In vivo two-photon microscopy of CSD in awake mice
This is a modified continuation of FOTS 12095. In this project we want to further study the dynamics of intra- and extracellular ion changes and signaling under the electrophysiological phenomenon "cortical spreading depression" (CSD) in awake head-fixed mice. CSD is an important part of the pathophysiology of migraine, strokes and epilepsy. To detect changes in ion concentrations and signaling substances, we will use genetically coded nanosensors delivered using recombinant adeno-associated viruses and electrophysiology. To understand the influence of the different mechanisms on the CSD phenomenon, we will use different types of genetically modified mice and genetic tools.
Adverse effects: The experiments involve implantation of chronic cranial windows that enable two-photon microscopy in the awake state. In the experiments themselves, the CSD phenomenon will be triggered using light-sensitive ion channels that are illuminated with LED lights (optogenetics - a completely non-invasive and painless intervention), and the CSD waves will subsequently be imaged through the cranial window. The total load (surgery and experiments) for the individual mouse should be categorized as moderate.
Expected utility: We were the first in the world to microscopy CSD waves using two-photon microscopy and genetically coded activity sensors, and our studies have clarified important controversies in the field (Enger et al, Cerebral Cortex 2015, Enger et al Cerebral Cortex 2017, Rosic et al. Glia, 2019). We expect that using this unique model to further study CSD can provide new knowledge about basic mechanisms that can identify new molecular targets in the treatment of a number of conditions in the brain, such as migraine, strokes and epilepsy.
Number of animals and species: A total of 280 mice of a total of 4 different genotypes are applied for.
How to comply with 3R: Our laboratory has nearly 10 years of experience with the techniques described here (especially relevant FOTS 7118 and 12095), and has optimized the procedures over time to improve animal welfare. The validity and value of our experiments depends on the animals' well-being, as illness or stress must be assumed to have a negative effect on the results. Because the mice have chronic implants, a lot of data can be collected per mouse, and we will thus be able to manage with significantly fewer mice than with other models for CSD. The planned studies cannot be replaced by experiments in reduced model systems (cells, brain slices) as the phenomena we study depend on intact connectivity and blood flow. Computer modeling can be used as a good supplement to experimental studies of CSD, but there is still far too much uncertainty about most of the parameters included in such modeling for it to provide new reliable knowledge.
Adverse effects: The experiments involve implantation of chronic cranial windows that enable two-photon microscopy in the awake state. In the experiments themselves, the CSD phenomenon will be triggered using light-sensitive ion channels that are illuminated with LED lights (optogenetics - a completely non-invasive and painless intervention), and the CSD waves will subsequently be imaged through the cranial window. The total load (surgery and experiments) for the individual mouse should be categorized as moderate.
Expected utility: We were the first in the world to microscopy CSD waves using two-photon microscopy and genetically coded activity sensors, and our studies have clarified important controversies in the field (Enger et al, Cerebral Cortex 2015, Enger et al Cerebral Cortex 2017, Rosic et al. Glia, 2019). We expect that using this unique model to further study CSD can provide new knowledge about basic mechanisms that can identify new molecular targets in the treatment of a number of conditions in the brain, such as migraine, strokes and epilepsy.
Number of animals and species: A total of 280 mice of a total of 4 different genotypes are applied for.
How to comply with 3R: Our laboratory has nearly 10 years of experience with the techniques described here (especially relevant FOTS 7118 and 12095), and has optimized the procedures over time to improve animal welfare. The validity and value of our experiments depends on the animals' well-being, as illness or stress must be assumed to have a negative effect on the results. Because the mice have chronic implants, a lot of data can be collected per mouse, and we will thus be able to manage with significantly fewer mice than with other models for CSD. The planned studies cannot be replaced by experiments in reduced model systems (cells, brain slices) as the phenomena we study depend on intact connectivity and blood flow. Computer modeling can be used as a good supplement to experimental studies of CSD, but there is still far too much uncertainty about most of the parameters included in such modeling for it to provide new reliable knowledge.