SE triggers a transient decrease in Map2 immunoreactivity and dendritic spine density in the CA1 hippocampal region that reaches a minimum at 14 days post-SE and partially recovers by 35 days. Microglial accumulation, as indicated by IBA1 staining, increases in the CA1 region in parallel with maximal levels at 14 days post-SE. The spatiotemporal profiles of SE-induced Map2 decline and microglial changes parallel each other in the CA1 area. This suggests microglia may play a role in the dendritic pathology associated with SE and epilepsy.
Caspase activation contributes to astrogliosis without inducing apoptosis in astrocytes. The study found that treating cultured neonatal rat astrocytes with dibutryl cAMP or beta-amyloid peptide, stimuli known to induce astrogliosis, led to increased caspase activity and expression of active caspase-3 without cell death. Inhibition of caspases attenuated the increased expression of glutamine synthetase and fibroblast growth factor-2, markers of astrogliosis. The results suggest caspases play a non-apoptotic role in regulating astrogliosis in astrocytes following brain injury.
LY2886721 is a potent inhibitor of BACE1 that was studied in mice, dogs, and humans. It showed high selectivity against other proteases. In mice and dogs, it efficiently lowered amyloid beta levels in the brain and cerebrospinal fluid after oral administration. Similar amyloid beta lowering was seen in plasma and cerebrospinal fluid of healthy humans given single or multiple doses of LY2886721, supporting its potential as a disease-modifying therapy for Alzheimer's disease. Crystallography confirmed LY2886721 bound tightly to the active site of BACE1.
GABAB receptor-mediated selective peripheral analgesia by the non-proteinogen...Igor Putrenko
1) The document examines the hypothesis that the amino acid isovaline produces peripheral analgesia through GABAB receptors without affecting the central nervous system.
2) Experiments in mice found that isovaline, like the GABAB agonists baclofen and GABA, reduced pain-related behavior induced by prostaglandin E2 injection into the paw in a GABAB receptor-dependent manner, but unlike baclofen, isovaline did not produce sedation or hypothermia indicating a lack of central effects.
3) Immunohistochemistry revealed co-localization of GABAB1 and GABAB2 receptor subunits on fine nerve endings and keratinocytes in skin
1) Mice lacking the inhibitory synapse cell adhesion molecule neuroligin 2 (NL2) were found to exhibit increased anxiety-like behavior.
2) While these NL2-deficient mice appeared to have a decrease in the density of inhibitory synaptic puncta, electron microscopy revealed no actual change in inhibitory synapse numbers.
3) This suggests that NL2 deletion impairs the function of inhibitory synapses without decreasing their numbers, and this decrease in inhibitory synaptic function correlates with increased anxiety in the mice.
An inhibitory pull–push circuit in frontal cortexTaruna Ikrar
Push–pull is a canonical computation of excitatory cortical circuits. By contrast, we identify a pull–push inhibitory circuit in frontal cortex that originates in vasoactive intestinal polypeptide (VIP)-expressing interneurons. During arousal, VIP cells rapidly and directly inhibit pyramidal neurons; VIP cells also indirectly excite these pyramidal neurons via parallel disinhibition. Thus, arousal exerts a feedback pull–push influence on excitatory neurons—an inversion of the canonical push–pull of feedforward input.
Directed research spring 2016 Daniel SvedbergDan Svedberg
1) The study genetically inhibited microglial NF-κB activity in mouse models of Spinocerebellar Ataxia Type 1 (SCA1) to determine if microglia contribute to SCA1 pathology. 2) Partial inhibition of microglial NF-κB was shown to significantly worsen SCA1 pathology in mice, while complete inhibition needs further study with more animals. 3) Inhibiting microglial NF-κB may also cause deficient synaptic pruning in the cerebellum of wild-type mice.
1) The document examines how stabilizing ryanodine receptors after traumatic brain injury can reduce tau pathology and prevent progression to Alzheimer's disease in mouse models.
2) Tests on Alzheimer's disease mice and healthy controls found that traumatic brain injury increased tau deposits and plaque propagation, with more severe effects in AD mice.
3) Treatment with ryanodine receptor stabilizers like dantrolene after injury significantly reduced tau pathology and plaque loads, showing potential for preventing long-term cognitive deficits and conversion to Alzheimer's disease.
BDNF stabilizes synapses and maintains the structural complexity of optic axons in vivo. The study used time-lapse imaging to examine how neutralizing endogenous BDNF or blocking NMDA receptors in the optic tectum affects synapse and axon branch stability in Xenopus retinal ganglion cell axons. Neutralizing BDNF enhanced synapse and branch elimination, indicating BDNF stabilizes both. Blocking NMDA receptors transiently eliminated synapses, but BDNF treatment rescued synapses by maintaining addition and rapid stabilization. Thus, BDNF promotes synaptic connectivity by stabilizing synapses and maintaining axon complexity.
Caspase activation contributes to astrogliosis without inducing apoptosis in astrocytes. The study found that treating cultured neonatal rat astrocytes with dibutryl cAMP or beta-amyloid peptide, stimuli known to induce astrogliosis, led to increased caspase activity and expression of active caspase-3 without cell death. Inhibition of caspases attenuated the increased expression of glutamine synthetase and fibroblast growth factor-2, markers of astrogliosis. The results suggest caspases play a non-apoptotic role in regulating astrogliosis in astrocytes following brain injury.
LY2886721 is a potent inhibitor of BACE1 that was studied in mice, dogs, and humans. It showed high selectivity against other proteases. In mice and dogs, it efficiently lowered amyloid beta levels in the brain and cerebrospinal fluid after oral administration. Similar amyloid beta lowering was seen in plasma and cerebrospinal fluid of healthy humans given single or multiple doses of LY2886721, supporting its potential as a disease-modifying therapy for Alzheimer's disease. Crystallography confirmed LY2886721 bound tightly to the active site of BACE1.
GABAB receptor-mediated selective peripheral analgesia by the non-proteinogen...Igor Putrenko
1) The document examines the hypothesis that the amino acid isovaline produces peripheral analgesia through GABAB receptors without affecting the central nervous system.
2) Experiments in mice found that isovaline, like the GABAB agonists baclofen and GABA, reduced pain-related behavior induced by prostaglandin E2 injection into the paw in a GABAB receptor-dependent manner, but unlike baclofen, isovaline did not produce sedation or hypothermia indicating a lack of central effects.
3) Immunohistochemistry revealed co-localization of GABAB1 and GABAB2 receptor subunits on fine nerve endings and keratinocytes in skin
1) Mice lacking the inhibitory synapse cell adhesion molecule neuroligin 2 (NL2) were found to exhibit increased anxiety-like behavior.
2) While these NL2-deficient mice appeared to have a decrease in the density of inhibitory synaptic puncta, electron microscopy revealed no actual change in inhibitory synapse numbers.
3) This suggests that NL2 deletion impairs the function of inhibitory synapses without decreasing their numbers, and this decrease in inhibitory synaptic function correlates with increased anxiety in the mice.
An inhibitory pull–push circuit in frontal cortexTaruna Ikrar
Push–pull is a canonical computation of excitatory cortical circuits. By contrast, we identify a pull–push inhibitory circuit in frontal cortex that originates in vasoactive intestinal polypeptide (VIP)-expressing interneurons. During arousal, VIP cells rapidly and directly inhibit pyramidal neurons; VIP cells also indirectly excite these pyramidal neurons via parallel disinhibition. Thus, arousal exerts a feedback pull–push influence on excitatory neurons—an inversion of the canonical push–pull of feedforward input.
Directed research spring 2016 Daniel SvedbergDan Svedberg
1) The study genetically inhibited microglial NF-κB activity in mouse models of Spinocerebellar Ataxia Type 1 (SCA1) to determine if microglia contribute to SCA1 pathology. 2) Partial inhibition of microglial NF-κB was shown to significantly worsen SCA1 pathology in mice, while complete inhibition needs further study with more animals. 3) Inhibiting microglial NF-κB may also cause deficient synaptic pruning in the cerebellum of wild-type mice.
1) The document examines how stabilizing ryanodine receptors after traumatic brain injury can reduce tau pathology and prevent progression to Alzheimer's disease in mouse models.
2) Tests on Alzheimer's disease mice and healthy controls found that traumatic brain injury increased tau deposits and plaque propagation, with more severe effects in AD mice.
3) Treatment with ryanodine receptor stabilizers like dantrolene after injury significantly reduced tau pathology and plaque loads, showing potential for preventing long-term cognitive deficits and conversion to Alzheimer's disease.
BDNF stabilizes synapses and maintains the structural complexity of optic axons in vivo. The study used time-lapse imaging to examine how neutralizing endogenous BDNF or blocking NMDA receptors in the optic tectum affects synapse and axon branch stability in Xenopus retinal ganglion cell axons. Neutralizing BDNF enhanced synapse and branch elimination, indicating BDNF stabilizes both. Blocking NMDA receptors transiently eliminated synapses, but BDNF treatment rescued synapses by maintaining addition and rapid stabilization. Thus, BDNF promotes synaptic connectivity by stabilizing synapses and maintaining axon complexity.
1) Zebrafish have an remarkable ability to regenerate their spinal cord after injury unlike mammals which have limited repair.
2) The study found that Fgf signaling induces zebrafish glial cells to form elongated bridges between the two sides of an injured spinal cord, allowing regenerating axons to cross.
3) Blocking Fgf signaling prevented glial bridge formation and axon regeneration. Mammalian astrocytes activated by Fgf took on a similar elongated morphology as zebrafish glia.
Using Pathway Studio in Neurodegenerative diseaseAnn-Marie Roche
Dr. Gabor Juhasz of ELTE University in Budapest discusses use of Pathway Studio in the study of neurodegenerative diseases such as Alzheimer’s Disease.
N-acetyl-D-glucosamine kinase (NAGK) interacts with dynein light-chain roadblock type 1 (DYNLRB1) at dendritic branch points in neurons. Immunocytochemistry and proximity ligation assays showed colocalization of NAGK and DYNLRB1 on microtubule fibers at dendritic branches. NAGK was also found to interact with Golgi outposts and DYNLRB1 at branch points, indicating a tripartite interaction between NAGK, dynein, and Golgi that regulates dendritic growth and branching. Introduction of a peptide derived from DYNLRB1 stunted dendrite development in cultured neurons.
Effect of D1 receptor agonist within the mPFCSorley O'Neill
The study tested the effects of administering a D1 receptor agonist, SKF81297, into the infralimbic (IL) and prelimbic (PL) cortices on an appetitive trace conditioning task in rats. Rats were trained with either a 2-second or 10-second trace interval between a conditioned stimulus (CS, noise) and unconditioned stimulus (UCS, food). On days 2 and 4 of training, rats that received saline responded significantly more during the pre-stimulus period compared to rats that received SKF81297 in the PL or IL cortices, suggesting the drug impaired learning. There were no significant effects observed on days 1 and 3. The findings did not demonstrate any functional
Efficiency of Stem Cell After Spinal Cord Injury with Clip-Compression_ Crims...Crimsonpublisherssmoaj
Efficiency of Stem Cell After Spinal Cord Injury with Clip-Compression by Tae Hoon Lee* in Crimson Publishers: Surgery Journal Impact Factor
Our experiment grafted mouse embryonic stem cell (mESC) to influence behavioral deficiency in rodent animal models of clip compressive surgery inducing spinal cord injury (SCI) of central nervous system. Our research proved the effect of grafted stem cells to the injured spinal cord region, focusing the application of mouse embryonic stem cells for regeneration of spinal cord nervous injury. Therefore, our research suggests manifest results that implantation of mouse embryonic stem cell could show behavioral improvement after severe spinal cord damage.
https://crimsonpublishers.com/smoaj/fulltext/SMOAJ.000533.php
For more open access journals in Crimson Publishers
Please click on: https://crimsonpublishers.com/
For more articles on Surgery Journal Impact Factor
Please click on link: https://crimsonpublishers.com/smoaj/index.php
Please follow the below link for our LinkedIn page
https://www.linkedin.com/company/crimsonpublishers
This study investigated cellular and molecular factors influencing axonal degeneration in neonatal mice. In part 1, ex-vivo sciatic nerves from wild type mice ages P9-P22 were examined and found that older mice (P22) had significantly more axonal fragmentation than younger mice (P9) after 72 hours. Part 2 analyzed protein expression of ROCK2, Prp, and SMN in brachial plexus nerves and found their levels changed as the mice aged. Part 3 discovered no significant difference in axonal degeneration between SMN deficient and control mice models at 72 hours. Overall, the study provided evidence that age-dependent axonal vulnerability exists in peripheral nerves and certain proteins' expression levels fluctuate
1) The study examines the role of tissue plasminogen activator (tPA) and plasmin in promoting axonal regrowth after spinal cord injury (SCI) via degradation of chondroitin sulfate proteoglycans (CSPGs).
