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Expression of Nogo receptor and RhoA during the development of rat brain
Poster Session 1 Abstracts 2 June 2008 / Int. J. Devl Neuroscience 26 (2008) 841–866
that members of the FGF receptor (FGFR) family are expressed in the diencephalon (bordering this turn site) throughout the period of optic tract development, suggesting that FGF signalling in the neuroepithelium could maintain the pattern of guidance factors for axons in the developing optic tract. Furthermore, we found that exposing the developing optic tract and neuroepithelium to an FGFR inhibitor (SU5402) just prior to when RGC axons reach the turn site in the diencephalon has two effects: (1) RGC axons stall at the turn in the mid-diencephalon, and (2) the patterning in the diencephalon, adjacent to the turn site, is specifically and rapidly disrupted (as evidenced through in situ hybridization of postmitotic transcription factors, xLhx9, xLhx2, xLhx1, xLhx5, and xDll3). This suggests that FGF signalling is required both for RGC axons to navigate this guidance decision and to maintain the pattern of expression in the surrounding tissue. Moreover, the rapidity of this effect argues that the patterned cues required to guide axons in the developing optic tract are not stably expressed in that they must be maintained with the continued presence of signalling molecules. doi: 10.1016/j.ijdevneu.2008.09.096 [P1.47] NADPH-diaphorase expression in the mauthner cells during postembryonic development of the teleost fish, Labeo rohita A.G. Jadhao *, N.V. Palande
857
[P1.48] Wnt and Shh cooperate in postcommissural axon guidance A.M. Wacker *, E. Domanitskaya, E.T. Stoeckli University of Zurich, Switzerland *Corresponding author. Keywords: Sonic hedgehog; Wnt; Sfrp; Axon guidance
After crossing the midline, axons of dorsolateral commissural neurons turn into the longitudinal axis of the spinal cord and extend rostrally. In the mouse, a rostrocaudal gradient of Wnt4 expressed in the floor plate was found to be responsible for this rostral turn of postcommissural axons (Lyuksyutova et al., 2003). In contrast, in the chicken embryo, a repellent activity of Shh mediated by Hedgehog-interacting protein was identified for this pathfinding decision (Bourikas et al., 2005). Based on these observations, we tested for a cooperation between Wnts and Shh in postcommissural axon guidance. Indeed, we found that Wnt5A and Wnt7A acted as guidance cues for postcommissural axons also in the chicken spinal cord. However, in contrast to mouse, no transcriptional WNT gradient was detected in the chicken floor plate. Rather, Wnt activity was regulated by a graded distribution of Sfrps (Secreted frizzled-related proteins), known Wnt antagonists. The expression of Sfrps was regulated by Shh. Therefore, we conclude that in the chicken spinal cord postcommissural axons are guided rostrally along the longitudinal axis by Shh that acts on these axons both directly and indirectly by regulating Wnt activity.
