Nitric oxide from inflammatory origin impairs neural stem cell proliferation by inhibiting epidermal growth factor receptor signaling

Neuroinflammation is characterized by activation of microglial cells, followed by production of nitric oxide (NO), which may have different outcomes on neurogenesis, favoring or inhibiting this process. In the present study, we investigated how the inflammatory mediator NO can affect proliferation of neural stem cells (NSCs), and explored possible mechanisms underlying this effect. We investigated which mechanisms are involved in the regulation of NSC proliferation following treatment with an inflammatory stimulus (lipopolysaccharide plus IFN-gamma), using a culture system of subventricular zone (SVZ)-derived NSCs mixed with microglia cells obtained from wild-type mice (iNOS(+/+)) or from iNOS knockout mice (iNOS(-/-)). We found an impairment of NSC cell proliferation in iNOS(+/+) mixed cultures, which was not observed in iNOS(-/-) mixed cultures. Furthermore, the increased release of NO by activated iNOS(+/+) microglial cells decreased the activation of the ERK/MAPK signaling pathway, which was concomitant with an enhanced nitration of the EGF receptor. Preventing nitrogen reactive species formation with MnTBAP, a scavenger of peroxynitrite (ONOO-), or using the ONOO- degradation catalyst FeTMPyP cell proliferation and ERK signaling were restored to basal levels in iNOS(+/+) mixed cultures. Moreover, exposure to the NO donor NOC-18 (100 mu M), for 48 h, inhibited SVZ-derived NSC proliferation. Regarding the antiproliferative effect of NO, we found that NOC-18 caused the impairment of signaling through the ERK/MAPK pathway, which may be related to increased nitration of the EGF receptor in NSC. Using MnTBAP nitration was prevented, maintaining ERK signaling, rescuing NSC proliferation. We show that NO from inflammatory origin leads to a decreased function of the EGF receptor, which compromised proliferation of NSC. We also demonstrated that NO-mediated nitration of the EGF receptor caused a decrease in its phosphorylation, thus preventing regular proliferation signaling through the ERK/MAPK pathway.Foundation for Science and Technology, (FCT, Portugal); COMPETE; FEDER [PEst-C/SAU/LA0001/2013-2014, PEst-OE/EQB/LA0023/2013-2014, PTDC/SAU-NEU/102612/2008, PTDC/NEU-OSD/0473/2012]; FCT, Portugal [SERH/BPD/78901/2011, SERH/BD/38127/2007, SFRH/BD/77903/2011, SFRH/BD/79308/2011]info:eu-repo/semantics/publishedVersio

The shape of the olfactory bulb influences axon targeting

Each primary olfactory neuron in the mouse expresses a single type of odorant receptor. All neurons expressing the same odorant receptor gene typically project to two topographically fixed glomeruli, one each on the medial and lateral surfaces of the olfactory bulb. While topographic gradients of guidance receptors and their ligands help to establish the retinotectal projection, similar orthogonal distributions of cues have not yet been detected within the olfactory system. While odorant receptors are crucial for the final targeting of axons to glomeruli, it is unclear whether the olfactory bulb itself provides instructive cues for the establishment of the topographic map. To begin to understand the role of the olfactory bulb in the formation of the olfactory nerve pathway, we developed a model whereby the gross shape of the bulb in the P2-IRES-tau-LacZ line of mice was radically altered during postnatal development. We have shown here that the topography of axons expressing the P2 odorant receptor is dependent on the shape of the olfactory bulb. When the dorsoventral axis of the olfactory bulb was compressed during the early postnatal period, newly developing P2 axons projected to multiple inappropriate glomeruli surrounding their normal target site. These results suggest that the distribution of local guidance cues within the olfactory bulb is influenced by the shape of the olfactory bulb and that these cues contribute to the topographic positioning of glomeruli. Crown Copyright (C) 2007 Published by Elsevier B.V. All rights reserved

The cell surface carbohydrate blood group A regulates the selective fasciculation of regenerating accessory olfactory axons

