1. Volume 85, Issue 2, Pages 377-389 (January 2015)
Coexistence of Two Forms of LTP in ACC Provides a Synaptic Mechanism for the Interactions between Anxiety and Chronic Pain 
Kohei Koga, Giannina Descalzi, Tao Chen, Hyoung-Gon Ko, Jinshan Lu, Shermaine Li, Junehee Son, TaeHyun Kim, Chuljung Kwak, Richard L. Huganir, Ming-gao Zhao, Bong-Kiun Kaang, Graham L. Collingridge, Min Zhuo 
Neuron 
Volume 85, Issue 2, Pages (January 2015)
DOI: /j.neuron
Copyright © 2015 Elsevier Inc. Terms and Conditions

2. Figure 1 Presynaptic NMDAR-Independent LTP in the ACC
(A) Top: sample traces of eEPSCs with paired-pulse stimulation at 50 ms interstimulus interval during baseline (1) and 60 min after the pre-LTP (2) at a holding membrane potential of −60 mV. Middle: a time course plot of a representative single example. Bottom: time course plot of PPR for this neuron. The arrow donates the time of LTP induction.
(B) Pooled data to illustrate the time course of pre-LTP and changes in PPR. Top: pre-LTP (red, n = 20 neurons/16 mice) and control (white, n = 13/9). Bottom: PPR for these neurons. ∗p < 0.05 for PPR change estimated for the 10 min intervals baseline and between 50 and 60 min after the induction stimulus.
(C) An NMDA receptor antagonist, AP-5 (50 μM), did not affect pre-LTP (n = 12/9).
(D) BAPTA (20 mM) in the recording pipette did not block pre-LTP (n = 11/8).
(E) A PKMζ inhibitor, ZIP (5 μM), did not affect maintenance of pre-LTP, applied 30 min after the pre-LTP induction (n = 10/8).
(F) Summary of the effects of an NMDA receptor antagonist, postsynaptic injection of BATPA, or a PKMζ inhibitor on pre-LTP. There was statistical difference when comparing control with pre-LTP, BAPTA, AP-5, or ZIP groups (one-way ANOVA, F4,61 = 3.45, ∗p < 0.05). There was no difference comparing pre-LTP with BAPTA, AP-5, or ZIP groups (NS, p > 0.05). The mean amplitudes of eEPSCs were determined at 50–60 min after the pre-LTP induction stimulus. Error bars represent SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 2 Kainate Receptors Mediate the Induction of pre-LTP
(A) Top: in GluK1−/− mice, sample traces of eEPSCs with paired-pulse stimulation at 50 ms during baseline (1) and 60 min after the induction stimulus (2) at a holding membrane potential of −60 mV. Middle: GluK2−/− mice showed normal pre-LTP (black, n = 8 neurons/6 mice). GluK1−/− mice showed greatly reduced pre-LTP (red, n = 14/8). Bottom: PPR for the GluK2−/− (black) and GluK1−/− (red) groups.
(B) A specific GluK1 antagonist, UBP310 (10 μM), completely blocked pre-LTP (n = 8/6).
(C) Top: a GluK1 agonist, ATPA (1 μM for 10 min), induced long-lasting potentiation, recorded for 2 hr (n = 8/7). Bottom: averaged data of PPR change before and after ATPA application.
(D) Summary of the effects of GluK2−/−, GluK1−/−, a GluK1 antagonist, or a GluK1 agonist on pre-LTP. The amplitudes of eEPSCs in GluK1−/− or UBP310 groups were significantly decreased compared with control pre-LTP (one-way ANOVA, F5,60 = 5.18, ∗p < 0.05). There was no difference among control pre-LTP, GluK2−/−, and ATPA. Error bars represent SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2015 Elsevier Inc. Terms and Conditions

4. Figure 3 AC1 and PKA Are Required for Pre-LTP
(A) Top: sample traces of eEPSCs with paired-pulse stimulation at 50 ms during baseline (1) and 60 min after the induction (2). Bottom: pre-LTP was absent in AC1−/− mice (red, n = 13 neurons/8 mice). AC8−/− mice showed normal pre-LTP (gray, n = 9/7).
(B) A specific AC1 inhibitor, NB001 (50 μM), blocked pre-LTP (red circle, n = 12/8).
(C) A PKA inhibitor, KT5720 (1 μM), abolished pre-LTP (n = 11/8).
(D) Pre-LTP was unaffected in GluA1 S845A KI mice (n = 10/7).
(E) Summary of the effects of AC8−/−, AC1−/−, an AC1 inhibitor, a PKA inhibitor, or GluA1 S845A on pre-LTP. The amplitudes of eEPSCs in AC1−/−, NB001, or KT5720 groups were significantly decreased compared to control pre-LTP (one-way ANOVA, F5,69 = 5.69, ∗p < 0.05). There was no difference among pre-LTP, AC8−/−, and GluA1 S845A groups. Error bars represent SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2015 Elsevier Inc. Terms and Conditions

