Journal: bioRxiv

Article Title: CS - plasticity and behavioral-state integration in the posterior insula during auditory fear learning

doi: 10.1101/2025.09.25.678609

Figure Lengend Snippet: A, Schematic of the auditory fear learning protocol. B, Schematic of an optrode assembly ( left ), and an example brain section for post-hoc localization of a tetrode recording tip ( right ; arrow). C, Individual (thin grey lines) and average (thick black lines) freezing curves (10 s time bin) of N = 20 mice from the in-vivo optrode recordings in , . D, Histogram of the waveform width of all the recorded units (n = 420, in N=20 mice). Units with widths above- and below 0.35 ms were regarded as putative principal neurons (n = 374) and putative FSIs (n = 46), respectively. E, Example of a CS learner unit that also responded to the US. CS- (blue shading) and US-evoked (pink shading) AP activity during the training session ( left ) and the recall session ( right ). Insets on the left show the unit’s average waveform on each tetrode channel. The trace on the bottom shows the average US response. F, Raster plot showing the average PSTH for the US responses of all putative principal neurons (n = 374), sorted in decreasing order of z-score values. Dashed lines correspond to z-score values above and below which the units were classified as positive US-onset responders ( top , n = 134) or US-inhibited units ( bottom , n = 41), respectively. The inset on the right shows the average footshock-evoked activity of all positive US-onset responders (red trace) and of all US-inhibited units (blue trace). G , Re-sorting the units for response maxima from 1-1.5 s after US-onset reveals n = 22 US-offset responders ( top ); the average trace is also shown ( bottom ). H, Results of unsupervised hierarchical clustering of CS-plasticity patterns observed across all putative principal neuron units (n=374). I, Raster plots of average CS-aligned activity of the units, sorted according to the cluster identity (see the color-coded bar, rightt ). J, Individual units and average plasticity plots of cluster #8 ( top , “Naive tone responders”) and of the pooled clusters #3, #4, #9 (bottom; “CS-learners”). K, Left , coronal view of the percentage of CS-learners on each tetrode, reconstructed from post-hoc histology data. Right , histogram showing the percentage of the CS-learners binned along five dorsoventral areas: S2, ventral S2 (vS2), pInsCS-GI (GI), pInsCx-DI (DI), pInsCx-AI (AIP). Note the predominant location of CS-learner units in the vS2 and GI. L, Data presentation as in K, now for the percentage of positive US-onset responders on each tetrode. For statistical parameters in K and L, see Results text.

Article Snippet: On day 2 (training session), six tone blocks (CS, as above), immediately followed by a 1s footshock (US; 0.6 mA AC, from a shock generator ENV-414S, Med Associates Inc) were presented.

Techniques: In Vivo, Activity Assay

Journal: bioRxiv

Article Title: CS - plasticity and behavioral-state integration in the posterior insula during auditory fear learning

doi: 10.1101/2025.09.25.678609

Figure Lengend Snippet: The footshock-evoked AP-firing response amplitude (z-score) is shown for individual units (markers) and as average ± SEM values (bars), for the six subsequent US applications during the training session (n = 134 positive US-onset responders out of n = 374 putative principal neurons; see ). Friedman test found an effect of footshock number (p = 0.011). A Dunn’s multiple comparison test reported a significantly smaller US-response to footshock #1, as compared to footshock #5 (p = 0.0049); all other comparisons were not significant.

Article Snippet: On day 2 (training session), six tone blocks (CS, as above), immediately followed by a 1s footshock (US; 0.6 mA AC, from a shock generator ENV-414S, Med Associates Inc) were presented.

Techniques: Comparison

Journal: bioRxiv

Article Title: CS - plasticity and behavioral-state integration in the posterior insula during auditory fear learning

doi: 10.1101/2025.09.25.678609

Figure Lengend Snippet: A, Schematic of the experimental approach which combines optrode recordings in the pInsCx with optogenetic inhibition of incoming A1 axons. B, Left , image of the virus injection site in A1 (green channel, eNpHR3.0-eYFP; blue channel, DAPI). Right, image on the level of the pInsCx - S2 border with eNpHR3.0-eYFP expressing A1 axons (green channel) and the trace of a tetrode tip (arrow). The track of the optic fiber is marked by yellow box. C, Schematic of optogenetic axon silencing during every second tone beep during the habituation - and recall sessions. D, Venn diagram showing the sub-populations of pInsCx units with different response types in this experiment (N = 6 mice; n = 74 units). See also for comparison to the larger dataset in and . E, Raster plot of average aligned z-score responses to the footshock (US) presentation during the training session, sorted in decreasing order of response value (n = 74 units; N = 6 mice). Dashed line shows cut-off for positive US responders (n = 14). F, z-score responses aligned to the onset of movement (n = 21 movement-up responders, see dashed line). G, Left : Raster plot of aligned average responses to non-silenced tone beep presentations during the habituation session, sorted in decreasing order of response amplitude. Right : corresponding time-averaged z-score values observed during the tone beep. H, Display as in G, but shown for the tone beeps that received a 300 ms light pulse (see green line). The unit identities are matched to the ones in panel G. I, Differential z-score values (Δz) computed for each neuron as a difference in the z-score between optogenetically silenced CS beeps (H) and non-silenced control beeps (G). J, Histogram of Δz values from the data in panel I. K, Left : Tone responses of naïve CS responders during the habituation session, plotted separately for the non-silenced (black) and silenced beeps (green). Shading is ± s.e.m. Right : quantification of peak amplitudes of the tone responses for light, and no light conditions. L-M, Data shown as in panels G-I, here for the recall session. Note that the Δz plot reveals more negative values, and more scattering than during the habituation session (compare panels N and I). O , Histogram of Δz values from the data in panel N. P, Data as in panel K, here shown for the recall session. As during the habituation session (see panel K), optogenetic inhibition of A1 axons had no effect on the naïve tone responders (n = 8, p = 0.74; Wilcoxon test). Q, Left : Tone responses of CS learners during the recall session, plotted for the non-silenced (red) and silenced tone beeps (green). Shading is ± s.e.m. Right : quantification of peak amplitudes of the tone responses for light, and no light conditions. Note the significant reduction of tone beep responses (n = 25, p = 0.038, paired t-test). R, Scatter plots of the modulation by light (Δz values) as a function of the tone beep response in the absence of light, both for the habituation session ( left ) and the recall session ( right ). Linear regression and Pearsońs correlation test revealed no significant correlation during habituation, but a significant negative correlation during the recall session (see Results for statistical parameters). Scale bars in B, 500 μm.

Article Snippet: On day 2 (training session), six tone blocks (CS, as above), immediately followed by a 1s footshock (US; 0.6 mA AC, from a shock generator ENV-414S, Med Associates Inc) were presented.

Techniques: Inhibition, Virus, Injection, Expressing, Comparison, Control