2019 Fiscal Year Research-status Report
Hippocampal Organization of Spatial Map
| Project/Area Number |
18K03168
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| Research Institution | University of Tsukuba |
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Principal Investigator |
Pavlides C 筑波大学, 人間系, 教授 (50712808)
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| Project Period (FY) |
2018-04-01 – 2021-03-31
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| Keywords | hippocampus / memory / immediate early genes / Zif268 / functional organization / sleep / spatial navigation / place cells |
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| Outline of Annual Research Achievements |
The first aim of the proposed studies was to determine functional neuronal organization in the hippocampus for different types of memory - spatial, sequential, fear. We used a sequential order discrimination (SQD) or a simple discrimination (SD) task. Neuronal activity was determined through the immediate early gene Zif268. We observed an increase of Zif268 expression in both the CA1 and CA3 hippocampal fields in SQD, in comparison to SD animals. In the CA3 field, there was an increase of Zif268 expression in the experimental animals in comparison to controls. Further we observed more clustering of Zif268 immunoreactive cells in the CA3 of SQD experimental animals.
The second aim was to determine mechanisms involved in functional neuronal organization in the hippocampus by optogenetically inhibiting the main afferents arising from the entorhinal cortex (EC) and determining effects on topographic distribution. We developed a new fear conditioning paradigm which combines a contextual (spatial) task with an object recognition task - the former involves the medial entorhinal cortex (MEC) while the latter involves the lateral entorhinal cortex LEC). Thus far, we have confirmed that animals can discriminate both the spatial and object components of the task. We have also determined virus injection parameters and brain sites along with coordinates for implanting of optogenetic stimulation probes for the inhibition of EC. We are currently in the process of testing the effects of inhibiting EC on behavior and neuronal functional organization in the hippocampus.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Progress has been more or less as expected, although the study would require additional time to complete. The first hurdle was to develop a behavioral task with a dissociation of brain regions (MEC/LEC) involved and to build a maze to test its efficacy. This has been completed and we have collected sufficient data to show that our paradigm works. The second hurdle was to perform the viral transfections in the MEC/LEC and also to implant light stimulation probes for the inhibition of these areas. Targeting the right spot turned out to be more difficult than expected, especially for the LEC which is located both deep in the brain and next to the skull. Finally we have succeeded to get the right coordinates and virus injection parameters that would infect one but not the other region. We are currently in the process of testing the effects of inhibiting MEC/LEC on spatial/object fear memory. We simply need to include a sufficient number of animals. The last part of the study would require immunohistochemical processing of the brains and analysis of the distribution of Zif268 immunoreactive cells in the hippocampus. This is a very time consuming process, but we have sufficient experience from our previous studies to ensure that it will proceed smoothly.
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| Strategy for Future Research Activity |
Completion of the studies will require analysis of the Zif268 immunoreactive cell distribution in different parts of the hippocampus (e.g., CA1, CA3, DG, proximal-distal, etc., to which the EC projects. Performing the immunohistochemical/anatomical analysis within different hippocampal areas will require a major undertaking. If indeed we determine behavioral dissociation following MEC/LEC inhibition, we will then proceed to stimulate (rather than inhibit MEC/LEC) and determine that we can produce the differential distribution of Zif268 immuroreactive cells. This will constitute proof positive that the hippocampus is organized in clusters, which we have identified previously and hopefully in the ongoing experiments. This would require double labeling of Zif268 cells using CatFISH immunohistochemistry. We will undertake this project in collaboration with Dr. N. Nakamura. We have collaborated with Dr. Nakamura previously and he has developed the probes and procedures necessary to perform the studies.
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| Causes of Carryover |
A substantial amount is allocated for purchase of animals and IHC/image analysis. Thus far, we have worked on determining brain injection sites, optogenetic probe implants, etc, but have not performed the anatomical analysis yet. Funds have also been set aside for doing the Cat-FISH immunohistochemistry which will require additional costs both for supplies as well as travel to Hoygo College of Medicine for doing some of the procedures. We are also anticipating a number of publications which should be coming to fruition during next year's budget which will require publication costs.
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[Journal Article] Hippocampal functional organization: A microstructure of the place cell network encoding space.2019
Author(s)
Pavlides, C., Donishi, T., Ribeiro, S., Mello, C.V., Blanco, W., and Ogawa, S.
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Journal Title
Neurobiology of Learning and Memory
Volume: 161
Pages: 122-134
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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