Overlapping in two cellular ensembles in the hippocampal CA1 region is important for behavioral tagging◯Masanori NOMOTO1,2, Noriaki OHKAWA1,2, Hirofumi NISHIZONO2,3, Mina Matsuo3, Jun YOKOSE1,2, Yukari TAKAHASHI4, Masashi NAGASE4, Akinobu SUZUKI1,2, Ayako M. WATABE4,5, Fusao KATO4,5, Kaoru INOKUCHI1,2
1 Department of Biochemistry, Faculty of Medicine, Graduate School of Medicine & Pharmaceutical Sciences, University of Toyama, 2 CREST, Japan Science and Technology Agency
3 Division of Animal Experimental Laboratory, Life Science Research Center, University of Toyama, 4 Department of Neuroscience, Jikei University School of Medicine, 5 Graduate School of Medicine, Nagoya University

3. Achievement of behavioral tagging required novelty and hippocampal de novo
protein synthesis.

1. Abstract
Behavioral tagging is one form of interactions between two independent memories in which short-term memory (STM) is transformed into a long-termmemory (LTM) when the STM-training and another novel experience occur at short time interval. Previous studies showed that behavioral tagging
resembles synaptic tagging in the interactions between short-term and long-term events, their requirement for de novo protein synthesis, and time
windows between two events (~hours), suggesting that synaptic tagging mechanism may underlie the behavioral tagging.
We previously showed that the number of neurons activated at both novel object recognition (NOR) and novel experience, denoted here overlapping
neurons, increased in the CA1 region when the behavioral tagging was successfully achieved. In this study, we examined the role of the overlapping cell
ensemble in behavioral tagging. Arc catFISH analysis showed that the increase in the number of overlapping neurons, which in this case were activatedduring both the retrieval of NOR and the exposure to the initial context, was also observed. Importantly, optogenetic experiment with lentiviral vector
expressing ArchT3.0 and cfos-tTA transgenic mice showed that optical silencing of the cell ensemble in the hippocampal CA1 region related to the novel
experience, but not different experience, led to an impairment in the NOR memory retrieval. These results suggest that inputs from two different piecesof information, NOR and novel experience, converged on the same neuronal ensemble in the hippocampal CA1 region after achievement of behavioral
tagging. Together, our results suggest that overlapping in two cellular ensembles in the hippocampal CA1 region is required for behavioral tagging andsynaptic tagging mechanism may be involved in the behavioral tagging.

Behavioral tagging resembles synaptic tagging in the interactions
between short-term and long-term events.
Synaptic tagging mechanism may underlie the behavioral tagging.

5. Optical silencing of the CA1 cell ensemble related to the original place experience, but not to different experience, led to an impairment in the NOR memory
retrieval and a decrease in the ratio of Egr1/zif268 expressing cells in ArchT3.0+light ON cells.

a-b. Anisomycin microinjection into hippocampus immediately after novelty impaired NOR-LTM.

a. Experimental design using Tettag(cfos-tTA) mice, lentivirus vector and optogenetics

c. Familiarization to novel context inhibited behavioral tagging.

b. ArchT3.0-EYFP expression was induced in OFF-Dox- and context-exposure-dependent manners

c-f. Optogenetic experiment followed by immunohistochemistry?

It is expected that these two units of information may be processed in the same neuronal circuit.
However, there is no evidence about the dynamics of cell assemblies
that correspond to these two independent information when the behavioral tagging is achieved.

2. NOR-STM was transformed into LTM by exposing mice to a novel place
at a short interval of ~1 hour.
Experimental design of novel
object recognition (NOR) test

a-f. Fifteen min but not five min NOR-training formed NOR-LTM, and five min training formed
NOR-STM in a hippocampal protein synthesis independent manner.

4. Novelty group showed an increase in the number of overlapping neurons
in the hippocampal CA1 region compared with the control groups.

6. Arc catFISH analysis showed that the ratio of overlapping neurons were also larger
compared with an control group during both NOR and context exposure.

a. Experimental design of Arc mRNA catFISH and dynamics of Arc mRNA after its transcription

a-d. Experimental design and results of Arc mRNA catFISH

7. Blockade of dopamine D1/D5 receptor during behavioral tagging training
affected NOR-LTM and ratio of overlapping neurons.
a-f. Experimental design using dopamine receptor antagonist SCH23390 and results of Arc mRNA catFISH

c-d. Results of CA1 region and Venn diagram of ratio of overlapping

e-f. Results of CA3 and DG regions: the number of overlapping neurons were comparable
between familiarized and non-familiarized groups.
h-j. Establishment of behavioral tagging paradigm using NOR and place memory test which has time window

7. Conclusion
NOR-STM is transformed into LTM by exposing mice to a novel place (Novelty)
at a short interval of ~1h in a protein synthesis-dependent manner.
Arc catFISH analysis showed that...
the number of neurons activated at both NOR and novel experience, overlapping neurons,
increases in the CA1 region when the behavioral tagging was successfully achieved.
Optogenetic experiment with lentiviral vector expressing ArchT3.0 and cfos-tTA transgenic mice showed that...
optical silencing of the cell ensemble in the hippocampal CA1 region related to the novel experience, but not different
experience, led to an impairment in the NOR memory retrieval.

These results suggest that...
inputs from two different pieces of information, NOR and novel experience, converged on the same neuronal ensemble
in the hippocampal CA1 region during behavioral tagging.

Together, our results suggest thatsynaptic tagging mechanism
may be involved in the behavioral tagging.