Cell surface carbohydrates are differentially expressed by discrete subpopulations of primary sensory axons in the mammalian main and accessory olfactory systems. It has been proposed that these carbohydrates provide a glycocode which mediates the sorting of these sensory axons as they project from the olfactory neuroepithelium to their central targets in the main and accessory olfactory bulbs during development. As the differential expression of cell surface carbohydrates on olfactory axons persists in the adult we have now investigated their role during regeneration. We have recently generated a line of transgenic mice, BGAT-Tg, that mis-express the blood group A (BGA) carbohydrate on all primary olfactory axons rather than just on accessory olfactory axons as in wild-type mice. Following unilateral bulbectomy, accessory and main olfactory axons regenerate and grow into the frontal cortex where they fill the cavity which remains after the olfactory bulb ablation. In wild-type mice, the regenerating BGA-expressing accessory olfactory axons selectively aggregated with each other in large bundles but clearly separated from the BGA-negative main olfactory axons. In contrast, in the BGAT-Tg transgenic mice in which all main and accessory axons express the BGA carbohydrate, the accessory olfactory axons failed to correctly separate from the main olfactory axons. Instead, these axons formed numerous small bundles interspersed with main olfactory axons. These data provide strong evidence that the restricted expression of BGA is in part responsible for the selective segregation of accessory olfactory axons. (c) 2008 Elsevier B.V. All rights reserved

Nerve growth in embryonic mice: the events regulating axonal overextension in the olfactory bulb

The olfactory system has become a popular model for studying neural regeneration and the underlying mechanisms for developing neural circuits. By utilising transgenic mice (S100β-DsRed and OMP-ZsGreen) we have the ability to visualise olfactory neurons (OMP-ZsGreen) and glial cells (S100β-DsRed). During development olfactory axons travel a considerable distance to the developing olfactory bulb. In this period overextension of axons can be seen. For proper development axons need to be guided to their target glomeruli and overextending axons need to be degraded. In E13.5 embryos the olfactory bulb houses S100β-DsRed positive cells that appear to play a role in the formation of the olfactory bulb. Before the glomeruli are formed these cells do not allow axons to protrude into the olfactory bulb. When overextending axons do enter the olfactory bulb they do so in locations where these cells are minimal. As development progresses the DsRed “barrier” cells are now located deeper in the olfactory bulb (E15.5), which coincides with the establishment of the glomerular layer. As overextending axons enter the olfactory bulb via the DsRed “barrier” gaps they are met by the radial glia filament scaffold. The axons travel caudally along the filament branches until they are eventually degraded. We also show evidence to indicate that after degradation, OMP-ZsGreen positive debris is taken up by the radial glia cells lining the ventricular lumen. These findings illustrate new events for the establishment of the olfactory bulb and show crucial cellular interactions for proper topographical development.Faculty of Science, Environment, Engineering and TechnologyNo Full Tex

Nerve Growth in Embryonic Mice: The Events Regulating Axonal Overextension in the Olfactory Bulb

The olfactory system has become a popular model for studying neural regeneration and the underlying mechanisms for developing neural circuits. By utilising transgenic mice (S100β-DsRed and OMP-ZsGreen) we have the ability to visualise olfactory neurons (OMPZsGreen) and glial cells (S100β-DsRed). During development olfactory axons travel a considerable distance to the developing olfactory bulb. In this period overextension of axons can be seen. For proper development axons need to be guided to their target glomeruli and overextending axons need to be degraded. In E13.5 embryos the olfactory bulb houses S100β-DsRed positive cells that appear to play a role in the formation of the olfactory bulb. Before the glomeruli are formed these cells do not allow axons to protrude into the olfactory bulb. When overextending axons do enter the olfactory bulb they do so in locations where these cells are minimal. As development progresses the DsRed “barrier” cells are now located deeper in the olfactory bulb (E15.5), which coincides with the establishment of the glomerular layer. As overextending axons enter the olfactory bulb via the DsRed “barrier” gaps they are met by the radial glia filament scaffold. The axons travel caudally along the filament branches until they are eventually degraded. We also show evidence to indicate that after degradation, OMP-ZsGreen positive debris is taken up by the radial glia cells lining the ventricular lumen. These findings illustrate new events for the establishment of the olfactory bulb and show crucial cellular interactions for proper topographical development.Faculty of Science, Environment, Engineering and TechnologyNo Full Tex

Olfactory ensheathing cells proliferate from local OECs and from precursors in the olfactory mucosa after different type of injuries

Olfactory ensheathing cells (OECs) support the regeneration of olfactory sensory neurons throughout life. However, it remains unclear how OECs respond to a major injury and whether OEC precursors within the olfactory mucosa give rise to new OECs in those conditions. We examined the proliferation and migration of OECs in two postnatal animal models of olfactory axon degeneration. In the first model, we surgically removed an olfactory bulb including the nerve fibre layer which contains OECs. In the second model, intraperitoneal injection of methimazole was used to selectively destroy the entire olfactory epithelium while not directly affecting OECs. Proliferating cells were labelled by the thymidine analogue, ethynyl deoxyuridine (EdU). In the unilateral bulbectomy model, there was a large stimulation of OEC proliferation throughout the olfactory nerve up to 14 days after bulbectomy. Using a pulse-chase EdU application, we tracked cells that had proliferated and found that OEC precursors lining the basal layer of the olfactory epithelium also gave rise to OECs that subsequently migrated along the length of the olfactory nerve. In the methimazole model, a small but significant proliferation of OECs was induced 7-10 days after axon death, with proliferation occurring in all regions of the olfactory nerve. These results demonstrate that OECs actively respond to widespread degeneration of olfactory axons and that OECs arise from both local proliferation as well as from OEC precursors. These results have important implications for selecting the source of OECs for neural regeneration therapies.Faculty of Science, Environment, Engineering and TechnologyNo Full Tex

