Enhanced long-term potentiation in the anterior cingulate cortex of tree shrew

Synaptic plasticity is a key cellular model for learning, memory and chronic pain. Most previous studies were carried out in rats and mice, and less is known about synaptic plasticity in non-human primates. In the present study, we used integrative experimental approaches to study long-term potentiation (LTP) in the anterior cingulate cortex (ACC) of adult tree shrews. We found that glutamate is the major excitatory transmitter and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid (AMPA) receptors mediate postsynaptic responses. LTP in tree shrews was greater than that in adult mice and lasted for at least 5 h. N-methyl-d-aspartic acid (NMDA) receptors, Ca2+ influx and adenylyl cyclase 1 (AC1) contributed to tree shrew LTP. Our results suggest that LTP is a major form of synaptic plasticity in the ACC of primate-like animals. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.


Introduction
Synaptic plasticity is a well-studied phenomenon for regulating the intensity of synaptic transmission and is considered to be the cellular model for learning and memory, chronic pain, anxiety and fear [1][2][3][4][5][6][7].However, most recent studies of long-term potentiation (LTP) have been carried out on rodents, while the information on LTP in primates is still scarce.Recent studies of LTP in monkeys have focused on imaging or behaviours [8,9], and less is known about cortical synaptic transmission and LTP [10].Compared with rodents, the tree shrew (Tupaia belangeri) shares much more similar genome sequences with primates and is considered to have a close affinity to primates [11,12].Considering its small body size, low cost of breeding and short reproductive cycle, the tree shrew provides a potentially useful animal model for investigating different physiological and pathological functions in primates (vision; spatial learning, stress and emotion adjustment) [13][14][15][16][17][18][19].There are few studies of synaptic physiology and plasticity in the adult tree shrews.
The anterior cingulate cortex (ACC) is an important cortical brain area for regulating chronic pain, fear memory and anxiety [1][2][3][20][21][22][23][24][25][26].In primates, ACC can be activated by noxious stimulation, pleasant touch, odours and taste [27][28][29].In patients with frontal lobotomies or cingulotomies, the perception of pain and the ability to identify voice and facial expressions are abolished, accompanied by changes to social behaviour and subjectively experienced emotions [30,31].LTP is considered to play a pivotal role in regulating these physiological and pathological processes.Controlling LTP in the ACC can reduce behavioural hyperalgesia [32].Therefore, it is important to investigate the features of LTP in tree shrews.
In the present study, which aimed to reveal the character of cortical LTP in the tree shrew, we used a 64-channel multi-electrode dish (MED64) recording system, whole-cell patch recording and calcium imaging to record the LTP and the network property of LTP in the ACC of tree shrew.We found that theta burst stimulation (TBS) induced a long-lasting LTP in the ACC of adult tree shrews.The amplitude of LTP in the adult tree shrew is greater than that of adult mice.Similar to the ACC of adult mice, N-methyl-d-aspartic acid (NMDA) receptors, especially the GluN2B receptor, and L-type Ca 2+ channels, are important for excitatory synaptic transmission.

Methods (a) Animals
Experiments were performed with adult male tree shrews (purchased from Kunming Institute of Zoology) and C57BL/6 J mice.All tree shrews and mice were maintained on a 12 h light/dark cycle with food and water provided ad libitum.All experimental protocols were approved by the Animal Care and Use Committee of the University of Xi'an Jiaotong University.

