Expression profiles of BDNF splice variants in cultured DRG neurons stimulated with NGF

Expression of brain-derived neurotrophic factor (BDNF) mRNA is increased in the dorsal root ganglion (DRG) in response to peripheral inflammation. Nerve growth factor (NGF) from inflammatory tissue is thought to induce expression of BDNF. Recently, it was reported that the BDNF gene has eight non-coding exons that are transcribed independently into several splice variants. Expression of these splice variants in DRG neurons stimulated with NGF has not been studied. We examined changes in expression of BDNF splice variants in a rat model of peripheral inflammation and in cultured DRG neurons exposed to NGF. Total BDNF mRNA was increased by inflammation in vivo and by NGF in vitro. Among all splice variants, exon 1-9 showed the greatest increase in expression in both experiments. Our results indicate that exon 1-9 contributes to changes in total BDNF levels and may play an important role in the acute response of DRG to NGF.     

Genome-wide population genetic analysis identifies evolutionary forces establishing continuous population divergence

Elucidating the mechanism shaping the spatial variations of traits has long been a central concern of evolutionary biologists. Geographic clines of allele/morph frequencies along environmental gradients are suggested to be established and maintained by the balancing of two opposing evolutionary forces, namely selection that generates spatial differentiation in morph frequencies, and selection and/or stochastic factors that lead to the coexistence of multiple morphs within a population. Thus, testing for both selection and stochastic factors is necessary for a comprehensive understanding of the mechanism underlying clinal variation in morph/allele frequency in natural populations. Here, I identified the evolutionary forces responsible for clinal variation of color morph frequency in Ischnura senegalensis by comparing the population divergence of putatively neutral loci generated by high-throughput next-generation sequencing (F _STn) with that of the putative color locus (F _STc). No strong correlation was observed between F _STn and F _STc, suggesting that stochastic factors contribute less to color-locus population divergence. F _STc was less than F _STn between populations exposed to similar environmental conditions, but greater than F _STn between populations exposed to different environmental conditions, suggesting that both balancing selection and divergent selection act on the color locus. Therefore, two antagonistic selection factors rather than stochastic and historical factors contribute to establishing the clinal variation of morph frequency in I. senegalensis.     

Expression of NGF receptor and GAP-43 mRNA in DRG neurons during collateral sprouting and regeneration of dorsal cutaneous nerves

The collateral sprouting of intact sensory axons and the regenertion of damaged ones differ in a number of respects. Regeneration is triggered by axotomy-induced damage, probably involves the loss of a peripheral signal, and appears to occur independently of NGF, while collateral sprouting is evoked and sustained by an increase in a target-driven signal, namely NGF. New findings strengthen the distinction between these two phenomena. Nerve growth factor receptor (NGFR) mRNA is increased in undamaged DRG neurons whose axons are sprouting into denervated skin. This response is related to an increased availablity of target-derived NGF, a proposal supported by a number of findings including increased NGF mRNA in the denervated target. In contrast, we observed little or no change in the NGFR mRNA levels in regenerating neurons, consistent with the observations that NGF does not play a role in this process. However, increases in neuronal GAP-43 mRNA are found during both regeneration and collateral sprouting, a result in keeping with the proposal that GAP-43 is primarily associated with nerve growth, and the observation that GAP-43 expression is not especially influenced by NGF. 1994 John Wiley & Sons, Inc.     

Simple molecular networks that respond optimally to time-periodic stimulation

Background  

Bacteria or cells receive many signals from their environment and from other organisms. In order to process this large amount of information, Systems Biology shows that a central role is played by regulatory networks composed of genes and proteins. The objective of this paper is to present and to discuss simple regulatory network motifs having the property to maximize their responses under time-periodic stimulations. In elucidating the mechanisms underlying these responses through simple networks the goal is to pinpoint general principles which optimize the oscillatory responses of molecular networks.     

A morphological analysis of growth cones of DRG neurons combining Atomic Force and Confocal Microscopy

We have analyzed the morphology of growth cones of differentiating neurons from rat dorsal root ganglia (DRG) with conventional Laser Scanning Confocal Microscopy (LSCM) and Atomic Force Microscopy (AFM). Images of immunofluorescent DRG growth cones colabeled for actin and tubulin were superimposed to images obtained with AFM at different scanning forces. In order to reduce changes of the image surface caused by the pressure of the AFM tip, we have developed a procedure to obtain 0 pN AFM images. Further analysis of these images revealed topographical structures with nanoscale dimensions, referred to as “invaginations” or “holes”. These holes had an area varying from 0.01 to 3.5 μm² with a depth varying from 2 to 178 nm. Comparative analysis with LSCM images showed that these holes correspond to regions where staining of both actin and tubulin was negligible. Filopodia height varied from 40 to 270 nm and their diameter from 113 to 887 nm. These results show that the combination of LSCM and AFM reveal structural details with a nanoscale dimension of DRG growth cones, difficult to resolve with conventional microscopy.     