2) It finds that tPA and plasmin are upregulated after SCI and can degrade CSPG core proteins. Mice lacking tPA show attenuated neurite outgrowth and recovery after SCI, even with chondroitinase ABC (ChABC) treatment to degrade CSPG sugar chains.
3) Coadministration of ChABC and plasmin enhanced recovery in tPA-deficient mice and further supported recovery in wild-type mice with S
This study investigated combining deep brain stimulation (DBS), PTEN suppression, and Schwann cell transplantation as a treatment for spinal cord injury in rats. Rats received either DBS, PTEN suppression via AAV-shPTEN injection, or a combination of both, along with Schwann cell transplantation at the injury site. Locomotor activity was assessed over 8 weeks and showed no significant differences between groups. Preliminary immunohistochemistry results found that the combination of PTEN suppression and DBS resulted in higher volumes of white matter and serotonin compared to other groups. This suggests the combined approach may produce better tissue repair than individual methods alone. Further studies are needed to confirm these findings.
Place Cell Mapping and Stress Monitoring in Head-Fixed Mice Navigating an Air...InsideScientific
In this webinar sponsored by Neurotar, experts present their research on 2-photon imaging of hippocampal place cells and on stress monitoring in head-fixed awake behaving mice. Dr. Konrad Juczewski from the National Institutes of Health (NIH)/National Institute on Alcohol Abuse and Alcoholism (NIAAA) discusses the impact of head fixation on animal’s stress, locomotion and performance in classical behavioral paradigms.
Dr. Mary Ann Go from the Laboratory of Neural Coding and Neurodegenerative Disease at Imperial College London led by Prof. Simon Schultz presents her research using 2-photon microscopy aimed at place cell mapping in the hippocampus during exploration and navigation of a circular linear track.
Key Discussion Topics Include:
- Stress reduction in head-fixed rodents
- Improving data reproducibility and translational value of the data acquired from head-fixed rodents
- Effects of head fixation on blood corticosterone concentration, locomotion patterns and performance in stress-associated behavioral tests
- Optimizing habituation protocol for head-fixed mice
- Monitoring neural activity and mapping of place cells using 2-photon microscopy during navigation and exploration behavior
- Automating the experiments using a closed-loop approach and behavior-triggered reward systems
Elevation of Brain Magnesium Prevents and Reverses Cognitive Deficits and Syn...Balogun Wasiu Gbolahan
This study investigated the effects of elevated brain magnesium on cognitive deficits and synaptic loss in an Alzheimer's disease mouse model. The study found that elevating brain magnesium levels through supplementation prevented and reversed cognitive impairment and synaptic loss in transgenic Alzheimer's mouse models. Specifically, magnesium treatment prevented learning and memory deficits, preserved synapse density, and reversed impairments in NMDA receptor signaling pathways involved in learning and memory. The treatment also reduced amyloid plaque levels and decreased expression of the amyloid precursor protein cleaving enzyme BACE1. The results suggest elevated brain magnesium as a potential therapeutic approach for Alzheimer's disease.
a short introduction about deep learning and how we can use deep neural networks in different biological problems such as protein function prediction and gene expression inference.
This document summarizes experiments exploring the neuroprotective effects of progranulin in two models of environmental neurotoxicity: PC12 cells exposed to MPTP and mice exposed to BSSG. In PC12 cells, progranulin protected against cell death induced by the neurotoxin MPTP. In mice, BSSG exposure induced gait disturbances and locomotor changes that were partially attenuated by progranulin treatment via lentiviral delivery, though muscle strength was not affected. Further studies are needed to better understand the mechanisms and long-term effects of progranulin neuroprotection in these models.
Physical activity is associated with increased brain-derived neurotrophic factor (BDNF) levels and neurogenesis. Multiple studies from the 1980s onward show that both acute and long-term exercise increase BDNF in the bloodstream and various brain regions. This suggests physical activity may help treat or prevent psychiatric conditions linked to reduced BDNF levels like depression. Exercise programs incorporating 3-5 sessions per week of 30-60 minutes each seem to be effective at improving mood and increasing BDNF levels in the brain.
Mobile Apps Presentation - Sanjay Chaudhary, Supertron InfotechSanjay Chaudhary
Presentation on Mobile Apps developed by Supertron Infotech , - a leading Mobile Apps Development agency
This presentation was made to Prudent Chapter Members of BNI Kolkata.
Presentation describes
Members; Konnect App - Digital Roster
Profile App - Digital Product Cataogue
Custom Developed Mobile Apps
This document summarizes the financial highlights and results of a company for 4Q 2015 and full year 2015. It reported 61% revenue growth in 4Q 2015 and 52% for full year 2015. Gross margins expanded 110 bps in 4Q and 150 bps for the year. Adjusted EBITDA grew 168% in 4Q and 170% for 2015. Revenue was driven by growth in oncology, hepatitis, immunology and other therapeutic classes. The company expects continued strong revenue growth of 32% and adjusted EBITDA growth of 26% in 2016.
El taller práctico: 10 claves para la implementación de tendencias y enfoques innovadores, tiene como propósito que los docentes identifiquen el cambio paradigmático que se requiere para atender al desafío pedagógico que implica incorporar las Tecnologías de la Información y la Comunicación (TIC) al aula y al currículo escolar.
This document summarizes the academic record of Syed Ebad Raza Abidi, including the degrees and honors awarded, courses taken each term, grades received, grade point averages, academic standing, and current course enrollments. It shows that he received a 2014 Dalhousie Entrance Scholarship, has maintained good academic standing and earned Dean's List recognition, and is currently enrolled in the Bachelor of Commerce program with a focus on finance.
Este documento presenta un taller práctico sobre 10 claves para la implementación de tendencias y enfoques innovadores en la enseñanza. El taller busca que los docentes identifiquen el cambio necesario para incorporar las TIC al aula y currículo. Se describen tres temáticas clave: nuevas habilidades del siglo XXI, políticas de acceso a TIC, y desafíos de la educación actual. Finalmente, el docente debe analizar cómo una tendencia pedagógica se refleja en su práctica y concluir sobre buenas pr
Woodfield School UK is a primary school located in the UK. It provides education for students from ages 5 to 11 years old, teaching them core subjects like English, math, science and history. The school aims to nurture students' growth through a well-rounded curriculum and emphasis on character development.
El taller práctico: 10 claves para la implementación de tendencias y enfoques innovadores, tiene como propósito que los docentes identifiquen el cambio paradigmático que se requiere para atender al desafío pedagógico que implica incorporar las Tecnologías de la Información y la Comunicación (TIC) al aula y al currículo escolar.
Qubes OS is an open source operating system that provides strong security for desktop computing using the principle of security by compartmentalization. It runs Fedora in a special administrative domain (dom0) and allows other template virtual machines like Debian and Arch Linux. Rather than providing process-level isolation like other operating systems, Qubes OS takes advantage of virtual machine isolation to compartmentalize different tasks and restrict what software can access. This allows it to address security issues with allowing applications to access keystrokes, screenshots, clipboard contents and devices on traditional desktop operating systems.
1) Zebrafish have an remarkable ability to regenerate their spinal cord after injury unlike mammals which have limited repair.
2) The study found that Fgf signaling induces zebrafish glial cells to form elongated bridges between the two sides of an injured spinal cord, allowing regenerating axons to cross.
3) Blocking Fgf signaling prevented glial bridge formation and axon regeneration. Mammalian astrocytes activated by Fgf took on a similar elongated morphology as zebrafish glia.
Using Pathway Studio in Neurodegenerative diseaseAnn-Marie Roche
Dr. Gabor Juhasz of ELTE University in Budapest discusses use of Pathway Studio in the study of neurodegenerative diseases such as Alzheimer’s Disease.
N-acetyl-D-glucosamine kinase (NAGK) interacts with dynein light-chain roadblock type 1 (DYNLRB1) at dendritic branch points in neurons. Immunocytochemistry and proximity ligation assays showed colocalization of NAGK and DYNLRB1 on microtubule fibers at dendritic branches. NAGK was also found to interact with Golgi outposts and DYNLRB1 at branch points, indicating a tripartite interaction between NAGK, dynein, and Golgi that regulates dendritic growth and branching. Introduction of a peptide derived from DYNLRB1 stunted dendrite development in cultured neurons.
Effect of D1 receptor agonist within the mPFCSorley O'Neill
The study tested the effects of administering a D1 receptor agonist, SKF81297, into the infralimbic (IL) and prelimbic (PL) cortices on an appetitive trace conditioning task in rats. Rats were trained with either a 2-second or 10-second trace interval between a conditioned stimulus (CS, noise) and unconditioned stimulus (UCS, food). On days 2 and 4 of training, rats that received saline responded significantly more during the pre-stimulus period compared to rats that received SKF81297 in the PL or IL cortices, suggesting the drug impaired learning. There were no significant effects observed on days 1 and 3. The findings did not demonstrate any functional
Efficiency of Stem Cell After Spinal Cord Injury with Clip-Compression_ Crims...Crimsonpublisherssmoaj
Efficiency of Stem Cell After Spinal Cord Injury with Clip-Compression by Tae Hoon Lee* in Crimson Publishers: Surgery Journal Impact Factor
Our experiment grafted mouse embryonic stem cell (mESC) to influence behavioral deficiency in rodent animal models of clip compressive surgery inducing spinal cord injury (SCI) of central nervous system. Our research proved the effect of grafted stem cells to the injured spinal cord region, focusing the application of mouse embryonic stem cells for regeneration of spinal cord nervous injury. Therefore, our research suggests manifest results that implantation of mouse embryonic stem cell could show behavioral improvement after severe spinal cord damage.
https://crimsonpublishers.com/smoaj/fulltext/SMOAJ.000533.php
For more open access journals in Crimson Publishers
Please click on: https://crimsonpublishers.com/
For more articles on Surgery Journal Impact Factor
Please click on link: https://crimsonpublishers.com/smoaj/index.php
Please follow the below link for our LinkedIn page
https://www.linkedin.com/company/crimsonpublishers
This study investigated cellular and molecular factors influencing axonal degeneration in neonatal mice. In part 1, ex-vivo sciatic nerves from wild type mice ages P9-P22 were examined and found that older mice (P22) had significantly more axonal fragmentation than younger mice (P9) after 72 hours. Part 2 analyzed protein expression of ROCK2, Prp, and SMN in brachial plexus nerves and found their levels changed as the mice aged. Part 3 discovered no significant difference in axonal degeneration between SMN deficient and control mice models at 72 hours. Overall, the study provided evidence that age-dependent axonal vulnerability exists in peripheral nerves and certain proteins' expression levels fluctuate
1) The study examines the role of tissue plasminogen activator (tPA) and plasmin in promoting axonal regrowth after spinal cord injury (SCI) via degradation of chondroitin sulfate proteoglycans (CSPGs).
2) It finds that tPA and plasmin are upregulated after SCI and can degrade CSPG core proteins. Mice lacking tPA show attenuated neurite outgrowth and recovery after SCI, even with chondroitinase ABC (ChABC) treatment to degrade CSPG sugar chains.
3) Coadministration of ChABC and plasmin enhanced recovery in tPA-deficient mice and further supported recovery in wild-type mice with S
This study investigated combining deep brain stimulation (DBS), PTEN suppression, and Schwann cell transplantation as a treatment for spinal cord injury in rats. Rats received either DBS, PTEN suppression via AAV-shPTEN injection, or a combination of both, along with Schwann cell transplantation at the injury site. Locomotor activity was assessed over 8 weeks and showed no significant differences between groups. Preliminary immunohistochemistry results found that the combination of PTEN suppression and DBS resulted in higher volumes of white matter and serotonin compared to other groups. This suggests the combined approach may produce better tissue repair than individual methods alone. Further studies are needed to confirm these findings.