(b) Slice preparation
The general methods for preparing ACC slices of tree shrew were similar to those previously described for mice [23,33,34].Adult male tree shrews and mice were anaesthetized with isoflurane, the whole brain was quickly removed from the skull and submerged in ice-cold, oxygenated (95% O 2 and 5% CO 2 ) cutting solution containing (in mM) 252 sucrose, 2.5 KCl, 6 MgSO 4 , 0.5 CaCl 2 , 25 NaHCO 3 , 1.2 NaH 2 PO 4 and 10 glucose, pH 7.3-7.4.After cooling in the cutting solution for a few minutes, the whole brain was trimmed to create an appropriate plane to glue onto the ice-cold platform of a vibrating tissue slicer (VT1200S, Leica).Coronal brain slices (300 µM), containing the ACC, were prepared after the corpus callosum connected.After cutting, slices were then incubated in a submerged recovery chamber with the artificial cerebrospinal fluid (ACSF) containing the following (in mM): 124 NaCl, 4.4 KCl, 2 CaCl 2 , 1 MgSO 4 , 25 NaHCO 3 , 1 NaH 2 PO 4 and 10 glucose, pH 7.3-7.4,for at least 1 h at room temperature.

(c) Multi-channel field potential recordings
After incubation for 1 h at room temperature, one slice was positioned on the MED64 probe and the ACC area was entirely covered by the recording dish, which was mounted on the stage of an inverted microscope (CKX41, Olympus).A fine mesh anchor (Warner Instruments, Harvard) was carefully positioned on the settled slice to ensure slice stability during recording.The slice was continuously perfused with oxygenated ACSF, 2 ml min -1 with the aid of a peristaltic pump (Minipuls 3, Gilson) throughout the experimental period.
During the recording period, one of the channels located in the deep layer (V-VI) of the ACC was chosen as the stimulation site.Monopolar and biphasic constant current pulses (10-20 µA, 0.2 ms) were applied to the stimulation site and field excitatory postsynaptic potentials (fEPSPs) evoked at a both superficial layer (II-III) and a deep layer (V-VI) of the ACC using MED Mobius software.The fEPSP was displayed on the monitor screen, amplified by a 64-channel amplifier and stored on the microcomputer for analysis.After the baseline was stabilized for 1 h, a weak TBS protocol (five bursts at 5 Hz, four pulses at 100 Hz for each burst) was given once time or a TBS protocol (five bursts at 5 Hz, four pulses at 100 Hz for each burst) was given five times (10 s interval) to induce post-LTP at the stimulation intensity, which was adjusted to elicit 40-60% of the maximal response [23,33].To test the compound of LTP, an NMDA receptor antagonist (AP5, 100 µM, 60 min), L-type Ca 2+ channel blocker (Nimodipine, 30 µM, 60 min) and adenylyl cyclase 1 (AC1) antagonist (NB001, 1 µM, 60 min) were applied 30 min before and after the weak TBS or TBS protocol, respectively.The synaptic responses were monitored for 5 h after stimulation or drug application to see the time course of post-LTP.The percentages of the fEPSP slopes were normalized by the averaged value of the baseline.A channel was denoted as displaying LTP if the response was increased by at least 15% of the baseline during this period.

(d) Two-photon calcium imaging
In vitro calcium imaging was performed using a two-photon laser scanning microscope (Olympus FV1000-MPE system, BX61WI microscope) based on a pulsed Ti-sapphire laser (MaiTai HP DeepSee, 690-1040 nm wavelength, 2.5 W average power, 100 fs pulse width, 80 MHz repetition rate; New Port Spectra-Physics, Santa Clara, CA, USA).The laser was focused through a ×40 water-immersion objective lens (LUMPLFL/IR40XW, N.A.: 0.8, Olympus) and the average power was set to <15 mW (measured under the objective).Neurons were filled with indicators via the patch pipette for 20-30 min to allow diffusion of the dye into the cells.Fluorescent imaging of Cal-520 K + salt (200 µM) and Alexa594 K + salt (20 µM) was separated into green and red 2 royalsocietypublishing.org/journal/rstb Phil.Trans.R. Soc.B 379: 20230240 channels by a dichroic mirror and emission filters (Chroma, Bellows Falls, VT, USA) and detected by a pair of photomultiplier tubes (Hamamatsu, Shizuoka, Japan) at 800 nm.To obtain time series of fluorescent signals from global soma images, images were collected with the following parameters [35][36][37][38]: 512 × 512-pixel images, digital zoom 3× with ×40 objective (N.A. 0.8), 2-µs pixel dwell time, 50 ms/frame for frame scan model with different recording times for different recording frames.Bidirectional scanning and line-scanning models were used to increase scan speed.Each trial was repeated at least three times and the mean value was collected.Fluorescence changes were quantified as increases in green fluorescence from the baseline of ΔF/F = (F − F 0 )/F 0 .

