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Oxygen resuscitation exacerbates hypoxic ischemic outcomes in rat cortex
884
Poster Session 2 Abstracts 3 June 2008 / Int. J. Devl Neuroscience 26 (2008) 867–892
Slit1/2 and Robo1/2 knockout mice when compared to heterozygous controls. Mutant SN-VTA axons spread out in the diencephalon to form a wider tract. Phenotypes included invasion of the ventral midline, consistent with Slit repulsion. However, individual axons also wandered in aberrant dorsal trajectories, which suggests an unexpected role for Slits. Based on these and previous results we suggest that Slit/Robo signaling is necessary for correct dorsal/ ventral positioning and precise pathfinding of dopaminergic longitudinal axons, as well as serving a repellant function. These results may provide important insights into treatment of degeneration of SN-VTA dopminergic fibers in Parkinson’s and Alzheimer’s disease. doi: 10.1016/j.ijdevneu.2008.09.176 [P2.52] Pioneer longitudinal axons navigate using floor plate and Slit/Robo signals W.T. Farmer 1,*, A.L. Altick 1, J.P. Dugan 1, T. Kidd 1, F. Charron 2, G.S. Mastick 1 1
University of Nevada, USA Institut de recherches cliniques de Montre´al (IRCM), Canada *Corresponding author. 2
Keywords: Slit; Robo; Floor plate; Longitudinal axon
Longitudinal axons transmit all signals between the brain and spinal cord. Their axon tracts through the brain stem are established by a simple set of pioneer axons. These axons navigate through several brain regions with precise trajectories parallel to the floor plate. To identify longitudinal guidance mechanisms in vivo, the overall role of floor plate tissue, and the specific roles of Slit/Robo signals were tested. Ectopic induction or genetic deletion of floor plate diverted longitudinal axons into abnormal trajectories, providing in vivo evidence for the importance of floor plate signals. The expression patterns of the diffusible cues of the Slit family were altered in the floor plate experiments, suggesting their involvement in longitudinal guidance. Genetic tests of Slit1 and 2, and the Slit receptors Robo1 and 2 were carried out in mutant mice. Slit1; Slit2 double mutants had severe longitudinal errors, particularly for ventral axons, including midline crossing and wandering longitudinal trajectories. Robo1 and Robo2 were largely genetically redundant, and neither appeared to specify specific tract positions. However, combined Robo1 and Robo2 mutations strongly disrupted each pioneer tract. Our results indicate that Slit/ Robo signalling may have at least two functions: a repulsive activity that prevents ventral axons from entering the floor plate and a second activity that maintains straight trajectories. Thus, pioneer axons depend on long-range floor plate cues, with Slit/ Robo signalling required for precise longitudinal trajectories. doi: 10.1016/j.ijdevneu.2008.09.177 [P2.53] A forward genetic screen in the mouse to identify novel genes that affect motor neuron development K.F. Liem *, K.V. Anderson Sloan-Kettering Institute, USA *Corresponding author. Keywords: Motor neuron; Genetics; Axon guidance; Cell fate
Introduction: The mammalian spinal motor system is an excellent model system to study the formation of a simple
Fig. 1. The HB9-GFP transgenic mouse labels motor neurons and motor axons.
topographical neuronal projection. Although we know much about how motor neurons develop, we know less about how various classes of motor neurons find and select their specific muscle targets. We have taken a forward genetic approach to identify novel genes that control motor neuron subtype identity and motor axon guidance and target specificity. Methods: Mice were mutagenized with ENU and outcrossed to a HB9-GFP transgenic reporter line which expresses GFP in spinal motor neurons and their axons. Litters were analyzed at e12.5 by fluorescence microscopy for recessive mutations that affect the motor axon projection pattern (see Fig. 1). Mutations were mapped by positional cloning techniques. Results: We have screened 137 F1 lines and identified and mapped or cloned 4 lines that have striking motor axon projection defects in the absence of gross morphological defects. These mutations affect various aspects of motor neuron development including motor column identity and motor axon guidance and fasciculation. Discussion: Forward genetics coupled with a transgenic reporter line is an unbiased approach for identifying novel genetic pathways involved in axon guidance and target specificity in mammals. doi: 10.1016/j.ijdevneu.2008.09.178 [P2.54] Oxygen resuscitation exacerbates hypoxic ischemic outcomes in rat cortex C. Rea *, C.B. Reilly, M.B. Gill, D.C. Ferrari, K.H. Bockhurs, P. Narayana, J.R. Perez-Polo University of Texas, USA *Corresponding author.