Place Cell Mapping and Stress Monitoring in Head-Fixed Mice Navigating an Air...InsideScientific
In this webinar sponsored by Neurotar, experts present their research on 2-photon imaging of hippocampal place cells and on stress monitoring in head-fixed awake behaving mice. Dr. Konrad Juczewski from the National Institutes of Health (NIH)/National Institute on Alcohol Abuse and Alcoholism (NIAAA) discusses the impact of head fixation on animal’s stress, locomotion and performance in classical behavioral paradigms.
Dr. Mary Ann Go from the Laboratory of Neural Coding and Neurodegenerative Disease at Imperial College London led by Prof. Simon Schultz presents her research using 2-photon microscopy aimed at place cell mapping in the hippocampus during exploration and navigation of a circular linear track.
Key Discussion Topics Include:
- Stress reduction in head-fixed rodents
- Improving data reproducibility and translational value of the data acquired from head-fixed rodents
- Effects of head fixation on blood corticosterone concentration, locomotion patterns and performance in stress-associated behavioral tests
- Optimizing habituation protocol for head-fixed mice
- Monitoring neural activity and mapping of place cells using 2-photon microscopy during navigation and exploration behavior
- Automating the experiments using a closed-loop approach and behavior-triggered reward systems
Elevation of Brain Magnesium Prevents and Reverses Cognitive Deficits and Syn...Balogun Wasiu Gbolahan
This study investigated the effects of elevated brain magnesium on cognitive deficits and synaptic loss in an Alzheimer's disease mouse model. The study found that elevating brain magnesium levels through supplementation prevented and reversed cognitive impairment and synaptic loss in transgenic Alzheimer's mouse models. Specifically, magnesium treatment prevented learning and memory deficits, preserved synapse density, and reversed impairments in NMDA receptor signaling pathways involved in learning and memory. The treatment also reduced amyloid plaque levels and decreased expression of the amyloid precursor protein cleaving enzyme BACE1. The results suggest elevated brain magnesium as a potential therapeutic approach for Alzheimer's disease.
a short introduction about deep learning and how we can use deep neural networks in different biological problems such as protein function prediction and gene expression inference.
This document summarizes experiments exploring the neuroprotective effects of progranulin in two models of environmental neurotoxicity: PC12 cells exposed to MPTP and mice exposed to BSSG. In PC12 cells, progranulin protected against cell death induced by the neurotoxin MPTP. In mice, BSSG exposure induced gait disturbances and locomotor changes that were partially attenuated by progranulin treatment via lentiviral delivery, though muscle strength was not affected. Further studies are needed to better understand the mechanisms and long-term effects of progranulin neuroprotection in these models.
Physical activity is associated with increased brain-derived neurotrophic factor (BDNF) levels and neurogenesis. Multiple studies from the 1980s onward show that both acute and long-term exercise increase BDNF in the bloodstream and various brain regions. This suggests physical activity may help treat or prevent psychiatric conditions linked to reduced BDNF levels like depression. Exercise programs incorporating 3-5 sessions per week of 30-60 minutes each seem to be effective at improving mood and increasing BDNF levels in the brain.
Mobile Apps Presentation - Sanjay Chaudhary, Supertron InfotechSanjay Chaudhary
Presentation on Mobile Apps developed by Supertron Infotech , - a leading Mobile Apps Development agency
This presentation was made to Prudent Chapter Members of BNI Kolkata.
Presentation describes
Members; Konnect App - Digital Roster
Profile App - Digital Product Cataogue
Custom Developed Mobile Apps
This document summarizes the financial highlights and results of a company for 4Q 2015 and full year 2015. It reported 61% revenue growth in 4Q 2015 and 52% for full year 2015. Gross margins expanded 110 bps in 4Q and 150 bps for the year. Adjusted EBITDA grew 168% in 4Q and 170% for 2015. Revenue was driven by growth in oncology, hepatitis, immunology and other therapeutic classes. The company expects continued strong revenue growth of 32% and adjusted EBITDA growth of 26% in 2016.
El taller práctico: 10 claves para la implementación de tendencias y enfoques innovadores, tiene como propósito que los docentes identifiquen el cambio paradigmático que se requiere para atender al desafío pedagógico que implica incorporar las Tecnologías de la Información y la Comunicación (TIC) al aula y al currículo escolar.
This document summarizes the academic record of Syed Ebad Raza Abidi, including the degrees and honors awarded, courses taken each term, grades received, grade point averages, academic standing, and current course enrollments. It shows that he received a 2014 Dalhousie Entrance Scholarship, has maintained good academic standing and earned Dean's List recognition, and is currently enrolled in the Bachelor of Commerce program with a focus on finance.
Este documento presenta un taller práctico sobre 10 claves para la implementación de tendencias y enfoques innovadores en la enseñanza. El taller busca que los docentes identifiquen el cambio necesario para incorporar las TIC al aula y currículo. Se describen tres temáticas clave: nuevas habilidades del siglo XXI, políticas de acceso a TIC, y desafíos de la educación actual. Finalmente, el docente debe analizar cómo una tendencia pedagógica se refleja en su práctica y concluir sobre buenas pr
Woodfield School UK is a primary school located in the UK. It provides education for students from ages 5 to 11 years old, teaching them core subjects like English, math, science and history. The school aims to nurture students' growth through a well-rounded curriculum and emphasis on character development.
El taller práctico: 10 claves para la implementación de tendencias y enfoques innovadores, tiene como propósito que los docentes identifiquen el cambio paradigmático que se requiere para atender al desafío pedagógico que implica incorporar las Tecnologías de la Información y la Comunicación (TIC) al aula y al currículo escolar.
Qubes OS is an open source operating system that provides strong security for desktop computing using the principle of security by compartmentalization. It runs Fedora in a special administrative domain (dom0) and allows other template virtual machines like Debian and Arch Linux. Rather than providing process-level isolation like other operating systems, Qubes OS takes advantage of virtual machine isolation to compartmentalize different tasks and restrict what software can access. This allows it to address security issues with allowing applications to access keystrokes, screenshots, clipboard contents and devices on traditional desktop operating systems.
The document discusses a conference titled "Intellectual Capital Method in the Slovak Context" focused on developing the Košice region in Slovakia as a node in the greater Danube Knowledge Region. It outlines sessions on developing regional innovation systems in Slovakia and using explicit knowledge methodology-based public projects. It also discusses the European strategy for forming a consistent Danube Region, academic projects using VAIC methodology, and politics in favor of developing the Danube Region, with Slovakia catching up and partnering with Germany.
The British celebrate Christmas through several traditions: sending Christmas cards, watching nativity plays, having family gatherings, singing carols, writing letters to Santa, and making snowmen. They decorate trees with lights, tinsel, and baubles. On Christmas Eve, children leave mince pies and milk for Santa. On Christmas Day, families open presents under the tree and have a traditional meal of roast turkey, roast potatoes, cranberry sauce, and Christmas pudding.
This document discusses integrating OSS, BSS, and workforce management (MWFM) systems to reduce costs and improve the customer experience. It describes Amdocs and TOA Technologies, who provide these solutions. A case study is presented on their joint work with TIM Brazil to implement a new fiber broadband service in just 7 months. Integrating Amdocs' BSS and OSS solutions with TOA's workforce management platform enabled TIM Brazil to rapidly launch the new service, improve network visibility, and reduce operating expenses.
"Инфузионная и нутритивная терапия больных с острым панкреатитом" Соботка Л....rnw-aspen
Доклад с XVI Межрегиональной научно-практической конференции "Искусственное питание и инфузионная терапия больных в медицине критических состояний" 21-22 апреля 2016 г.
Доклад с XVI Межрегиональной научно-практической конференции "Искусственное питание и инфузионная терапия больных в медицине критических состояний" 21-22 апреля 2016 г.
"Распространенность и выраженность недостаточности питания среди пациентов ФН...rnw-aspen
Доклад с XVI Межрегиональной научно-практической конференции "Искусственное питание и инфузионная терапия больных в медицине критических состояний" 21-22 апреля 2016 г.
The best way to achieve a flat stomach is through a healthy, low-calorie diet and plenty of exercise. But if you want to speed the process along, try incorporating some of these foods into your meals. Not only do they relieve water retention, but they also help to stave off cravings, boost your metabolism and keep you feeling fuller for longer. Bonus!
A disinhibitory microcircuit initiates critical period plasticity in the visu...Taruna Ikrar
Early sensory experience instructs the maturation of neural circuitry in the cortex1, 2. This has been studied extensively in the primary visual cortex, in which loss of vision to one eye permanently degrades cortical responsiveness to that eye3, 4, a phenomenon known as ocular dominance plasticity (ODP). Cortical inhibition mediates this process4, 5, 6, but the precise role of specific classes of inhibitory neurons in ODP is controversial. Here we report that evoked firing rates of binocular excitatory neurons in the primary visual cortex immediately drop by half when vision is restricted to one eye, but gradually return to normal over the following twenty-four hours, despite the fact that vision remains restricted to one eye. This restoration of binocular-like excitatory firing rates after monocular deprivation results from a rapid, although transient, reduction in the firing rates of fast-spiking, parvalbumin-positive (PV) interneurons, which in turn can be attributed to a decrease in local excitatory circuit input onto PV interneurons. This reduction in PV-cell-evoked responses after monocular lid suture is restricted to the critical period for ODP and appears to be necessary for subsequent shifts in excitatory ODP. Pharmacologically enhancing inhibition at the time of sight deprivation blocks ODP and, conversely, pharmacogenetic reduction of PV cell firing rates can extend the critical period for ODP. These findings define the microcircuit changes initiating competitive plasticity during critical periods of cortical development. Moreover, they show that the restoration of evoked firing rates of layer 2/3 pyramidal neurons by PV-specific disinhibition is a key step in the progression of ODP.
A disinhibitory microcircuit initiates critical period plasticity in the visu...Taruna Ikrar
Earlysensoryexperienceinstructsthematurationofneuralcircuitry in the cortex1,2. This has been studied extensively in the primary visualcortex,inwhichlossofvisiontooneeyepermanentlydegrades corticalresponsivenesstothateye3,4,aphenomenonknownasocular dominance plasticity (ODP). Cortical inhibition mediates this process4–6,butthepreciseroleofspecificclassesofinhibitoryneurons in ODP is controversial. Here we report that evoked firing rates of binocular excitatory neurons in the primary visual cortex immediatelydropbyhalfwhenvisionisrestrictedtooneeye,butgradually return to normal over the followingtwenty-four hours, despite the fact that vision remains restricted to one eye. This restoration of binocular-like excitatory firing rates after monocular deprivation resultsfromarapid,althoughtransient,reductioninthefiringrates of fast-spiking, parvalbumin-positive (PV) interneurons, which in turncanbeattributedtoadecreaseinlocalexcitatorycircuitinput onto PV interneurons.This reduction in PV-cell-evoked responses after monocular lid suture is restricted to the critical period for ODPandappearstobenecessaryforsubsequentshiftsinexcitatory ODP. Pharmacologically enhancing inhibition at the time of sight deprivation blocks ODP and, conversely, pharmacogenetic reduction of PV cell firing rates can extend the critical period for ODP. Thesefindingsdefinethemicrocircuitchangesinitiatingcompetitive
plasticityduringcriticalperiodsofcorticaldevelopment.Moreover, they show that the restoration of evoked firing rates of layer 2/3 pyramidal neurons by PV-specific disinhibition is a key step in the progression of ODP.
1) Rapamycin treatment significantly increased survival and delayed disease progression in a mouse model of Leigh syndrome (mitochondrial disease) that was deficient in the Ndufs4 gene.
2) Rapamycin treated mice did not develop neurological lesions in the brain that were present in untreated mice, and had reduced neurological symptoms and inflammation.
3) While rapamycin did not improve mitochondrial function directly, it induced metabolic changes in the mice including increased amino acid catabolism and decreased glycolysis, which helped alleviate symptoms of the mitochondrial disease.
Hippocampal ltp and contextual learning require surface diffusion of ampa rec...Masuma Sani
1) The study investigated how AMPA receptor (AMPAR) surface diffusion contributes to long-term potentiation (LTP) and learning.
2) Using techniques to immobilize surface AMPARs, the study found that interfering with AMPAR diffusion impaired LTP in hippocampal slices and fear learning in mice.
3) The results provide direct evidence that recruitment of AMPARs to synapses through surface diffusion is critical for early LTP expression and hippocampal-dependent learning.
