Fig 2: BDNF is enriched in glutamatergic corticostriatal presynaptic terminals. A, Confocal (top) and SIM (bottom) microscopic images showing BDNF-IR in the same section in glutamatergic (left) versus dopaminergic terminals (right) in the dorsal striatum. B, BDNF-IR is present in VGluT1-positive terminals (magenta arrows). Single BDNF-IR signals overlap with TH (white arrows). VGluT1- and TH-positive terminals reside in direct regional proximity but do not overlap. C, Quantification of BDNF signals in VGluT1-positive terminals and TH-positive terminals. True colocalization between BDNF/VGluT1 was confirmed by Costes p value (Costes p > 0.95) but not between BDNF/TH (Costes p « 0.95). D, Representative Western blots showing recombinant BDNF (lanes 1, 2) versus endogenous BDNF derived from anterior cortex or striatum of P21 NFL-Cre BDNFfl/ko mice (lane 3), P21 sedentary mice (lanes 4, 5), and P21 runners after 72 h voluntary running-wheel exercise (lanes 6, 7); 30 µg of protein lysate was loaded for each sample. BDNF levels were normalized to cytochrome C. Band intensities were determined from extracts of 9 independent mice and presented in % of P21 sedentary mice. Statistical analysis reveals significant increase in BDNF protein levels in both brain areas after running-wheel exercise. Statistical analysis: unpaired t test (anterior CTX: t = 5,312, p < 0.0001; striatum: t = 2,784, p = 0.0133). E, SIM images showing BDNF-IR in VGluT1-positive terminals in the dorsal striatum in sedentary mice (top row) and after 72 h of voluntary running-wheel exercise (bottom row). Data are presented as box and whiskers (Tukey). +, Mean. Vertical line indicates median. Black dots indicate outliers. n, number indicated below. Raw data are provided in Extended Data Figure 7-1 and Table 2. Scale bars: A, 2.5 µm; B, 1.5 µm; E, Overview, 2 µm; Detail, 1 µm. *p < 0.05; ****p < 0.0001.

Fig 3: Quantitative Analysis of Pre- and Postsynaptic Molecules following Presynaptic [Ca2+] Measurements with Two-Photon Microscopy(A) Two-photon maximum intensity projection image of a local axon collateral of a CA1 PC filled with Alexa594 (25 µM), Fluo5F (100 µM), and biocytin. The scanned boutons (#1–8) are indicated.(B) Fluorescent [Ca2+] transients in individual boutons evoked by single APs (top panel) or 5 APs (at 40 Hz, bottom panel; arrows indicate APs). Note that single AP-evoked rapid-rising [Ca2+] transients were not detected in boutons #2, 3, 5, and 7.(C) Confocal image stack of the same axon collateral as in (A) after fixing and embedding in epoxy resin.(D) High-magnification confocal images of the boutons after resectioning at 200 nm. Bouton numbers correspond to those in (A) and (C). Images are from either single sections (middle and right panels) or maximum intensity projections of 2 consecutive sections (left panel).(E–G) Immunolabeling for Munc13-1 (E), PSD95 (F), and vGluT1 (G). Open white ROIs indicate the position of the boutons.(H) Normalized integrated fluorescent intensity values show that boutons with [Ca2+] transients (filled symbols, n = 4) have higher Munc13-1 and PSD95 labeling than boutons without single AP-evoked [Ca2+] transients (open symbols, n = 4), whereas all boutons contain vGluT1.

Fig 4: Loss of GABAergic transmission in individual neurons did not change glutamatergic transmission. (A) mEPSCs recording showed loss of GABAARs in individual neurons did not change glutamatergic transmission. Hippocampal neurons were transfected with ß1-3 gRNAs at DIV3 and recorded at DIV14-17 (n = 25 for GFP and ß1-3 gRNAs; p > 0.05 for mEPSC frequency and amplitude, t-test; Kolmogorov-Smirnov test was used for cumulative graphs, p > 0.05 for both conditions, N = 4). Scale bar, 100 ms, 20 pA. (B) Single-cell genetic deletion of GABAARs did not change the expression levels of surface GluA1 (GFP, n = 17; ß1-3 gRNAs, n = 19; p > 0.05 for both conditions, t-test; N = 3). GFP was not immunolabeled with anti-GFP antibodies. Scale bar, 5 µm. (C) Single-cell genetic deletion of GABAARs did not change the vGluT1 puncta (GFP, n = 24; ß1-3 gRNAs, n = 28; p > 0.05 for both conditions, t-test; N = 3). GFP was not immunolabeled with anti-GFP antibodies. Scale bar, 5 µm. (D) The density of dendritic spines was not altered in hippocampal neuron expressing ß1-3 gRNAs at DIV 18 (GFP, n = 26; ß1-3 gRNAs, n = 35; p > 0.05, t-test; N = 3). GFP was immunolabeled with anti-GFP antibodies to boost the fluorescence (green). Scale bar, 5 µm. (E) Normal spine types in hippocampal neurons expressing ß1-3 gRNAs (GFP, n = 26; ß1-3 gRNAs, n = 28; p > 0.05, t-test; N = 3). GFP was immunolabeled with anti-GFP antibodies to boost the fluorescence (green). Scale bar 1 µm. n represents the number of cells analyzed and N represents the number of independent experiments.

Fig 5: Subcortical brain targets of PFC-SERT+ neurons and their large contribution to the PFC-to-DRN synaptic circuit. a Schematic illustration of the injection site in the PFC. The AAV2/1-CAG-LSL-EGFP-bGH virus was used for conditional anterograde tracing in SERTCre/+ mice. Injection was done at P4–5 and histology 3 weeks after. b Effective recombination is visible in PFC neurons that express EGFP (indicated by arrows). c-e Main brain targets of PFC-SERT+ neuron axons: c Thalamic nuclei including the paraventricular (PV), mediodorsal (MD), rhomboid (Rh), ventromedial (VM), and ventrolateral (VL) nuclei; (d) the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA); (e) the dorsal raphe nucleus (DRN) and periaqueductal gray (PAG). Laterodorsal nucleus (LD), aqueduct (Aq), substantia nigra pars reticulata (SNpr). f AAV1-CAG.FLEX-TdTomato-WPRE-bGH was bilaterally injected in the PFC of P4-P5 SERTCre/+ mice used for the array tomographic anterograde tracing analysis in the DRN. g Array tomography image of seven serial-ultrathin 70-nm sections immunolabeled against synapsin (green), VGLUT1 (blue), and TdTomato (red), after anterograde viral tracing from the PFC. Asterisks indicate the same PFC TdTomato-positive axon terminal across the multiple serial sections often colabeled for VGLUT1 and synapsin. The arrow indicates a cortical axon bouton negative for TdTomato. The pie chart shows the percentages of cortical axon boutons present in the DRN that exclusively originated from PFC-SERT neurons (67 ± 8 %, red) in comparison with those arising from other cortical neurons (33 ± 8 %, blue)