(h) Data analysis
Results are expressed as mean ± s.e.m.Statistical analyses were conducted using GraphPad Prism (GraphPad Software).Unpaired Student's t-test was used for statistical comparisons.The level of significance was set at *p < 0.05.

(a) AMPA receptor mediates basal synaptic transmission in the ACC of tree shrew
We used the whole-cell patch in vitro to test the characteristics of synaptic transmission in the ACC of tree shrew.The EPSCs were recorded in pyramidal neurons in layer II/III of ACC using Alexa594-labelled recording electrodes (figure 2a,b).The input (stimulation intensity)-output (EPSCs amplitude) (I-O curves) were recorded in ACC neurons of tree shrews (figure 2c, n = 8 neurons/4 tree shrews).The EPSCs were recorded stabilized for at least 10 mins as the baseline.After bath application of the CNQX (25 µM) for 10 min, the EPSCs were reduced severely and a small residual current remained as 13.2 ± 2.1% of baseline that could be blocked as 9.1 ± 2.0% of baseline by application of CNQX and AP5 (50 µM) together (figure 2d,e).These results indicate that the synaptic transmission in the ACC was mediated by glutamate AMPA/kainate receptors.
To characterize whether there is a similar neuronal network connection, we used the MED64 recording system to map the cortical circuit responses within the ACC of tree shrew according to focal electrical stimulation (figure 2f).One channel in the deep layer of the ACC (layer V) was then chosen as the stimulation site, and the other 63 channels were used for measuring evoked responses.We found that local stimulation induced widespread fEPSPs, which could be observed both in superficial and deep layers and concentrated at the stimulation site.The number of channels with evoked fEPSPs (active channels) was dependent on the intensities of stimulation and reached a maximum of 49.6 ± 3.1 (n = 12 slices/6 tree shrews; figure 2g).After bath application of the AMPA/kainate receptor antagonist CNQX (25 µM), the fEPSPs of one channel, one slice and all 10 slices from 3 tree shrews were almost abolished (figure 2h,i).These results also showed that the synaptic transmission in the ACC was mediated mostly by glutamate AMPA/kainate receptors.