This study investigated the effects of chronic diabetes mellitus type 1 on the ventral and dorsal zones of the hippocampus. Rats were induced with diabetes through streptozotocin injection. After 8 weeks, the brains were analyzed. The number of dead neurons in the CA1 and CA3 regions of the ventral hippocampus were significantly higher than in the dorsal hippocampus. This provides evidence that the ventral zone is more vulnerable to neuronal degeneration from diabetes mellitus type 1 compared to the dorsal zone. The ventral hippocampus is involved in emotional processes, so greater neuronal loss could impact those functions.
This study examined the effects of chronic diabetes mellitus type 1 on the dorsal and ventral zones of the hippocampus in rats. Diabetes was induced in rats using streptozotocin injections. After 8 weeks, the brains were analyzed. The number of dead neurons was significantly higher in the CA1 and CA3 regions of the ventral hippocampus compared to the dorsal hippocampus. This provides evidence that the ventral hippocampus is more vulnerable to neuronal degeneration from diabetes mellitus type 1 than the dorsal hippocampus. The ventral hippocampus plays a role in emotion and stress responses, so greater neuronal loss could impact those functions.
This study examined the effects of chronic diabetes mellitus type 1 on the dorsal and ventral zones of the hippocampus in rats. Diabetes was induced in rats using streptozotocin injections. After 8 weeks, the brains were analyzed. The number of dead neurons was significantly higher in the CA1 and CA3 regions of the ventral hippocampus compared to the dorsal hippocampus. This provides evidence that the ventral hippocampus is more vulnerable to neuronal degeneration from diabetes mellitus type 1 than the dorsal hippocampus. The ventral hippocampus plays a role in emotion and stress responses, so greater neuronal loss could impact those functions.
and lifelong absence of FXR as occurs inthe FXR-null model, .docxjustine1simpson78276
and lifelong absence of FXR as occurs in
the FXR-null model, can result in
abnormal metabolic effects that are quite
different from those caused by acute,
transient antagonism of this receptor.
Because the FXR-null mouse was
produced using Cre–loxP technology,
conditional disruption of this allele after
normal development has occurred can
now be used to help resolve this issue. An
alternative explanation is that the site(s)
of pharmacological action of
guggulsterone do not include all of the
tissues in which FXR is functional, such as
the liver and gut (i.e. although FXR
synthesis is uniformly absent from all
tissues of the FXR-null mouse model,
guggulsterone might antagonize FXR only
within a subset of these sites). In the
absence of in vivo data regarding the
modulation of FXR target gene expression
by guggulsterone, this is difficult to judge.
Thus, it remains a possibility that the
effects of orally-administered
guggulsterone occur primarily at the level
of the gut (i.e. versus gut and liver), for
instance, by affecting cholesterol
absorption and bile-acid reuptake
processes regulated by FXR, rather than
the hepatic biosynthesis and transport of
bile acids. Again, the conditional nature of
the strategy used to create the FXR-null
mouse model allows for tissue-specific
deletion of the FXR gene and might help
resolve this issue.
As reinforced by the recent work of
Urizar et al. [3], as well as by the present
therapeutic use of bile-acid binding
resins for hypercholesterolemia, there
exists an intimate linkage between bile
acid and cholesterol metabolism. Recent
demonstrations that FXR is also involved
in the regulation of genes (e.g. encoding
apolipoprotein A-I, apolipoprotein C-II
and phospholipids transfer protein) [4–6]
more closely linked with lipid rather
than bile-acid homeostasis, presents
additional avenues by which FXR
ligands could be beneficial for the
treatment of disorders of lipid
metabolism. As suggested by the work of
Urizar et al. [3] and others (e.g. [7]),
careful and comprehensive study of the
effects of natural products, such as
guggulsterone, on the function of nuclear
hormone receptors, is likely to yield
additional agents with desirable
therapeutic effects.
References
1 Sinal, C.J. et al. (2000) Targeted disruption of the
nuclear receptor FXR/BAR impairs bile acid and
lipid homeostasis. Cell 102, 731–744
2 Singh, R.B. et al. (1994) Hypolipidemic and
antioxidant effects of Commiphora mukul as an
adjunct to dietary therapy in patients with
hypercholesterolemia. Cardiovasc. Drugs Ther. 8,
659–664
3 Urizar, N.L. et al. (2002) A natural product that
lowers cholesterol as an antagonist ligand for
FXR. Science 296, 1703–1706
4 Claudel, T. et al. (2002) Bile acid-activated nuclear
receptor FXR suppresses apolipoprotein A-I
transcription via a negative FXR response
element. J. Clin. Invest. 109, 961–971
5 Kast, H.R. et al. (2001) Farnesoid X-activated
receptor induces apolipoprotein C-II
transcription: a molecular mech.
1) The document discusses theoretical models of pattern formation in cells and tissues through reaction-diffusion equations, which can be used to model processes like morphogen gradient formation and cell-cell interactions.
2) Reaction-diffusion equations incorporate both the diffusion of substances over time and space as well as reactions between substances, and can generate concentration gradients that provide positional information.
3) These gradients form when a factor is produced in a localized region, diffuses out, and is degraded, with the characteristic length of the gradient determined by the diffusion coefficient and degradation rate.
1) Dopamine transmission in the basal ganglia controls motor behavior through two pathways - the direct pathway which stimulates motion, and the indirect pathway which inhibits motion.
2) CK1δ over-expression disrupts this system, possibly through dopamine deficiency caused by downregulation of D1 and D2 receptors.
3) This study found an increase in calretinin-containing neurons in the striatum of CK1δ over-expressing mice, suggesting this imbalance contributes to their ADHD-like behaviors.
1) The expression of clock genes in the avian premammillary nucleus (PMM) was examined under short and long photoperiods to understand photoperiodic time measurement and its effects on seasonal reproduction.
2) Under long photoperiods, the Per3 gene was phase-delayed by 16 hours compared to short photoperiods. Cry1 and Per3 genes were also induced by light pulses at Zeitgeber time 14, the photosensitive phase.
3) In contrast, clock genes in the pineal gland and suprachiasmatic nucleus showed different patterns of expression depending on photoperiod and light pulses, suggesting they may not be directly associated with photosensitivity and reproduction
This document summarizes a study on phototransduction in a rabbit model of retinitis pigmentosa (RP). The study found that:
1) Transgenic rabbits with a mutation associated with RP showed decreased pupil response and electroretinogram amplitudes with age compared to wild-type rabbits, losing response to red light by 12 months but retaining some response to blue light.
2) Examination of retinal tissue found loss of photoreceptor cells over time, with pyknotic nuclei and disappearing rods in the periphery by 8-12 months.
3) The retained response to blue light in late-stage RP is believed to be mediated by intrinsically photosensitive retinal ganglion cells that contain melanopsin and
Austin Journal of Clinical Ophthalmology is an open access, peer reviewed, scholarly journal dedicated to publish articles in all areas of ophthalmology and visual sciences.
The aim of the journal is to provide a forum for ophthalmologists, researchers, physicians, and other health professionals to find most recent advances in the areas of clinical and experimental ophthalmology. Austin Journal of Clinical Ophthalmology accepts original research articles, review articles, case reports, clinical images and rapid communication on all the aspects of ophthalmology and eye diseases.
Austin Journal of Clinical Ophthalmology strongly supports the scientific upgradation and fortification in related scientific research community by enhancing access to peer reviewed scientific literary works. Austin Publishing Group also brings universally peer reviewed journals under one roof thereby promoting knowledge sharing, mutual promotion of multidisciplinary science.
Austin Journal of Clinical Ophthalmology is an open access, peer reviewed, scholarly journal dedicated to publish articles in all areas of ophthalmology and visual sciences.
hemichannel makes it a major contributor toionic dysregulaSusanaFurman449
hemichannel makes it a major contributor to
ionic dysregulation in ischemia. Second, Px1
hemichannel opening may result in efflux of
glucose and adenosine triphosphate (ATP),
further compromising the neuron_s recovery
from an ischemic insult. Consistent with this
was our observation that fluorescent dyes
became membrane-permeable only during
OGD. Hemichannels are putative conduits for
ATP release from astrocytes (21) and in the
cochlea (22). Third, the large amplitude of
the Px1 hemichannel current at holding po-
tentials near the neuron_s resting membrane
potential (È –60 mV) indicates that these
currents likely contribute substantially to
Banoxic depolarization,[ a poorly understood
but well-recognized and key component of
ischemic neuronal death (2, 23, 24). There-
fore, hemichannel opening may be an impor-
tant new pharmacological target to prevent
neuronal death in stroke.
References and Notes
1. A. J. Hansen, Physiol. Rev. 65, 101 (1985).
2. P. Lipton, Physiol. Rev. 79, 1431 (1999).
3. M. Kamermans et al., Science 292, 1178 (2001).
4. J. E. Contreras et al., Proc. Natl. Acad. Sci. U.S.A. 99, 495
(2002).
5. R. P. Kondo, S. Y. Wang, S. A. John, J. N. Weiss,
J. I. Goldhaber, J. Mol. Cell. Cardiol. 32, 1859 (2000).
6. H. Li et al., J. Cell Biol. 134, 1019 (1996).
7. L. Bao, S. Locovei, G. Dahl, FEBS Lett. 572, 65
(2004).
8. R. Bruzzone, M. T. Barbe, N. J. Jakob, H. Monyer,
J. Neurochem. 92, 1033 (2005).
9. R. Bruzzone, S. G. Hormuzdi, M. T. Barbe, A. Herb,
H. Monyer, Proc. Natl. Acad. Sci. U.S.A. 100, 13644
(2003).
10. See supporting material on Science Online.
11. J. Gao et al., Neuron 48, 635 (2005).
12. M. Aarts et al., Cell 115, 863 (2003).
13. C. Tomasetto, M. J. Neveu, J. Daley, P. K. Horan, R. Sager,
J. Cell Biol. 122, 157 (1993).
14. G. Feng et al., Neuron 28, 41 (2000).
15. A. Nimmerjahn, F. Kirchhoff, J. N. Kerr, F. Helmchen,
Nat. Methods 1, 31 (2004).
16. G. Sohl, S. Maxeiner, K. Willecke, Nat. Rev. Neurosci. 6,
191 (2005).
17. J. C. Saez, M. A. Retamal, D. Basilio, F. F. Bukauskas,
M. V. Bennett, Biochim. Biophys. Acta 1711, 215 (2005).
18. R. J. Thompson, M. H. Nordeen, K. E. Howell,
J. H. Caldwell, Biophys. J. 83, 278 (2002).
19. M. L. Fung, G. G. Haddad, Brain Res. 762, 97 (1997).
20. H. Benveniste, J. Drejer, A. Schousboe, N. H. Diemer,
J. Neurochem. 43, 1369 (1984).
21. C. E. Stout, J. L. Costantin, C. C. Naus, A. C. Charles,
J. Biol. Chem. 277, 10482 (2002).
22. H. B. Zhao, N. Yu, C. R. Fleming, Proc. Natl. Acad. Sci.
U.S.A. 102, 18724 (2005).
23. T. R. Anderson, C. R. Jarvis, A. J. Biedermann, C. Molnar,
R. D. Andrew, J. Neurophysiol. 93, 963 (2005).
24. G. G. Somjen, Physiol. Rev. 81, 1065 (2001).
25. Supported by the Canadian Institutes for Health Research
and the Canadian Stroke Network. B.A.M. has a Tier 1
Canada Research Chair in Neuroscience and a Michael
Smith Foundation for Health Research distinguished
scholar award. We thank Y.-T. Wang, C. C. Naus, and
T. Snutch for critical re ...
This study investigated the role of peptidergic input in modulating respiratory rhythm and frequency in the preBötzinger Complex (preBötC), the hypothesized site of respiratory rhythm generation in mammals. The key findings were:
1) Application of NK1R and μ-opioid receptor agonists into the preBötC increased and decreased respiratory frequency, respectively, and these effects were independent of GABA and glycine transmission.
2) NK1R and μ-opioid receptor expression was concentrated in the preBötC region, defining its anatomical boundaries.
3) Subsets of preBötC neurons expressed both NK1Rs and μ-opioid
Dietary Administration of Diquat for 13 Weeks Does Not Result in a Loss of Do...EPL, Inc.
Dietary Administration of Diquat for 13 Weeks Does Not Result in a Loss of Dopaminergic Neurons in the Substantia Nigra Pars Compacta (SNpc) of C57BL/6J Mice
1) Deletion of the SCN1A gene in GABAergic interneurons mediated by the Dlx5,6Cre locus leads to increased susceptibility to seizures induced by pentylenetetrazol (PTZ) in mice, including shorter latency to seizures, longer duration of seizures, and increased fatality.
