Adenylyl cyclase subtype 1 is essential for late-phase long term potentiation and spatial propagation of synaptic responses in the anterior cingulate cortex of adult mice

A MED64 recording system has been recently used to map synaptic responses from different layers of adult mouse ACC [11, 16, 33]. In the present study, we first examined the network L-LTP in the ACC of adult C57 mice. The location of the 8x8 array MED64 probe electrodes within the ACC slice is shown in Figure  1A and B. One channel (Figure  1B, yellow circle) that located on the deep layer V of the ACC was chosen as the stimulation site and field excitatory post-synaptic potentials (fEPSPs) recorded from the other channels around the stimulation site were recorded. Figure  1C showed an example of such experiments and fEPSPs were recorded from 18 channels in both deep (V/VI) and superficial layers (II/III) of the ACC. After one hour of stable baseline recording, theta burst stimulation (TBS) was applied to induce L-LTP. We found that TBS induced stable L-LTP that lasted for 3 h in most of the active channels (Figure  1D). The fEPSPs slopes and the total number of activated channels including those with or without L-LTP in both superficial and deep layers were then analyzed. Figure  1E indicates the slope of two channels both located in the superficial layers: Ch. 35 showed a long-lasting LTP (141.9% of baseline at 3 h after TBS) but Ch. 34 did not undergo any potentiation (94.6% of baseline at 3 h after TBS). The averaged data from 6 channels in superficial layers of the sample slice was plotted in Figure  1F (LTP: 146.3 ± 9.2% of baseline at 3 h after TBS). In deep layers, similar results were observed and the slope of two channels (Ch. 34 and 36) were plotted in Figure  1G: Ch. 36 showed a long-lasting LTP (141.2% of baseline at 3 h after TBS) but Ch. 34 did not show any potentiation (98.3% of baseline at 3 h after TBS). The averaged data from 12 channels in deep layers of the same slice was plotted in Figure  1H (LTP: 145.3 ± 6.2% of baseline at 3 h after TBS). L-LTP was similar within both the superficial and deep layer. We found the fEPSP from 77.8% of all active channels were significantly potentiated (77 from 99 active channels; 6 slices/6 mice). The mean percentage of potentiation was 155.2 ± 4.4% in the 77 channels. In other 22 channels, no obvious potentiation was detected. The overall changes of all 99 channels was 140.4 ± 10.3% of the baseline at 3 h after TBS (P < 0.001, paired t-test, Figure  1I).

A previous study has shown that an AC1 inhibitor NB001 blocks E-LTP in the ACC [21]. Here we tested whether bath application of NB001 could induce the similar blocking effect on the network L-LTP in the ACC slices. In the presence of NB001 (0.1 μM), we found that L-LTP induction was totally blocked both in superficial and deep layers (Figure  2A-G). Figure  2C showed an example of two active channels (Ch. 27 and 35) in the superficial layers with NB001 application. After TBS, they all failed to undergo any potentiation (Ch. 27 was 102.0% and Ch. 35 was 98.3% of baseline at 3 h after TBS). In a total of 9 channels in the sample slice, TBS did not induce L-LTP (101.4 ± 6.3% of baseline at 3 h after TBS) in the superficial layers (Figure  2D). Similar results were found in deep layers (Figure  2E-F). In two randomly selected active channels (Ch. 29 and 36), no potentiation was observed (Ch. 29 was 103.1%; Ch. 36 was 100.2% of baseline at 3 h after TBS) (Figure  2E). TBS did not induce LTP in all 13 channels (106.2 ± 10.7% of baseline at 3 h after TBS) (Figure  2F). The mean slope of fEPSPs reached 98.5 ± 4.5% of the baseline at 3 h after TBS (n = 6 slices/6 mice; P = 0.085, paired t-test, Figure  2G). Meanwhile, bath application of NB001 did not change the basal synaptic transmission (103.0 ± 6.0% of the baseline after application for 1 h, n = 6 slices/6 mice, Figure  2H).