Pavlovian olfactory fear conditioning: Its neural circuity and importance for understanding clinical fear-based disorders

Odors have proven to be the most resilient trigger for memories of high emotional saliency. Fear associated olfactory memories pose a detrimental threat of potentially transforming into severe mental illness such as fear and anxiety-related disorders. Many studies have deliberated on auditory, visual and general contextual fear memory (CFC) processes; however, fewer studies have investigated mechanisms of olfactory fear memory. Evidence strongly suggests that the neuroanatomical representation of olfactory fear memory differs from that of auditory and visual fear memory. The aim of this review article is to revisit the literature regarding the understanding of the neurobiological process of fear conditioning and to illustrate the circuitry of olfactory fear memory

Microtopography of fear memory consolidation and extinction retrieval within prefrontal cortex and amygdala

Rationale: The precise neural circuitry that encodes fear memory and its extinction within the brain are not yet fully understood. Fearful memories can be persistent, resistant to extinction, and associated with psychiatric disorders, especially post-traumatic stress disorder (PTSD). Here, we investigated the microtopography of neurons activated during the recall of an extinguished fear memory, as well as the influence of time on this microtopography. Methods: We used the plasticity-related phosphorylated mitogen-activated protein kinase (pMAPK) to identify neurons activated in the recall of consolidated and extinguished auditory Pavlovian fear memories in rats. Quantitatively matched brain regions were used to investigate activity in the amygdala and prefrontal cortex. Results: Recall of a consolidated, nonextinguished auditory fear memory resulted in a significantly greater number of activated neurons located in the dorsolateral subdivision of the lateral amygdala (LADL) when recalled 24 h after consolidation but not when recalled 7 days later. We found that the recall of an extinction memory was associated with pMAPK activation in the ventrolateral subdivision of the lateral amygdala (LAVL). Next, we showed that the pattern of pMAPK expression in the prelimbic cortex differed spatially following temporal variation in the recall of that memory. The deep and superficial layers of the pre-limbic cortex were engaged in recent recall of a fear memory, but only the superficial layers were recruited if the recall occurred 7 days later. Conclusions: Collectively, our findings demonstrate a functional microtopography of auditory fear memory during consolidation and extinction at the microanatomical level within the lateral amygdala and medial prefrontal cortex.</p

Contextual fear conditioning alter microglia number and morphology in the rat dorsal hippocampus

Contextual fear conditioning is a Pavlovian conditioning paradigm capable of rapidly creating fear memories to contexts, such as rooms or chambers. Contextual fear conditioning protocols have long been utilized to evaluate how fear memories are consolidated, maintained, expressed, recalled, and extinguished within the brain. These studies have identified the lateral portion of the amygdala and the dorsal portion of the hippocampus as essential for contextual fear memory consolidation. The current study was designed to evaluate how two different contextual fear memories alter amygdala and hippocampus microglia, brain derived neurotrophic factor (BDNF), and phosphorylated cyclic-AMP response element binding (pCREB). We find rats provided with standard contextual fear conditioning to have more microglia and more cells expressing BDNF in the dentate gyrus as compared to a context only control group. Additionally, standard contextual fear conditioning altered microglia morphology to become amoeboid in shape - a common response to central nervous system insult, such as traumatic brain injury, infection, ischemia, and more. The unpaired fear conditioning procedure (whereby non-reinforced and non-overlapping auditory tones were provided at random intervals during conditioning), despite producing equivalent levels of fear as the standard procedure, did not alter microglia, BDNF or pCREB number in any dorsal hippocampus or lateral amygdala brain regions. Despite this, the unpaired fear conditioning protocol produced some alterations in microglia morphology, but less compared to rats provided with standard contextual fear conditioning. Results from this study demonstrate that contextual fear conditioning is capable of producing large alterations to dentate gyrus plasticity and microglia, whereas unpaired fear conditioning only produces minor changes to microglia morphology. These data show, for the first time, that Pavlovian fear conditioning protocols can induce similar responses as trauma, infection or other insults within the central nervous system