Using a P7 rat model of hypoxic-ischemic (HI) brain injury we have shown that exposure to hypoxia and hyperoxia (HHI) results in generation of reactive oxygen species, inflammation and cell death—all risk factors for subsequent deficits in neuronal development and function. Hyperoxia increases oxidative stress that can trigger inflammatory cascades, neutrophil activation, and brain microvascular injury. Our experiments utilize a modified version of the Rice–Vanucci model of perinatal hypoxia-ischemia in Wistar rat pups that undergo exposure to 100% oxygen. By using T2-weighted magnetic resonance imaging we documented enhanced cortical lesions in HHI animals. Electrochemical monitoring showed bursts of superoxide during 100% oxygen resuscitation. Western blot analyses showed increased cell death measured by cleaved caspase 3 and cytosolic oligonucleosomes,
Poster Session 2 Abstracts 3 June 2008 / Int. J. Devl Neuroscience 26 (2008) 867–892
and augmented IL-1 signalling above and beyond levels reported for HI alone. Multiplex antibody cytokine assay demonstrates a spectrum of pro-inflammatory cytokines elevated at 24 h post injury. Consistent with the observed increases in oxidative stress components in the HI rat pups resuscitated with 100% oxygen, there was an increase in COX-2 levels likely to account in part for the increase in oxidative stress. In 80% of preterm infants, the common view is that the benefits of oxygen therapy outweigh the risks. We have shown deficits in attention, motor, visual-motor, and executive processing skills in preterm infants otherwise considered ‘‘healthy,’’ but these are not apparent before entry into formal learning settings [Smith et al., 2008. Int. J. Dev. Neurosci. 26(1): 125–131]. Few clinical studies have attempted to target improving outcomes in this large preterm population. These deficits may be accounted for by inflammation caused by the use of 100% oxygen resuscitation. Our long-term goal is to guide clinical trials of oxygen resuscitation by exploring its consequences in the P7 rat model of HI and developing novel interventions that will enhance benefits while decreasing inflammatory consequences. doi: 10.1016/j.ijdevneu.2008.09.179 [P2.55] Expansion of somatosensory activity onto visual structures in the congenital anophthalmic mouse R. Tremblay *, D. Boire, G. Bronchti Universite du Quebec a Trois-Rivieres, Canada *Corresponding author. Keywords: Blindness; Cross-modal compensation; 2-Deoxyglucose; Neuronal tracer
Blind peoples perform better in tactile tasks than sighted individuals. Imaging experiences on early blinds have shown that the somatosensory system can recruit visual areas. In early blind animal models, anatomical invasion and functional activation of primary visual structures by non-visual modalities have been demonstrated. In our laboratory, we use the congenital anophthalmic ZRDCT/An mutant mouse, in which neither retina nor optic nerve develop. We previously demonstrated the auditory-evoked c-Fos activation of primary visual cortex (V1) and dorsal lateral geniculate nucleus (LGNd), the main visual thalamic nucleus. We here study, in this animal model of early blindness, the hypothetical expansion of the somatosensory system onto visual territories. The C57Bl/6 mouse served as sighted control. After a [14]C-2-deoxyglucose injection, the animals explored an object filled box placed in a light- and noise-proof chamber to avoid visual and auditory stimulation. On autoradiograms of tangential and coronal sections, we observed that in sighted controls the whiskerrelated cortical activation was restricted within the primary somatosensory cortex (SI). In contrast, in blind mice the cortical activation expanded posteriorly and encompassed parietal association cortex (Par) and V1 (Fig. 1). At the thalamic level the LGNd was also correspondingly activated. In search for the origin of this activation, we injected neuronal tracer (BDA) in SI of both blind and control mice. Numerous fibres and cells were labelled in the mutant V1. Though at a lesser extent, a similar connectivity was observed in the control mice. However, only ZRDCT/An mutant mice had somatosensory projections entering LGNd. As we have reported that the auditory system also activates the LGNd and V1 in the anophthalmic mutant, it will be interesting to investigate whether both non-visual
885
Fig. 1. [14]C-2-glucose uptake following whisker stimulation.