Dopaminergic amacrine neurons of rat retinas with photoreceptor degeneration continue to respond to light

Exposure of albino rats to continuous light of low intensity (350–700 lux) for 4 months produces massive degeneration of the photoreceptor segments and cell bodies of the outer nuclear layer of the retina. Only a few heterochromatic, receptor cell nuclei remain, and no photoreceptor segments are present. On the other hand, the inner layers of these retinas remain morphologically intact. The inner nuclear layer of the normal rat retina contains a group of amacrine cells which contain the putative neurotransmitter, dopamine (DA). Short term exposure to light (30 or 60 min) markedly stimulates the rate of DA turnover in these cells in normal, previously dark-adapted rats. Such enhancement of the rate of neurotransmitter turnover in the brain has been correlated with an increase in nerve impulse activity. The present study was undertaken to determine if the dopaminergic amacrine cells of the inner nuclear layer were still responsive to light in the retinas of rats whose photoreceptors were previously destroyed by long term exposure to continuous illumination. One week before sacrifice, the animals which had been housed in continuous light for 4 months were returned to normal 14 hr light: 10 hr dark lighting conditions. At the end of this time they and a group of control rats which had been housed in cyclic lighting conditions for the entire 4 months were dark adapted for approximately 15 hr. Then the rate of retinal DA turnover was estimated from the depletion of DA following inhibition of DA synthesis by α methyl para-tyrosine. The turnover of DA in the dark-adapted retinas of the control rats and of experimental rats with photoreceptor degeneration was dramatically enhanced 2–4 fold by short term exposure (up to 1 hr) to light. Since rats are nocturnal and avoid light, we tested the light aversion of another group of rats which had been exposed to light for 4 months and then returned to cyclic lighting conditions for one week. These rats and control animals which had been maintained in cyclic lighting conditions for 4 months both chose the dark side of a light-dark box over 80% of the time. This behavior of the rats with retinal degeneration was taken as a crude indication of their continued ability to detect light. The light-induced increase in DA activity in retinas with photoreceptor degeneration may play a role in the continued ability of these rats to perceive light.     

Prospero maintains the mitotic potential of glial precursors enabling them to respond to neurons

During central nervous system development, glial cells need to be in the correct number and location, at the correct time, to enable axon guidance and neuropile formation. Repair of the injured or diseased central nervous system will require the manipulation of glial precursors, so that the number of glial cells is adjusted to that of neurons, enabling axonal tracts to be rebuilt, remyelinated and functional. Unfortunately, the molecular mechanisms controlling glial precursor proliferative potential are unknown. We show here that glial proliferation is regulated by interactions with axons and that the Drosophila gene prospero is required to maintain the mitotic potential of glia. During growth cone guidance, Prospero positively regulates cycE promoting cell proliferation. Neuronal Vein activates the MAPKinase signalling pathway in the glia with highest Prospero levels, coupling axon extension with glial proliferation. Later on, Prospero maintains glial precursors in an undifferentiated state by activating Notch and antagonising the p27/p21 homologue Dacapo. This enables prospero-expressing cells alone to divide further upon elimination of neurons and to adjust glial number to axons during development.     

Increased expression of GAP-43 in small sensory neurons after stimulation by NGF indicative of neuroregeneration in capsaicin-treated rats.

Intraplantar injections of human recombinant nerve growth factor (rhNGF-beta) into the hind paw of capsaicin-treated adult rats are known to lead to a recovery of depleted peptide transmitter substances, to the immunohistochemical reappearance of peptidergic innervation in the skin and in the dorsal horn of the spinal cord, as well as to a recovery of the function of capsaicin-lesioned neurons. In the present study a marker peptide for neuronal regeneration and outgrowth, growth associated protein 43 (GAP-43), was investigated in lumbar dorsal root ganglia (DRGs) and in the hindpaw skin, in order to differentiate which population of the sensory neurons responds with a neuroregenerative behaviour. In situ hybridization histochemistry (ISH) revealed that at day 8 after the capsaicin treatment GAP-43 expression was significantly increased in small DRG cells as compared to control animals, and treatment with NGF in capsaicinized rats lead to an even more pronounced increase of GAP-43 expression in the small-sized cell population. Intraepidermal labelling of GAP-43 peptide was observed in the skin of control animals, but was markedly reduced in the animals that were treated with capsaicin alone. However, intraepidermal GAP-43 immunoreactive (GAP-43-IR) fibres nearly fully recovered in the capsaicin + NGF-treated group. These results indicate that the population of small DRG cells shows spontaneous regenerative activity after a capsaicin lesion which does not lead to a successful recovery of nerve terminals in the skin. Only after an additional NGF treatment small DRG cells show an even stronger regenerative response which now also involves structural reorganization of neuron membranes and axogenesis in the periphery.     

