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. 2022 Jan 17;13:339. doi: 10.1038/s41467-022-27977-7

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Top left, Schematic showing experimental setup. ChR2 was injected into contralateral CA3 and retrobeads injected into PFC. 2 weeks later input-specific connectivity was assessed using paired recordings of superficial and deep vH-PFC neurons in acute slices. Bottom left, Average light-evoked responses in pairs of superficial (blue) and deep (black) layer PFC-projecting hippocampal neurons in response to cCA3 input. Scale bar = 10 ms, 5 mV. Purple tick represents the light stimulus. Middle, summary of amplitude of Sup and Deep responses to increasing durations of light pulse (top), and amplitudes of individual pairs at 5 ms (bottom). Right, summary of the ratio of superficial: deep neuron EPSP amplitude. Higher values mean input is biased to superficial neurons, low values towards deep layer neurons. Note log scale. CA3 input is equivalent onto superficial and deep layer neurons (two- sided Wilcoxon paired test, W = 27, ^N.S.p = 1.0, n = 10 pairs of neurons from 3 mice). As in (a) but for ENT (b), ATh (c) and DBB (d) input. Scale bar = 10 ms, 2 mV (b), 0.5 mV (c), 1 mV (d). ENT input is biased towards superficial layer neurons (two- sided Wilcoxon paired test, W = 44, *p = 0.007, n = 9 pairs of neurons from 3 mice), while both ATh and DBB are biased towards deep layer neurons (ATh: two- sided Wilcoxon paired test, W = 12, *p = 0.034, n = 7 pairs of neurons from 4 mice; DBB: two- sided Wilcoxon paired test, W = 24, *p = 0.041, n = 15 pairs of neurons from 7 mice). e As in (a) but for local PV interneuron input. Scale bar = 10 ms, 500 pA. PV + inhibitory input is equivalent onto superficial and deep layer PFC-projecting neurons (two-sided Wilcoxon paired test, W = 57, *p = 0.38, n = 17 pairs of neurons from 4 mice). f As in (e) but in neighboring, unlabelled vH neurons from superficial and deep layers. Scale bar = 10 ms, 200 pA. PV + inhibitory input is biased towards non-retrogradely labelled deep layer neurons (two-sided Wilcoxon paired test, W = 10, *p = 0.042, n = 11 pairs of neurons from 3 mice). g Strategy to investigate superficial and deep vH neuron connectivity onto local interneurons. h Focused light allows activation of neurons expressing soCoChR2 with high spatial resolution. Scale bar = 10 mV, 20 ms. i Connectivity of superficial and deep PFC-projecting vH neurons onto neighboring dlx + interneurons. Probability of connectivity and amplitude is equivalent for both layers (connectivity: Fishers Exact test, Odds ratio = 0.95, ^N.S.p = 1.0, n = 19 (sup) and 18 (deep) neurons from 4 mice; amplitude: two-sided Mann Whitney, U = 8, ^N.S.p = 0.42, n = 5 (sup and deep) connected neurons from 4 mice). Scale bar = 10 pA, 20 ms. Box plots show median, 75^th (box) and 95^th (whiskers) percentile. Line plots show mean (line) and SEM (shading and error bars).