A Cooperative Mechanism Involving Ca2+-Permeable AMPA Receptors and Retrograde Activation of GABAB Receptors in Interpeduncular Nucleus Plasticity  Peter Koppensteiner, Riccardo Melani, Ipe Ninan  Cell Reports  Volume 20, Issue 5, Pages 1111-1122 (August 2017) DOI: 10.1016/j.celrep.2017.07.013 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 20, 1111-1122DOI: (10.1016/j.celrep.2017.07.013) Copyright © 2017 The Authors Terms and Conditions

Figure 1 Activity-Dependent Potentiation of Glutamatergic Transmission in IPN Neurons (A) Schematic presentations of the positions of the stimulating and recording electrodes in the brain slice preparation containing the IPN (left) and the MHb-IPN synapse (right). (B) Example traces before and 20 min after the 20- and 100-Hz stimulation or at the same time points in control group (scale bar 25 ms/10 pA). (C) Examples of EPSC measurements at 0.1 Hz from one cell each of control, 20-Hz, and 100-Hz groups. Horizontal dashed lines represent average EPSC amplitude during a 5-min baseline, and vertical dashed lines represent the time point of stimulation. (D) Average EPSC amplitude in control, 20-Hz, and 100-Hz groups. A 20-Hz (5 s) or 100-Hz (1 s) stimulation produced a long-lasting enhancement of EPSC amplitude (n = 9 neurons/5 mice for both groups) compared to the control group that did not receive the high-frequency stimulation (n = 9 neurons/4 mice). Arrow represents the application of the 20- or 100-Hz stimulation. Error bars reflect SEM. (E) Paired pulse ratio before and after the 100-Hz stimulation. Left panel shows example traces before and 20 min after the 100-Hz stimulation (scale bar 25 ms/50 pA). Cell Reports 2017 20, 1111-1122DOI: (10.1016/j.celrep.2017.07.013) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Activity-Dependent Potentiation of Glutamatergic Transmission in the MHb-IPN Synaptic Pathway (A) Schematic presentation of optogenetically evoked EPSC recording in IPN neurons of ChAT-ChR2-EYFP mice. (B) Examples of control and 100-Hz stimulation experiments in slices from ChAT-ChR2-EYFP mice. Horizontal dashed lines represent average EPSC amplitude during a 5-min baseline, and vertical dashed lines represent the time point of stimulation. (C) Left panel shows example traces before and 20 min after the 100-Hz stimulation or at the same time points in control group (scale bar 25 ms/10 pA). Blue bar indicates light activation. Right panel shows average EPSC amplitude in control and 100-Hz groups. A 100-Hz (1 s) stimulation produced a long-lasting enhancement of EPSC amplitude (n = 9 neurons/6 mice) compared to the control group that did not receive the high-frequency stimulation (n = 9 neurons/5 mice). Arrow represents the application of the 100-Hz stimulation. Error bars reflect SEM. Cell Reports 2017 20, 1111-1122DOI: (10.1016/j.celrep.2017.07.013) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Induction, but Not the Expression, of 100-Hz-Stimulation-Induced Potentiation of Glutamatergic Transmission in the IPN Neurons Depends upon GABAB Receptors (A) Inclusion of the GABAB antagonist CGP 55845 in the bath solution blocked the 100-Hz stimulation-induced potentiation of glutamatergic transmission (10 neurons/7 mice). However, CGP 55845 failed to block this potentiation when perfused 5 min after the 100-Hz stimulation (11 neurons/6 mice). 100 Hz group is the same data presented in the Figure 1. Upper panels show examples of experiments from 100 Hz, CGP+100 Hz, and CGP after 100 Hz groups. Horizontal dashed lines represent average EPSC amplitude during a 5-min baseline, and vertical dashed lines represent the time point of stimulation. Lower left panels show examples of traces for 100 Hz (scale bar 25 ms/50 pA), CGP+100 Hz (scale bar 25 ms/10 pA), and CGP after 100 Hz (scale bar 25 ms/50 pA) groups. Lower right panel shows the average EPSC amplitude in 100 Hz, CGP+100 Hz, and CGP after 100 Hz groups. Arrow represents the application of 100-Hz stimulation. Dashed gray line represents perfusion of CGP 55845 after the 100-Hz stimulation. (B) Right panel shows the effect of 100 Hz stimulation on IPSC amplitude in IPN neurons (n = 9 neurons/6 mice). Middle panel shows examples of traces of IPSCs before and at 20 min after 100 Hz stimulation (scale bar 25 ms/100 pA). Left panel shows an example of a single experiment. Arrow represents application of the 100-Hz stimulation. Error bars reflect SEM. Cell Reports 2017 20, 1111-1122DOI: (10.1016/j.celrep.2017.07.013) Copyright © 2017 The Authors Terms and Conditions

