Fig. 3: Withdrawal selectively decreases post-synaptic glutamate transmission in mPFCCRF1+ neurons, which is partly mediated by the basolateral amygdala.
A Representative traces of average spontaneous excitatory post-synaptic currents (sEPSC) recorded with a holding potential of −70 mV in mPFCCRF1− pyramidal neurons from naïve, dependent, and withdrawn mice. B, C Average sEPSC frequency and amplitude in mPFCCRF1− neurons from naïve (white), dependent (black), and withdrawn (grey) mice. n = 12–22 cells from N = 6–10 male mice; *p < 0.05, **p < 0.01 by one-way ANOVA and post hoc multiple comparisons compared to naive. D Representative traces of average sEPSCs recorded with a holding potential of −70 mV in mPFCCRF1+ neurons from naïve, dependent, and withdrawn mice. E, F Average sEPSC frequency and amplitude in mPFCCRF1+ pyramidal neurons from naïve (green), dependent (white), and withdrawn (grey) mice. n = 10–21 cells from N = 6–10 male mice; *p < 0.05, **p < 0.01 by one-way ANOVA and post hoc multiple comparisons compared to naive. G Schematic of viral strategy for ex vivo optogenetic circuit dissection of glutamate transmission in the basolateral amygdala (BLA) to mPFCCRF1+ pathway. Representative 4X magnification, brightfield and mCherry fluorescence images of injection site expression of channelrhodopsin-2 (ChR2)-mCherry in the BLA (right, top) and corresponding ChR2-mCherry BLA terminal in the mPFC (right, bottom). H ChR2-mediated photocurrents in mCherry expressing BLA neurons elicited by pulses and increasing trains of blue-light stimulation from a holding potential of −70 mV. I Representative BLA-mediated AMPA currents in mPFCCRF1+ neurons elicited by paired pulse stimulation (traces scaled to first EPSC) of two consecutive blue light pulses (3 ms, 470 nm) with an interstimulus interval of 200 ms from a holding potential −80 mV. J Average paired pulse ratio (i.e., amplitude of the second response normalized to that of the first) in mPFCCRF1+ neurons from naïve, dependent, and withdrawn mice with a significant F(2,57) = 4.27, p = 0.01 effect by one-way ANOVA, and post hoc significance *p < 0.05 compared to naïve is represented in panel. n = 12–27 cells from N = 5–6 male mice. K Average ± SEM BLA-mediated NMDA (top row) and AMPA (bottom row) currents in mPFCCRF1+ neurons from naïve (green), dependent (black), and withdrawn (grey) mice. L Average AMPA/NMDA ratio in mPFCCRF1+ neurons from naïve, dependent, and withdrawn mice with a significant F(2,86) = 6.79, p = 0.001 effect by one-way ANOVA, and post hoc significance *p < 0.05 compared to naïve is represented in panel. n = 17–39 cells from N = 5–6 male mice. M Average ± SEM BLA-mediated AMPA currents elicited by blue light pulses during baseline and following 200 nM CRF application from a holding potential of −80 mV in mPFCCRF1+ neurons from naive, dependent, and withdrawn mice. N Average baseline normalized AMPA amplitude elicited by optical stimulation of BLA terminals with a significant F(2,16) = 3.55, p = 0.05 effect of CRF across groups by one-way ANOVA and post hoc multiple comparisons, and significant *p < 0.05 and **p < 0.01 effects of CRF by one sample t-test. n = 4–11 cells from N = 3–6 male mice per group.