University of Pune, India *Corresponding author. doi: 10.1016/j.ijdevneu.2008.09.098
Keywords: Mauthner Cell; Brainstem neurons; Startle response; NADPH-d histochemistry
The information on the motor activity of the Mauthner cells (MC) function and related brainstem neurons have been well reviewed and these cells activate fast-start responses such as seen in fishes escaping from predatory attacks. Many studies have shown NADPH-d activity in the CNS of fishes, but it has been reported only in the MC of adult swoardtail and goldfish and larval lamprey. However, no reports are on NADPH-diaphorase (NADPHd) mapping in the fish brain during ontogeny. For this study various developing stages and adult carp (Labeo rohita) were obtained from the government fish farm, Pune. The brains were fixed in 4% paraformaldehyde and cut at 10–12 mm in transverse or sagittal planes. The NADPH-d histochemistry was performed according to a modification of the original method by Scherer-Singler et al (1983). In the hindbrain, MC was seen intensely labeled with NADPH-d situated ventrally on either side of the ventricle, in the hatchling (12 mm), fry (16–30 mm), semifingerling (45–70 mm), fingerling (85–90 mm) and moderately stained in adult (350–370 mm) L. rohita. The gradual increase in cell diameter was evident from hatchling to adult, while the nuclear diameter remained nearly unchanged. It is generally considered that NADPH-d expressing cells in the brain are responsible for the production of nitric oxide (NO). Presuming that NADPH-d containing MC may be involved in the production of the NO, and therefore, NO might have a role in the initiation of startle response to escape from the predators and unfavorable environment in L. rohita. This study is supported by the grants from the DST and UGC Govt. of India to AGJ. doi: 10.1016/j.ijdevneu.2008.09.097
[P1.49] Expression of Nogo receptor and RhoA during the development of rat brain B.Y. Su *, Q. Guo, S.R. Li Chengdu Medical College, China *Corresponding author. Tel.: +86 28 68289158. Keywords: Nogo receptor; RhoA; Rat brain; Development
Nogo receptor (NgR) is a mambrane receptor through which Nogo, myelin associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) transmit their regeneration inhibitory signal into neurons. RhoA is the downstream factor of NgR which can lead to the collapse of cytoskeleton and the failure of regeneration. In the present study, we investigated the expression of NgR and RhoA in rat brain during development by means of immunohistochemistry and Western blot. Both the expression of NgR and RhoA could be detected at embryonic day 15, but the expression level was very low before birth. However, at postnatal day 14, neurons in cerebral cortex, hippocampus and some nuclei showed high NgR expression level. It peaked at postnatal day 42 and subsequently declined to medium intensity until 1 year. The expression of RhoA peaked at postnatal day 14 with distribution regions mainly in neurons in cerebral cortex and hippocampus and subsequently declined to medium intensity until 1 year, while its expression in nuclei was weak at postnatal day 14. It peaked at postnatal day 70 and declined subsequently to very weak at 1 year. In conclusion, although the distribution region is almost the same, the time point when the expression peak occurs and the duration when the expression level is high between NgR and RhoA are different. NgR and RhoA are in the same inhibitory signal pathway, but they do not have the same expression profile, which may be useful to the design of regeneration strategy. Further investigation
858
Poster Session 1 Abstracts 2 June 2008 / Int. J. Devl Neuroscience 26 (2008) 841–866
should be done in order to elucidate the significance of the expression.
[P1.51]
Acknowledgement
M. Tamura 1,*, S. Nakahara 1, A. Kakita 2, N. Matsuki 1, R. Koyama 1
This study was supported by the Major State Basic Research Development Program of China (973 Program, NO.2003CB515300)
1
doi: 10.1016/j.ijdevneu.2008.09.099 [P1.50] Atrazine delays Purkinje cell development in vitro as evidenced by Golgi method in primary organotypic cultures of mouse cerebellar cortex ˜ a-Contreras *, D. Davila-Vera, R.V. Mendoza-Bricen ˜o Z. Pen University of Los Andes, Venezuela *Corresponding author. Keywords: Purkinje cell development; Primary organotypic cultures; Atrazine; Mouse cerebellar cortex
