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).
Reduced presynaptic type III Nrg1 signaling decreases depolarization-induced FM1-43 destaining along vHipp axons. Cultures of vHipp microslices from WT (Nrg+/+) or heterozygous (Nrg+/−) mice were loaded with FM1-43. Representative micrographs of WT (Nrg+/+; A, left) and Nrg+/− (A, right) vHipp axons (loaded with FM1-43, green) before (A, top) and after (A, bottom) depolarization with elevated extracellular K+ are shown. Scale bar, 10μm. B, Bar graph showed no difference in the initial FM1-43 fluorescence intensities after K+-dependent loading between +/+ and +/− vHipp axons. The efficacy of depolarization in eliciting release was assayed from determinations of FM1-43 staining including measures of the % decrease with depolarization as well as any differences in the number and/or size of the FM1-43 clusters (C--E). C, The overall FM1-43 fluorescence intensity decreased along vHipp axons after depolarization in both Nrg+/+ and Nrg+/− vHipp axons, the magnitude of the effect is significantly lower for the Nrg+/− vHipp axons compared with control whether assessed as the percentage of total fluorescence intensity (C) or the percentage decrease in cluster number (D) or in the size of the FM1-43 clusters (E). Bar graphs of mean ± SEM of eight independent experiments. The effect of depolarization on transmitter release (assayed as FM1-43 destaining) significantly depressed in the Nrg+/− vHipp compared with the Nrg+/+ controls (**p < 0.01).
Reduced presynaptic type III Nrg1 slows depolarization-induced FM1-43 release. A, Spinning disk confocal images of FM1-43-loaded axons from either +/+ or +/− vHipp were collected every 1.5 s for 5 min, and FM1-43 fluorescence intensities were calculated and quantified as a normalized integrated intensity at each time point. The percentage decrease of normalized integrated intensity at an individual 1-µm spot (3 pixels) along vHipp axons were plotted versus time. Representative plots of the release time course from Nrg+/+ (red) and Nrg+/− (green) vHipp axon are shown before and after depolarization. B, Box plot of pooled data shows slower decay time constant (τ) of high K+ depolarization-induced FM1-43 destaining along axons from Nrg+/− (∼8.5 s, n = 20, 8) compared with Nrg+/+ (∼4.8 s, n = 36, 10 experiments; **p < 0.01).
ErbB4-induced back-signaling rescues presynaptic phenotype of type III Nrg+/− vHipp axons. A, Representative micrographs of Nrg+/+ (top), Nrg+/− (middle) and Nrg+/− with 24 h of treatment of soluble ErbB4 (bottom) vHipp axons loaded with FM1-43 (green) to visualize sites of vesicle clusters (scale bar, 10μm). B, Box plot of pooled data shows a significant decrease in the number of FM1-43-stained clusters along axons from Nrg+/− compared with Nrg+/+ vhipp axons. Following 24 h of treatment of Nrg+/− axons with 2 ng/ml soluble ErbB4-ECD, the number of FM1-43-stained clusters along Nrg+/− axons was rescued to Nrg +/+ levels; **p < 0.01. C, Representative micrographs of Nrg+/+ (top), Nrg+/− (bottom) vHipp axons stained with antibodies recognizing vGluT1 (red), and a pan axonal marker (green) are shown. Scale bar, 10μm. D, Quantification of vGluT1 cluster numbers along vHipp axons from Nrg+/+ (28 ± 4 per 100 μm) or Nrg+/− (28.5 ± 5.5 per 100 μm) shows that there is no significant difference between conditions. E, Box plot of pooled data shows no significant difference in the size of vGluT1 clusters along axons from Nrg+/− compared with Nrg+/+.
ErbB4-induced back-signaling reverses ultrastructural defects in the number of SV clusters. A, Representative electron micrographs of control (nonphotoconverted, PC-, top) and photoconverted (PC+; bottom) SVs along vHipp axons are shown (scale bar, 500 nm). The arrows indicate electron-dense material resulting from photoconversion of FM1-43 dye that is confined to the lumen of photoconverted s SVs. B, Representative electron micrographs of Nrg+/+ (top), Nrg+/− (middle), and Nrg+/− after 24 h of treatment with soluble ErbB4 (bottom) vHipp axons are shown. The arrows indicate the location of vesicle clusters (defined as ≥15 vesicles within less than a vesicle diameter of one another; scale bar, 500 nm). C, Box plot of pooled data (>40 axons from 2 separate experiments) shows a significant decrease in the number of vesicle clusters along axons from Nrg+/− compared with Nrg+/+; **p < 0.01. Following 24 h of treatment with 2-ng/ml soluble ErbB4-ECD, the total number of vesicle clusters per 100-µm axon length along Nrg+/− vHipp axons was rescued to levels comparable to Nrg+/+ control (**p < 0.01).