(b) NMDAR-mediated synaptic transmission in the ACC of tree shrew
Considering that the NMDAR is critical for synaptic transmission and LTP, we then tested the composition of the NMDAR subtype that was measured in the tree shrew.By using western blot, we found that GluN1, GluN2A and GluN2B subtypes can be found in the ACC, as well as in the PFC, insular cortex (IC), primary somatosensory cortex (S1), motor cortex (M), hippocampus (Hippo) and SC.d (figure 3a).Using subcellular fractionation of the ACC, we found that GluN1, GluN2A and GluN2B receptors were located at the synaptic sites of the ACC (figure 3b).In addition, the GluN2A and GluN2B receptor-mediated EPSCs were measured in the ACC neuron of tree shrew by performing whole-cell patch recording.As shown in figure 3c,d, the NMDAR-mediated EPSCs were significantly blocked by bath application of GluN2A antagonist PEAQX (1 µM) and continued reduced by application of GluN2B antagonist Ro-256981 (3 µM) (n = 6 neurons/3 tree shrews).The GluN2A antagonist reduced around 60.2 ± 5.2% of NMDAR-mediated EPSCs, and the application of GluN2B antagonist further decreased the EPSCs to around 6.7 ± 2.1%.It is worth noting that a small residual current was exited even after AP5 was applied, suggesting AP5 may not be enough to block the NMDAR-mediated EPSCs in the tree shrew neurons.
Considering that the NMDA receptor-dependent Ca 2+ signal is critical for synaptic transmission and LTP, we then tested the global Ca 2+ signal by combining whole-cell patch recording and two-photon Ca 2+ imaging in the ACC pyramidal neurons of tree shrew.As shown in figure 1a, the neuronal morphology was well labelled by Alexa594 and Cal-520 K + salt after 30 min diffusion in the recording pipette.We found that global calcium transients were clearly observed when single action potentials (APs) occurred, which could be induced by injecting depolarizing currents into the soma of cells (figure 1b).The ΔF/F values of Ca 2+ signals were significantly increased in an intensity-and frequency-dependent manners (five APs at 20 Hz; figure 1c).Next, we tested Ca 2+ influx evoked in the tree shrew neuron by the puff-application of exogenous glutamate (Glu, 1 mM).As shown in figure 1d,e, puff-application of Glu-evoked currents was significantly reduced by AMPA receptor antagonist CNQX and almost completely blocked by additional NMDA receptor antagonist AP5, while puff-application of Glu was associated with a remarkable subthreshold soma calcium influx, and only a small reduction by CNQX even though puff-application of Glu-evoked currents was almost inhibited.The application of AP5 significantly reduced the Ca 2+ signals.These results suggest that the NMDA receptor in the ACC neuron of tree shrew is a major contributor to the Ca 2+ signal, with the residual Ca 2+ signal possibly mediated by voltage-gated calcium channels or the calcium-permeable AMPA receptor.

(c) Multi-channel recordings of TBS and weak TBS-induced LTP in the ACC circuit of tree shrew
In previous studies, by applying a multi-channel recording system, we have tested the intercellular connections in the ACC of mice [23,40].To characterize the possibility of L-LTP induction within the ACC of tree shrew, we then applied TBS (five trains of burst with four pulses at 100 Hz, at 200 ms intervals; figure 4a) to induce LTP in the stimulation site in the deep layer of ACC from tree shrew, after 1 h baseline recording.When comparing with mice, both the occurrence ratio and degree of potentiation were larger in tree shrew.In one typical sample slice of tree shrew with 28 active channels, 20 channels showed potentiation of the slope lasting for 5 h (212.0 ± 12.4% of the baseline), 5 channels showed short-term potentiation and 3 channels remained stable throughout the entire recording period (figure 4c).The final averaged slope for all 28 active channels was 182.5 ± 13.1% of the baseline at 5 h after TBS stimulation (figure 4b).In total, in 112 active channels (averaged 14.0 ±1.7%) from 8 slices/4 mice,82 channels (73.2 ± 1.2%) showed L-LTP that lasted for 5 h, 13 channels (11.6 ± 1.9%) showed short-term potentiation and 17 channels (15.2 ± 1.8%) showed no potentiation.In total, in 230 active channels from 11 slices/5 tree shrews, 181 channels (78.7 ± 1.2%) showed L-LTP, 29 channels (12.6 ± 2.0%) showed short-term potentiation and 20 channels (8.7 ± 0.4%) showed no potentiation (figure 4k).Meanwhile, the TBS-induced potentiation degree was larger in tree shrew than in C57 mice at different time points after L-LTP induction (table 1, p < 0.05 in each time point, figure 4f,g).
It has been proved that weak TBS activates relatively few afferent fibers, and cannot trigger LTP in the hippocampus in mice [41].We thus wanted to test if weak TBS could induce the LTP in the ACC of tree shrews.After 1 h baseline recording, weak TBS (five trains of burst with four pulses at 100 Hz, at 200 ms intervals) was applied in the stimulation site in the deep layer of ACC from tree shrew and mice.It was found that the fEPSPs of most of the active channels (50.5 ± 2.1% from 6 slices/4 mice) could not be potentiated in mice.It was found that the fEPSPs of most of the active channels (65.5%) could be potentiated in tree shrews.Interestingly, around half of the active channels (48%) showed L-LTP of the fEPSPs in tree shrew and the slope of fEPSP in one typical channel was enhanced to 140.7 ± 7.5% of the baseline.In one sample slice of tree shrew with 24 active responses, 12 showed potentiation of the fEPSP slope lasting for 5 h (152.8 ± 11.8% of the baseline), 3 showed short-term potentiation and 9 remained stable throughout the entire recording period (figure 4e).The final averaged slope for all 24 active channels was 129.9 ± 1.6% of the baseline at 5 h after weak TBS stimulation (figure 4d).In total, 109 active channels (averaged 18.2 ± 2.9%) from 6 slices/4 mice, 41 channels (37.6 ± 2.3%) showed L-LTP that lasted for 5 h, 13 channels (11.9 ± 2.8%) showed short-term potentiation and 55 channels (50.5 ± 2.1%) showed no potentiation.In total, in 200 active channels from 9 slices/5 tree shrews, 96 channels (48 ± 3.0%) showed L-LTP, 35 channels (17.5 ± 1.5%) showed short-term potentiation and 69 channels (34.5 ± 2.0%) showed no potentiation (figure 4k).Meanwhile, the weak TBS-induced potentiation degree was larger in tree shrew than in C57 mice at different time points after L-LTP induction (table 2, p < 0.01 in 1 h after TBS, p < 0.05 in other time point, figure 4h,i).The results showed that the weak TBS can induce the L-LTP in tree shrew, but the slope of the fEPSP potentiation degree was lower than TBS in tree shrew (figure 4j).

