cell viability. Cultures without IL-7 showed high rates of apoptosis resulting in 50% cell death by day 5 of culture. Upon phorbol 12-myristate 13-acetate + ionomycin stimulation, accumulation of cytoplasmic IL-2 was similar to that observed in freshly isolated cells, but no IL-4- or IFN-gamma-positive cells were detected. IL-7 maintained the naive T cells in a quiescent state expressing the CD45RA antigen. A significant finding was the loss of CD38 antigen expression on the naive cord blood T cells to levels similar to that observed on adult naive T cells. In contrast to the reduced proliferative response of fresh cord blood T cells to anti-CD2 + CD28 stimulation, the proliferative response of IL-7-treated cells was similar to that of adult naive T cells. This study shows that as well as maintaining the naive T cell pool by enhancing cell survival and up-regulating bcl-2 expression, IL-7 also functions as a maturation factor for post-thymic naive T cells.     

Calcitonin gene-related peptide innervation of A2-catecholamine cells in the nucleus of the solitary tract of the rat

By using double immunolabeling light and electron microscopic techniques, dense neuronal network of calcitonin-related peptide (CGRP) has been visualized in the nucleus of the solitary tract with complete overlapping of the tyrosine hydroxylase (TH)-containing cells. TH-immunoreactive perikarya and dendrites were seen in synaptic contact with CGRP-immunopositive fibers, indicating that CGRP, by carrying sensory signals may influence autonomic regulatory mechanisms in the NTS through local catecholaminergic neurons.     

Calcitonin gene-related peptide is necessary for ultraviolet B-impaired induction of contact hypersensitivity

Since nerve termini on Langerhans cells (LC) contain calcitonin gene-related peptide (CGRP), and since ultraviolet B radiation (UVR) causes CGRP to be released from cutaneous nerve endings, we examined whether CGRP participates in the immune aberrations caused in skin by UVR. First, intradermally injected CGRP, in a dose-dependent manner, reduced LC density and impaired CH induction when hapten was painted on the injected site. Second, CGRP antagonist restored CH induction after UVR. Third, anti-TNF-alpha Abs injected before CGRP prevented the loss of LC density and restored CH induction. Fourth, CGRP failed to impair CH induction in mast cell-deficient mice. Fifth, CGRP induced mast cells to release TNF-alpha. We conclude that CGRP plays an essential role in the loss of CH induction after UVR. These data indicate that UVR, by causing the release of CGRP from cutaneous nerve endings, triggers mast cell release of TNF-alpha, which impairs CH induction.     

Basic fibroblast growth factor promotes the generation and differentiation of calretinin neurons in the rat cerebral cortex in vitro

Calretinin-expressing neurons are some of the earliest postmitotic cells to appear in the developing cerebral cortex. Lineage studies have shown that the expression of this calcium-binding protein in cortical neurons is not genetically programmed and is likely to be induced by external factors. A number of studies have clearly shown that basic fibroblast growth factor (bFGF) and a number of neurotrophins promote the proliferation and differentiation of cortical progenitor cells to a particular lineage. Here, using a culture system of dissociated rat cortical cells, we found that brain-derived neurotrophic factor and neurotrophin-3 promoted the morphological differentiation of one of the calretinin-containing neuronal subpopulations, the Cajal-Retzius cells. Another subpopulation of calretinin-expressing cells of smaller size and bipolar form was generated when cultures were treated with bFGF. The progenitors of these neurons were stimulated by bFGF to divide a number of times before initiating their differentiation programme. The number of calretinin-expressing neurons increased further when cultures were treated with a combination of bFGF and retinoic acid.     

Trophic actions of transforming growth factor alpha on mesencephalic dopaminergic neurons developing in culture

Transforming growth factor alpha messenger RNA and protein levels are highest in the striatum, the target area of mesencephalic dopaminergic neurons of the substantia nigra, suggesting a role as a target-derived neurotrophic factor for these cells. To test this hypothesis, we characterized the actions of transforming growth factor alpha on fetal rat dopaminergic neurons in culture. Transforming growth factor alpha promoted dopamine uptake in a dose- and time-dependent manner. Administration of transforming growth factor alpha at the time of plating for 2 h produced a significant increase in dopamine uptake after five days of growth in vitro. As cultures aged they became less responsive to transforming growth factor alpha, such that longer times of exposure were required to elicit a similar, but weaker, response. Dopaminergic cell survival was selectively promoted by transforming growth factor alpha, since there was an increase in the number of tyrosine hydroxylase-immunostained cells without a parallel increase in the total number of neuron-specific enolase-immunopositive cells. Neurite length, branch number and soma area of tyrosine hydroxylase-immunopositive cells also were enhanced by transforming growth factor alpha treatment. Increases in each of the dopaminergic parameters due to transforming growth factor alpha were accompanied by a rise in glial cell number, making it possible that these effects were mediated by this cell population. The neurotrophin antagonist, K252b, failed to inhibit the transforming growth factor alpha-induced increase in dopamine uptake, indicating that transforming growth factor alpha's effects were not mediated by neurotrophin mechanisms. The actions of transforming growth factor alpha on the differentiation of dopaminergic neurons only partially overlapped with those of epidermal growth factor. Thus, while transforming growth factor alpha and epidermal growth factor are believed to share the same receptor they differentially affect dopaminergic cell development in vitro. These results indicate that transforming growth factor alpha is a trophic factor for mesencephalic cells in culture and suggests that transforming growth factor alpha plays a physiological role in the development of these cells in vivo.     