5. Figure 4 HCN Channels Are Critical for the Induction of Pre-LTP
(A) Top: a model of a retrograde labeling dye, DiI, injected into the ACC. Middle and bottom: DiI-negative and -positive neurons. Yellow color (right) indicates overlap of DiI positive (left) and biocytin (middle), indicating thalamocoritcal neurons. Scale bar, 20 μm.
(B) Top: single traces of Ih currents in a thalamic neuron (unlabeled). Middle: an HCN channel inhibitor, ZD7288 (10 μM) completely blocked the Ih currents. Bottom: voltage steps from −140 mV to −60 mV with 10 mV steps.
(C) Sample traces of Ih currents in a thalamocortical neuron (red) and an ACC neuron (blue).
(D) Expanded traces of Ih currents in (B) and (C).
(E) Summary of Ih currents in thalamocortical, unlabeled thalamus, and ACC neurons (thalamocortical neurons: n = 11 neurons/6 mice, unlabeled thalamus neurons: n = 11/6, ACC neurons: n = 14/8, two-way ANOVA, F2,297 = 33.16, ∗p < 0.05 at each voltage comparing thalamocortical neurons with ACC neurons, #p < 0.05 at each voltage comparing thalamocortical neurons with unlableled thalamus neurons).
(F) Western blot analysis of HCN1–HCN4 channel proteins in thalamus and the ACC.
(G) Within the asymmetric synapses, HCN1, HCN2, and HCN4 immunostained nano-gold particles were observed in both pre- and postsynaptic regions.
(H) The distribution ratio of HCN immunostained particles in the cytoplasm and membrane of the pre- and postsynaptic profiles. Error bars represent SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2015 Elsevier Inc. Terms and Conditions

6. Figure 5 HCN Channels Are Involved in the Maintenance of Pre-LTP
(A) An HCN channel inhibitor, ZD7288 (10 μM), blocked the induction of pre-LTP (red, n = 9 neurons/6 mice). Open circles showed normal pre-LTP as a control (n = 8/6).
(B) Post-LTP was insensitive to ZD7288 (n = 7/7).
(C) ZD7288 blocked the maintenance of pre-LTP, applied 30 min after the pre-LTP induction (n = 9/9).
(D) Summary of pre-LTP and post-LTP experiments investigating HCN channels. The amplitudes of eEPSCs in the two ZD7288 pre-LTP groups were significantly decreased compared with pre-LTP without ZD7288 (one-way ANOVA, F3,30 = 5.09, ∗p < 0.05).
(E) ZD7288 (10 μM) did not alter the amplitudes of eEPSCs (n = 6/6, paired t test, t5 = −1.320, NS; top). The PPR was not changed by ZD7288 (n = 6/6, paired t test, t5 = 0.845, NS; bottom). Traces 1 and 2 show recordings before and in the presence of ZD7288 (10 μM) for 20 min, respectively.
(F) Recordings of eEPSCs from FosGFP-negative neurons in sham group (black), FosGFP-positive neurons in nerve injury (red), and FosGFP-negative neurons in nerve injury (gray) from FosGFP transgenic mice. Traces 1 and 2 show recordings before and in the presence of ZD7288 for 20 min, respectively.
(G) ZD7288 reduced the amplitudes of eEPSCs in FosGFP-positive neurons (n = 13/12), but not in FosGFP-negative neurons of nerve injury (n = 12/10) or in FosGFP-negative neurons of the sham-operated mice (n = 10/8).
(H) ZD7288 altered the PPR in FosGFP-positive neurons of nerve injury (n = 13/12), but not in FosGFP-negative neurons of nerve injury (n = 12/10) or in FosGFP-negative neurons of the sham mice (n = 10/8).
(I) Summary of the effects of ZD7288 on the amplitude of eEPSCs in FosGFP transgenic mice. The FosGFP-positive group, activated by nerve injury, were statistically different from the sham or nerve injury groups (FosGFP-negative with sham: n = 10, FosGFP-negative with injury: n = 12, FosGFP-positive with injury: n = 13, one-way ANOVA, F2,32 = 6.93, ∗p < 0.05, FosGFP-positive in injury versus FosGFP-negative in sham or FosGFP-negative in injury).
(J) Summary of the effects of ZD7288 on the PPR of the same neurons (one-way ANOVA, F2,32 = 7.79, ∗p < 0.05, FosGFP-positive in injury versus FosGFP-negative in sham or FosGFP-negative in injury). Error bars represent SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2015 Elsevier Inc. Terms and Conditions

