Extended Data Fig. 10: Reciprocal inhibitory connections between ITC clusters.
From: Intercalated amygdala clusters orchestrate a switch in fear state
a, Inter-cluster virus-based tracing (ITCdm-targeted example shown, same strategy for ITCvm targeting). AAV encoding Cre-dependent synaptophysin–GFP targeted to ITCdm or ITCvm neurons in Foxp2-Cre mice. b, c, Confocal images from ITCdm (b) or ITCvm (c) injections. FOXP2 expression visualized using immunohistochemistry (IHC). Injections repeated in five (ITCdm), and four (ITCvm) mice. Scale bars: 500 μm (b1, c1); 200 μm (b2, 3, c2, 3). d, e, Histological validation of selective ChR2 expression in ITCdm (d1–3) and ITCvm (e1–3). Scale bars: 500 μm (d1, e1); 50 μm (d3, e3). Repeated for all mice shown in Fig. 4. f, Representative example of IPSCs recorded at different holding potentials from an ITCdm neuron upon photostimulation of ChR2-EYFP fibers from ITCvm. g, i, Schematics illustrating ex vivo slice electrophysiological experiments with direct cluster stimulation. The ITCvm (g) or ITCdm (i) cluster was targeted with an AAV encoding Cre-dependent ChR2. Recordings were performed in the other cluster while the ChR2-positive cluster or the surrounding area was selectively stimulated with aperture-restricted stimulation (see Methods). h, j, Images taken from the recording setup illustrating positions of on- and off-cluster stimulation for ITCvm while recording in ITCdm and vice versa. k, Summary of PTX application experiments (n = 3 from 3 mice). Magenta lines represent ITCvm→ITCdm connections and green lines represent ITCdm→ITCvm connections. *P = 0.005, paired t-test. l, Summary of experiments shown in g–j. On-cluster photostimulation evoked significantly larger IPSCs than off-cluster stimulation. Note that we used the highest value out of the four off-cluster stimulation sites. *P = 0.0016, paired t-test.