There is limited information on neurotoxic effects of atrazine in developing nervous system. Atrazine, an extensively used chloros-triazine herbicide, has been identified as an endocrine disruptor in amphibians. Considering the importance of endocrine influence for normal brain development, in this study we examined the effects of atrazine on the development of Purkinje cells (PC) in vitro, by employing the Golgi method to study the morphological changes that occur during PC differentiation. Primary organotypic cultures of mouse cerebellar cortex were prepared from recently born Mus musculus mice (P0) and at postnatal days 3 (P3), P5, P7 and P9. Explanted cerebellar cortices from each age were divided into two groups: control and atrazine-exposed. A dosis of 15 mg atrazine/kg wet weight was added into the incubation medium every 48 h during the 6 day-period of incubation. Cultured tissues were harvested, fixed and stained using a modified Golgi method5 and then observed under Polyvar high resolution light microscopy. After 6 days of incubation, PC from control cultures obtained from mice at P0 until P9 showed a normal sequence of development in vitro. Immature morphological characteristics of PC were observed in P0 cultures, such as cell bodies that are highly irregular in shape and the presence of numerous prolongations from their soma. Cultures obtained at P3 showed PC with few cellular prolongations and cell body of more regular shape. At this stage, the emergence of PC primary dendrite and initial segment of the axon can already be observed. Cultures from P5 and P7 animals showed more developed axons and dendritic trees with secondary dendrite ramifications. In P9 cerebellar cultures, the PCs demonstrated phenotypic characteristics as in situ mature PCs. On the other hand, administration of atrazine during incubation not only delayed the development of PCs but also hindered the acquirement of mature phenotypic characteristics. Atrazine-exposed cultures from P0 until P3 showed highly immature PCs and it was not until P5 that the emergence of the primary dendrite and axon initial segment was observed. These results demonstrate the endocrinedisrupting effects of atrazine in the developing nervous system. doi: 10.1016/j.ijdevneu.2008.09.100
Sex differences in dendritic maturation of hippocampal granule cells after prenatal stress
The University of Tokyo, Japan University of Niigata, Japan *Corresponding author. 2
Keywords: Glucocorticoid; Neurogenesis; Antidepressant; Time-lapse imaging
Exposure of pregnant mothers to stress increases the risk of depressive disorders in their offspring later in life. However, cellular and molecular mechanisms underlying the phenomena are not clear. Here, we investigated the effect of antenatal stress on the morphological maturation of dentate granule cells in the hippocampus, which is strongly involved in the development of depressive disorders. Pregnant rats were subjected daily to three sessions of 45 min of restraint stress from day 15 to 21 of gestation, and subsequently hippocampal slices were prepared from their offspring at postnatal day 0–1 and cultured. After 14 days in vitro, the branch number and the total length of granule cell dendrites, which were visualized by biocytin injection, significantly decreased compared with control. Importantly, the effect was more remarkable in female than in male. This is the first study that reveals the sex differences in the prenatal stressinduced suppression of dendritic maturation of granule cells. doi: 10.1016/j.ijdevneu.2008.09.101 [P1.52] IgCAMs redundantly control different aspects of axon guidance in C. elegans V. Schwarz 1, J. Pan 2, S. Voltmer-Irsch 1, H. Hutter 2,* 1
Max Planck Institute for Medical Research, Germany Simon Fraser University, Canada *Corresponding author. 2
Keywords: IgCAM; Axon guidance; C. elegans
Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) form one of the largest and oldest families of adhesion molecules and receptors with a role in neuronal development. We systematically analyzed previously uncharacterized members of this family in C. elegans. Expression studies of 12 IgCAMs revealed that most of them are expressed in the nervous system. However, expression for none of these genes is confined to a single tissue and all tissues express at least one of the IgCAMs. Neuronal expression was analyzed at the single cell level. Significant overlap in expression was found in central components of the motor circuit like command interneurons and ventral cord motoneurons as well as motoneurons innervating head muscles. Sensory neurons are underrepresented among the cells expressing these IgCAMs. Mutations were obtained for eight genes showing significant neuronal expression. Phenotypic analysis of single mutants revealed only limited axonal defects in neurons belonging to the motor circuit in a few of the mutants. The majority of the mutants had no apparent defects in the neurons studied. Systematic genetic interaction studies identified two sets of interactions affecting interneuron navigation and commissure outgrowth. A quadruple mutant of rig-1, rig-3, rig-5 and rig-6 show significant interneuron navigation defects. None of the four possible triple mutant combinations shows significant defects, indicating that all four genes act redundantly to guide interneuron axons. Similarly, significant defects in motoneuron commissure outgrowth are apparent only in a triple mutant combination of syg-1, wrk-1 and
that members of the FGF receptor (FGFR) family are expressed in the diencephalon (bordering this turn site) throughout the period of optic tract development, suggesting that FGF signalling in the neuroepithelium could maintain the pattern of guidance factors for axons in the developing optic tract. Furthermore, we found that exposing the developing optic tract and neuroepithelium to an FGFR inhibitor (SU5402) just prior to when RGC axons reach the turn site in the diencephalon has two effects: (1) RGC axons stall at the turn in the mid-diencephalon, and (2) the patterning in the diencephalon, adjacent to the turn site, is specifically and rapidly disrupted (as evidenced through in situ hybridization of postmitotic transcription factors, xLhx9, xLhx2, xLhx1, xLhx5, and xDll3). This suggests that FGF signalling is required both for RGC axons to navigate this guidance decision and to maintain the pattern of expression in the surrounding tissue. Moreover, the rapidity of this effect argues that the patterned cues required to guide axons in the developing optic tract are not stably expressed in that they must be maintained with the continued presence of signalling molecules. doi: 10.1016/j.ijdevneu.2008.09.096 [P1.47] NADPH-diaphorase expression in the mauthner cells during postembryonic development of the teleost fish, Labeo rohita A.G. Jadhao *, N.V. Palande
857
[P1.48] Wnt and Shh cooperate in postcommissural axon guidance A.M. Wacker *, E. Domanitskaya, E.T. Stoeckli University of Zurich, Switzerland *Corresponding author. Keywords: Sonic hedgehog; Wnt; Sfrp; Axon guidance
After crossing the midline, axons of dorsolateral commissural neurons turn into the longitudinal axis of the spinal cord and extend rostrally. In the mouse, a rostrocaudal gradient of Wnt4 expressed in the floor plate was found to be responsible for this rostral turn of postcommissural axons (Lyuksyutova et al., 2003). In contrast, in the chicken embryo, a repellent activity of Shh mediated by Hedgehog-interacting protein was identified for this pathfinding decision (Bourikas et al., 2005). Based on these observations, we tested for a cooperation between Wnts and Shh in postcommissural axon guidance. Indeed, we found that Wnt5A and Wnt7A acted as guidance cues for postcommissural axons also in the chicken spinal cord. However, in contrast to mouse, no transcriptional WNT gradient was detected in the chicken floor plate. Rather, Wnt activity was regulated by a graded distribution of Sfrps (Secreted frizzled-related proteins), known Wnt antagonists. The expression of Sfrps was regulated by Shh. Therefore, we conclude that in the chicken spinal cord postcommissural axons are guided rostrally along the longitudinal axis by Shh that acts on these axons both directly and indirectly by regulating Wnt activity.
