Neural computation of space and time addressed by calcium imaging in vivo
A goal for 21st century neuroscience is to understand neural computation in high-end association cortices. This can be challenging because neural codes in these systems are multiplexed and intermixed. A rare exception is the spatial-representation system of the hippocampal and entorhinal cortices. In these regions, neural firing correlates are so evident that cells have been given simple, descriptive names, such as place cells, grid cells, head direction cells, and border cells. The presence of functionally dedicated cell types opens a window for studies aiming to identify computational algorithms at the peak of the cortical hierarchy. Taking advantage of this opportunity, we have uncovered many of the functional elements of spatial computation, at the nuts-and-bolts level. However, how the firing patterns are generated, integrated and read out is poorly understood. Since such processes likely take place in distributed neural circuits, we need parallel data from many hundreds of entorhinal-hippocampal cells, of multiple categories, to decipher mechanisms. The aim of the research program for which we seek approval with the present application, is to introduce new microscopy tools to obtain simultaneous single-cell-resolution activity data from up to 1,000 entorhinal-hippocampal neurons in freely behaving rodents, and to use these tools to understand local-circuit mechanisms of pattern formation and pattern organization. The proposed studies will pioneer our understanding of neural-circuit computation in non-sensory association cortices. Because representation of spatial location has appeared as one of the first cognitive functions to be understood in mechanistic detail, the project opens possibilities for understanding general principles of computation in cortical microcircuits, which we see as a prerequisite for comprehending and treating any disease that affects the brain – along the entire spectrum of neurological and psychiatric diseases, but with Alzheimer´s disease as a prime example, given the early damage of hippocampal and entorhinal systems in this disease.
The project is an extension of past or existing FOTS projects (6008, 6021 and 7163) but introduces improved microscopy techniques for collecting parallel data from hundreds of labelled neurons simultaneously during the next 4 years, an achievement that we expect will put us on the track of some of the core mechanisms of brain computation. All major new technologies have been piloted in ongoing FOTS projects.
We expect to use 2,160 mice and less than 216 rats over a period of 4 years. The animals will receive implants of miniature lenses attached to miniature head-fixed microscopes, weighing no more than 2.8 g, for recording calcium-based electrical activity in the hippocampus or cortex of the brain, using previously approved fast-recovery anaesthesia and anaelgesia protocols. Over the subsequent weeks, activity is recorded while animals rest or search for food rewards, with minimal food deprivation. No pain or discomfort is expected during behavioural testing; discomfort would be detectable instantly from the animal's behaviour. Numbers of animals are reduced by recording as many cells as possible simultaneously. Animals are housed in enriched environments and receive daily handling. Experiments are supplemented by computational modelling to minimize needs for experiment and maximize conceptual steps between experiments.
The project is an extension of past or existing FOTS projects (6008, 6021 and 7163) but introduces improved microscopy techniques for collecting parallel data from hundreds of labelled neurons simultaneously during the next 4 years, an achievement that we expect will put us on the track of some of the core mechanisms of brain computation. All major new technologies have been piloted in ongoing FOTS projects.
We expect to use 2,160 mice and less than 216 rats over a period of 4 years. The animals will receive implants of miniature lenses attached to miniature head-fixed microscopes, weighing no more than 2.8 g, for recording calcium-based electrical activity in the hippocampus or cortex of the brain, using previously approved fast-recovery anaesthesia and anaelgesia protocols. Over the subsequent weeks, activity is recorded while animals rest or search for food rewards, with minimal food deprivation. No pain or discomfort is expected during behavioural testing; discomfort would be detectable instantly from the animal's behaviour. Numbers of animals are reduced by recording as many cells as possible simultaneously. Animals are housed in enriched environments and receive daily handling. Experiments are supplemented by computational modelling to minimize needs for experiment and maximize conceptual steps between experiments.
Etterevaluering
Etter godkjenningen av endringen 7/12-20 vurderer vi at den kumulative belastningen for de mest utsatte dyrene kan bli betydelig, jf. forsøksdyrforskriften vedlegg B.
Mattilsynet er forpliktet etter regelverket til å etterevaluere alle forsøk som er betydelig belastende for forsøksdyrene.
Mattilsynet er forpliktet etter regelverket til å etterevaluere alle forsøk som er betydelig belastende for forsøksdyrene.
Begrunnelse for etterevalueringen
This evaluation is based on a sub-project in FOTS 18013. The sub-project is related to a change application sent December 4th, 2020, and the following decision from the Food Safety Authorities requiring a retrospective evaluation.
The objective of this sub-project was to reduce the number of mice used for aspiration surgeries by re-aspirating animals the experimenters did not have success getting any data from. The idea behind the experiment was that the lack of success was due to not having aspirated enough in the former surgery, so while training and optimizing the first aspiration surgery the experimenters could make use of the animals already included in the main project.
This experiment was tested out on two pilot animals, and from this it became apparent that the re-aspiration surgery came with problems of itself that prevented any improvement in success rate.