7. Figure 6
Loss of Pre-LTP in Chronic Inflammatory and Nerve Injury Models
(A) Experimental models of pain and fear: nociceptive heat stimulation by hot plate (55°C, tested immediately after withdrawal), 2–3 days after chronic inflammatory pain models, 7–8 days after nerve injury models, and fear conditioning (tested immediately and 2–3 days after the conditioning).
(B) Mice exposed to the hot plate showed normal pre-LTP (n = 9 neurons/6 mice).
(C) Mice exposed to the chronic inflammatory pain model did not exhibit pre-LTP after the injury (red, n = 11/7). Sample traces of eEPSCs before (1) and 60 min after (2) the induction stimulus.
(D) Mice exposed to the nerve injury model did not exhibit pre-LTP after the injury (red, n = 15/9). Sample traces of eEPSCs before (1) and 60 min after (2) the induction stimulus.
(E) Mice exposed to trace fear conditioning immediately (blue, n = 10/8) and 2–3 days after the conditioning (black, n = 10/8) showed normal pre-LTP in the ACC. Single traces of evoked EPSCs before (1) and 60 min after (2) after the pre-LTP (immediately after the conditioning).
(F) Summary of pre-LTP experiments in acute nociceptive, chronic pain models, or fear conditioning models. The levels of pre-LTP in the inflammation and nerve injury groups were significantly decreased compared with control pre-LTP group (one-way ANOVA, F5,69 = 5.52, ∗p < 0.05 compared to control pre-LTP). There was no difference among control pre-LTP, hot plate, or fear conditioning groups (immediately or 2–3 days after the conditioning). Error bars represent SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2015 Elsevier Inc. Terms and Conditions

8. Figure 7
HCN Activity in the ACC Is Required for Nerve Injury-Triggered Anxiety-like Behavior
(A) Mice exposed to the raised open platform did not exhibit pre-LTP in the ACC (red, n = 14 neurons/8 mice). Post-LTP in the ACC was normal in mice exposed to the raised open platform (gray, n = 8/6). Red traces indicate EPSCs during baseline (1) and 60 min after the pre-LTP induction protocol (2). Gray traces show eEPSCs during baseline (1) and 60 min after the pairing protocol (2).
(B) Summary of pre-LTP and post-LTP experiments in the mice exposed to a raised open platform. The levels of pre-LTP were significantly smaller after the EPM compared with pre-LTP in naive mice (one-way ANOVA, F4,37 = 4.34, ∗p < 0.05). The mice exposed in the raised open platform for 5 min and placed in the home cage for 9 hr showed normal pre-LTP compared to control pre-LTP (166% ± 11%, n = 4/3, NS).
(C) Sample traces of pre-LTP in mice following 5 min exposure to closed arms (blue) and normal EPM (orange). Traces 1 and 2 show baseline and 60 min after pre-LTP induction, respectively.
(D) Mice from the closed arm group showed normal pre-LTP (159% ± 12%, n = 8/7). Mice tested in the normal EPM for 5 min displayed a smaller pre-LTP (125% ± 15%, n = 9/8).
(E) ZD7288 increased the time spent in the open arms of the EPM for the nerve injury group but had no effect on the sham injury group (Sham treated with saline: n = 7 mice, Sham treated with ZD7288: n = 6, Nerve injury treated with saline: n = 8, Nerve injury treated with ZD7288: n = 8, two-way ANOVA, F1,25 = 5.55, ∗p < 0.05, sham with saline versus injury with saline, saline versus ZD7288 in injury groups).
(F) In the EPM, total crossings were not significantly different among all four groups (F1,25 = 1.55, NS).
(G) ZD7288 increased the distance traveled in the center of the open field test in the nerve injury group but had no effect on the sham group (F1,25 = 18.40; ∗p < 0.05, sham with saline versus injury with saline, saline versus ZD7288 in injury groups).
(H) Total distance traveled in the open field test was not significant different between all four groups (F3,150 = 0.77, NS).
(I) ZD7288 microinjected bilaterally into the ACC of the nerve injury group significantly reduced mechanical allodynia (sham with saline: n = 7 mice, sham with ZD7288: n = 6, nerve injury with saline: n = 7, nerve injury with ZD7288: n = 7, two-way ANOVA, F1,23 = 10.99, ∗p < 0.05, sham with saline versus nerve injury with saline, saline versus ZD7288 in injury groups). Error bars represent SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2015 Elsevier Inc. Terms and Conditions

9. Figure 8
A Hypothetical Scheme for How Synaptic Plasticity May Encode Synergistic Interactions between Chronic Pain and Anxiety
(A) Basal transmission: the synaptic response, evoked by low-frequency stimulation, is due to glutamate activating AMPARs in the ACC.
(B) Chronic pain: peripheral nerve injury enhances excitatory synaptic transmission through postsynaptic LTP. The activation of NMDARs leads to an increase in postsynaptic Ca2+ influx. The Ca2+ binds to calmodulin (CaM) and leads to activation of Ca2+-stimulated signaling pathways via AC1. The pathway leads to the enhancement of postsynaptic AMPARs containing the GluA1 subunit and produces post-LTP.
(C) Anxiety: normal anxiety enhances the release of glutamate, which feeds back onto presynaptic GluK1 containing KARs. The presynaptic Ca2+ influx via GluK1 KARs leads to activation of the AC1-PKA pathway, which then results in modulation of HCN channels resulting in a long-lasting increase in glutamate release (pre-LTP).
(D) Chronic pain and anxiety: nerve injuries and the associated anxiety activate both pre-LTP and postsynaptic LTP of excitatory transmission in the ACC. The synergistic interaction between pre-LTP and post-LTP underlies the heightened behavioral response.
Neuron  , DOI: ( /j.neuron )
Copyright © 2015 Elsevier Inc. Terms and Conditions