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bioRxiv · 10.1101/2025.01.15.633173

Optical recordings of unitary synaptic connections reveal high and random local connectivity between CA3 pyramidal cells

Layous, R.·Tamas, B.·Mike, A.·Sipos, E.·Arszovszki, A.·Brunner, J.·Szatai, A.·Yaseen, F.·Andrasi, T.·Szabadics, J.
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Abstract

The hippocampal CA3 region is thought to play crucial roles in episodic memory functions because of the extensive recurrent connections between CA3 pyramidal cells (CA3PCs). However, different methods provided contradicting observations about the synaptic connectivity between CA3PCs. Therefore, we estimated the connectivity rate between individual CA3PCs using a new approach that is not affected by the confounds of conventional methods. Specifically, we used voltage imaging with the Voltron sensor in acute slices from rats of both sexes to test CA3PC connections by detecting spontaneous spiking and subthreshold responses in anatomically identified neurons. We detected 164 monosynaptic excitatory connections in 3078 tested CA3PC-CA3PC pairs. This 5.3% connectivity rate was much higher than that we observed with the theoretically more sensitive patch clamp method in similar experimental conditions, but it remained below the anatomically observed number of CA3PC-CA3PC contacts. We verified that the imaged excitatory connections were mediated by AMPA receptors. Our results also showed that the recurrent connections did not enrich into preferred connectivity motifs and followed a distribution that was consistent with random connectivity in general. Moreover, voltage imaging revealed CA3PCs with distinct firing properties and somatic locations corresponding to previously established heterogeneity and showed that specific connectivity rules create preferred information routes among these subpopulations. Finally, we showed that there is at least one condition, influencing patch clamp recordings but not voltage imaging, that affects the observable functional connections between CA3PCs. Altogether, our results obtained with a new voltage imaging approach argue for high local connectivity rates between CA3PCs. Significance StatementVoltage imaging offers new opportunities to measure synaptic connectivity between individual identified neurons. We employed the Voltron sensor to measure the monosynaptic connectivity between local CA3 pyramidal cells (CA3PCs) in acute slices from rats. Recurrent CA3PC connections are thought to play crucial roles in storing memory. However, the estimated connectivity rates between CA3PCs differ depending on the methods used. We showed that in spite of having a lower signal-to-noise ratio, voltage imaging results showed a much higher connectivity between CA3PCs than patch clamp recordings. Our results also suggest that the recurrent excitatory connections between CA3PCs are random, with preferred connectivity between and within subpopulations of CA3PCs.

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Layous, R., Tamas, B., Mike, A., Sipos, E., Arszovszki, A., Brunner, J., Szatai, A., Yaseen, F., Andrasi, T., Szabadics, J.. 2025-01-17. Optical recordings of unitary synaptic connections reveal high and random local connectivity between CA3 pyramidal cells. https://doi.org/10.1101/2025.01.15.633173

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