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. 2018 Nov 7;100(3):684–699.e6. doi: 10.1016/j.neuron.2018.09.001

Figure 2.

Figure 2

Output Connectivity of Layer 1 NDNF-Interneurons in the Auditory Cortex

(A) AAV-mediated expression of tdTomato and synaptophysin-GFP in the Ndnf-Ires-CreERT2 mouse auditory cortex (left). Synaptophysin-GFP fluorescence is strongly enriched in L1, suggesting that L1 is the primary output location of L1 NDNF-INs (right) (19 slices, 3 animals).

(B) Optogenetic identification of the postsynaptic partners of L1 NDNF-INs in acute slices (top left). L1 and L2/3 INs were identified by nuclear mCherry expression (Peron et al., 2015) and L2/3 PNs by morphology. Calibration of ChR-2 expressing L1 NDNF-IN stimulation (bottom; top right shows an example trace). The chosen irradiance (gray lines, 45 mW/mm2) elicited 1.2 action potentials per pulse (0.5 ms, n = 10).

(C) Average IPSCs in ChR-2 negative L1 INs (gray, n = 12), L2/3 PNs (black, n = 24), and L2/3 INs (red, n = 11).

(D) Comparison of L1 NDNF-IN-mediated IPSCs in the different postsynaptic populations. Note the greater amplitude and charge of IPSCs in L2/3 PNs and the faster rise and decay in L2/3 INs (Kruskal-Wallis H-test with Dunn’s multiple comparison).

(E) Optogenetic activation of L1 NDNF-IN synapse selectively in L1 under action potential block (1 μM tetrodotoxin [TTX], 100 μM 4-AP).

(F) Input to L2/3 PNs is targeted to their distal dendrites located in L1.

(G) Comparison of NDNF and SST-IN input to the distal dendrites of L2/3 PNs. Note that the two datasets are from different experiments.

(H) Average IPSCs evoked by SST- (green, n = 13) and NDNF-IN stimulation (blue, n = 24).

(I) IPSCs mediated by L1 NDNF-INs showed greater charge transfer and longer rise and decay times compared to SST inhibition (Mann-Whitney test).

(J) IPSCs from SST- (green, n = 7) and L1 NDNF-INs (blue, n = 9) in baseline and after bath application of the selective GABAB receptor antagonist CGP 55845 (3 μM, brown, normalized).

(K) GABAB receptor block accelerated the decay time of IPSCs mediated by L1 NDNF-INs but left SST-IN inhibition unaffected (Mann-Whitney test).

(L) Kinetic differences between NDNF- and SST-IN IPSCs persist under GABAB receptor block, indicating additional sources (Mann-Whitney test).

Data in (B), (C), (F), (H), and (J) represent mean ± SEM; other plots show range, quartiles, and median. (D, I, K, and L) ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.