O-linked N-acetylglucosamine levels in cerebellar neurons respond reciprocally to pertubations of phosphorylation.

The novel intracellular carbohydrate O-linked N-acetylglucosamine (O-GlcNAc) is present on proteins ranging from those of viruses to those of humans and include cytosolic, nuclear and plasma-membrane proteins. In this report we have examined the effect of manipulation of phosphorylation on the levels of O-GlcNAc in cerebellar neurons from early postnatal mice. Our results indicate a reciprocal response of O-GlcNAc levels to phosphorylation. Activation of protein kinase A or C, for example, results in reduced levels of O-GlcNAc specifically in the fraction of cytoskeletal and cytoskeleton-associated proteins, while inhibition of the same kinases results in increased levels of O-GlcNAc. These data are in keeping with a reciprocal action of O-GlcNAc with respect to phosphorylation and suggest that this modification may have a role in signal transduction.     

Orally tolerant CD4 T cells respond poorly to antigenic stimulation but strongly to direct stimulation of intracellular signaling pathways

The response of splenic CD4 T cells from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic mice after long-term feeding of a diet containing this antigen was examined. These CD4 T cells exhibited a decreased response to OVA peptide stimulation, in terms of proliferation, interleukin-2 secretion, and CD40 ligand expression, compared to those from mice fed a control diet lacking OVA, demonstrating that oral tolerance of T cells had been induced through oral intake of the antigen. We investigated the intracellular signaling pathways, which were Ca/CN cascade and Ras/MAPK cascade, of these tolerant CD4 T cells using phorbol-12-myristate-13-acetate (PMA) and ionomycin, which are known to directly stimulate these pathways. In contrast to the decreased response to TCR stimulation by OVA peptide, it was shown that the response of splenic CD4 T cells to these reagents in the state of oral tolerance was stronger. These results suggest that splenic CD4 T cells in the state of oral tolerance have an impairment in signaling, in which signals are not transmitted from the TCR to downstream signaling pathways, and have impairments in the vicinity of TCR. This revised version was published online in July 2006 with corrections to the Cover Date.     

Import of a mutant mitochondrial precursor fails to respond to stimulation by a cytosolic factor

Import of the precursor to ornithine carbamyltransferase is stimulated by a partially-purified, NEM-sensitive soluble factor from rabbit reticulocyte lysate. A mutant in which the carboxy-terminal 73 amino acids were deleted, had a sharply reduced response to this factor. The NEM-sensitive, import-stimulating factor interacts with the surface of mitochondria in the absence of precursor protein. Thus reticulocyte lysate contains an NEM-sensitive, import stimulating factor which interacts both with the surface of mitochondria and whose activity appears to be dependent upon the structure of the mature portion of the precursor.     

Mesencephalic dopaminergic neurons express a repertoire of olfactory receptors and respond to odorant-like molecules

Background

The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the Substantia Nigra (SN) (A9 neurons) and the Ventral Tegmental Area (VTA) (A10 cells). Selective degeneration of A9 neurons occurs in Parkinson’s disease (PD) while abnormal function of A10 cells has been linked to schizophrenia, attention deficit and addiction. The molecular basis that underlies selective vulnerability of A9 and A10 neurons is presently unknown.

Results

By taking advantage of transgenic labeling, laser capture microdissection coupled to nano Cap-Analysis of Gene Expression (nanoCAGE) technology on isolated A9 and A10 cells, we found that a subset of Olfactory Receptors (OR)s is expressed in mDA neurons. Gene expression analysis was integrated with the FANTOM5 Helicos CAGE sequencing datasets, showing the presence of these ORs in selected tissues and brain areas outside of the olfactory epithelium. OR expression in the mesencephalon was validated by RT-PCR and in situ hybridization. By screening 16 potential ligands on 5 mDA ORs recombinantly expressed in an heterologous in vitro system, we identified carvone enantiomers as agonists at Olfr287 and able to evoke an intracellular Ca²⁺ increase in solitary mDA neurons. ORs were found expressed in human SN and down-regulated in PD post mortem brains.

Conclusions

Our study indicates that mDA neurons express ORs and respond to odor-like molecules providing new opportunities for pharmacological intervention in disease.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-729) contains supplementary material, which is available to authorized users.     

