Fig 1: Reduced presynaptic type III Nrg1 signaling impairs glutamatergic synaptic transmission at vHipp-nAcc synapses. A, Schematic of genotype-specific in vitro circuits. Glutamatergic transmission at vHipp-nAcc synapses was examined in gene chimeric cocultures. Experiments show ventral hippocampus/subiculum slices from an individual Nrg+/+ or Nrg+/- mouse plated as micro thinned explants. Dispersed nucleus accumbens neurons from WT mice were added 24 h later. B, Representative traces of spontaneous glutamate-receptor mediated synaptic activity (Glu mEPSCs: bicuculline and TTX resistant; CNQX-APV sensitive) recorded from +/+ vHipp to +/+ nAcc synapses (top) and from +/- vHipp to +/+ nAcc synapses (bottom). C, Box plots of mEPSC frequency data from +/+ vHipp to +/+ nAcc and from +/- vHipp to +/+ nAcc reveal more than a 3-fold difference (**p < 0.01, n = 8 for each condition) at gene chimeric synapses compared with WT- WT synapses. D, Box plots of mEPSC amplitude data from +/+ vHipp to +/+ nAcc and from +/- vHipp to +/+ nAcc reveal a 2-fold difference (**p < 0.01, n = 8 for each condition) at gene chimeric synapses compared with WT- WT synapses. E, Examination of pre and postsynaptic markers in gene chimeric vHipp-nAcc cocultures. After 5-7 d in vitro, cocultures were fixed, permeabilized, and stained with antibodies targeted to vesicular glutamate transporter 1 (vGluT1; red) and to PSD95 (green). Red “clusters” of vGluT1 are colocalized with PSD95 (green) on neurites of dispersed nAcc neurons innervated by +/+ vHipp (E, top left) or +/- vHipp (E, bottom left; scale bar, 10µm). The arrows indicate colocalization of PSD95 with vGLuT1 (yellow puncta) along neurites of nAcc MSNs. The number of PSD95 positive/vGluT1 clusters along neurites of dispersed nAcc neurons innervated by +/+ vHipp were significantly greater than those innervated by +/- vHipp inputs (28 ± 4 per 100 µm, n = 9, 5 vs 17 ± 3 per 100 µm n = 6, 3; where n= the number of samples/experiment and the number of separate experiments; **p < 0.01; E, middle, left). The number of PSD95 clusters along neurites of nAcc neurons were counted and the bar graph (E, middle, right) showed no difference between +/+ and +/- vHipp innervation (+/+vHipp to +/+nAcc: 60 ± 10, n = 9, 5 vs +/-vHipp to +/+nAcc: 54 ± 10, n = 6, 3; p > 0.05). The number of vGluT1 clusters along neurites of nAcc neurons were also counted and the bar graph (E, right) showed no difference between +/+ and +/- vHipp innervation (+/+ vHipp to +/+ nAcc: 31 ± 4, n = 9, 5 vs +/- vHipp to +/+ nAcc: 29 ± 8, n = 6, 3; p > 0.05). F, Examination of surface versus total glutamate A2/A3 receptor subtypes (GluA2) in gene chimeric vHipp-nAcc cocultures. After 5-7 d in vitro, cocultures were stained with antibodies targeted to GluA2. For labeling of sGluA2 (green), the cultures were incubated with anti-GluR2 antibody, extracellular, for 45 min before fixation, and then, cultures were fixed, permeabilized, and total GluA2 (red) were recognized with anti-GluR2 + GluR3 antibody, C terminal. Clusters of surface (green) vs total GluA2/3 (red) can be found on neurites of dispersed nAcc neurons innervated by vHipp axons (F, left panel; scale bar, 5 µm). The ratio of integrated intensities of sGluA2 versus total GluA2 between gene chimeric conditions differed in a statistically significant manner (+/+ vHipp to +/+ nAcc: 0.36 ± 0.07, n = 10, 4 vs +/- vHipp to +/+nAcc: 0.18 ± 0.02, n = 8, 3; **p < 0.01; F, right-hand panel). G, Examination of synapse formation of sGluA2 (red) and vGluT1 (green) along nAcc neurites in vHipp-nAcc cocultures. After 5-7 d in vitro, cocultures were incubated with anti-GluR2 antibody recognizing an extracellular epitope, for 45 min to label sGluA2, and then, cultures were fixed, permeabilized, and stained with antibody targeted to vGluT1. Representative micrographs of vGluT1 and sGluA2 colocalization are shown, the arrows are examples of those colocalized sites (yellow puncta) of sGluA2 (red) and vGLuT1 (green) along neurites of nAcc MSNs (G; scale bar, 5 µm).