Journal: bioRxiv
Article Title: Temporal Coding rather than Circuit Wiring allows Hippocampal CA3 Neurons to Dynamically Distinguish Different Cortical Inputs
doi: 10.64898/2026.02.20.707083
Figure Lengend Snippet: a, Schematic of viral injection strategy in MEC and LEC. Sagittal view of mouse brain showing MEC (red), LEC (green), and Hippocampus (orange). The dotted line represents the plane of the section. AAV2/5.Syn.Flex.ChrimsonR.tdTomato and AAV2/5.Syn.Flex.Chronos.eGFP were injected into MEC and LEC of CaMKII-Cre mice to drive excitatory opsin expression selectively in glutamatergic neurons. We alternated the opsins between the two regions to avoid confounds due to opsin-specific effects. This enabled input-specific dual-optogenetic circuit mapping of MEC and LEC inputs to hippocampal area CA3. b, (top) Representative confocal images of injection sites in a 100 µm horizontal section through the dorsal hippocampus and entorhinal cortex showing restricted expression of fluorophores in MEC and LEC. This brain slice was stained for DAPI, a nuclear marker. (bottom) Expanded view of the area enclosed in the dotted box in the top image shows axonal expression of fluorophores in stratum lacunosum moleculare (SLM) of CA3. MEC and LEC projections show typical patterning, MEC axons more proximal to stratum pyramidale (SP) than LEC axons. c, (left) Input-specific, dual-optogenetic functional circuit mapping in hippocampal area CA3 in acute mouse brain slice experimental design. Optogenetic stimulation of MEC glu and LEC glu axons via 470 nm and 625 nm light while performing whole-cell voltage-clamp recordings of CA3 pyramidal neurons (PN). (right) Schematic of connectivity hypothesis. d, Confocal image of CA3 region of 400 µm transverse hippocampal, mouse brain slice showing recorded CA3 a/b PN filled with Biocytin (white) and MEC (red) and LEC axons (green). The slice is stained with DAPI and streptavidin-AF647 (Biocytin counterstain). e, Sample traces of MEC glu -(blue) and LEC glu -driven (green) responses in CA3 PN voltage-clamped at −80 mV in 1 µM TTX & 10 µM 4-AP (top) and ACSF (bottom). f, Fraction of CA3 PN that receives MEC glu (blue) and LEC glu (green) inputs in ACSF and TTX & 4-AP. g, Input-output function curves of MEC glu (blue) and LEC glu (green) light-evoked CA3 PN EPSCs in TTX & 4-AP across low (<10%, 0.31 mW/mm 2 ) light intensities (n = 19 neurons, N = 10 mice, *p = 0.0429 Mixed-Effects Model w/ Geisser-Greenhouse Correction). h, Peak amplitudes of LEC glu (green) and MEC glu (blue) light-evoked CA3 PN EPSC responses at 8.8% light intensity in TTX & 4-AP (left, n = 19 neurons, N = 10 mice) and ACSF (right, n = 15 neurons, N = 8 mice) (TTX & 4-AP **p = 0.0039 Wilcoxon matched-pairs two-tailed,, ACSF NS p = 0.3591 Wilcoxon matched-pairs two-tailed). Each data point represents the mean peak EPSC amplitude of an individual cell. All error bars represent SEM.
Article Snippet: Lastly, slices were washed five times for 15 min in PBS and mounted in Vectashield Hard Set Mounting Medium with DAPI (Vector Laboratories).
Techniques: Injection, Expressing, Slice Preparation, Staining, Marker, Functional Assay, Two Tailed Test
Vector Laboratories is a verified supplier 
Vector Laboratories manufactures this product 