Figure 4 100-Hz-Stimulation-Induced Plasticity in IPN Neurons Is Independent of NMDA and Muscarinic Receptors and Is Absent during Adolescence (A) An inclusion of the NMDA receptor antagonist, AP5 (9 neurons/6 mice), or muscarinic receptor antagonist, atropine (11 neurons/6 mice), in the bath solution did not affect 100-Hz-stimulation-induced potentiation of glutamatergic transmission in IPN neurons. A 100-Hz stimulation for 1 s produced a long-lasting enhancement of EPSC amplitude (n = 10 neurons/5 mice). Upper panels show examples of experiments from 100 Hz, AP5+100 Hz, and atropine+100 Hz groups. Lower left panels show example traces before and 20 min after the 100-Hz stimulation in 100 Hz (scale bar 30 ms/10 pA), AP5+100 Hz (scale bar 25 ms/20 pA), and atropine+100 Hz (scale bar 25 ms/10 pA) groups. Lower right panel shows the average EPSC amplitude in 100 Hz, AP5+100 Hz, and atropine+100 Hz groups. (B) 100-Hz-stimulation-induced potentiation of glutamatergic transmission in IPN neurons is absent in adolescent mice (n = 10 neurons/6 mice). Left panel shows an example of a single experiment. Middle panel shows example traces before and 20 min after the 100-Hz stimulation (scale bar 30 ms/20 pA). Right panel shows the average EPSC amplitude. Arrow represents the application of 100 Hz stimulation. Error bars reflect SEM. See also Figure S1. Cell Reports 2017 20, 1111-1122DOI: (10.1016/j.celrep.2017.07.013) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Lack of Synaptic CPARs at the vMHb-IPN Glutamatergic Synapses of Adolescent Mice (A) Left panels show example traces of EPSCs recorded at −60, −40, −20, 0, +20, +40, and +60 mV in IPN neurons of adult and adolescent mice. Blue bar indicates light activation. Current/voltage relationship (I-V) plots for synaptic currents show inward rectification in adults, but not in adolescent group. (B) Slopes of the lines connecting EPSC amplitudes at −60–0 and at 0–+60 mV in adult (16 cells/4 mice) and adolescent (16 cells/4 mice) groups. (C) Rectification index (RI) in adult and adolescent mice. (D) Inclusion of CPAR antagonist, NASPM, in the bath solution blocked 100-Hz-induced potentiation of glutamatergic transmission in the adult IPN neurons (9 neurons/5 mice) compared to the 100-Hz group (n = 9 neurons/7 mice). Arrow represents the application of 100 Hz stimulation. (E) Example traces of electrically evoked IPSCs before, during, and after the application of NASPM (scale bar 10 ms/50 pA) and examples of experiments on the effect of NASPM on IPSC amplitudes in adult and adolescent groups. (F) NASPM produced a significantly stronger reduction of IPSC amplitudes in adult IPN neurons (9 neurons/6 mice) compared to adolescent IPN neurons (9 neurons/5 mice). In (A), (C), (D), and (F), error bars reflect SEM. See also Figure S2. Cell Reports 2017 20, 1111-1122DOI: (10.1016/j.celrep.2017.07.013) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Optogenetically Induced Release of GABA from IPN Neurons Potentiates Glutamate Release by Activation of GABAB Receptors and Occludes 100-Hz-Stimulation-Induced Synaptic Potentiation (A) Optogenetic activation (10 Hz for 15 s) of IPN neurons in VGAT-ChR2-EYFP mice resulted in long-lasting enhancement of glutamatergic transmission, which occluded 100-Hz-electrical-stimulation-induced synaptic plasticity (9 neurons/8 mice, right panel). Left panel shows an example of a single experiment. Middle panel shows example traces of EPSCs before and after optogenetic and 100 Hz electrical stimulation, respectively (scale bar 10 ms/75 pA). (B) CGP 55845, but not NASPM, blocked optogenetically induced long-lasting enhancement of glutamatergic transmission. Left panel shows examples for experiments from CGP-55845- and NASPM-treated slices. Right panel shows average EPSC amplitude in CGP 55845 (10 neurons/6 mice)- and NASPM (9 neurons/5 mice)-treated slices that received 10-Hz stimulation. (C) Schematic presentation of potential mechanism in activity-dependent potentiation of glutamatergic transmission at the MHb-IPN synapses. Activation of CPARs enhances Ca2+ levels in the post-synaptic IPN neurons, leading to the release of GABA, which acts on the pre-synaptic GABAB receptor on the MHb terminals to enhance glutamate release. Lack of CPARs in IPN neurons might be responsible for the impaired activity-dependent plasticity in adolescent mice. CIAR, Ca2+-impermeable AMPA receptors. In (A) and (B), error bars reflect SEM. See also Figures S3 and S4. Cell Reports 2017 20, 1111-1122DOI: (10.1016/j.celrep.2017.07.013) Copyright © 2017 The Authors Terms and Conditions

Figure 7 Suppression of Activity-Dependent Plasticity after Fear Conditioning and Its Reversal by Fear Extinction (A) Example traces of sEPSCs in tone alone (TA), fear-conditioned (FC), and fear-extinguished (FE) groups (scale bar 500 ms/10 pA). (B) Average sEPSC frequency and amplitude in TA (36 neurons/21 mice), FC (29 neurons/15 mice), and FE (39 neurons/20 mice) groups. (C) Examples of 100-Hz-stimulation-induced plasticity experiments from TA, FC, and FE groups. (D) Example EPSC traces before and 20 min after 100 Hz stimulation in TA (scale bar 25 ms/20 pA), FC (scale bar 25 ms/25 pA), and FE (scale bar 25 ms/75 pA) groups. (E) Average EPSC amplitude before and after 100 Hz stimulation in TA (15 neurons/12 mice), FC (14 neurons/11 mice), and FE (13 neurons/11 mice) groups. In (B) and (E), error bars reflect SEM. See also Figure S5. Cell Reports 2017 20, 1111-1122DOI: (10.1016/j.celrep.2017.07.013) Copyright © 2017 The Authors Terms and Conditions