2) Preliminary analysis found an association between increased attenuation of EEG waves after seizures and sudden unexpected death in epilepsy (SUDEP) in a Dravet syndrome mouse model, suggesting EEG attenuation could be a predictive biomarker for SUDEP risk.
3) The study aims to further compare the Dravet syndrome mouse model to a focal cortical dysplasia model to examine shared physiological biomarkers and mechanisms of
1. 1Scientific Reports | 6:24988 | DOI: 10.1038/srep24988
www.nature.com/scientificreports
Spatiotemporal profile of Map2
and microglial changes in the
hippocampalCA1 region following
pilocarpine-induced status
epilepticus
Nicole D. Schartz1
, Seth A. Herr1
, Lauren Madsen1
, Sarah J. Butts1
,Ceidy Torres2
,
Loyda B. Mendez2
&Amy L. Brewster1,3
Status epilepticus (SE) triggers pathological changes to hippocampal dendrites that may promote
epileptogenesis.The microtubule associated protein 2 (Map2) helps stabilize microtubules of the
dendritic cytoskeleton. Recently, we reported a substantial decline in Map2 that coincided with robust
microglia accumulation in theCA1 hippocampal region after an episode of SE.A spatial correlation
between Map2 loss and reactive microglia was also reported in human cortex from refractory epilepsy.
New evidence supports that microglia modulate dendritic structures.Thus, to identify a potential
association between SE-induced Map2 and microglial changes, a spatiotemporal profile of these
events is necessary.We used immunohistochemistry to determine the distribution of Map2 and the
microglia marker IBA1 in the hippocampus after pilocarpine-induced SE from 4 hrs to 35 days.We found
a decline in Map2 immunoreactivity in theCA1 area that reached minimal levels at 14 days post-SE and
partially increased thereafter. In contrast, maximal microglia accumulation occurred in theCA1 area at
14 days post-SE.Our data indicate that SE-induced Map2 and microglial changes parallel each other’s
spatiotemporal profiles.These findings may lay the foundation for future mechanistic studies to help
identify potential roles for microglia in the dendritic pathology associated with SE and epilepsy.
Epilepsy is a neurological disorder characterized by spontaneous recurrent seizures. Evidence from experimental
models support that episodes of prolonged and uninterrupted seizure activity (status epilepticus; SE) increase
the risk for the generation of future unprovoked recurrent seizures (epilepsy)1
. Mechanisms underlying neuronal
hyperexcitability after SE include injury to the hippocampal network where dendrites are vulnerable to struc-
tural and morphological alterations2–4
. SE-induced decreases in spine densities and dendritic branching are often
observed in parallel to epileptogenesis and also are associated with the development of hippocampal-dependent
cognitive deficits2,3,5,6
.
Dendritic structural plasticity is in part controlled by cytoskeletal components such as those from the family
of microtubule associated protein 2 (Map2) along with their phosphorylation status7–9
. Map2 associates with
microtubules to provide support to the neuronal cytoskeleton and is important for dendritic structural stability
and neurite growth7–9
. In addition, Map2 is essential for cellular functions such as the integration of synaptic
inputs, local signal transduction, protein trafficking, and synaptic plasticity7,9–11
. In both human and experimen-
tal models of epilepsy, decreases in cortical and hippocampal Map2 immunoreactivity (IR), along with Map2
dephosphorylation, have been reported5,12–14
. While it is likely that a degree of Map2 loss is attributable to the
neuronal death that is often seen after SE and in epilepsy15
, altered Map2 levels in remaining neurons may impact
the dynamics of dendritic structures. In order to understand the potential contribution that SE-induced Map2
changes may have on hippocampal dendritic stability, it is important to define their spatiotemporal evolution. To
1
Department of Psychological Sciences, West Lafayette, IN 47907, USA. 2
School of Science and Technology,
Universidad del Este, Carolina, PR 00984, Puerto Rico. 3
Weldon School of Biomedical Engineering,West Lafayette,
IN 47907,USA.Correspondence and requests for materials should be addressed toA.L.B. (email: abrewst@purdue.
edu)
received: 11October 2015
accepted: 06April 2016
Published: 04 May 2016
OPEN
2. www.nature.com/scientificreports/
2Scientific Reports | 6:24988 | DOI: 10.1038/srep24988
our knowledge, spatiotemporal analyses of SE-induced Map2 changes have only been reported in the developing
brain16
. Therefore, the first objective of this study is to characterize the temporal profile of the distribution of
Map2 in the hippocampus of mature animals after an episode of SE.
Previously, we reported a substantial decline of Map2 IR in the hippocampal CA1 region after an episode
SE that correlated with a robust accumulation of hypertrophied microglial cells5
, the resident immune cells of
the brain. Interestingly, following treatment with the drug rapamycin, an inhibitor of the mechanistic target of
rapamycin (mTOR), the SE-induced Map2 and microglial changes were largely attenuated5
. Because rapamy-
cin modulates microglial properties in epilepsy5,17,18
as well as other injury models19–21
, these data suggest that
microglia may contribute to the observed Map2 dysregulation. Extensive evidence support microglia activation
in the hippocampus in association with seizures in human and experimental models5,22–29
. Interestingly, recent
studies exposed new roles for microglia in the modulation of dendritic and axonal structures including synapse
pruning30–35
. Given the potential impact that SE-induced microglial changes may have on hippocampal dendritic
stability, a second objective of this study is to characterize the spatiotemporal progression of SE-induced micro-
glial changes in parallel to Map2. Therefore, here we used immunohistochemistry to assess the distribution of
Map2 and the microglia marker IBA1 in the hippocampal formation of mature rats at various time points (4 hrs
to 35 days) after an episode of pilocarpine-induced SE.
Results
SE triggers a transient decrease in Map2 immunoreactivity in the CA1 hippocampal area. We
determined the temporal progression in the distribution of Map2 immunostaining in the mature hippocampal
formation at 4 hrs, 1-, 3-, 14-, and 35-days after an episode of pilocarpine-induced SE (Fig. 1). Densitometry
analysis followed by Analysis of Variance (ANOVA) revealed significant group effects for the intensity of Map2 IR
signal in the CA1 pyramidal cell layer (pcl) and stratum radiatum (sr) [CA1 pcl, F (6, 39) = 6.40, p < 0.01; CA1 sr,
F (6, 39) = 6.12, p < 0.01], as well as the molecular layer (ml) of the Dentate Gyrus (DG) [DG ml, F (6, 39) = 3.35,
p < 0.01] (Fig. 1I). In contrast, the intensity of Map2 IR localized in the CA3 region was not significantly altered
by SE (4 hrs to 35 days) when compared to the control group (CA3 pcl [F (6, 39) = 1.79, p = 0.13], CA3 sr [F (6,
39) = 1.26, p = 0.30]). In control hippocampi, a homogenous distribution of Map2 IR was evident throughout the
dendritic fields of CA1-3 sr (Fig. 1A). High magnification images of the CA1 area showed a continuous Map2
staining pattern within the labeled dendritic structures of control hippocampi (Fig. 1A). At 4 hrs post-SE, the
intensity of Map2 IR over the CA1 pcl and sr regions was comparable to that of the control group (pcl, p = 0.61;
sr, p = 0.10) (Fig. 1B). Between 1 and 14 days post-SE, the Map2 signal was gradually and significantly less intense
than the control group in the CA1 pcl (ctl vs 1d, p = 0.04; ctl vs 3d, p = 0.01; ctl vs 14d, p < 0.01) and sr (ctl vs 1d,
p = 0.02; ctl vs 3d, p < 0.01; ctl vs 14d, p < 0.01) regions (Fig. 1C–F). Note that a number of dendrites displayed
prominent punctated Map2 staining by day 14. The significant decrease in Map2 IR in the CA1 area seen at two
weeks post-SE was specific to the SE event. Rats that received the same dose of pilocarpine but did not develop
class 5 seizures showed a hippocampal distribution of Map2 similar to controls (14 days pilo-non SE; Fig. 1G)
(CA1 pcl, p = 0.61; CA1 sr, p = 0.10). Furthermore, the SE-induced decrease in Map2 IR was evident throughout
the dorsoventral axis of the hippocampus (Supplementary Fig. 1). At 35 days following SE onset, the Map2 IR lev-
els in CA1 pcl were significantly decreased compared to controls (p = 0.01) (Fig. 1H) and resembled that observed
at 1–3 days post-SE (1d vs 35d, p = 0.67; 3d vs 35d, p = 0.92). However, a significant increase in the intensity of
Map2 IR was observed between 14 and 35 days after SE (p < 0.01) suggesting a partial recovery of Map2 IR in the
CA1 region.
Because phosphorylation of Map2 is an important modification that regulates microtubule assembly and den-
dritic stability8
, we investigated the distribution of phosphorylated Map2 at the time points when total Map2 lev-
els were significantly decreased (3-, 14- and 35-days post-SE; Fig. 1) (Supplementary Fig. 2). ANOVA revealed a
group effect for the intensity of phospho-Map2 IR [F (4, 34) = 2.79, p = 0.04]. We found a significant reduction in
the intensity of phospho-Map2 IR over the CA1 region at all time points after SE relative to the control group (ctl
vs 3d, p = 0.02; ctl vs 14d, p = 0.01; ctl vs 35d, p < 0.01). This finding is consistent with a previous study showing
that in human and experimental epilepsy dephosphorization of Map2 occurred in association with epileptiform
activity13
.
To determine whether dendritic arborizations were present in the CA1 region at the time points when Map2
was significantly decreased, we performed golgi staining in controls and at 3-, 14- and 35-days post-SE (Fig. 2).
While golgi impregnation was evident throughout all hippocampal regions, the structural analysis of dendritic
arborizations was largely obstructed by the presence of golgi impregnated glial cells in all SE groups. Thus, we quan-
tified the spine density of second order CA1 dendritic branches and performed an ANOVA to determine a group
effect [F (3, 1361) = 10.01, p < 0.01]. We found that in parallel to Map2 decline, spine density was significantly
decreased at 3-, 14- and 35-days post-SE when compared to the control group (ctl vs 3d, 14d, or 35d, p < 0.01).
SE triggers a transient increase in microgliosis that is prominent in the CA1 region. We previ-
ously showed that Map2 loss in the CA1 area correlated with accumulation of hypertrophied microglia at 2 and 3
weeks after SE5
. However, little is known about the temporal progression between these events in the same tissues.
Therefore, in parallel to Map2 and in consecutive brain sections, we mapped the temporal profile of SE-induced
microglial changes in the hippocampus using IBA1 to identify this cells (Fig. 3). Densitometry analysis of IBA1
signal followed by ANOVA showed a significant group effect in areas CA1, CA3 and hilus [CA1, F (6, 42) = 11.28,
p < 0.01; CA3, F (6, 42) = 5.99, p < 0.01; hilus, F (6, 42) = 6.22, p < 0.01] (Fig. 3I). In the control group, immu-
nostaining showed a homogeneous distribution of IBA1-positive microglial cells throughout the hippocampal
regions CA1, CA3, and DG (Fig. 3A). Higher magnification images from the CA1, CA3, and DG areas showed
that in control hippocampi the morphological features of microglial cells included small cell bodies with highly
branched and elongated processes (Fig. 3A, arrows). We found that SE triggered changes in the morphology
3. www.nature.com/scientificreports/
3Scientific Reports | 6:24988 | DOI: 10.1038/srep24988
and accumulation of IBA1-stained microglial cells in the hippocampus that progressed between 4 hrs and 35
days (Fig. 3B–H). Four hrs after SE onset, the processes of microglial cells localized throughout all hippocampal
regions (CA1, CA3 and DG) were hypertrophied compared to those of control hippocampi. Furthermore, the
levels of IBA1 IR at 4 hrs after SE were significantly increased throughout all hippocampal regions compared to
the control group (CA1, p < 0.01; CA3, p < 0.01; hilus, p < 0.01). By day 1 after SE, the morphology of microglial
cells and IBA1 IR levels were similar to the control group (CA1, p = 0.32; CA3, p = 0.13; DG, p = 0.34) (Fig. 3C).