modalities converge or are segregated at the cellular level. In other words, is the primary visual system multimodal in the early blind? doi: 10.1016/j.ijdevneu.2008.09.180 [P2.56] Expansion of auditory and somatosensory cortices in the blind mutant mouse zrdct in comparison with sighted controls S. Guillemette *, R. Tremblay, K. Arsenault, N. Chabot, D. Boire, G. Bronchti Universite du Quebec a Trois-Rivieres, Canada *Corresponding author. Keywords: Blindness; Cross-modal compensation; Serotonin transporter; Primary sensory cortices
Following sensory deprivation there is a reorganization of both spared and deprived cortices. We want here to determine the effect of early visual deprivation on the areal extent of the primary sensory cortices. We surmise a smaller primary visual cortex (V1) in the blind compared to the sighted, but are auditory (AC) and somato sensory (SI) cortices correspondingly enlarged? In our laboratory, we used the ZRDCT/An anophthalmic mouse and ZRDCT/RAX/Chumd.j or C57Bl/6 mice as controls. Since the entire cortical volume is larger in the sighted strains than in the blind, all volume and surface values were reported to the volume (or surface) of the entire cortex. In adults, the relative size of V1 and of V2 delimited on Nissl-stained coronal serial sections were surprisingly similar in blind and controls, and so was AC. The areal extent of the cortical barrelfield, measured on flat mounted cytochrome oxydase stained sections, was not different between the strains. Therefore the relative size devoted to SI is larger in the blind. Are these differences present in the early postnatal period? Using serotonin transporter immunochemistry which delineates precisely the primary sensory areas in the young brain, we measured the volume of the auditory, visual and barrel cortices in mutant and control pups at the postnatal (P) day 9. As in adults, blind pups had a smaller overall cortical volume than sighted ones. But, in contrast with what we found in adults, at P9 the relative size of V1 was significantly smaller in anophthalmic mice than in controls whereas AC and S1 were considerably enlarged in the blind pups. At this age, the absence of spontaneous activity in the visual system seems to affect the development of V1. We suggest that the relatively ‘‘normal’’ size of V1 at adulthood reflects its later activation by auditory and somatosensory inputs. doi: 10.1016/j.ijdevneu.2008.09.181
Poster Session 2 Abstracts 3 June 2008 / Int. J. Devl Neuroscience 26 (2008) 867–892
Slit1/2 and Robo1/2 knockout mice when compared to heterozygous controls. Mutant SN-VTA axons spread out in the diencephalon to form a wider tract. Phenotypes included invasion of the ventral midline, consistent with Slit repulsion. However, individual axons also wandered in aberrant dorsal trajectories, which suggests an unexpected role for Slits. Based on these and previous results we suggest that Slit/Robo signaling is necessary for correct dorsal/ ventral positioning and precise pathfinding of dopaminergic longitudinal axons, as well as serving a repellant function. These results may provide important insights into treatment of degeneration of SN-VTA dopminergic fibers in Parkinson’s and Alzheimer’s disease. doi: 10.1016/j.ijdevneu.2008.09.176 [P2.52] Pioneer longitudinal axons navigate using floor plate and Slit/Robo signals W.T. Farmer 1,*, A.L. Altick 1, J.P. Dugan 1, T. Kidd 1, F. Charron 2, G.S. Mastick 1 1
University of Nevada, USA Institut de recherches cliniques de Montre´al (IRCM), Canada *Corresponding author. 2
Keywords: Slit; Robo; Floor plate; Longitudinal axon
Longitudinal axons transmit all signals between the brain and spinal cord. Their axon tracts through the brain stem are established by a simple set of pioneer axons. These axons navigate through several brain regions with precise trajectories parallel to the floor plate. To identify longitudinal guidance mechanisms in vivo, the overall role of floor plate tissue, and the specific roles of Slit/Robo signals were tested. Ectopic induction or genetic deletion of floor plate diverted longitudinal axons into abnormal trajectories, providing in vivo evidence for the importance of floor plate signals. The expression patterns of the diffusible cues of the Slit family were altered in the floor plate experiments, suggesting their involvement in longitudinal guidance. Genetic tests of Slit1 and 2, and the Slit receptors Robo1 and 2 were carried out in mutant mice. Slit1; Slit2 double mutants had severe longitudinal errors, particularly for ventral axons, including midline crossing and wandering longitudinal trajectories. Robo1 and Robo2 were largely genetically redundant, and neither appeared to specify specific tract positions. However, combined Robo1 and Robo2 mutations strongly disrupted each pioneer tract. Our results indicate that Slit/ Robo signalling may have at least two functions: a repulsive activity that prevents ventral axons from entering the floor plate and a second activity that maintains straight trajectories. Thus, pioneer axons depend on long-range floor plate cues, with Slit/ Robo signalling required for precise longitudinal trajectories. doi: 10.1016/j.ijdevneu.2008.09.177 [P2.53] A forward genetic screen in the mouse to identify novel genes that affect motor neuron development K.F. Liem *, K.V. Anderson Sloan-Kettering Institute, USA *Corresponding author. Keywords: Motor neuron; Genetics; Axon guidance; Cell fate
Introduction: The mammalian spinal motor system is an excellent model system to study the formation of a simple
Fig. 1. The HB9-GFP transgenic mouse labels motor neurons and motor axons.