Synchronization of neuronal activity promotes survival of individual rat neocortical neurons in early development

Neural activity is thought to play a significant role during the development of the cerebral cortex. In this study, we examined the effects of global activity block or enhancement and the effects of patterned firing on the ability of cultured rat neocortical neurons to survive during the second week in vitro, beyond the beginning of synaptogenesis. Blockade of neuronal activity by adding tetrodotoxin (TTX) and increasing magnesium concentration in the medium strongly reduced the survival of cortical cells. Increasing neuronal activity by raising the external potassium concentration significantly improved the survival of cortical neurons. We postulated that in a developing neuronal network the survival of nerve cells is regulated by synaptically mediated events that involve changes in the intracellular calcium concentration. To examine this question further, we monitored the activity of the developing network by optically recording the intracellular calcium signals of many neurons simultaneously. These recordings show that in low magnesium neocortical neurons express synchronized oscillation of their intracellular calcium concentration. The ability of a network to synchronize the changes in intracellular calcium of multiple cells appeared gradually during the second week in culture, paralleled by both an increase in the synaptic density and a decline in the number of surviving neurons. By examining the fate of identified cells several days after a recording session, we found that those nerve cells that were co-activated with other neurons had a significantly higher chance to survive than cells that did not participate in synchronized events. These experiments demonstrate that during early cortical network development cortical neurons show synchronized firing activity and that the survival of neurons is at least partially dependent on this pattern of neuronal activity.     

Stimulatory effect of human, but not bovine, growth hormone expression on numbers of tuberoinfundibular dopaminergic neurons in transgenic mice

Mice transgenic for heterologous and ectopic GH expression serve as models for studying the feedback effects of elevated nonregulated GH on hypothalamic hypophysiotropic neurons as well as on peripheral function. For example, hypothalamic somatostatin expression has been shown to be increased markedly in mice bearing either bovine (b) or human (h) GH transgenes. Human, but not bovine, GH has lactogenic properties in mice, and appears to stimulate PRL-inhibiting tuberoinfundibular dopaminergic (TIDA) neurons. The present study was designed to determine the effect of a lifelong excess of hGH on dopamine (DA) expression in and numbers of TIDA neurons. Male mice of four transgenic lines were examined. The transgenic animals bore constructs of either bGH or hGH fused to either metallothionein (MT) or phosphoenolpyruvate carboxykinase (PEPCK) promoters; brains of transgenic mice were compared morphologically with those of nontransgenic littermates. Formaldehyde-induced catecholamine histofluorescence and tyrosine hydroxylase (TH) immunocytochemistry were examined in alternate brain sections; cell number was quantified for TIDA neurons (area A12) and a nonhypophysiotropic diencephalic DA area, the medial zona incerta (A13). Body weights were higher (P < 0.01) in PEPCK-GH than in MT-GH transgenic mice, as were serum levels of heterologous GH in those lines. In MT-hGH, but not MT-bGH or PEPCK-bGH, transgenic mice, A12 perikaryal fluorescence was enhanced, and ME fluorescence was reduced compared with those in control animals. The reduced ME DA is likely to reflect stimulation of TIDA neurons, because A12 TH-immunoreactive neuron number was increased by 34% in MT-hGH mice compared with that in controls (P < 0.05). In mice bearing the PEPCK-hGH construct, A12 TH neuron number was increased 47% (P < 0.001) compared with that in littermate controls. There were no differences in A13 cell number among animals, and A12 cell numbers in mice expressing bGH did not differ from control values. These results suggest that although extremely high levels of circulating bGH do not stimulate TIDA neurons, lifelong high levels of hGH have a stimulatory and graded effect on developmental differentiation of these cells for TH and DA production, supporting the concept of PRL as a trophic factor for TIDA neurons.     

Neurochemical regulation of feeding in the rat: Facilitation by a-noradrenergic, but not dopaminergic, receptor stimulants.