University of Pune, India *Corresponding author. doi: 10.1016/j.ijdevneu.2008.09.098
Keywords: Mauthner Cell; Brainstem neurons; Startle response; NADPH-d histochemistry
The information on the motor activity of the Mauthner cells (MC) function and related brainstem neurons have been well reviewed and these cells activate fast-start responses such as seen in fishes escaping from predatory attacks. Many studies have shown NADPH-d activity in the CNS of fishes, but it has been reported only in the MC of adult swoardtail and goldfish and larval lamprey. However, no reports are on NADPH-diaphorase (NADPHd) mapping in the fish brain during ontogeny. For this study various developing stages and adult carp (Labeo rohita) were obtained from the government fish farm, Pune. The brains were fixed in 4% paraformaldehyde and cut at 10–12 mm in transverse or sagittal planes. The NADPH-d histochemistry was performed according to a modification of the original method by Scherer-Singler et al (1983). In the hindbrain, MC was seen intensely labeled with NADPH-d situated ventrally on either side of the ventricle, in the hatchling (12 mm), fry (16–30 mm), semifingerling (45–70 mm), fingerling (85–90 mm) and moderately stained in adult (350–370 mm) L. rohita. The gradual increase in cell diameter was evident from hatchling to adult, while the nuclear diameter remained nearly unchanged. It is generally considered that NADPH-d expressing cells in the brain are responsible for the production of nitric oxide (NO). Presuming that NADPH-d containing MC may be involved in the production of the NO, and therefore, NO might have a role in the initiation of startle response to escape from the predators and unfavorable environment in L. rohita. This study is supported by the grants from the DST and UGC Govt. of India to AGJ. doi: 10.1016/j.ijdevneu.2008.09.097
[P1.49] Expression of Nogo receptor and RhoA during the development of rat brain B.Y. Su *, Q. Guo, S.R. Li Chengdu Medical College, China *Corresponding author. Tel.: +86 28 68289158. Keywords: Nogo receptor; RhoA; Rat brain; Development
Nogo receptor (NgR) is a mambrane receptor through which Nogo, myelin associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) transmit their regeneration inhibitory signal into neurons. RhoA is the downstream factor of NgR which can lead to the collapse of cytoskeleton and the failure of regeneration. In the present study, we investigated the expression of NgR and RhoA in rat brain during development by means of immunohistochemistry and Western blot. Both the expression of NgR and RhoA could be detected at embryonic day 15, but the expression level was very low before birth. However, at postnatal day 14, neurons in cerebral cortex, hippocampus and some nuclei showed high NgR expression level. It peaked at postnatal day 42 and subsequently declined to medium intensity until 1 year. The expression of RhoA peaked at postnatal day 14 with distribution regions mainly in neurons in cerebral cortex and hippocampus and subsequently declined to medium intensity until 1 year, while its expression in nuclei was weak at postnatal day 14. It peaked at postnatal day 70 and declined subsequently to very weak at 1 year. In conclusion, although the distribution region is almost the same, the time point when the expression peak occurs and the duration when the expression level is high between NgR and RhoA are different. NgR and RhoA are in the same inhibitory signal pathway, but they do not have the same expression profile, which may be useful to the design of regeneration strategy. Further investigation
858
Poster Session 1 Abstracts 2 June 2008 / Int. J. Devl Neuroscience 26 (2008) 841–866
should be done in order to elucidate the significance of the expression.
[P1.51]
Acknowledgement
M. Tamura 1,*, S. Nakahara 1, A. Kakita 2, N. Matsuki 1, R. Koyama 1
This study was supported by the Major State Basic Research Development Program of China (973 Program, NO.2003CB515300)
1
doi: 10.1016/j.ijdevneu.2008.09.099 [P1.50] Atrazine delays Purkinje cell development in vitro as evidenced by Golgi method in primary organotypic cultures of mouse cerebellar cortex ˜ a-Contreras *, D. Davila-Vera, R.V. Mendoza-Bricen ˜o Z. Pen University of Los Andes, Venezuela *Corresponding author. Keywords: Purkinje cell development; Primary organotypic cultures; Atrazine; Mouse cerebellar cortex