At 3 days post-SE, drastic changes were evident in the morphology of microglial cells from highly branched to
amoeboid (Fig. 3D). Throughout the hippocampus, smaller amoeboid microglia displayed shortened processes
at 3 days after SE compared to the earlier time points and to the control group (Fig. 3A–D). By two weeks post-SE,
a robust immunostaining for IBA1-positive amoeboid microglia was concentrated within the pcl, sr, and slm of
the CA1 region (Fig. 3E,F). At this time point the presence of amoeboid microglia also was evident in the CA3
Figure 1. Temporal profile of Map2 immunostaining in the hippocampus after status epilepticus (SE).
(A) shows a representative image of the Map2 staining (brown) from a control hippocampus with a high
magnification image of the CA1 area (boxed). (B–H) show representative hippocampal images with high
magnification of boxed CA1 areas at different time points after an episode of SE ((B)4 hrs; (C) 1 day (d); (D) 3d;
(E,F) 14d; (H) 35d). A representative image of a hippocampus from a rat that was given pilocarpine but failed to
develop SE (pilo-non SE; Pilo in graphs) is shown in (G). (I) shows the densitometry analysis as relative mean
pixel intensity for the different hippocampal sub-regions CA1 pyramidal cell layer (pcl) and stratum radiatum
(sr), CA3 pcl and sr, and the molecular layer (ml) of the dentate gyrus (DG). Note that significant differences
in the intensity of Map2 immunoreactivity are evident within the CA1 region between the control group and
1–35d post-SE groups. Data are shown as mean ± standard error of the mean. *p < 0.05, **p < 0.01 compared to
the control group. #
p < 0.05, comparison between 14d and 35d groups (n = 3–9/group). ANOVA with Fishers
LSD post hoc test.
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Figure 2. Temporal profile of changes in spine density in hippocampal CA1 cells after status epilepticus
(SE). (A–D) shows representative images of golgi impregnated hippocampal dendrites from a control rat
(A) and at different time points following an episode of SE ((B) 3 days (d); (C) 14d; (D) 35d). (E) shows
representative images of 20 μ m sections of CA1 dendritic branches from a control, and 3-, 14- and 35- days
post-SE. (F) shows the quantitative analysis of spine density in all groups. Data are shown as mean ± standard
error of the mean. **p < 0.01 compared to the control group. ANOVA with Fishers LSD post hoc test.
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Figure 3. Temporal profile of IBA1 immunostaining in the hippocampus after status epilepticus (SE).
(A–H) show representative images of the IBA1 staining (brown) from a control hippocampus (A) and from
hippocampi collected at different time points after an episode of SE ((B) 4 hrs; (C) 1 day (d); (D) 3d; (E,F) 14d;
(H) 35d). A representative hippocampus from a rat that was given pilocarpine but failed to develop SE (pilo-non
SE; Pilo in graphs) is shown in (G). Right panels show high magnification images of boxed CA1, CA3, and the
hilus of the dentate gyrus (DG). High magnification images with IBA1-labeled microglia (arrows) are also shown.
Nissl stained cellular nuclei are shown in blue. Abbreviations: pcl, pyramidal cell layer; sr, stratum radiatum;
slm, stratum lacunosum-moleculare. (I) shows the densitometry analysis as relative mean pixel intensity for the
different hippocampal sub-regions CA1, CA3, and hilus. Significant differences in the intensity of IBA1-stained
microglia are evident in the CA1, CA3, and hilar regions at 4 hrs and 14d after SE compared to the control group.
Data are shown as mean ± standard error of the mean. **p < 0.01 compared to the control group. #
p < 0.05,
comparison between 14d and 35d groups (n = 4–11/group). ANOVA with Fishers LSD post hoc test.
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pcl and in the hilar region of DG, albeit at a lesser extent to that observed in the entire CA1 area. Statistical anal-
yses showed a significant increase in the intensity of IBA1 IR at 14 days post-SE compared to the control group
in all hippocampal regions (CA1, p < 0.01; CA3, p < 0.01; DG, p < 0.01). The intensity of IBA1 signal in the 14
day pilo-non SE group was not different from controls in all hippocampal areas (CA1, p = 0.66; CA3, p = 0.76;
DG, p = 0.64) (Fig. 3G). Furthermore, we found that the drastic SE-induced changes on microglial morphology
and accumulation in the CA1 hippocampus were nearly resolved by 35 days after SE (Fig. 3H). Note that at this
time point IBA1 IR was significantly less intense when compared to the compared to the 14 day post SE-time
point in all hippocampal regions (CA1, p < 0.01; CA3, p < 0.01; DG, p < 0.01) and not significantly different to
controls (CA1, p = 0.85; CA3, p = 0.81; DG, p = 0.77). Because microglial morphological changes are associated
with inflammatory activation of these cells36
, and neuroinflammation is often linked to the neuropathology of
epilepsy37
, we determined the temporal profile of a number of cytokines, chemokines, and tropic factors in the
hippocampus (Supplementary Fig. S3). Consistent with several studies37–40
, we found higher concentration of
cytokines such as TNFα along with chemokines such as GRO/KC, MCP-1, and MIP-1α acutely after SE (4 hrs to
3 days post-SE). However, no significant differences in the levels of these inflammatory molecules were found at
14 days post-SE, when microgliosis was most prominent in the hippocampus. Taken together these data suggest
that SE triggered a transient accumulation of microglia within the hippocampus that peaked at two weeks after
SE and decreased thereafter.
SE triggers a transient decrease in NeuN immunoreactivity and apoptosis in CA1 cells.
Neuronal loss induced by SE may contribute to the decreased levels of Map2 as well as increased microglio-
sis. Therefore, to assess potential neuronal changes we used the marker NeuN to identify neurons (Fig. 4).
Densitometry analysis of the NeuN signal localized within the CA1 pcl, CA3 pcl, and DG gcl showed significant
group effects [CA1 pcl, F (6, 37) = 3.94, p < 0.01; CA3 pcl, F (6, 37) = 3.15, p = 0.01; DG gcl, F (6, 37) = 3.75,
p < 0.01] (Fig. 4I). In controls, NeuN positive neurons outlined the regional architecture of the hippocampal
principal cell layers and showed a homogenous signal within the soma of the CA1-3 pyramidal cells and the gcl
(Fig. 4A). We found that at 14 days post-SE the intensity of NeuN IR was significantly decreased in the CA1 pcl
when compared to controls (p = 0.02) (Fig. 4E,F). Albeit the levels of NeuN signal were drastically declined at
14 days post-SE, high magnification images showed that CA1 neurons contained weak NeuN IR (Fig. 4E,F). In
contrast to the observations in CA1 pcl, ANOVA revealed a significant increase in the levels of NeuN IR at 3 days
post-SE in the CA3 pcl and in the gcl (CA3 pcl, p = 0.03; DG gcl, p = 0.02). Unexpectedly, we also found that the
intensity of NeuN signal in the hippocampal CA1 pcl, CA3 pcl and gcl of the pilo-non SE group was significantly
increased when compared to controls (CA1 pcl, p = 0.04; CA3 pcl, p = 0.02; DG gcl, p = 0.01). At 35 days-post
SE the NeuN signal was not different from the control or 14 days post-SE groups (CA1 pcl, p = 0.40; CA3 pcl,
p = 0.30; DG gcl, p = 0.86).
SE-induced cell loss in the hippocampus is widely reported after SE and one associated mechanism for this cell
death is apoptosis15,41
. Therefore, we used antibodies against cleaved-caspase 3 to identify and quantify apoptotic
cells in the CA1 pcl (Fig. 5). We found a large number of cleaved-caspase 3 positive cells in controls and all SE
groups and significant changes in the group comparison analysis [F (6, 34) = 3.54, p < 0.01] (Fig. 5H). The num-
ber of cleaved-caspase 3 positive cells at 4 hrs and 1 day after SE onset was not different from the control group (ctl
vs 4 hrs, p = 0.11; ctl vs 1d, p = 0.46). However, at 3 days post-SE a significant increase in the number of cleaved
caspase 3-positive cells was evident in the CA1 pcl when compared to controls (p < 0.01). Despite the drastic
SE-induced loss of NeuN and Map2 IR within CA1 at 14 days post-SE, the number of cells positive for cleaved
caspase-3 was not different from controls at this time point or at 35 days after SE (ctl vs 14d, p = 0.82; ctl vs 35d,
p = 0.16). Taken together these data indicate that a maximal number of cells underwent apoptosis during the first
week after SE and did not correlate with the temporal profile of SE-induced Map2, NeuN, or microglial changes.
Discussion
The main findings of this study describe the spatial and temporal correlation between the SE-induced changes in
Map2 IR and microglial accumulation in the hippocampal formation at various time points following an episode
of SE. Specifically, we found that: (1) SE triggered a decrease in the intensity of Map2 IR in the CA1 hippocam-
pal pcl and sr areas that was evident as early as 1 day after SE, reached minimal expression at 14 days, and was
partially increased by day 35 post-SE (Fig. 1); (2) SE induced changes in the morphology of microglial cells that
were evident as early as 4 hrs post-SE in all hippocampal regions. This was followed by a maximal accumulation
of hypertrophied/amoeboid microglia mainly localized within the CA1 area at two weeks post-SE (Fig. 3). Even
though alterations in Map2 and microglia within the hippocampal formation are often seen subsequent to SE and
in epilepsy5,12,16,22–27,38
, this study is the first to describe that the evolution of these events follow similar spatial and
temporal profiles in an experimental model of SE and acquired temporal lobe epilepsy.
Spatiotemporal analyses of SE-induced Map2 changes in the hippocampus have been previously reported in
the developing brain16
. SE in the immature brain triggers a transient increase in the levels of the high molecular
weight Map2 protein, which is the dominant Map2 isoform in the adult brain8,16
. In contrast, we found that SE
in the mature brain triggered a decline in the intensity of Map2 IR that was prominent in the hippocampal CA1
pcl and sr areas (Fig. 1). Studies showing that Map2 deficient mice display reduced dendritic lengths along with
decreased microtubule densities7
suggest that the observed SE-induced Map2 loss may contribute to the altered
dendritic arborizations and dendritic structural instability often seen in epilepsy3,42
. Note that we found a signif-
icant decline in spine density (Fig. 2) and in the levels of phospho-Map2 IR (Supplementary Fig. 2) suggesting
a correlation between these events. Since phosphorylation of Map2 at Ser136 destabilizes its association with
microtubules8
, we speculate that a decrease in the phospho-Map2 levels after SE may help stabilize microtubule
assembly when Map2 levels are low.
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In addition, it is possible that Map2 decline after SE may disrupt cellular processes such as dendritic traffick-
ing7,9–11
. Interestingly, the timeline of SE-induced Map2 changes is similar to the one described for the distribu-
tion of the dendritic HCN channels, which following SE concentrate in the somatic region of CA1 cells until at
least day 30 when their localization within CA1 sr is restored43
. While it is not known if the Map2 decline directly
contributes to the altered HCN channel distribution after SE, we speculate that the transient Map2 dysregulation
may contribute to homeostatic plasticity in the CA1 hippocampus. A number of studies support that when neu-
rons are challenged with abnormal activity (i.e. seizures) their physiological responses adapt to the initial imposed
changes, most likely trying to restore normal activity patterns (homeostatic plasticity)44
. However, determining
whether the transient Map2 loss is an attempt to preserve neuronal and dendritic homeostasis in response to SE,
and whether it is detrimental or beneficial for neuronal stability, requires future investigation.
It is also expected that some of the SE-induced Map2 loss is a direct consequence of neuronal death and
injury12
. We found a significant increase in the number of apoptotic cells positive for cleaved-caspase 3 during
Figure 4. Temporal profile of NeuN immunostaining in the hippocampus after status epilepticus (SE).
(A–H) show representative images of the NeuN staining (brown) from a control hippocampus (A) and from
hippocampi collected at different time points after an episode of SE ((B) 4 hrs; (C) 1 day (d); (D) 3d; (E,F) 14d;
(H) 35d). A representative hippocampus from a rat that was given pilocarpine but failed to develop SE (pilo-non
SE; Pilo in graphs) is shown in (G). Right panels show high magnification images of boxed CA1 pyramidal cell
layer (pcl) sections for each group. Nissl stained cellular nuclei are shown in blue. (I) shows the densitometry
analysis as relative mean pixel intensity for the hippocampal CA1 pcl, CA3 pcl, the granule cell layer (gcl) of the
dentate gyrus (DG). Significant differences in the intensity of NeuN-stained neurons are evident in the CA1
pcl at 14d post SE, and in the CA3 pcl and DG gcl at 3d post-SE compared to the control group. Note that the
intensity of the NeuN signal in the CA1 pcl of the Pilo group is significantly elevated compared to the control
group (n = 3–9/group). Data are shown as mean ± standard error of the mean. *p < 0.05 compared to the
control group. ANOVA with Fishers LSD post hoc test.
