Fig 1: Activating IL–pBLACalb1 circuit ameliorates anxiety- and depression-like behaviours in CUMS mice.a Schematic of IL–pBLA photostimulation and pBLACalb1 inhibition in CUMS mice during OFT, EPM, and SPT. AAV-CaMKII-ChR2 and AAV-DIO-hM4Di were injected into the IL and pBLA of Calb1-Cre mice, respectively. At the 5th week of CUMS training, fibres were implanted in the pBLA. 30-32 min before behaviour tests, CNO was intraperitoneally injected. b, c, e–g, h ChR2-CUMS mice were tested in a single 9 min session in OFT or EPM with three 3 min epochs. ChR2-CUMS mice showed less anxiety- and depression-like behaviours during the illumination epoch, evidenced by greater centre entries (b) and time staying in the centre (c) in OFT, more open-arm exploration (e, f) and shorter open-arm entry latency (g) in EPM, and higher sucrose preference in SPT (h), while the suppression on anxiety- and depression-like behaviours were reversed when Calb1+ neurons in the pBLA were inhibited by CNO injection. No difference on travelled distance was detected during and after light stimulation or CNO injection (d). Two-way ANOVA group × epoch interaction. In OFT: n = 11 mice per group, [Centre entries]: F(4,90) = 3.611, P = 0.0089; [Time in centre]: F(4,90) = 5.541, P = 0.0005; [Distance moved]: F(2,60) = 0.3377, P = 0.7174. In EPM: n = 9 (ChR2:IL-pBLA, HM4D: pBLA+ChR2:IL-pBLA) or 10 (eYFP:IL-pBLA) mice, [Open arm entries]: F(4,75) = 2.651, P = 0.0396; [Time in open arm]: F(4,75) =9.704, P < 0.0001; [open-arm entry latency]: F(4,75) = 3.344, P = 0.0142. [Sucrose preference]: n = 9 mice per group, F(2,48) = 34.86, P < 0.0001. Bonferroni post hoc analysis, **P < 0.01 vs eYFP:IL-pBLA+veh:pBLA; #P < 0.05, ##P < 0.01 vs ChR2:IL-pBLA+veh:pBLA. Data were presented as mean ± SEM. i Proposed working model. The excitatory IL–pBLACalb1 circuit drives reward generalization for ambiguous information in a reversible manner. Activating IL–pBLACalb1 pathway promotes reward generalization for the ambiguity, reducing anxiety- and depression-like behaviours. Source data are provided as a Source Data file.
Fig 2: Expression of the H-latch mimic R207C-I138C in organotypic cultured slices leads to dose-dependent neurotoxicity.(a) Flow cytometry gating strategy of PI positive CAD5 cells. (b) PNGase-F digestion of cell lysates induced a shift in both murine wild-type PrPC and mPrP2cys, indicating that both moieties had undergone N-linked glycosylation to a similar extent. Non-adjacent lanes were merged from the same gel. (c) CAD5 Prnp-/- cells expressing mPrP2cys did not show an upregulation of the unfolded protein response, suggesting that mPrP2cys did not undergo pathological degradation. Values are given as percentage of empty control vector (p-eIF2a / eIF2a / actin). One datapoint per group corresponds to a different cell culture passage. Two-sided, unpaired t-test. (d) A POM2/POM3 sandwich ELISA of COCS transduced with empty control, mPrPC, mPrP2cys and buffer control shows robust mPrP2cys expression in transduced COCS, albeit significantly less than wild-type mPrPC. Slices were harvested at 28 days post-transduction. One datapoint corresponds to an independent, biological replicate of 6–9 pooled slices. Ordinary, one-way Anova with Šídák’s multiple comparisons test, *: adjusted p-value = 0.039 (e) Reduced levels of mNG in Prnp-/- (ZH3) COCS expressing mPrP2cys. mNG immunoreactivity values are divided by actin immunoreactivity and expressed as percentages of empty control. Slices were harvested at 28 days post-transduction. One datapoint corresponds to an independent, biological replicate of 6–9 pooled slices. Ordinary, one-way Anova with Šídák’s multiple comparisons test. Raw, uncropped blots can be found in the Source Data supplement. (f) Quantification of mNG and Calb1 fluorescence intensity from experiments shown in Fig. 3d-f. One datapoint corresponds to a biological replicate, e.g. one organotypic cultured slice. Unpaired, two-tailed t-test without adjustment for multiple testing. P-values are as follows: 31 dpt, 5.2x1010 vg*ml-1, mNG: 0.001; 31 dpt, 5.2x1010 vg*ml-1, Calb1: 0.0496; 15 dpt, 1.4x1011 vg*ml-1, mNG: 0.0065; 15 dpt, 1.4x1011 vg*ml-1, Calb1: 0.001. ***: p = 0.001, **: p < 0.01, * p < 0.05. Source data
Fig 3: Robust expression and conformation of the PrPR207A point mutant.(a) Representative images of expression levels of Synapsin 1 (Syn1, upper) and Calbindin 1 (Calb1, lower) show predominant (Syn1) or almost exclusive (Calb1) expression in Purkinje cells (pc) in the cerebellar cortex. Image credit: Allen Institute. Scale bar = 100 µm. (b) Fluorescent micrographs of PrnpZH3/ZH3 COCS transduced with the AAV outlined in panel (A) show mNeonGreen expression predominantly in calbindin 1-expressing Purkinje cells. Scale bar = 50 µm. cgl = cerebellar internal granular layer, pc = Purkinje cell layer, ml = molecular layer. These findings were repeated in three independent experiments. (c) Left panel: Stably transfected CAD5-mPrPC and CAD5-mPrPR207A cells show similar PrPC expression levels. epresentative PrPC levels of one cell culture passage are shown. Right panel: POM19 immunoreactivity is divided by actin immunoreactivity, values are given as percentages of PrPC. One datapoint corresponds to one passage of CAD5 cells. (d) Surface plasmon resonance (SPR) traces showing binding of POM1 to recombinant mPrPR207A (rmPrPR207A, ka=3.8E+05 1/Ms, kd=1.8E-04 1/s, KD=4.7E-10 M; for comparison binding to recombinant wild-type murine PrP showed ka=3.6E+05 1/Ms; kd=9.1E-05 1/s; KD=2.5E-10 M). (e) Immunohistochemistry of CAD5 Prnp-/- cells stably transfected with pcDNA3.1 vector expressing wild-type murine PrPC (mPrPC), mPrPR207A and mPrP2cys. Monoclonal anti-PrPC antibodies targeting distinct conformational epitopes on the globular domain of PrPC were incubated to assess conformational changes in mPrPR207A (POM1: a1-a3, POM5: ß2-a2, POM8: a1-a2, POM19: ß1-a3). Except for diminished staining of POM1 in mPrPR207A, we observed robust detection of mPrPR207A by POM5, POM8 and POM19 and mPrP2cys by POM8 and POM19. Parts of this experiment, for example POM1 and POM19, were repeated twice. Scale bar = 20 µm. Source data
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