topographical neuronal projection. Although we know much about how motor neurons develop, we know less about how various classes of motor neurons find and select their specific muscle targets. We have taken a forward genetic approach to identify novel genes that control motor neuron subtype identity and motor axon guidance and target specificity. Methods: Mice were mutagenized with ENU and outcrossed to a HB9-GFP transgenic reporter line which expresses GFP in spinal motor neurons and their axons. Litters were analyzed at e12.5 by fluorescence microscopy for recessive mutations that affect the motor axon projection pattern (see Fig. 1). Mutations were mapped by positional cloning techniques. Results: We have screened 137 F1 lines and identified and mapped or cloned 4 lines that have striking motor axon projection defects in the absence of gross morphological defects. These mutations affect various aspects of motor neuron development including motor column identity and motor axon guidance and fasciculation. Discussion: Forward genetics coupled with a transgenic reporter line is an unbiased approach for identifying novel genetic pathways involved in axon guidance and target specificity in mammals. doi: 10.1016/j.ijdevneu.2008.09.178 [P2.54] Oxygen resuscitation exacerbates hypoxic ischemic outcomes in rat cortex C. Rea *, C.B. Reilly, M.B. Gill, D.C. Ferrari, K.H. Bockhurs, P. Narayana, J.R. Perez-Polo University of Texas, USA *Corresponding author.
Using a P7 rat model of hypoxic-ischemic (HI) brain injury we have shown that exposure to hypoxia and hyperoxia (HHI) results in generation of reactive oxygen species, inflammation and cell death—all risk factors for subsequent deficits in neuronal development and function. Hyperoxia increases oxidative stress that can trigger inflammatory cascades, neutrophil activation, and brain microvascular injury. Our experiments utilize a modified version of the Rice–Vanucci model of perinatal hypoxia-ischemia in Wistar rat pups that undergo exposure to 100% oxygen. By using T2-weighted magnetic resonance imaging we documented enhanced cortical lesions in HHI animals. Electrochemical monitoring showed bursts of superoxide during 100% oxygen resuscitation. Western blot analyses showed increased cell death measured by cleaved caspase 3 and cytosolic oligonucleosomes,
Poster Session 2 Abstracts 3 June 2008 / Int. J. Devl Neuroscience 26 (2008) 867–892
and augmented IL-1 signalling above and beyond levels reported for HI alone. Multiplex antibody cytokine assay demonstrates a spectrum of pro-inflammatory cytokines elevated at 24 h post injury. Consistent with the observed increases in oxidative stress components in the HI rat pups resuscitated with 100% oxygen, there was an increase in COX-2 levels likely to account in part for the increase in oxidative stress. In 80% of preterm infants, the common view is that the benefits of oxygen therapy outweigh the risks. We have shown deficits in attention, motor, visual-motor, and executive processing skills in preterm infants otherwise considered ‘‘healthy,’’ but these are not apparent before entry into formal learning settings [Smith et al., 2008. Int. J. Dev. Neurosci. 26(1): 125–131]. Few clinical studies have attempted to target improving outcomes in this large preterm population. These deficits may be accounted for by inflammation caused by the use of 100% oxygen resuscitation. Our long-term goal is to guide clinical trials of oxygen resuscitation by exploring its consequences in the P7 rat model of HI and developing novel interventions that will enhance benefits while decreasing inflammatory consequences. doi: 10.1016/j.ijdevneu.2008.09.179 [P2.55] Expansion of somatosensory activity onto visual structures in the congenital anophthalmic mouse R. Tremblay *, D. Boire, G. Bronchti Universite du Quebec a Trois-Rivieres, Canada *Corresponding author. Keywords: Blindness; Cross-modal compensation; 2-Deoxyglucose; Neuronal tracer