(d) Recruitment of synaptic responses within the ACC network after TBS and weak TBS induction
The MED64 recording system provides a convenient way to study the cortical network L-LTP.The distribution of all activated channels during the whole recording was displayed by a polygonal graph (the blue lines represent the activated channels during the baseline and the red lines represent the activated channels after TBS or weak TBS).In our previous studies from mice, we have confirmed that some channels that were inactive during baseline recordings showed evoked fEPSPs after TBS induction (recruited channels) [23].From our research, we found that both TBS and weak TBS can induce the recruited responses in tree shrew (figure 5a).After TBS or weak TBS induction, the average amplitude of fEPSPs of recruited channels gradually increased (finally reached as large as −21.2 ± 1.0 µV with TBS and −18.2 ± 1.7 µV with weak TBS) and remained stable for 5 h (figure 5b,d).At 5 h after TBS or weak TBS, the number of recruited channels reached 3.4 ± 0.4 with TBS (n = 37 channels from total 11 slices/5 tree shrews, figure 5c) and 1.4 ± 0.4 with weak TBS (n = 13 channels from total 9 slices/3 tree shrews, figure 5e).Such recruitment was obvious in most recorded slices (n = 11 slices/5 tree shrews with TBS; n = 7 slices/3 tree shrews with weak TBS), but some slices did not show any recruitment (n = 2 slices/2 tree shrews with weak TBS).Such recruited fEPSP could be accounted for in all slices and only distributed in scattered channels on the edge of the active area.However, most of the edge channels could not be activated by TBS or weak TBS induction, indicating that the recruitment is unlikely owing to changed stimulation intensities or unstable recordings.
(e) The involvement of NMDAR, L-type Ca 2+ channel and adenylyl cyclase 1 in the induction of LTP The direction of the plasticity is largely controlled by the kinetics and amount of Ca 2+ influx through the NMDA receptors on the synapses [42].The L-type Ca 2+ channel takes an important role in mediating Ca 2+ influx in the ACC [43], where calmodulin-stimulated AC1 is critical for LTP.To test whether NMDA receptor, L-type Ca 2+ channels and AC1 are required for the induction of cingulate network L-LTP in tree shrew, we applied the NMDA receptor, L-type Ca 2+ channels and AC1 antagonist AP5, nimodipine and NB001, respectively, both before and after TBS or weak TBS protocol.After the AP5 (100 µM) application, the baseline remains stable, indicating that the AP5 does not influence the basic transmission in tree shrew.The slope of all fEPSP showed little potentiation (115.4 ± 1.1% of baseline for 5 slices/4 tree shrews) with TBS and no potentiation (106.0 ± 0.5% of baseline for 6 slices/6 tree shrews, figure 6a,d) with weak TBS.With the same tendency, the nimodipine does not influence the basic transmission in tree shrew.The slope of fEPSP still showed potentiation (123.5 ± 0.7% of baseline for 7 slices/5 tree shrews) with TBS and no potentiation (102.3 ± 4.8% of baseline for 5 slices/4 tree shrews, p < 0.05, figure 6b,d) with weak TBS.For the NB001 application, the baseline remains stable, indicating that AC1 does not influence the basic transmission in tree shrew.The slope of all fEPSP showed little potentiation (116.6 ± 11.1% of baseline for 6 slices/6 tree shrews) with TBS and no potentiation (102.3 ± 2.0% of baseline for 5 slices/4 tree shrews, figure 6c,d) with weak TBS.The results showed that AP5, nimodipine and NB001 all have an effective, although not complete, attenuation of synaptic potentiation in tree shrew with TBS and weak TBS (both p < 0.01 and p < 0.05 in comparison with the control group, unpaired t-test, data not shown).The results demonstrate that NMDA receptor, L-type Ca 2+ channels and AC1 are important for LTP induction in tree shrew.

