Fig 2: Exosomal PRR7 is sufficient for the removal of excitatory synapses. a Representative immunofluorescent images of PSD-95/vGLUT and vGAT/GABAAR?2 in hippocampal neurons treated with CS harvested from GFP control-transfected or HA-PRR7-transfected neurons. b Quantitation of the CS effect on excitatory and inhibitory synapses. c, d Quantitation of the CS effects on PSD-95 harvested from GFP-transfected, HA-PRR7-transfected or PRR7-GFP-transfected neurons. Cluster density (c) and intensity (d). n = 12 per condition. e Exosomal secretion of various PRR7 mutants. CB Coomassie blue staining. f Reversal of PSD-95 clusters by replacing PRR7-overexpressing CS with normal conditioned medium at 12 h. g Time course of the effect of HA-PRR7-exosomes on PSD-95. Top panels show representative images and quantified data are shown bottom. n = 15 each. h–j Uptake of exosomal PRR7 by neurons. Representative images of hippocampal neurons treated with exosomes purified from either GFP-transfected (control) or HA-PRR7-transfected neuron culture. Total staining of HA and PSD-95 in recipient neurons after the spot application of exosomes (h). Total and surface staining of HA and MAP2 after the application of evenly mixed HA-PRR7-containing exosomes (i). Western blotting analyses of TCLs prepared from Txf and exosome-treated recipient (Rec.) neurons (j). Scale bars, 5 (a), 10 (g), and 20 µm (h, i). All data are mean ± SEM. Two-way (b) or one-way ANOVA (c, d) Post hoc Tukey’s test, F1,56 = 30.71 (b), F2,33 = 78.78 (c), and F2,33 = 14.66 (d). ***P < 0.0001. Mr relative molecular weight

Fig 3: TMTC3 is expressed in hypothalamus and piriform cortex of rat brain and co-localizes with vesicular GABA transporter (VGAT) but not vesicular glutamate transporter 2 (Vglut2). (A,B) Low magnification images of TMTC3 immunoreactivity in hypothalamus adjacent to the third ventricle (A) and in piriform cortex (layers 1-3 are indicated) (B). Both images show punta surrounding immunonegative cell bodies; examples of which are marked with white arrows (scale bar is 100 µm). (C–E) Confocal images of the rat hypothalamus showing that TMTC3 does not co-localize with Vglut2, a marker of excitatory nerve terminals. (C) shows Vglut2 staining while panel (D) shows TMTC3 expression. (E) is a merged image stack. (F–H) VGAT is co-localized with TMTC3 in a subpopulation of VGAT positive puncta in the hypothalamus, along the third ventricle. (F) shows VGAT immunoreactivity while (G) shows TMTC3 immunoreactivity. (H) Merged image showing that TMTC3 and VGAT are co-localized in puncta surrounding cell bodies. White represents voxels, where TMTC3 and VGAT are colocalized in three dimensions (60X mag, scale bar is 20 µm). In (I), we show merged channels and co-localization of TMTC3/VGAT at 60 X magnification in piriform cortex. (layers 1–3 are indicated).