Gabapentin is an anti-epileptic agent and recommended as the first line agent for neuropathic pain treatment [34, 35]. It is believed that gabapentin binds to α2δ-1, a subunit of voltage gated calcium channel, to produce analgesia [36]. However, the effect of gabapentin on synaptic plasticity has not been reported yet. We then tested whether gabapentin affect the network L-LTP in the ACC. We found that even at a high concentration (100 μM), gabapentin had no effect on the L-LTP and most of the active channels in superficial and deep layers showed potentiation (Figure  3A-G). Ch. 42 and 43 were two randomly selected channels in the superficial layers. After TBS, Ch. 43 showed a long-lasting LTP (132.4% of baseline at 3 h after TBS) but Ch. 42 did not undergo any potentiation (95.9% of baseline at 3 h after TBS) (Figure  3C). The averaged data from 8 channels in superficial layers of the sample slice reached 137.6 ± 4.0% of baseline at 3 h after TBS) (Figure  3D). Similar results were observed in the deep layers. In Figure  3E, Ch. 36 showed a long-lasting LTP (137.8% of baseline at 3 h after TBS) but Ch. 12 did not have any potentiation (96.3% of baseline at 3 h after TBS). The averaged data from 11 channels in deep layers was 135.2 ± 10.0% of baseline at 3 h after TBS. In a total of 6 slices from 6 mice, the mean fEPSP slope of 97 active channels (76 channels with L-LTP, 78.4% of all activated channels) was 136.2 ± 9.0% of the baseline (P < 0.001, paired t-test, Figure  3G). However, gabapentin application directly decreased the basal synaptic transmission (80.9 ± 7.4% of the baseline, after application for 1 h, n = 6 slices/6 mice; P < 0.001, paired t-test; Figure  3H).

Multi-channels recording provides a convenient way to study the cortical network L-LTP. We then mapped the spatial distribution of the active responses in the ACC before and after TBS by using the previously method [11, 12, 30, 33]. The distribution of all observed activated channels during the whole recording progress was displayed by a polygonal graph on a grid representing the channels (the blue lines represent the activated channels during the baseline and the red lines represent the activated channels after TBS). When we stimulated the local site in deep layer, the spread of active response was observed in both deep and superficial layers around the stimulation site. As described before, closer to the stimulation site, more channels could be activated. Moreover, the spreading leaned to the superficial layers (Figure  4A). During the baseline recording, 99 channels from 6 slices/6 mice were activated (26.2% of all 378 channels, 16.5 ± 1.5 per slice in average). However, sixteen channels that had no responses during baseline recording were recruited and showed responses at 3 h after TBS (4.2% of all 378 channels at 3h after TBS, Figure  4A-B, G). Similar to our previous results of Fmr1 WT mice [11], the recruited channels were located on the edge of the originally active area (Figures  1D, 4A).

We then tested the effect of NB001 and gabapentin on the spatial distribution and network recruitment in the ACC. We found that bath application of NB001 and gabapentin did not affect the number of activated channels (NB001:101 activated channels, 16.8 ± 1.5 per slice in average; gabapentin: 97 activated channels, 16.2 ± 1.8 per slice in average; n = 6 slices/6 mice in each group, P > 0.05 in comparison with the control group, unpaired t-test). However, NB001 but not gabapentin blocked the TBS induced channel recruitment (NB001, -1 recruited channels in total; gabapentin, 14 recruited channels in total, n = 6 slices/6 mice, Figure  4C-F, G). When standardizing the area of one grid as 1, the sum area of all channels in control slices was counted as 49. The average activated areas were 10.5 ± 1.1 and 11.8 ± 1.3 before and after TBS, whereas the increased area was 1.3 ± 0.0% per slice (P < 0.001, paired t-test, Figure  4H). Similarly, NB001 but not gabapentin blocked the increasing areas 3 h after TBS (NB001:10.8 ± 1.3 and 10.6 ± 1.4 before and after TBS, P = 0.175, paired t-test; gabapentin: 10.1 ± 1.4 and 11.3 ± 1.4 before and after TBS, P < 0.001, paired t-test. Figure  4H).

The time course of the recruitment changes in control group were shown in Figure  5. The number of recruited channels gradually increased across the extended time scale after TBS induction and finally reached 2.7 ± 0.2 per slice at 3 h after induction. The averaged amplitude of the recruited responses also gradually potentiated and finally reached as large as 16.7 ± 2.3 μV. NB001 but not gabapentin blocked the TBS induced channel recruitment (NB001: -0.2 ± 0.2 recruited channels per slice at 3 h after TBS, P = 0.363, paired t-test; gabapentin: 2.3 ± 0.2 recruited channels per slice at 3 h after TBS, P < 0.001, paired t-test). The averaged amplitude of the recruited responses finally reached as 1.9 ± 0.3 μV or 14.2 ± 0.2 μV in the presence of NB001 or gabapentin.