42 male Charles River rats ate voraciously after intraventricular injections of the a-noradrenergic receptor stimulant clonidine. Intraventricular administration of levonorepinephrine also facilitated feeding, but similar injections of dopamine and apomorphine (a dopamine receptor stimulant) were ineffective and even tended to suppress feeding. Clonidine was 100 times more potent than norepinephrine and increased the intake of both the ordinary diet of powdered food and a highly palatable wet food. The anorexic action of amphetamine was reversed by centrally administered clonidine. These observations suggest "respondent" rather tban "operant" regulation of feeding by noradrenergic systems. That is, in relation to noradrenergic mechanisms, feeding appeared to be a respondent which was sensitized or disinhibited by activation of a-noradrenergic receptors, rather than an operant which was reinforced by the release of norepinephrine. (26 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Chondroitin/dermatan sulphate promotes the survival of neurons from rat embryonic neocortex

Recently we have shown that biglycan, a small chondroitin sulphate proteoglycan of the extracellular matrix, supports the survival of cultured neurons from the developing neocortex of embryonic day 15 rats. Here we investigate the structure-function relationship of this neurotrophic proteoglycan and show that chondroitin/dermatan sulphate chains are the active moieties supporting survival. Heparin, a highly sulphated glucosaminoglycan, is less active than the galactosaminoglycans (chondroitin-4-sulphate, chondroitin-6-sulphate and dermatan sulphate), whereas hyaluronic acid, an unsulphated glucosaminoglycan, does not support neuron survival. Galactosaminoglycans must be in direct contact with neurons to cause survival. Experiments with elevated potassium concentrations and antagonists of voltage-gated calcium channels exclude the involvement of membrane depolarization. However, genistein and an erbstatin analogue, which are inhibitors of tyrosine kinases with low specificity, abolished neuron survival in the presence of chondroitin/dermatan sulphate, whereas a selective inhibitor of neurotrophin receptor kinases (K252a) had no suppressive effect. Thus, yet unidentified tyrosine kinases are involved in the chondroitin/dermatan sulphate-dependent survival of neocortical neurons. In the embryonic stages of rat neocortical development chondroitin sulphate is mainly located in layers I, V and VI and the subplate. Chondroitin sulphate expression is maintained after birth, extends up to cortical layer IV on postnatal day 7, and is down-regulated until postnatal day 21 concomitant with the period of naturally occurring cell death. The latter observation is consistent with a putative role of chondroitin sulphate in the control of neuron survival during cortical histogenesis.     

Calcitonin gene-related peptide- and substance P-like-immunoreactive innervation of the anterior pituitary in the rat

In our previous studies substantial amounts of substance P- and calcitonin gene-related peptide-like-immunoreactive nerve fibers have been identified in the anterior pituitary of the monkey and the dog. They were found to be in close proximity to the gland cells, even making synaptic contacts with some types of the gland cells. The present study investigated in detail the calcitonin gene-related peptide- and substance P-like immunoreactivities of the anterior pituitary in the rat. Though the immunoreactive fibers were not as abundant as in the anterior pituitary of the monkey and the dog, they still appeared in notable amounts. The calcitonin gene-related peptide- and substance P-like-immunoreactive nerve fibers occurred mostly as thin, tortuous, and densely varicose fibers, weaving among the gland cells. They are widely distributed, more in the central part of the gland. Double-immunostaining proved nearly complete co-localization of these two peptides in the nerve fibers. It is hypothesized that the anterior pituitary can be regulated by direct neural factors as well as humoral factors.     

The neuropeptide calcitonin gene-related peptide inhibits TNF-alpha but poorly induces IL-6 production by fetal rat osteoblasts

Tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) are potent inflammatory cytokines produced by osteoblasts and whose contribution to bone loss occurring in oestrogen deficiency is well documented. Calcitonin gene-related peptide (CGRP) is a neuropeptide abundantly concentrated in sensory nerve endings innervating bone metaphyses and periosteum suggesting that it controls bone homeostasis locally. Since CGRP was shown to inhibit TNF-alpha production by T cells and stimulate IL-6 expression by fibroblasts, this study was designed to investigate whether CGRP regulated TNF-alpha and IL-6 production by osteoblasts. We show that CGRP inhibits the production of TNF-alpha by both lipopolysaccharide (LPS)- and IL-1-stimulated fetal rat osteoblasts. Like CGRP, the cAMP agonists prostaglandin E2 (PGE2), dibutyryl cAMP (Bt2cAMP) and forskolin inhibit TNF-alpha production by osteoblasts. Exposure of osteoblasts to a high dose of phorbol myristoyl acetate (PMA) to deplete PKC activity abolished CGRP-mediated TNF-alpha suppression. In contrast with its potent inhibition of TNF-alpha production, we show that CGRP is a weak inducer of IL-6 when compared to PGE2, Bt2cAMP and forskolin. However, in presence of isobutylmethylxanthine (IBMX) CGRP stimulates the production of IL-6. Collectively, these data suggest that the inhibition of TNF-alpha CGRP is cAMP dependent and PMA sensitive and that the concentration of intracellular cAMP may be a regulatory mechanism for IL-6 expression in osteoblasts.