Discussion
In the present study, we demonstrate for the first time that TBS induced a long-lasting LTP in the ACC of adult tree shrews.The amplitude of LTP in the adult tree shrew is significantly greater than that of adult mice, suggesting that cortical LTP plays more important roles in primate-like animals.Tree shrew is an excellent model for the study of cortical transmission and plasticity.Similar to the ACC of adult mice, excitatory transmission is mostly mediated by glutamate, and postsynaptic AMPA receptors contribute to basal synaptic transmission.The MED64 system used in the present study allows us to investigate the ACC circuit at the same time.Our pharmacological results confirm the findings from our previous report that glutamate is the major excitatory transmitter [44].Both AMPA and Kainic acid (KA) receptors contribute to synaptic transmission, and it is quite likely that excitatory synapses are heterogenous.Our previous studies found that there are both pure AMPA receptor-containing synapses and synapses containing a moxture of AMPA and KA receptors in the ACC of adult mice [45,46].The nerve projections from different regions of cortical and subcortical areas to the ACC are one of the reasons for such heterogeneity.In addition, silent synapses have been reported in adult cortex of mice [47][48][49].In the present study, we found that some silent responses in the ACC can be recruited by LTP.Furthermore, this recruitment is long-lasting and persists for at least 5 h.Our results strongly suggest that silent glutamatergic synapses may exist in the brain of adult primate-like animals, and the recruitment of these silent synapses could contribute to learning and memory.
LTP is the popular cellular model for investigating molecular mechanisms for synaptic changes under physiological and pathological conditions [20,21,[50][51][52].In addition to commonly used mice, rats and guinea pigs, LTP has also been investigated in rabbit [53,54], cat [55,56] and pig [57,58].Although ACC has been thought to be important for many higher-order brain functions, previous studies of LTP in the ACC have only been carried out in rats and mice [20,21,52].Therefore, our work presents an important confirmation for the existence of cortical LTP in primate-like animals' brains.
It is difficult to obtain a stable recording of lasting LTP in adult neurons using the whole-cell patch clamp recording method.The MED64 system has overcome this problem.As we have shown before in adult mice and in the current work in tree shrew, TBS can induce long-lasting LTP in the ACC for at least 3-6 h [23,33].In the ACC of tree shrew, we found that TBS induced long-lasting LTP for at least 5 h.More importantly, the magnitude of LTP is much greater than that seen in mice, supporting the fact that tree shrew are much more intelligent and smarter than mice.According to this finding, it is quite likely that LTP in adult monkeys or human brains may be even more important.
The intracellular mechanism for ACC LTP has been well investigated in adult mice [20].In the present study, we found that tree shrews' LTP requires the activation of NMDARs, including GluN2B-containing NMDARs.This finding is similar to a previous report in adult mice [22].In addition, we found that L-type voltage-dependent calcium channels (L-VDCCs) also contribute to LTP, indicating that there are at least two possible mechanisms for triggering ACC LTP in the tree shrew.These results are consistent with previous findings in adult mice, suggesting that LTP in these two species shares similar mechanisms [20].Calcium-stimulated AC1 has been reported to contribute to ACC LTP in adult mice, both in the genetic deletion of the AC1 gene and a selective pharmacological inhibitor NB001 [59][60][61][62].In this study, we found that NB001 blocked LTP in the tree shrew ACC, indicating that NMDA (GluN2B) receptor-AC1-cAMP plays an important role in ACC LTP of the adult tree shrew.Considering the important roles of AC1 in chronic pain and pain-related emotional fear and anxiety [20,21,52], it is likely that it could play important roles in chronic pain and emotional disorders in primates and humans.In summary, our present results provide the first evidence to demonstrate the field excitatory postsynaptic potentials, induced the late-phase LTP and spatial propagation in the ACC of tree shrew.We also tested the mechanism of LTP regulation and found that the AC-cAMP signal cascade takes an important role in this process.This provides the foundation of the regulation mechanism of LTP in primates for our future research.
Ethics.This work did not require ethical approval from a human subject or animal welfare committee.

