Figure 6.

Plasticity of Layer 1 NDNF-Interneuron Responses after Associative Learning

(A) Discriminative auditory fear conditioning in combination with awake in vivo 2-photon imaging. Trains of frequency-modulated sweeps of opposite modulation direction (counterbalanced between experiments) were used as conditioned stimuli (CS).

(B) Freezing behavior of the fear-conditioned animals presented in (E)–(N) in a freely behaving memory retrieval session on day 3 or 4 indicates strong, discriminative fear memory (CS+: 8 animals up sweeps, 6 animals down sweeps, one-way ANOVA F(1.7, 21.7) = 56.1, p < 0.0001; Tukey’s multiple comparison test).

(C) Example pupil diameter response to CS presentation during habituation (green) and memory retrieval (red, sweep onset blue lines) in head fixation.

(D) The change in pupil response for the CSs (response integral retrieval minus integral habituation) correlated with freezing to the stimuli (both fear and pseudoconditioned mice shown), demonstrating that pupil responses can be used as a fear readout under the microscope.

(E) Field of view for in vivo imaging of NDNF-INs in the auditory cortex L1 of awake, head-fixed mice (conditional expression of GCaMP6s [green] and tdTomato [red] in Ndnf-Ires-CreERT2).

(F) Responses of an example L1 NDNF-IN before and after fear conditioning (thin traces represent single trials and thick traces averages).

(G) Average CS responses of all imaged L1 NDNF-INs (133 neurons in 8 mice, CS+: 58 neurons up sweeps, 75 neurons down sweeps) showing a modest increase for the CS− and strong potentiation of CS+ responses.

(H) Quantification of response integral. Both CS− and CS+ responses were significantly potentiated after fear conditioning, whereas no difference was observed during habituation (p = 0.93, Friedman test with Dunn’s multiple comparison).

(I) Freezing behavior of pseudoconditioned animals (n = 5). Note absence of CS evoked freezing (one-way ANOVA F(1.5, 5.8) = 3.2, p > 0.05).

(J) Average CS responses of all imaged L1 NDNF-IN in mice from (I) (84 neurons in 5 mice, CS1 and CS2 combined) showing a decrease in responses for these stimuli.

(K) Correlation between the response change in L1 NDNF-INs due to fear conditioning (response integral retrieval minus integral habituation) and the change in pupil response elicited by that stimulus for fear and pseudoconditioned animals indicates that potentiation of L1 NDNF-IN correlates with learned stimulus relevance.

(L) Field of view during imaging of axons derived from SST-INs in the auditory cortex L1 (GCaMP6s, green; tdTomato, red).

(M) Average CS responses of all imaged SST axons (6 regions in 6 mice, CS+: 4 animal up sweeps, 2 animals down sweeps) showing no change with fear conditioning.

(N) Quantification of response integral (p > 0.05, Friedman test with Dunn’s multiple comparison).

Data are shown as mean ± SEM. (B and H) ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.