Blind peoples perform better in tactile tasks than sighted individuals. Imaging experiences on early blinds have shown that the somatosensory system can recruit visual areas. In early blind animal models, anatomical invasion and functional activation of primary visual structures by non-visual modalities have been demonstrated. In our laboratory, we use the congenital anophthalmic ZRDCT/An mutant mouse, in which neither retina nor optic nerve develop. We previously demonstrated the auditory-evoked c-Fos activation of primary visual cortex (V1) and dorsal lateral geniculate nucleus (LGNd), the main visual thalamic nucleus. We here study, in this animal model of early blindness, the hypothetical expansion of the somatosensory system onto visual territories. The C57Bl/6 mouse served as sighted control. After a [14]C-2-deoxyglucose injection, the animals explored an object filled box placed in a light- and noise-proof chamber to avoid visual and auditory stimulation. On autoradiograms of tangential and coronal sections, we observed that in sighted controls the whiskerrelated cortical activation was restricted within the primary somatosensory cortex (SI). In contrast, in blind mice the cortical activation expanded posteriorly and encompassed parietal association cortex (Par) and V1 (Fig. 1). At the thalamic level the LGNd was also correspondingly activated. In search for the origin of this activation, we injected neuronal tracer (BDA) in SI of both blind and control mice. Numerous fibres and cells were labelled in the mutant V1. Though at a lesser extent, a similar connectivity was observed in the control mice. However, only ZRDCT/An mutant mice had somatosensory projections entering LGNd. As we have reported that the auditory system also activates the LGNd and V1 in the anophthalmic mutant, it will be interesting to investigate whether both non-visual
885
Fig. 1. [14]C-2-glucose uptake following whisker stimulation.
modalities converge or are segregated at the cellular level. In other words, is the primary visual system multimodal in the early blind? doi: 10.1016/j.ijdevneu.2008.09.180 [P2.56] Expansion of auditory and somatosensory cortices in the blind mutant mouse zrdct in comparison with sighted controls S. Guillemette *, R. Tremblay, K. Arsenault, N. Chabot, D. Boire, G. Bronchti Universite du Quebec a Trois-Rivieres, Canada *Corresponding author. Keywords: Blindness; Cross-modal compensation; Serotonin transporter; Primary sensory cortices
Following sensory deprivation there is a reorganization of both spared and deprived cortices. We want here to determine the effect of early visual deprivation on the areal extent of the primary sensory cortices. We surmise a smaller primary visual cortex (V1) in the blind compared to the sighted, but are auditory (AC) and somato sensory (SI) cortices correspondingly enlarged? In our laboratory, we used the ZRDCT/An anophthalmic mouse and ZRDCT/RAX/Chumd.j or C57Bl/6 mice as controls. Since the entire cortical volume is larger in the sighted strains than in the blind, all volume and surface values were reported to the volume (or surface) of the entire cortex. In adults, the relative size of V1 and of V2 delimited on Nissl-stained coronal serial sections were surprisingly similar in blind and controls, and so was AC. The areal extent of the cortical barrelfield, measured on flat mounted cytochrome oxydase stained sections, was not different between the strains. Therefore the relative size devoted to SI is larger in the blind. Are these differences present in the early postnatal period? Using serotonin transporter immunochemistry which delineates precisely the primary sensory areas in the young brain, we measured the volume of the auditory, visual and barrel cortices in mutant and control pups at the postnatal (P) day 9. As in adults, blind pups had a smaller overall cortical volume than sighted ones. But, in contrast with what we found in adults, at P9 the relative size of V1 was significantly smaller in anophthalmic mice than in controls whereas AC and S1 were considerably enlarged in the blind pups. At this age, the absence of spontaneous activity in the visual system seems to affect the development of V1. We suggest that the relatively ‘‘normal’’ size of V1 at adulthood reflects its later activation by auditory and somatosensory inputs. doi: 10.1016/j.ijdevneu.2008.09.181