To confirm the results from the multi-channel recording, the effects of NB001 and gabapentin were also tested by using whole-cell patch-clamp recording method. One site in deep layer V was stimulated and the evoked excitatory postsynaptic currents (eEPSCs) in superficial layers (II/III) were recorded (Figure  6A). Ten min after a stable baseline recording, spike timing protocol [14] were applied and an obvious LTP of the synaptic responses were induced. The averaged amplitude of EPSCs increased to 143.4 ± 10.9% of the baseline at 30 min after induction (P < 0.001, paired t-test; n = 6 neurons/6 slices, Figure  6B, E). In accordance with the field recording results, incubation with NB001 (0.1 μM) blocked the potentiation (102.2 ± 13.3% of the baseline at 30 min after induction, P = 0.143, n = 6 neurons/6 slices, Figure  6C, E). However, bath application of gabapentin (100 μM) did not block the spike timing induced potentiation (155.5 ± 24.2% of baseline at 30 min after induction, P < 0.001, n = 6 neurons/6 slices, paired t-test, Figure  6D, E).

We then tested the effects of NB001 and gabapentin on the basal synaptic transmission. Similar to the field recording results, we found that NB001 did not affect the amplitude of the evoked EPSCs (106.8 ± 15.5% of the baseline 20 min after bath application, P = 0.359, n = 6 neurons/6 slices, paired t-test; Figure  6G, I). However, gabapentin significantly reduced the amplitude of the eEPSCs (68.4 ± 9.9% of the baseline 20 min after bath application, P < 0.001, n = 6 neurons/6 slices; paired t-test; Figure  6H, I). To determine whether the synaptic changes observed with NB001 and gabapentin are due to presynaptic or postsynaptic mechanisms, AMPA receptor-mediated miniature EPSCs (mEPSCs), paired-pulse ratio (PPR) and fast blockade of NMDA receptor-mediated eEPSCs were tested [19, 20]. According to the quantal analysis, changes in the amplitude of mEPSCs are related to the postsynaptic alteration, while changes in the frequency typically reflect the alteration in presynaptic glutamate release [16, 37, 38]. Next we tested whether bath application of NB001 and gabapentin affect the amplitude or the frequency. We found that NB001 had no effect on both the frequency (Baseline: 1.2 ± 0.3 Hz, NB001: 1.3 ± 0.3 Hz, P = 0.135, paired t-test, n = 8 neurons/8 slices) and amplitude (Baseline: 15.5 ± 0.8 pA, NB001: 15.8 ± 0.7 pA, P = 0.673, paired t-test, n = 8 neurons/8 slices) of the mEPSCs (Figure  7A). However, gabapentin application caused a significant decrease in the amplitude of mEPSCs (Baseline: 15.8 ± 0.3pA, gabapentin: 14.8 ± 0.4 pA, P = 0.01, paired t-test, n = 7 neurons/7 slices), without affecting the frequency (Baseline: 1.1 ± 0.2 Hz, gabapentin: 1.1 ± 0.1 Hz, n = 7 neurons/7 slices, P = 0.99, paired t-test; Figure  7B). These results indicate that gabapentin may decrease the basal transmission through postsynaptic mechanisms.

Similar with the mEPSCs frequency analyses, paired-pulse ratio is a simple form of presynaptic plasticity, in which the response to the second stimulus is enhanced as a result of residual calcium in the presynaptic terminal after the first stimulus [38, 39]. Thus, to further determine the possible presynaptic change after NB001 and gabapentin application, we examined the PPR at different stimulus intervals of 35, 50, 75, 100 and 150 ms. We found that neither NB001 nor gabapentin changed the PPR in the ACC neurons (NB001: F _{(1, 70)}  = 0.012, P = 0.913; n = 8 neurons/8 slices; Gabapentin:F _{(1, 60)}  = 0.006, P = 0.937, n = 7 neurons/7 slices, Two-way ANOVA; Figure  7C-D). These results further indicate that the effect of NB001 and gabapentin are not associated with presynaptic release, although they have different effect on the basal synaptic transmission.

It has been widely reported that the blocking rate of NMDA receptor-mediated synaptic current by MK-801, a selective and non-competitive NMDA receptor antagonist, is correlated with glutamate release probability [40, 41]. Thus, testing the blocking rate of NMDA current could reflect whether the release probability was changed [19]. In slices with or without NB001 or gabapentin, we found that MK-801 could progressively block NMDA EPSCs and completely inhibited the current in 20 min (Figure  7E). By analyzing the time required for peak amplitude of NMDA EPSC to 50% decay of initial value in MK-801, we found no significant differences of the blocking rates among NB001-, gabapentin-treated and control groups (control: 5.85 ± 0.41 min; NB001: 5.78 ± 0.55 min; gabapentin: 5.98 ± 0.55 min. NB001 vs. control, P = 0.998; gabapentin vs. control, P = 0.694, One-way ANOVA, both from 7 neurons/7 slices). These results indicate that neither NB001 nor gabapentin affect the probability of presynaptic neurotransmitter release. Taken together, all these results suggest that NB001 has no effect on the basal synaptic transmission, whereas gabapentin may decrease the synaptic transmission through inhibiting the postsynaptic elements, instead of inhibiting the presynaptic glutamate release in the ACC.

