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Volga Neuroscience School 2016 Astroglial control of rhythm genesis in the brain Aims
The main goal of this research is to create adenoassociated viral vector for expressing of neurotrophic factor BDNF in neuronal cells. For this study we created the adenoassotiated viral vector with fragment of BDNF gene and EGFP and infected transgenic cell line HEK293T and primary neuronal culture to estimate the viral expression.
Methods
We used standard cloning techniques to create adenoassotiated viral vector with fragment of BDNF gene. We extracted mouse total RNA, made PCR with reverse transcription and amplify the cloning fragment of BDNF gene (900 base). Then we cloned the BDNF fragment into AAV-Syn-kid2 plasmid containing strong human synapsin promoter and Woodchuck hepatitis posttranscriptional regulatory element which enhances the synapsin promoter.
Afterwards we contransfected with this plasmid and helper plasmids the transgenic HEK293T cell culture and collected the virus. We had two different AAV packaging system - DJ and pDP5. Next we cleaned the virus using benzonase treatment and additionally cleaned and concentrated the virus on Amicon Ultra columns to achieve necessary purity of virus so far as the purity of viral sample is important for primary neuronal cultures. The last step was the infection of primary neuronal culture and transgenic HEK293T cell culture and estimating the viral expression using confocal microscopy. Primary neuronal culture was received from E18 mouse.
Results
Adenoasstiated virus containing fragment of BDNF gene has strong expression in HEK293T cell culture and primary neuronal culture. The expression of adenoassotiated virus is more efficient in case of using DJ AAV packaging system. Strong expression appeared on 3-4 day in case of HEK293T cell culture infection and on 5-7 day in case of primary neuronal culture infection. The expression was proved by confocal microscopy, immunocytochemistry and PCR methods.
Conclusions
Thereby it was created the adenoassotiated viral vector containing the fragment of BDNF gene and it was tested on primary neurons culture.
Acknowledgements
The research was supported by the Federal Target Program "Research and development in priority areas of the development of the scientific and technological complex of Russia for 2014-2020" of the Ministry of Education and Science of Russia, contract 14.581.21.0016 (Project ID RFMEFI58115X0016).
Effect of Epileptiform Activity on Hippocampal Astrocytes
A. Lebedeva1 *, O. Tyurikova1,2, A. Plata1, V. Tovpyga1, P. Denisov1, S. Makovkin1,I. Ivlev1, A. Semyanov1
1 Institute of Neuroscience, Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia;
2 Department of Clinical and Experimental Epilepsy, University College London, London, UK. * Presenting e-mail: [email protected]
Over decades, epilepsy has been considered a neurogenerative disease mainly manifested by abnormal neuronal firing. However, some recent findings suggest that electrically passive astrocytes strongly contribute to neural function in health and disease, owing to the release of a variety of signalling molecules (such as glutamate, ATP, D-serine) that target receptors in both neuronal and non-neuronal cells. Astrocytes also participate in neuronal signalling trough high-affinity uptake of neurotransmitters and spatial buffering of extracellular K+. Glutamate transporters deal with up to 80% of the released glutamate, as well as exchanging also Na+ H+ and K+. However, during strong neuronal activity, extracellular K+ concentration can significantly increase from 2.5 mM to 10-12 mM. In pathological condition, such as epileptiform activity, this concentration can even rise up to 30mM [Verkhratsky, Butt, 2007]. According to the kinetics of glutamate transporter, increase of extracellular K+ could interfere with local glutamate uptake. How synaptic activity is affected by epilepsy remains poorly understood. We investigated astrocytic activity in rat slices from control animals and lithium-pilocarpine epilepsy model animals. Ca2+ dynamics was monitored in astrocytes loaded with sulforhodamine 101 (200nM), a specific astrocytic marker, and Oregon Green BAPTA AM (7.95 |oM), a Ca2+ sensor. Transporter and K+ currents in astrocytes were measured with patch pipette in response to electric stimulation of Schaffer collaterals in baseline conditions and subsequently after pharmacological blockade of the excitatory amino acid transporters (EAATs) with TBOA (50 |oM). The present study demonstrates changes in astrocytes associated with epileptogenesis.
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Volga Neuroscience School 2016 Astroglial control of rhythm genesis in the brain Acknowledgements
This work was supported by the Russian Science Foundation (project 15-14-30000).
Identification of Novel Mutations Causing Malformations in Cortical Development by ENU Induced Mutagenesis in the Mouse
E.V. Borisova *, A.A. Babaev, M.V. Turovskaya, E.A. Turovsky, V.S. Tarabykin
Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia. * Presenting e-mail: [email protected]
Abstract. The cerebral cortex, is the seat of our higher cognitive capacity that distinguish humans from other species. The development of the cerebral cortex is a complex and highly orchestrated process whose disruption can result in a wide range of developmental disorders that are recognized as malformations of cortical development. Malformations of the cerebral cortex can frequently cause of epilepsy, developmental delay, neurological deficits, and mental retardation in humans. Intellectual disability and epilepsy caused by neurodevelopmental disorders has a high prevalence of about 2% in human population. Consequently identification of novel mouse mutants with malformations of cortical development and identification and characterization of the causal genes will improve our understanding of the genetic regulation of cortical development and pathogenesis of malfunctioning of the brain.
Aims
The main goal of this project is to identify novel genes that control proper establishment of the cortical structure and functions. For this study we generate mouse mutants by N-ethyl-N-nitrosurea (ENU) directed mutagenesis with disrupted structure and function of the cerebral cortex. We will identify mutants with epileptiform activity and then identify respective genes with positional cloning. Also we would like to characterize the behavior of these mutants and underlying molecular mechanisms.
Methods
To screen for recessive mutations we use back cross three generation scheme. Male C3H mice (12 weeks old) are treated with three intraperitoneal injections in a dose of 80, 90, 100, 120 and 150 mg/kg of ENU at weekly intervals. Following injections ENU induces a variable period (10-15 weeks) of sterility during which mutagenized spermatogonial stem cells repopulate the testis. The surviving males after that period were used for mating with the C3H females to produce G1 offspring.
On next step we will take males from G1 and mated them with C57BL6 females fron Satb2-LacZ line, their upperlayer neurons of brain cortex express LacZ as a reporter (Dobreva G. et al., 2006). This makes it easy to visualize changes cy-toarchitecture cortex and connections between cells. To detect the presence of LacZ transgene in line Satb2-LacZ mice and genotyping PCR protocol was developed.
Results
The optimal concentration of ENU was determined (100 mg/kg). Also we assume that a fertile period after injections of ENU longer than 12 weeks.
The protocol of genotyping Satb2-LacZ mice was developed. As a samples for PCR we use tail cuts of mice from this line, then lysing performed, we use the phenol-chloroform method for extraction of DNA and after that PCR. Besides brain drugs was optimized staining protocol mice Satb2-LacZ-reporters. Beta-galactosidase (p-gal), encoded by the gene LacZ, hydrolyses beta-galactosides, resulting in the appearance of a blue color. Females, genotyped and carrying the reporter Satb2-LacZ, were further crossed with G1 males.
Conclusions
Thus males after ENU injections were obtained and females with help of the developed genotyping protocol were selected for mating to produce G1 population.
Acknowledgements
Research carried out with the financial support of the grant of the Russian Scientific Foundation (project №15-14-10021)
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