1. Volume 21, Issue 7, Pages 1770-1782 (November 2017)
A Central Amygdala-Substantia Innominata Neural Circuitry Encodes Aversive Reinforcement Signals 
Yuting Cui, Guanghui Lv, Sen Jin, Jie Peng, Jing Yuan, Xiaobin He, Hui Gong, Fuqiang Xu, Tonghui Xu, Haohong Li 
Cell Reports 
Volume 21, Issue 7, Pages (November 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 1770-1782DOI: (10.1016/j.celrep.2017.10.062)
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3. Figure 1
Aversive Stimulus Increases Ca2+ Signals of CeLPKC-δ+ Neurons in Negative Reinforcement Learning
(A) Behavioral setup of the go/no-go task and definition of four behavioral outcomes.
(B) Paradigm of the go/no-go task. A tone (2 clicks/s) was used as the go cue. A light or tone (50 clicks/s) was used as the no-go cue to counterbalance the modality-specific activity of CeLPKC-δ+ neurons.
(C) Performances in the go/no-go task in 7 PKC-δ-Cre mice (4 PKC-δ-CreGCaMP5g mice and 3 PKC-δ-CreGFP mice; light was used as the no-go cue) and 3 PKC-δ-CreGCaMP5g mice (tone was used as the no-go cue). Data are presented as means ± SEM.
(D) Schematic of GCaMP5g viral targeting to CeLPKC-δ+ neurons (left). Confocal image of GCaMP5g expression in the CeL and location of optical fiber (right). Scale bar, 100 μm.
(E) Top: representative images of GCaMP5g (green) and cell-type-specific markers (red: PKC-δ+ neurons) expressed in the CeL. Scale bars, 100 μm. Bottom: enlargements of boxed areas. Scale bar, 10 μm.
(F) Heatmap of Ca2+ signals from mouse aligned to the initiation of water and air puff during the early phase of learning. Color scales on the right indicate ΔF/F.
(G−I) Mean Ca2+ transients from seven PKC-δ-CreGCaMP5g mice aligned to events starting with water and air puff during early learning (G), late learning (H), and expression of learning (I) (early phase of learning, 1–3 sessions; late phase of learning, 6−8 sessions; expression of learning, 9−11 sessions). ∗∗p = (G); ∗∗p = 0.002 (H); ∗∗p = (I).
(J) Mean Ca2+ transients from three PKC-δ-CreGFP mice aligned to events starting with air puff and water across whole phases.
A Mann-Whitney U test was used to identify differences between baseline (average of 0.5 s before cues) and mean peak values (average duration of water or air puff delivered). Thick lines indicate mean, and shaded areas indicate SEM.
See also Figure S1.
Cell Reports  , DOI: ( /j.celrep )
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4. Figure 2
Inhibition of CeLPKC-δ+ Neurons Impairs Negative Reinforcement Learning
(A) Bilateral stereotaxic delivery of virus and implantation of optical fibers into the CeL of PKC-δ-Cre mice.
(B) Confocal image of a coronal brain section taken from a PKC-δ-Cre mouse bilaterally injected with AAV-EF1α-Flex-ArchT-GFP in the CeL. Scale bar, 500 μm.
(C) Experimental timeline.
(D) Paradigm of go/no-go task.
(E–H) Compared with the control group (n = 8), ArchT-expressing mice (n = 8) showed significantly lower performance (E), discriminability (F), and correct-rejection rates (H), but not hit rates (G). Two-way analysis of variance with mixed design; F(1, 14) = 12.48, ∗∗p = (E); F(1, 14) = 6.41, ∗p = (F); F(1, 14) = 9.28, ∗∗p = 0.009, n.s., not significant (H). Data represent means ± SEM.
(I) Distribution of the response delay in go trials.
(J) Median response delay for each group.
(K and L) Performance (K) and correct-rejection (L) rate of mice in the last session of learning without laser (training) and with laser (expression). n.s., not significant (Mann-Whitney U test). Boxplots show the mean (solid square), median, quartiles (boxes), and range (whiskers).
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5. Figure 3 CeLPKC-δ+ Neurons Project to the SI
(A) Virus expression in CeL of PKC-δ-Cre mice. Scale bars represent 500 μm (left) and 100 μm (right). Approximate distance from the bregma is shown at the bottom.
(B) Representative histological images of the SI innervated by axonal terminals from CeLPKC-δ+ neurons. Scale bars represent 500 μm (left) and 200 μm (right). Similar results were confirmed in two other mice. Approximate distance from the bregma is shown at the bottom. CPu, caudate putamen (striatum); LGP, lateral globus pallidus; LH, lateral hypothalamic area.
(C) Virus injection procedures for rabies retrograde tracing. Helper viruses: AAV5-CAG-FLEX TVA66T-mCherry, AAV5-CAG-FLEX-RG (1:1). Rabies virus: EnvA-RV-YFP.
(D) Typical monosynaptic rabies tracing coronal sections of the anatomical localization of the BF and CeL. Scale bars represent 200 μm (BF), 20 μm (starter neuron), and 50 μm (CeL). The results were repeated in at least three mice per group.
(E) Top left: coronal brain section showing the SI injected with CTB-488 (green), scale bar, 500 μm. Top right: projecting neurons labeled with CTB (green). PKC-δ+ neurons stained with Alexa Fluor 594 (red). Scale bar, 50 μm. Bottom: higher-magnification of images of the noted region in the CeL. Scale bar, 10 μm.