Fig 4: ACC projection to the BLA is monosynaptic and glutamatergic. a Anterograde terminal tracing strategy based on expression of eGFP-Syb2. Confocal image shows mCherry-expressing cells selectively observed in the ACC. Scale bar, 500 µm. b Representative confocal images showing eGFP-Syb2 expression pattern in the amygdala sub-nuclei. Strong eGFP-Syb2 expression was observed in the BLA. Scale bar, 200 µm. c Quantitative analysis of the eGFP-Syb2 signals in the amygdala sub-nuclei (across three different mice). Fluorescence intensity was normalized to mean intensity value (0–255 scale) measured in the LA area. d Retrograde tracing strategy using CAV2-Cre-dependent expression of DIO-eYFP. e Representative confocal image showing eYFP-expressing cells in the ACC. Scale bar, 500 µm. f Quantitative analysis of the retrogradely labeled neurons (eYFP+) in the ipsilateral and contralateral ACC. g Representative confocal images showing VGLUT1 or VGAT immunoreactive signals with eGFP-Syb2 signals. Scale bar, 10 µm. h Bar graph showing co-localized puncta in the BLA. i Illustration of ex vivo patch-clamp recording experiments for monitoring light-evoked PSPs in ACC input-identified BLA neurons. j Representative epi-fluorescence image indicating the patch-clamp recording site. Bottom left image, magnification of the black square area. tdTomato-positive (tdT+) cells were selectively targeted, as indicated by red arrow. Scale bar, 500 µm. k Proportion of neurons displaying PSP responses to light stimulation. Total 17 tdT+BLA neurons were successful for whole-cell patch-clamp recording from 10 slices of 5 mice. 12 of 17 cells were PSP-positive (PSP+). l Representative recording traces indicating light-evoked PSPs in the ACSF (black), and in the presence of bicuculline (+bic; blue), bicuculline+4-AP+TTX (+4AP/TTX; green), or bicuculline+4-AP+TTX+NBQX+APV (+NBQX/APV; red). m PSP latency of the PSP+/tdT+BLA neurons recorded (n = 12 cells). n Summary of amplitudes of light-evoked PSPs measured from each drug treatment condition (n = 6 cells from 6 slices of 3 mice). Repeated measures one-way ANOVA (F(1.167, 5.837) = 6.900, P = 0.0376) with Dunnett’s post hoc test (ACSF vs.+NBQX/APV; P = 0.0106). Data are expressed as mean ± s.e.m. *P < 0.05. o Anterograde tracing strategy based on transneuronal delivery of AAV1-hSyn-Cre. p Representative confocal images showing the pattern of tdT+expression in the ACC (left, scale bar: 500 µm) and the amygdala (right, scale bar: 200 µm). q Quantitative analysis of the tdT+neurons in the BLA and CeA. BMA, basomedial nucleus; LA, lateral nucleus; CeA (CeM, CeL, and CeC), central nucleus (medial, lateral and capsular subdivisions) of the amygdala15

Fig 5: Fractional abundance of immunostained P2 events in different FSC ranges and in different sample buffers. These experiments were conducted using FM triggering on the Influx. A, Representative density plots of P2 samples immunostained for VGAT, VGLUT1, and VMAT2 and run in PBS. Top, Secondary-only controls in which primary antibodies were omitted. Bottom, Immunostained with indicated primary antibodies. B, Box and whiskers plot of immunostained events as indicated, expressed as a percentage of all events within the respective FSC gate. Central bar represents the median. Lower and upper edges correspond to 25th and 75th percentiles. Lower and upper whiskers extend to the smallest or largest value no greater than 1.5 times the interquartile range away from the corresponding edge. Data are derived from P2 samples triple stained and washed in PBS, and subsequently run in either PBS (n = 6) or SET (n = 6) as sample buffer. Two-way ANOVA was performed for each dataset. Left, For VGAT+ events, FSC gate (Df = 5, F = 21.51, p = 2.9e-12) and buffer (Df = 1, F = 4.70, p = 0.034) were both significant, with no significant interaction (Df = 5, F = 0.40, p = 0.85). Middle, For VGLUT1+ events, only FSC gate (Df = 5, F = 68.9, p < 2e-16) was significant, while buffer (Df = 1, F = 0.36, p = 0.55) and the interaction (Df = 5, F = 0.19, p = 0.97) were not. Right, For VMAT2+ events, FSC gate (Df = 5, F = 12.54, p = 2.4e-8) and buffer (Df = 1, F = 5.21, p = 0.026) were both significant, with no significant interaction (Df = 5, F = 0.27, p = 0.93). C, Box and whiskers plots of single-positive, double-positive, and triple-positive events expressed as a percentage of all fluorescent events within the respective FSC gate. Data are derived from the same immunostained P2 samples as in B. Two-way ANOVA was performed for each dataset. Left, For single-positive events, FSC gate (Df = 5, F = 19.83, p = 1.3e-11) and buffer (Df = 1, F = 6.74, p = 0.012) were both significant, with no significant interaction (Df = 5, F = 0.15, p = 0.98). Middle, For double-positive events, FSC gate (Df = 5, F = 23.25, p = 6.6e-13) and buffer (Df = 1, F = 8.00, p = 0.006) were both significant, with no significant interaction (Df = 5, F = 0.40, p = 0.85). Right, For triple-positive events, only FSC gate (Df = 5, F = 8.37, p = 4.7e-6) was significant, while buffer (Df = 1, F = 1.91, p = 0.17) and the interaction (Df = 5, F = 0.19, p = 0.97) were not. See also Extended Data Figures 7-1, 7-2, 7-3.