Responses of caudate neurons to acoustic stimulation in cats

Responses of 141 neurons of the caudate nucleus to acoustic stimuli — tones (500 and 2000 Hz) and clicks of different frequency (0.2 and 0.8/sec) and intensity (75, 80, 95 dB) — were recorded extracellularly in chronic experiments on cats. The responses recorded showed great variability with respect to character (phasic, tonic), structure (one or two phases of excitation), latent periods (from 7.5 to 300.0 msec), and burst discharge frequency (from 90 to 800 spikes/sec). Analysis of averaged poststimulus histograms and graphs of the dynamics of the responses showed that responses of 74% of neurons were much better expressed if less frequent stimuli were used: The regularity of the responses and the number of spikes in each response increased. Responses of neurons also increased and acquired a more distinct temporal structure if the intensity of the clicks increased. The character of responses to clicks and tones differed qualitatively in 17% of neurons studied: Phasic excitation arose in response to clicks, tonic changes in spike activity to tones. The particular features of responses of caudate neurons to acoustic stimulation with different parameters are discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 12, No. 6, pp. 588–595, November–December, 1980.     

Dorsal root ganglion neurons expressing trk are selectively sensitive to NGF deprivation in utero.

In utero immune deprivation of the neurotrophic molecule nerve growth factor (NGF) results in the death of most, but not all, mammalian dorsal root ganglion (DRG) neurons. The recent identification of trk, trkB, and trkC as the putative high affinity receptors for NGF, brain-derived neurotrophic factor, and neurotrophin-3, respectively, has allowed an examination of whether their expression by DRG neurons correlates with differential sensitivity to immune deprivation of NGF. In situ hybridization demonstrates that virtually all neurons expressing trk are lost during in utero NGF deprivation. Most, if not all, neurons expressing trkB and trkC survive this treatment. In contrast, the low affinity NGF receptor, p75NGFR, is expressed in both NGF deprivation-resistant and -sensitive neurons. These experiments show that DRG neurons expressing trk require NGF for survival. Furthermore, at least some of the DRG neurons that do not require NGF express the high affinity receptor for another neurotrophin. Finally, these experiments provide evidence that trk, and not p75NGFR, is the primary effector of NGF action in vivo.     

Contribution of neurons to habituation to mechanical stimulation in Caenorhabditis elegans

In Caenorhabditis elegans, a light touch induces a locomotor response. Repeated touches, however, result in an attenuation of response, that is, habituation. Withdrawal responses elicited by anterior touch are controlled by anterior mechanosensory neurons (AVM and ALMs), and by four pairs of interneurons (AVA, AVB, AVD, and PVC) (Chalfie et al., 1985; White et al., 1986). To identify the neurons that participate in habituation, we ablated these neurons with a laser microbeam and investigated the resulting habituation of the operated animals. The animals lacking both left and right homologues AVDLR were habituated more rapidly than intact animals. We propose that chemical synapses at AVD play a critical role in the habituation of intact animals.     

Bird population declines due to radiation exposure at Chernobyl are stronger in species with pheomelanin-based coloration

Eumelanin and pheomelanin are the most common pigments providing color to the integument of vertebrates. While pheomelanogenesis requires high levels of a key intracellular antioxidant (glutathione, GSH), eumelanogenesis is inhibited by GSH. This implies that species that possess the molecular basis to produce large amounts of pheomelanin might be more limited in coping with environmental conditions that generate oxidative stress than species that produce eumelanin. Exposure to ionizing radiation produces free radicals and depletes antioxidant resources. GSH is particularly susceptible to radiation, so that species with large proportions of pheomelanic integument may be limited by the availability of GSH to combat oxidative stress and may thus suffer more from radiation effects. We tested this hypothesis in 97 species of birds censused in areas with varying levels of radioactive contamination around Chernobyl. After controlling for the effects of carotenoid-based color, body mass and similarity among taxa due to common phylogenetic descent, the proportion of pheomelanic plumage was strongly negatively related to the slope estimates of the relationship between abundance and radiation levels, while no effect of eumelanic color proportion was found. This represents the first report of an effect of the expression of melanin-based coloration on the capacity to resist the effects of ionizing radiation. Population declines were also stronger in species that exhibit carotenoid-based coloration and have large body mass. The magnitude of population declines had a relatively high phylogenetic signal, indicating that certain groups of birds, especially non-corvid passeriforms, are particularly susceptible to suffer from the effects of radioactive contamination due to phylogenetic inertia.     

Human olfactory neurons respond to odor stimuli with an increase in cytoplasmic Ca2+.

The sense of smell allows terrestrial animals to collect information about the chemical nature of their environment through the detection of airborne molecules. In humans smell is believed to play an important role in protecting the organism from environmental hazards such as fire, gas leaks and spoiled food, in determining the flavor of foods, and perhaps in infant-parent bonding. In addition, the study of human olfaction is relevant to a number of medical problems that result in olfactory dysfunction, which can affect nutritional state, and to the study of the etiology of neurodegenerative diseases which manifest themselves in the olfactory epithelium. Although much is known about behavioral aspects of human olfaction, little is understood about the underlying cellular mechanisms in humans. Here we report that viable human olfactory neurons (HON) can be isolated from olfactory tissue biopsies, and we find that HON respond to odorants with an increase in intracellular calcium concentration ([Cai]).