(F) Confocal images of a coronal brain section from a Som-IRES-Cre; Ai3 mouse with CTB-647 injected into the SI. Scale bars represent 100 μm (top) and 10 μm (bottom, higher-magnification images).
(G) Statistical graphs showing co-labeling of CTB-labeled neurons and PKC-δ+ or SOM+ neurons in the CeL. n = 3 mice/group. Boxplots show mean (solid square), median, quartiles (boxes), and range (whiskers).
See also Figure S3.
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6. Figure 4
CeLPKC-δ+-SI Projections Modulate Negative Reinforcement Learning
(A) Schematic of the experimental approach.
(B) Top: confocal image of a coronal brain section taken from a PKC-δ-Cre mouse with AAV5-EF1α-DIO-hChR2-EYFP injected into the CeL. Scale bar, 200 μm. Bottom: ChR2-expresssion terminals were observed in the SI. Scale bar, 200 μm.
(C) Experimental design of the go/no-go task.
(D–G) Performance (D), discriminability (E), hit rate (F), and correct-rejection rate (G) of the three groups. Black line represents comparison of the ChR2-expressing group with laser as punishment (n = 6) and the control group with laser as punishment (n = 7); green line represents comparison of the ChR2-expressing group with laser as punishment and the control group with air puff as punishment (n = 6). n.s., not significant (two-way ANOVA with mixed design). (D) Black line: F(1, 11) = 17.26, ∗∗p = 0.002; green line: F(1, 10) = 8.23, ∗p = (G) Black line: F(1, 11) = 18.11, ∗∗p = 0.001; green line: F(1, 10) = 9.76, ∗p = Data represent means ± SEM.
(H) Schematic of targeted genetic inactivation of the CeLPKC-δ+-SI circuit.
(I) Representative images of the AAV injection site in the CeL (top) and ArchT-expressing terminals in the SI (bottom). Scale bar, 500 μm.
(J–M) Performance (J), discriminability (K), hit rate (L), and correct rejection rate (M) of ArchT (n = 8) and control mice (n = 8). n.s., not significant (two-way ANOVA with mixed design). (J) F(1, 14) = 37.90, ∗∗∗p < 0.001; (K) F(1, 14) = 16.81, ∗∗p = 0.001; (M) F(1, 14) = 42.35, ∗∗∗p < Data represent means ± SEM.
(N) Distribution of the response delay in the go trials.
(O) Median response delay of mice.
(P and Q) Performance (P) and correct-rejection rate (Q) of mice in the last session of learning without laser (training) and with laser (expression). n.s., not significant (Mann-Whitney U test). Boxplots show mean (solid square), median, quartiles (boxes), and range (whiskers).
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7. Figure 5 Activation of CeLPKC-δ+-SI Projections Drives CPA
(A) Procedure of CPP/A performance.
(B) Traces on day 1 (pre-test) and day 3 (post-test 1 and post-test 2).
(C) Post-test 1/pre-test and post-test 2/pre-test ratios of time spent in conditioned chamber. CeLPKC-δ+-SI-GFP (black), n = 7; CeLPKC-δ+-SI-ChR2 (blue), n = 7. Post-test 1, ∗p = 0.035; post-test 2, ∗∗p = (Mann-Whitney U test).
(D) Time spent in conditioned chamber on day 3 and day 1. Post-test 1: ∗p = 0.018; post-test 2: ∗∗p = (Mann-Whitney U test). Boxplots show mean (solid square), median, quartiles (boxes), and range (whiskers).
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8. Figure 6
SIVgluT2+ Neurons Are Indispensable for CPA Produced by CeLPKC-δ+ Neurons
(A) Procedure for RTPP performance.
(B) Examples of pre-test and real-time behavior traces accompanying photostimulation of SI VgluT2+ neurons.
(C–H) Aversion or preference for place was shown when SI VgluT2+ (C and D, VgluT2-GFP, n = 4; VgluT2-ChR2, n = 4), GABA+ (E and F, GABA-GFP, n = 5; GABA-ChR2, n = 4), and ChAT+ neurons (G and H, ChAT-GFP, n = 4; ChAT-ChR2, n = 5) were activated. n.s., not significant (Mann-Whitney U test). VgluT2+: RT/Pre, ∗p = 0.021; avoidance score, ∗p = GABA+: RT/Pre, ∗p = 0.027; avoidance score, ∗p =
(I) Diagram of viruses injected into the SI and CeL. Optical fiber was implanted above the SI for photostimulation of CeLPKC-δ+ outputs.
(J) Images show apoptosis neurons by TUNEL apoptosis detection after AAV-flex-taCasp3-TEVp or AAV2-EF1α-Flex-GFP was injected into the SI of VgluT2-Cre mice. Scale bar, 20 μm.
(K and L) Preference (K) and time spent (L) in the conditioned chamber after ablating SI VgluT2+ neurons compared with control (VgluT2-GFP, n = 7; VgluT2-taCasp3, n = 6). RT/Pre, VgluT2-GFP: RT/Pre, ∗p = 0.022; avoidance score, ∗p = (Mann-Whitney U test). Boxplots show mean (solid square), median, quartiles (boxes), and range (whiskers).
Cell Reports  , DOI: ( /j.celrep )
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