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the first week after SE (3 days; Fig. 5) that are consistent with a previous study45
. However, the finding that Map2
decline was maximal at two weeks and not 3 days post-SE suggests that additional mechanisms may be under-
lying the disruption of Map2 expression in remaining neurons. This could be due to alterations in the activation
of intracellular signaling cascades such as mTOR and MAPK/ERK pathways which are altered by SE and have
been shown to regulate protein synthesis of Map25,46,47
. In addition, we would have expected that the intensity of
NeuN IR over the CA1 pcl at 3 days post-SE (Fig. 4) would have been significantly reduced due to the increase in
the number of apoptotic cells at this time point. One possibility for the lack of a significant decline in NeuN IR
over CA1 pcl is that NeuN may be altered in the remaining neurons. Studies using immunofluorescence would
be better suited to address potential changes in the intensity of NeuN IR in individual cells. Nevertheless, a spatial
Figure 5. Temporal profile of cleaved-caspase 3 immunostaining in the hippocampus after status
epilepticus (SE). (A–G) Representative images for cleaved (clvd) caspase-3 immunostaining (brown) are shown
for control (A) and at different time points after SE onset ((B) 4 hrs; (C) 1 day (d); (D) 3d); (E) 14d; (G) 35d). A
representative hippocampus from a rat that was given pilocarpine but failed to develop SE (pilo-non SE; Pilo in
graphs) is shown in (F). Abbreviations: so, stratum oriens; pcl, pyramidal cell layer; sr, stratum radiatum.
(I) Quantitative analysis of Clvd-caspase-3 positive cells show a significant increase in the number of labeled
cells at 3 days post-SE compared to controls. No significant differences were evident other time points post-SE
relative to controls (n = 4–8/group). Data are shown as mean ± standard error of the mean. *p < 0.01 compared
to the control group. ANOVA with Fishers LSD post hoc test.
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correlation between reduced Map2 and NeuN IR along with presence of reactive microglia also was reported in
cortical tissue from human refractory epilepsy14
.
Previously, we reported that prominent microgliosis overlapped with decreased Map2 IR in CA1 sr area at 2
and 3 weeks after SE5
. In this study we showed that there is considerable spatial and temporal overlap between
the progression of SE-induced loss of Map2 IR and increased microgliosis in the CA1 area (dorsoventrally and
bilaterally) (Supplementary Fig. S1). Substantial evidence support that inflammatory activation of microglial
cells after SE contributes to some of the neuropathological changes associated with prolonged seizures37,38
. We
confirmed neuroinflammation at the acute time points following pilocarpine-induced SE39,40
, which correlated
with early changes in Map2. In contrast, at the 14 days post-SE time point when microgliosis was most prominent
(Fig. 3) no significant changes were evident in at least 20 inflammatory mediators (Supplementary Fig. S3). These
data support that the initial SE-induced microglial morphological changes, including hypertrophied processes,
observed between 4–24 hrs may be associated with their inflammatory activation. Microglia are phagocytic cells
that clear dead cells and cellular debris along with neuronal elements such as synapses48
. Thus, the vast accumula-
tion of amoeboid microglia with shorter processes in CA1 pcl may be associated with a phagocytic phenotype36
.
This may be in response to the increased number of apoptotic cells. We speculate that once the dead cells within
CA1 have been cleared, microglial cells recede from this area over time but not before possibly potentiating den-
dritic alterations in the remaining neurons.
Recent studies show that SE enhances the attraction of microglial processes toward neural elements49
and
increases the density of cell-to-cell contacts between activated microglia and CA1 dendrites5,50
. This is important
because a growing body of evidence supports the idea that microglia participate in shaping neuronal dendritic
and synaptic connectivity33,34,51
. For instance, microglial processes regularly survey their surrounding microenvi-
ronment making direct contacts with spines and synaptic structures which they can engulf and eliminate33,34,51,52
.
Thus, it is conceivable that the vast accumulation of microglial cells in CA1 may play a role in the disruption of
dendritic structures. In future studies we will investigate this possibility.
Taken together, our findings suggest that SE-induced Map2 and microglial changes mirror each other’s spa-
tiotemporal profiles. Given recently described novel functions for microglial cells in the regulation of neuronal
connectivity33
, our findings may lay the foundation for future mechanistic experiments to identify potential roles
for microglia in the modulation dendritic structures in epilepsy. In addition, a map of microgliosis and its asso-
ciation with other pathological SE-induced changes in the hippocampus (e.g. inflammation, astrogliosis, tran-
scriptional and/or translational dysregulation) may lead to the identification of more specific time windows for
pharmacological interventions using immunosuppressants. For instance, it is possible that the temporal profile of
SE-induced microglial alterations may contribute to the discrepant observations reported following early vs. late
rapamycin treatments in models of SE and acquired epilepsy5,17,53–55
.
Materials and Methods
Animals. Male Sprague Dawley rats (150–175 grams) (Harlan Laboratories) were housed at the Psychological
Sciences Building. Ambient temperature was constantly 22 °C, with diurnal cycles of a 12-hour (hr) light and
12-hr dark (8:00 to 20:00 hr). All animals had access to unlimited food and water.
Pilocarpine-induced status epilepticus. SE was induced using previously described protocols5
. Briefly,
rats were injected with scopolamine methylbromide (1 mg/kg) intraperitoneally (i.p.). Thirty minutes (min)
later, injections of saline (Control) or pilocarpine (280–300 mg/kg; Sigma Chemical Co., St Louis, MO, USA)
(SE group) were administered (i.p.). SE onset was determined by development of class 5 limbic motor seizures
(rearing and falling)56
. SE was allowed to continue for up to 1 hr, at which point seizure activity was stopped with
diazepam (10 mg/kg; i.p.; Sigma Chemical Co.). Two hours after, injections (i.p.) of sterile 0.9% saline (AddiPak)
were administered for hydration. Sliced peeled apples and Kellogg’s Fruit Loop cereal were placed in all rats’ cages
in addition to the rat chow, for up to one week after SE onset. All rats were monitored daily for adequate food/
water intake and for body weight. Animals were sacrificed at the following time points after SE: 4 hrs, (n = 4), 1
day (n = 6), 3 days (n = 9), 14 days (n = 7) and 35 days (n = 7) after SE. A sham (control) group (n = 11) and a
pilo-non SE group for the 14 day time point (n = 9) were analyzed in parallel.
Immunohistochemistry (IHC). Rats were profoundly anesthetized with Beuthanasia (200 mg/kg) and per-
fused with ice cold 1X phosphate buffered saline (PBS) (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4,
1.47 mM KH2PO4, pH 7.4) followed by 4% paraformaldehyde (PFA). After overnight post-fixation (4%-PFA) and
cryoprotection (30% sucrose), brains were frozen in pre-chilled isopentane, and stored at − 80 °C until used for
IHC. IHC was done following previously described protocols5,18,57
. Brains were sliced in coronal sections (50 μ m)
using a Leica CM1860 cryostat, and stored in 1XPBS + 0.1% Sodium Azide at 4 °C. For colorimetric IHC, we
used serial sections along the dorsoventral axis at approximately the following Bregma coordinates: − 3.00 mm,
− 3.48 mm, − 4.08 mm, − 4.36 mm, − 4.92 mm, and − 5.28 mm. These sections represent an equal sampling of the
hippocampus along its dorsoventral axis (see Supplementary Fig. 1). IHC was done in free floating sections. All
sections were washed in 1XPBS (5 mins), incubated in 3%H2O2 (30 min) and then in 1XPBS + 3% Triton (1XPBS-
3%T) (20 min). Sections were then placed in immuno buffer (5% goat serum, 0.3% BSA, 0.3% triton diluted
in 1XPBS) for a minimum of 1 hr at room temperature. Then, sections were incubated overnight on a rotating
platform at 4 °C with the following primary antibodies: anti-mouse Map2, anti-mouse NeuN (1:3K; Millipore,
Temecula, CA); anti-rabbit IBA1 (1:3K; Wako, Cambridge, MA); and anti-rabbit cleaved caspase 3 (1:1K; Cell
Signaling Technology; Boston, MA). Anti-rabbit phospho-Map2 (Ser 136) was also used (1:1K; Cell signaling)
(Supplementary Fig. 2). Following a series of washes in 1XPBS-0.1%T, sections were incubated in biotinylated
anti-mouse or anti-rabbit secondary antibodies (Vector labs, Burlingame, CA) (1 hr), washed, and incubated with
ABC Avidin/Biotin complex solution. Following washes in 1XPBS-0.1%T, signal was visualized using the DAB
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10Scientific Reports | 6:24988 | DOI: 10.1038/srep24988
Peroxidase (HRP) Substrate Kit, 3,3′ -diaminobenzidine (DAB) according to manufacturer’s instructions (Vector
Laboratories). Sections were mounted in gelatin-coated slides, air dried, Nissl stained, dehydrated through
increasing alcohol concentrations [50%, 70%, 95%, 100%], de-fatted in Xylene, and coverslipped using Permount
mounting media. All chemicals were obtained from Fisher Scientific unless otherwise indicated.
Golgi Staining. Rats were profoundly anesthetized with Beuthanasia and perfused with ice cold 1XPBS.
All brains were rapidly dissected and processed using the FD Rapid Golgi Stain kit following the manufactur-
er’s instructions (Neurodigitech, San Diego, CA, USA). Brains were incubated in golgi impregnating solutions
provided in the kit for a minimum of 4 weeks. Then, brains were cut into serial coronal sections (80 μ m thick),
mounted on gelatin-coated slides, stained following the FD Rapid Golgi Stain kit protocol. After staining, sec-
tions were dehydrated through increasing alcohol concentrations [50%, 70%, 95%, 100%], de-fatted in Xylene,
and coverslipped using Permount mounting media. Quantification of spine density was performed using a 100X
immersion (oil) objective with a Leica DM5500 microscope equipped with a high definition Leica DFC290 cam-
era and using the LASV4.6 software. Five representative sections were selected along the dorsoventral axis at
approximately the following Bregma coordinates: − 3.48 mm, − 4.08 mm, − 4.36 mm, − 4.92 mm, and − 5.28 mm.
Five CA1 neurons were randomly selected per section. From these, the number of spines was counted in 20 μ m
sections of five second order dendrites per neuron as previously described5
. Total dendritic branches analyzed per
group: Control: 375; 3 day post-SE: 375; 14 days post-SE: 375; 35 days post-SE: 240. Brains per group: Controls: 3;
3 day post-SE: 3; 14 days post-SE: 3; 35 days post-SE: 2.
Semi-quantitative densitometry analysis. Immunostaining was visualized using a Leica DM500
microscope and images for quantitative analyses were captured with high resolution digital camera (Leica MC120
HD) with 4X objectives using the LAS4.4 software. The relative mean pixel intensity of the immunostaining
signal was acquired using the Image J NIH software (V1.49) by investigators blinded to treatment group as previ-
ously described58
. Brain tissues that were damaged and the hippocampal anatomical landmarks were broken and
thereby unrecognizable following the free-floating IHC procedures were excluded from the quantitative analyses.
Therefore, between 4 and 6 sections were analyzed per brain. Densitometry analyses were performed bilaterally
over the hippocampus.
Cell counts. Semi-quantitative analyses of cells immunostained with cleaved-caspase 3 were performed by
investigators blinded to treatment groups using the cell counter available in the Image J software. Images were
captured using a 20X objective in a Leica DM5500 microscope equipped with a high definition Leica DFC290
camera and using the LASV4.6 software. Cleaved-caspase 3 positive cells contained within the visual field over
CA1 pcl53
and localized inside a rectangular box (80 μ m × 370 μ m) were counted in all sections. Between 4 and 6
sections were analyzed per brain. Cell counts were performed bilaterally over the hippocampus.
Statistical Analyses. IBM SPSS Statistics 22 software was used for statistical analyses and Analysis of
Variance (ANOVA) with Fishers LDS post hoc tests to determine statistical significance (α < 0.05) between the
control and experimental groups. Values are reported as means ± SEM. Figures were generated using Adobe
Photoshop (CS6).
Ethics Statement. All procedures concerning animals were approved by the Purdue Institutional Animal
Care and Use Committee and followed in accordance to the approved Institutional and NIH guidelines.