Figure 1 .
Figure 1.Action potential (AP) and puffing-Glu-induced calcium imaging in the ACC of tree shrew.(a) Representative two-photon fluorescent image of patch neuron loading by Alexa 594 and Cal-520 K + salt.(b and c) Single AP (b) and 5 APs (c) evoked Ca 2+ signals in soma.Waveforms of fluorescence change (ΔF/F) in response to a single AP (b) and 5 APs (c) in soma.(d) Average traces of puff application of Glu-evoked currents (left) and associated Ca 2+ signals (ΔF/F) (right) evoked by puff-application of 1 mM Glu (10 psi, 100 ms, n = 6 neurons from 3 mice).(e) Summary results showing the percentage of application of Glu-evoked currents (left) and Ca 2+ signals (ΔF/F, right) in the presence of CNQX (25 μM) and ᴅ-2-amino-5-phosphonovalerate (AP5) (50 μM).Arrows in (a-e) indicate a starting point of the pairing protocol application.Error bars in (e) represent s.e.m.

Figure 2 .
Figure 2. Glutamate receptor-mediated synaptic transmission in the ACC of tree shrew.(a) The photo shows the process of anaesthesia of tree shrew with 1-2% isoflurane (left).The schematic diagram showed slices including ACC area (from Bregma +1.68 to +0.47 mm) of tree shrew was used for the research (right).(b) Schematic diagram showing the placement of stimulating and recording electrodes in the ACC of tree shrew (top).Representative photomicrograph of an Alexa 594-labelled pyramidal neuron in layer II/III of ACC (bottom).Scale bar: 50 μm.(c) Sample traces and pooled data show the input-output relationship of basal EPSCs in the ACC of tree shrew (n = 8 neurons/4 tree shrews).(d) Sample time course points show the EPSCs in the presence of CNQX and AP5.EPSCs were recorded in the presence of picrotoxin (100 μM).After the perfusion of CNQX (25 μM) for 10 min, a small residual current remained that could be blocked by CNQX and AP5 (50 μM) together.(e) Statistical results show the percentage of EPSCs in the presence of CNQX and AP5 (n = 8 neurons/4 tree shrews).Insets in (d) are sample traces with the perfusion of CNQX and AP5, respectively.(f) The microphotograph and schematic diagram show one example of ACC fEPSP recording by using the MED64 system.A cortex slice containing the ACC of tree shrew was placed on a probe (MEDP515A, 8 × 8 array).One channel of the probe (red circle) was selected as the stimulation site.The evoked field potentials in all the other 63 channels were recorded 1 h before and 5 h after TBS.(g) The number of activated channels induced by different stimulation intensities (input-output) in tree shrew (left) and in sample traces (right) show one channel with enhanced fEPSP in response to different intensities of stimuli in tree shrew.(h) The fEPSP slope and the sample traces from one channel show that CNQX blocked the potential.(i) The summarized fEPSP slopes show that CNQX blocked all activated potentials from 10 slices in 3 tree shrews.Calibration: 100 µV, 10 ms.Error bars in (c), (e) and (i) represent s.e.m.Unpaired Student's t-test, ***p < 0.001.