Mice were anesthetized with ether and decapitated. The whole brain was rapidly removed and transferred to ice cold oxygenated (95% and 5%) artificial cerebrospinal fluid (ACSF) containing (in mM): 124NaCl, 25 NaHCO₃, 2.5 KCl, 1 KH₂PO₄, 2 CaCl₂, 2 MgSO₄ and10 glucose, pH 7.4. After cooling for about 1–2 min, appropriate portions of the brain were then trimmed and the remaining brain block was glued onto the stage of a vibrating tissue slicer (Leica VT1200S). Three coronal brain slices (300 μm), after the corpus callosum meets and contains ACC, were cut and transferred to a chamber with oxygenated ACSF at room temperature for at least 1.5 h [52].

The procedures for preparation of the MED64 probe were similar to those described previously [11]. The MED64 probe (MED-P515A, 8 × 8 array, inter-polar distance 150 μm) was perfused with ACSF at 28–30°C with the aid of a peristaltic pump (Minipuls 3, Gilson) during the whole experimental period of electrophysiological recording. Before use, the surface of the MED64 probe was treated with 0.1% polyethyleneimine (Sigma, St. Louis) in 25 mM borate buffer (pH 8.4) overnight at room temperature. In addition, the probe surface was rinsed 3–5 times with sterile distilled water before immediate use in each experiment.

After incubation, one slice was transferred to the recording chamber and suffused with ACSF at a 2 ml/min flow rate. The slices were positioned on the MED64 probe in such a way that the whole array of the electrodes can cover the different layers of the ACC, with middle part of the probe close to the central point of the ACC. One of the channels located in the layer V of the ACC, from which the best synaptic responses can be induced in the surrounding recording channels, was then chosen as the stimulation site. Channels in which field potentials can be induced were considered as active. A microphotograph of one ACC slice positioned on the MED64 probe was shown in Figure  1A-B. After the baseline responses were stabilized for at least 1 h, a TBS protocol (10 bursts at 5 Hz, 4 pulses at 100 Hz for each burst) was given 5 times (10 s interval) at the same stimulation site to induce L-LTP.

For whole cell patch clamp recording, slices were transferred to a submerged chamber and superfused (2 ml/min) with oxygenated ACSF at 28–30°C. Experiments were performed in a recording chamber on the stage of a BX51W1 (Olympus) microscope equipped with infrared DIC optics for visualization. Excitatory postsynaptic currents (EPSCs) were recorded from superficial layers neurons with an Axon 200B amplifier (Axon Instruments). Patch pipettes with resistances of 3–5 MΩ were filled with the following solution (in mM): 120 K-gluconate, 5 NaCl, 1 MgCl₂, 0.2 EGTA, 10 HEPES, 2 Mg-ATP, 0.1 Na₃-GTP and 10 phosphocreatine disodium (adjusted to pH 7.2 with KOH). The local stimulations were delivered by a bipolar tungsten stimulating electrode placed in deep layer (layer V/VI). AMPA receptor–mediated EPSCs were induced by repetitive stimulations at 0.05 Hz, and neurons were voltage-clamped at -60 mV. LTP was induced by spike timing protocol (three presynaptic stimuli at 30 Hz, which caused three EPSPs, were paired with three postsynaptic action potentials (APs), and did 15 times with an interval of 5 s. Presynaptic stimulus was delivered 10 ms before postsynaptic AP). For miniature EPSC (mEPSC) recording, 0.5 μM tetrodotoxin was added to the perfusion solution. Picrotoxin (100 μM) was always present to block GABA_A (γ-aminobutyric acid type A) receptor–mediated inhibitory synaptic currents both in AMPA receptor-mediated EPSCs and mEPSC. NMDA EPSCs were recorded for 20 min in MK-801 (35 μM) with 0.1 Hz stimulation [14].

The chemicals and drugs used in this study were as follows: gabapentin, MK-801, picrotoxin and tetrodotoxin were purchased from Sigma (St. Louis). Drugs were prepared as stock solutions for frozen aliquots at -20°C. All these drugs were diluted from the stock solutions to the final desired concentration in ACSF before used.

Whole-cell patch-clamp data were collected and analyzed with Clampex 10.3 and Clampfit 10.2 software (Axon Instruments). MED64 Mobius was used for data acquisition and analysis. The percentages of the fEPSP slopes were normalized by the averaged value of the baseline. We defined LTP in a channel if the response was increased by at least 15% of baseline during this period. Results are expressed as mean ± SEM. The student’s t-test, One-way ANOVA and two-way ANOVA were used for Statistical comparisons. The level of significance was set at P < 0.05.