References
1. Levesque, M., Avoli, M. & Bernard, C. Animal models of temporal lobe epilepsy following systemic chemoconvulsant
administration. J Neurosci Methods 15, 45–52 (2015).
2. Swann, J. W., Al-Noori, S., Jiang, M. & Lee, C. L. Spine loss and other dendritic abnormalities in epilepsy. Hippocampus 10, 617–25
(2000).
3. Wong, M. Modulation of dendritic spines in epilepsy: cellular mechanisms and functional implications. Epilepsy Behav 7, 569–77
(2005).
4. Zeng, L. H. et al. Kainate seizures cause acute dendritic injury and actin depolymerization in vivo. J Neurosci 27, 11604–13 (2007).
5. Brewster, A. L. et al. Rapamycin reverses status epilepticus-induced memory deficits and dendritic damage. PLos One 8, e57808
(2013).
6. Casanova, J. R., Nishimura, M., Owens, J. W. & Swann, J. W. Impact of seizures on developing dendrites: implications for intellectual
developmental disabilities. Epilepsia 53 Suppl 1, 116–24 (2012).
7. Harada, A., Teng, J., Takei, Y., Oguchi, K. & Hirokawa, N. MAP2 is required for dendrite elongation, PKA anchoring in dendrites,
and proper PKA signal transduction. J Cell Biol 158, 541–9 (2002).
8. Sanchez, C., Diaz-Nido, J. & Avila, J. Phosphorylation of microtubule-associated protein 2 (MAP2) and its relevance for the
regulation of the neuronal cytoskeleton function. Prog Neurobiol 61, 133–68 (2000).
9. Dehmelt, L. & Halpain, S. The MAP2/Tau family of microtubule-associated proteins. Genome Biol 6, 204 (2005).
10. Gardiner, J., Overall, R. & Marc, J. The microtubule cytoskeleton acts as a key downstream effector of neurotransmitter signaling.
Synapse 65, 249–56 (2011).
11. Urbanska, M., Blazejczyk, M. & Jaworski, J. Molecular basis of dendritic arborization. Acta Neurobiol Exp (Wars) 68, 264–88 (2008).
12. Ballough, G. P. et al. Microtubule-associated protein 2 (MAP-2): a sensitive marker of seizure-related brain damage. J Neurosci
Methods 61, 23–32 (1995).
13. Sanchez, C. et al. Microtubule-associated protein 2 phosphorylation is decreased in the human epileptic temporal lobe cortex.
Neuroscience 107, 25–33 (2001).
14. Dachet, F. et al. Predicting novel histopathological microlesions in human epileptic brain through transcriptional clustering. Brain
138, 356–70 (2015).
15. Henshall, D. C. & Meldrum, B. S. In Jasper’s Basic Mechanisms of the Epilepsies (eds. Noebels, J. L., Avoli, M., Rogawski, M. A., Olsen,
R. W. & Delgado-Escueta, A. V.) (Bethesda (MD), 2012).
11. www.nature.com/scientificreports/
11Scientific Reports | 6:24988 | DOI: 10.1038/srep24988
16. Jalava, N. S., Lopez-Picon, F. R., Kukko-Lukjanov, T. K. & Holopainen, I. E. Changes in microtubule-associated protein-2 (MAP2)
expression during development and after status epilepticus in the immature rat hippocampus. Int J Dev Neurosci 25, 121–31 (2007).
17. van Vliet, E. A. et al. Blood-brain barrier leakage after status epilepticus in rapamycin-treated rats II: Potential mechanisms. Epilepsia
57, 70–8 (2016).
18. Nguyen, L. H. et al. mTOR inhibition suppresses established epilepsy in a mouse model of cortical dysplasia. Epilepsia 56, 636–46
(2015).
19. Hailer, N. P. Immunosuppression after traumatic or ischemic CNS damage: it is neuroprotective and illuminates the role of
microglial cells. Prog Neurobiol 84, 211–33 (2008).
20. Srivastava, I. N., Shperdheja, J., Baybis, M., Ferguson, T. & Crino, P. B. mTOR pathway inhibition prevents neuroinflammation and
neuronal death in a mouse model of cerebral palsy. Neurobiol Dis 85, 144–54 (2016).
21. Song, Q., Xie, D., Pan, S. & Xu, W. Rapamycin protects neurons from brain contusioninduced inflammatory reaction via modulation
of microglial activation. Mol Med Rep 12, 7203–10 (2015).
22. Ravizza, T. et al. Inflammatory response and glia activation in developing rat hippocampus after status epilepticus. Epilepsia 46,
113–7 (2005).
23. De Simoni, M. G. et al. Inflammatory cytokines and related genes are induced in the rat hippocampus by limbic status epilepticus.
Eur J Neurosci 12, 2623–33 (2000).
24. Rizzi, M. et al. Glia activation and cytokine increase in rat hippocampus by kainic acid-induced status epilepticus during postnatal
development. Neurobiol Dis 14, 494–503 (2003).
25. Vezzani, A. & Granata, T. Brain inflammation in epilepsy: experimental and clinical evidence. Epilepsia 46, 1724–43 (2005).
26. Avignone, E., Ulmann, L., Levavasseur, F., Rassendren, F. & Audinat, E. Status epilepticus induces a particular microglial activation
state characterized by enhanced purinergic signaling. J Neurosci 28, 9133–44 (2008).
27. Shapiro, L. A., Wang, L. & Ribak, C. E. Rapid astrocyte and microglial activation following pilocarpine-induced seizures in rats.
Epilepsia 49, 33–41 (2008).
28. Amhaoul, H. et al. Brain inflammation in a chronic epilepsy model: Evolving pattern of the translocator protein during
epileptogenesis. Neurobiol Dis 82, 526–39 (2015).
29. Patterson, K. P. et al. Rapid, Coordinate Inflammatory Responses after Experimental Febrile Status Epilepticus: Implications for
Epileptogenesis. eNeuro 2, e0034-15.2015 (2015).
30. Eyupoglu, I. Y., Bechmann, I. & Nitsch, R. Modification of microglia function protects from lesion-induced neuronal alterations and
promotes sprouting in the hippocampus. FASEB J 17, 1110–1 (2003).
31. Horn, K. P., Busch, S. A., Hawthorne, A. L., van Rooijen, N. & Silver, J. Another barrier to regeneration in the CNS: activated
macrophages induce extensive retraction of dystrophic axons through direct physical interactions. J Neurosci 28, 9330–41 (2008).
32. Richwine, A. F. et al. Architectural changes to CA1 pyramidal neurons in adult and aged mice after peripheral immune stimulation.
Psychoneuroendocrinology 33, 1369–77 (2008).
33. Tremblay, M. E. et al. The role of microglia in the healthy brain. J Neurosci 31, 16064–9 (2011).
34. Schafer, D. P. et al. Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner. Neuron 74, 691–705
(2012).
35. Stevens, B. et al. The classical complement cascade mediates CNS synapse elimination. Cell 131, 1164–78 (2007).
36. Kettenmann, H., Hanisch, U. K., Noda, M. & Verkhratsky, A. Physiology of microglia. Physiol Rev 91, 461–553 (2011).
37. Vezzani, A., French, J., Bartfai, T. & Baram, T. Z. The role of inflammation in epilepsy. Nat Rev Neurol 7, 31–40 (2011).
38. Choi, J. & Koh, S. Role of brain inflammation in epileptogenesis. Yonsei Med J 49, 1–18 (2008).
39. Arisi, G. M., Foresti, M. L., Katki, K. & Shapiro, L. A. Increased CCL2, CCL3, CCL5, and IL-1beta cytokine concentration in
piriform cortex, hippocampus, and neocortex after pilocarpine-induced seizures. J Neuroinflammation 12, 129 (2015).
40. Benson, M. J., Manzanero, S. & Borges, K. Complex alterations in microglial M1/M2 markers during the development of epilepsy in
two mouse models. Epilepsia 56, 895–905 (2015).
41. Lopez-Meraz, M. L., Niquet, J. & Wasterlain, C. G. Distinct caspase pathways mediate necrosis and apoptosis in subpopulations of
hippocampal neurons after status epilepticus. Epilepsia 51 Suppl 3, 56–60 (2010).
42. Wong, M. & Guo, D. Dendritic spine pathology in epilepsy: cause or consequence? Neuroscience 251, 141–50 (2013).
43. Shin, M., Brager, D., Jaramillo, T. C., Johnston, D. & Chetkovich, D. M. Mislocalization of h channel subunits underlies h
channelopathy in temporal lobe epilepsy. Neurobiol Dis 32, 26–36 (2008).
44. Swann, J. W. & Rho, J. M. How is homeostatic plasticity important in epilepsy? Adv Exp Med Biol 813, 123–31 (2014).
45. do Nascimento, A. L. et al. Neuronal degeneration and gliosis time-course in the mouse hippocampal formation after pilocarpine-
induced status epilepticus. Brain Res 1470, 98–110 (2012).
46. Gong, R. & Tang, S. J. Mitogen-activated protein kinase signaling is essential for activity-dependent dendritic protein synthesis.
Neuroreport 17, 1575–8 (2006).
47. Lugo, J. N. et al. Altered phosphorylation and localization of the A-type channel, Kv4.2 in status epilepticus. J Neurochem 106,
1929–40 (2008).
48. Sierra, A., Abiega, O., Shahraz, A. & Neumann, H. Janus-faced microglia: beneficial and detrimental consequences of microglial
phagocytosis. Front Cell Neurosci 7, 6 (2013).
49. Eyo, U. B. et al. Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12 receptors
after status epilepticus. J Neurosci 34, 10528–40 (2014).
50. Hasegawa, S., Yamaguchi, M., Nagao, H., Mishina, M. & Mori, K. Enhanced cell-to-cell contacts between activated microglia and
pyramidal cell dendrites following kainic acid-induced neurotoxicity in the hippocampus. J Neuroimmunol 186, 75–85 (2007).
51. Paolicelli, R. C. et al. Synaptic pruning by microglia is necessary for normal brain development. Science 333, 1456–8 (2011).
52. Nimmerjahn, A., Kirchhoff, F. & Helmchen, F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.
Science 308, 1314–8 (2005).
53. Zeng, L. H., Rensing, N. R. & Wong, M. The mammalian target of rapamycin signaling pathway mediates epileptogenesis in a model
of temporal lobe epilepsy. J Neurosci 29, 6964–72 (2009).
54. Buckmaster, P. S. & Lew, F. H. Rapamycin suppresses mossy fiber sprouting but not seizure frequency in a mouse model of temporal
lobe epilepsy. J Neurosci 31, 2337–2347 (2011).
55. van Vliet, E. A. et al. Inhibition of mammalian target of rapamycin reduces epileptogenesis and blood-brain barrier leakage but not
microglia activation. Epilepsia 53, 1254–63 (2012).
56. Racine, R. J. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 32,
281–94 (1972).
57. Shapiro, L. A., Korn, M. J. & Ribak, C. E. Newly generated dentate granule cells from epileptic rats exhibit elongated hilar basal
dendrites that align along GFAP-immunolabeled processes. Neuroscience 136, 823–31 (2005).
58. Marcelin, B. et al. Differential dorso-ventral distributions of Kv4.2 and HCN proteins confer distinct integrative properties to
hippocampal CA1 pyramidal cell distal dendrites. J Biol Chem 287, 17656–61 (2012).
Acknowledgements
We would like to thank Melissa McCurley and Ricardo Murillo for technical assistance.
12. www.nature.com/scientificreports/
12Scientific Reports | 6:24988 | DOI: 10.1038/srep24988
AuthorContributions
A.L.B. designed and initiated the project; A.L.B. and N.D.S. conducted induction of SE, perfusions,
immunohistochemistry, imaging acquisition, analysis, and wrote the manuscript; N.D.S., L.M., S.A.H. and
S.J.B. conducted immunohistochemistry, imaging acquisition, and performed cell and spine counts along
with densitometry analyses blinded to treatment groups. C.T. and L.B.M. performed the enzyme-linked
immunosorbent assay for inflammatory mediators. All authors participated in the discussion of the experiments,
data, and manuscript.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Schartz, N. D. et al. Spatiotemporal profile of Map2 and microglial changes in the
hippocampal CA1 region following pilocarpine-induced status epilepticus. Sci. Rep. 6, 24988; doi: 10.1038/
srep24988 (2016).
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