There is limited information on neurotoxic effects of atrazine in developing nervous system. Atrazine, an extensively used chloros-triazine herbicide, has been identified as an endocrine disruptor in amphibians. Considering the importance of endocrine influence for normal brain development, in this study we examined the effects of atrazine on the development of Purkinje cells (PC) in vitro, by employing the Golgi method to study the morphological changes that occur during PC differentiation. Primary organotypic cultures of mouse cerebellar cortex were prepared from recently born Mus musculus mice (P0) and at postnatal days 3 (P3), P5, P7 and P9. Explanted cerebellar cortices from each age were divided into two groups: control and atrazine-exposed. A dosis of 15 mg atrazine/kg wet weight was added into the incubation medium every 48 h during the 6 day-period of incubation. Cultured tissues were harvested, fixed and stained using a modified Golgi method5 and then observed under Polyvar high resolution light microscopy. After 6 days of incubation, PC from control cultures obtained from mice at P0 until P9 showed a normal sequence of development in vitro. Immature morphological characteristics of PC were observed in P0 cultures, such as cell bodies that are highly irregular in shape and the presence of numerous prolongations from their soma. Cultures obtained at P3 showed PC with few cellular prolongations and cell body of more regular shape. At this stage, the emergence of PC primary dendrite and initial segment of the axon can already be observed. Cultures from P5 and P7 animals showed more developed axons and dendritic trees with secondary dendrite ramifications. In P9 cerebellar cultures, the PCs demonstrated phenotypic characteristics as in situ mature PCs. On the other hand, administration of atrazine during incubation not only delayed the development of PCs but also hindered the acquirement of mature phenotypic characteristics. Atrazine-exposed cultures from P0 until P3 showed highly immature PCs and it was not until P5 that the emergence of the primary dendrite and axon initial segment was observed. These results demonstrate the endocrinedisrupting effects of atrazine in the developing nervous system. doi: 10.1016/j.ijdevneu.2008.09.100
Sex differences in dendritic maturation of hippocampal granule cells after prenatal stress
The University of Tokyo, Japan University of Niigata, Japan *Corresponding author. 2
Keywords: Glucocorticoid; Neurogenesis; Antidepressant; Time-lapse imaging
Exposure of pregnant mothers to stress increases the risk of depressive disorders in their offspring later in life. However, cellular and molecular mechanisms underlying the phenomena are not clear. Here, we investigated the effect of antenatal stress on the morphological maturation of dentate granule cells in the hippocampus, which is strongly involved in the development of depressive disorders. Pregnant rats were subjected daily to three sessions of 45 min of restraint stress from day 15 to 21 of gestation, and subsequently hippocampal slices were prepared from their offspring at postnatal day 0–1 and cultured. After 14 days in vitro, the branch number and the total length of granule cell dendrites, which were visualized by biocytin injection, significantly decreased compared with control. Importantly, the effect was more remarkable in female than in male. This is the first study that reveals the sex differences in the prenatal stressinduced suppression of dendritic maturation of granule cells. doi: 10.1016/j.ijdevneu.2008.09.101 [P1.52] IgCAMs redundantly control different aspects of axon guidance in C. elegans V. Schwarz 1, J. Pan 2, S. Voltmer-Irsch 1, H. Hutter 2,* 1
Max Planck Institute for Medical Research, Germany Simon Fraser University, Canada *Corresponding author. 2
Keywords: IgCAM; Axon guidance; C. elegans
Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) form one of the largest and oldest families of adhesion molecules and receptors with a role in neuronal development. We systematically analyzed previously uncharacterized members of this family in C. elegans. Expression studies of 12 IgCAMs revealed that most of them are expressed in the nervous system. However, expression for none of these genes is confined to a single tissue and all tissues express at least one of the IgCAMs. Neuronal expression was analyzed at the single cell level. Significant overlap in expression was found in central components of the motor circuit like command interneurons and ventral cord motoneurons as well as motoneurons innervating head muscles. Sensory neurons are underrepresented among the cells expressing these IgCAMs. Mutations were obtained for eight genes showing significant neuronal expression. Phenotypic analysis of single mutants revealed only limited axonal defects in neurons belonging to the motor circuit in a few of the mutants. The majority of the mutants had no apparent defects in the neurons studied. Systematic genetic interaction studies identified two sets of interactions affecting interneuron navigation and commissure outgrowth. A quadruple mutant of rig-1, rig-3, rig-5 and rig-6 show significant interneuron navigation defects. None of the four possible triple mutant combinations shows significant defects, indicating that all four genes act redundantly to guide interneuron axons. Similarly, significant defects in motoneuron commissure outgrowth are apparent only in a triple mutant combination of syg-1, wrk-1 and