The re-aspiration surgery required that the already implanted lens was removed. The implant is fixed to the skull with three different adhesive agents to be completely stable. Although this is a desirable outcome, it makes for a challenge to remove it during the aspiration surgery. To remove the implant, all three layers of adhesive agents had to be drilled away, creating micro particles that were exceptionally challenging to flush away thoroughly before fully removing the implant from the brain. Any residual micro particles pose both an infection risk, but also a risk for degrading the image quality when the new lens is implanted. In addition to these issues, the tissue itself had gone through much (expected) reorganization after the first implantation, so re-aspiration based on anatomical landmarks proved to be very difficult. Furthermore, when removing the implanted lens, brain tissue previously surrounding the lens naturally collapsed into the newly created space, eventually leading to the need of aspirating more cortical tissue, which was what the experimenters wanted to avoid with the first surgeries in the first place.
In summary, the experimenters tried to re-aspirate animals already aspirated to save animal lives while optimizing the aspiration protocols, but the success rate did not improve with re-aspiration and the risks associated to these surgeries was not worth continuing the experiments.
The re-aspiration procedure was done with the purpose of improving the data yield per mouse, by optimizing the field of view to sample more high-quality imaging data per animal. The approach used in this sub-project turned out to not be successful.
However, in parallel with this sub-project, the experimenters have improved the type of viral vector used to express the signal needed to detect with our lenses and by rather increasing the size of the aspirated area during the initial implantation surgeries (notice of change, January 2021), the need for any re-aspiration in the future has been eliminated.
Two animals were used in the sub-project. One animal was allowed to survive the re-aspiration, making it the third surgery the animal had gone through and thus falling under severity category ‘severe’.
Due to concerns about the micro particles described above, the animal received postoperative antibiotics for three days and recovered well. When imaging 1 week after recovery, micro particles could be detected under the lens and there was no improvement of the imaging outcome. The animal was therefore perfused.
The other animal was never allowed to survive the re-aspiration, so it falls under category ‘moderate’, due to already having been both injected and chronically implanted before the acute re-aspiration.
The experimenters have now improved their initial aspirations, leading to a much larger overall success rate of the main experiments – reducing the number of animals needed in that way instead of by re-aspirations. This entails remaining on severity category moderate instead of severe, which the re-aspirations automatically would have fallen into.
The objective of this sub-project was to reduce the number of mice used for aspiration surgeries by re-aspirating animals the experimenters did not have success getting any data from. The idea behind the experiment was that the lack of success was due to not having aspirated enough in the former surgery, so while training and optimizing the first aspiration surgery the experimenters could make use of the animals already included in the main project.
This experiment was tested out on two pilot animals, and from this it became apparent that the re-aspiration surgery came with problems of itself that prevented any improvement in success rate.
The re-aspiration surgery required that the already implanted lens was removed. The implant is fixed to the skull with three different adhesive agents to be completely stable. Although this is a desirable outcome, it makes for a challenge to remove it during the aspiration surgery. To remove the implant, all three layers of adhesive agents had to be drilled away, creating micro particles that were exceptionally challenging to flush away thoroughly before fully removing the implant from the brain. Any residual micro particles pose both an infection risk, but also a risk for degrading the image quality when the new lens is implanted. In addition to these issues, the tissue itself had gone through much (expected) reorganization after the first implantation, so re-aspiration based on anatomical landmarks proved to be very difficult. Furthermore, when removing the implanted lens, brain tissue previously surrounding the lens naturally collapsed into the newly created space, eventually leading to the need of aspirating more cortical tissue, which was what the experimenters wanted to avoid with the first surgeries in the first place.
In summary, the experimenters tried to re-aspirate animals already aspirated to save animal lives while optimizing the aspiration protocols, but the success rate did not improve with re-aspiration and the risks associated to these surgeries was not worth continuing the experiments.
The re-aspiration procedure was done with the purpose of improving the data yield per mouse, by optimizing the field of view to sample more high-quality imaging data per animal. The approach used in this sub-project turned out to not be successful.
However, in parallel with this sub-project, the experimenters have improved the type of viral vector used to express the signal needed to detect with our lenses and by rather increasing the size of the aspirated area during the initial implantation surgeries (notice of change, January 2021), the need for any re-aspiration in the future has been eliminated.
Two animals were used in the sub-project. One animal was allowed to survive the re-aspiration, making it the third surgery the animal had gone through and thus falling under severity category ‘severe’.
Due to concerns about the micro particles described above, the animal received postoperative antibiotics for three days and recovered well. When imaging 1 week after recovery, micro particles could be detected under the lens and there was no improvement of the imaging outcome. The animal was therefore perfused.
The other animal was never allowed to survive the re-aspiration, so it falls under category ‘moderate’, due to already having been both injected and chronically implanted before the acute re-aspiration.
The experimenters have now improved their initial aspirations, leading to a much larger overall success rate of the main experiments – reducing the number of animals needed in that way instead of by re-aspirations. This entails remaining on severity category moderate instead of severe, which the re-aspirations automatically would have fallen into.