Figure 3 .Figure 4 .
Figure 3. GluN2B receptor-mediated synaptic transmission in the ACC of tree shrew.(a) Representative western blots for GluN1, GluN2A and GluN2B in different brain areas and SC.d of tree shrew.(b) Subcellular localization of GluN1, GluN2A and GluN2B in the ACC of tree shrew.(c) Time course of the amplitudes of NMDA receptors-mediated eEPSCs with application of PEAQX, Ro-256981 and AP5 in the ACC of tree shrew.(d) The summarized proportions of NMDA eEPSCs with PEAQX, Ro-256981 and AP5 application.Unpaired Student's t-test, ***p < 0.001.Error bars in (d) represent s.e.m.

5 Figure 5 .
Figure 5. Spatial analysis of post-LTP distribution in the ACC of tree shrew.(a) Polygonal diagram of the channels that are activated in the baseline (blue, left) and after TBS (red, right) in tree shrew.The blue and red circles and regions denote activated channels.The green circles indicate the recruitment channels in the slice.The stimulation sites are marked as yellow circles.(b)Summary of the typical trace and the amplitude of fEPSP were summarized from all recruited channels (n = 16 channels from 9 slices/4 tree shrews).(c) Summary of the number of recruited channels was summarized after TBS induction.(d) The typical trace and the summary graph of the amplitude of fEPSP of recruited channels (n = 13 channels from 9 slices/3 tree shrews) from tree shrew with weak TBS.(e) Summary or the number of recruited channels was summarized after weak TBS induction.Arrows in (b-e) indicate starting point of TBS or weak TBS application.Error bars in (c, e) represent s.e.m.

Figure 6 .
Figure 6.Weak TBS-induced the post-LTP depends on the activation of NMDA receptor, L-type voltage-gated calcium channels and adenylyl cyclase 1 in the ACC of tree shrew.(a) The final averaged slope for all 97 active channels from 6 slices of 6 tree shrews was 105.1 ± 2.0% of the baseline at 5 h after weak TBS application in AP5 (100 μM).(b) The final averaged slope for all 90 active channels from 5 slices of 4 tree shrews was 102.3 ± 4.8% of the baseline at 5 h after weak TBS application in nimodipine (30 μM).(c) The final averaged slope for all 150 active channels from 5 slices of 4 tree shrews was 97.1 ± 4.7% of the baseline at 5 h after weak TBS application in NB001 (1 μM).(d) Bar histogram summarizing quantified data within the last 30 mins of the 5 h recording.Unpaired Student's t-test, *p < 0.05, **p < 0.01.Arrows in (a-c) indicate the starting point of weak TBS application.Error bars in (a-d) represent s.e.m.

Table 1 .
Multi-channel recordings of TBS-induced LTP in the ACC of tree shrew and mice.

Table 2 .
Multi-channel recordings of weak TBS-induced LTP in the ACC of tree shrew.