Cholecystokinin release triggered by NMDA receptors produces LTP and sound-sound associative memory

Abstract

Memory is stored in neural networks via changes in synaptic strength mediated in part by NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here we show that a cholecystokinin (CCK)-B receptor (CCKBR) antagonist blocks high-frequency stimulation-induced neocortical LTP, whereas local infusion of CCK induces LTP. CCK^-/- mice lacked neocortical LTP and showed deficits in a cue-cue associative learning paradigm; and administration of CCK rescued associative learning deficits. High-frequency stimulation-induced neocortical LTP was completely blocked by either the NMDAR antagonist or the CCKBR antagonist, while application of either NMDA or CCK induced LTP after low-frequency stimulation. In the presence of CCK, LTP was still induced even after blockade of NMDARs. Local application of NMDA induced the release of CCK in the neocortex. These findings suggest that NMDARs control the release of CCK, which enables neocortical LTP and the formation of cue-cue associative memory.

Keywords: NMDA receptor; cholecystokinin; entorhinal cortex; long-term potentiation; memory.

Fig. 1.

Role of CCK in neocortical LTP induction on the rats. (A) Position of recording and stimulating electrodes in the auditory cortex of rats. (B) Representative relationship between input currents and evoked fEPSPs. (C) Normalized slopes of fEPSPs before and after HFS. HFS paradigm above the curve. (D) Representative single fEPSP traces before (1, just before HFS) and after HFS (2, just after HFS; 3, at the end of the recording, corresponding to the numbers indicated in C). (E) Diagram of stimulation electrode placements and microdialysis with a schedule showing time points of ACSF collection and HFS application. (F) A bar chart showing concentrations of CCK before and after HFS in the auditory cortex based on ELISA. At the baseline level, from −60 min to 0 min, the concentration of CCK was lower than the detection limit (1 pg/mL), set as 0. After HFS, the concentration of CCK increased to 5.37 ± 1.15 pg/mL (one-way RM ANOVA, *P = 0.021), but dropped quickly in the following half hour. (G) Positions of recording and stimulating electrodes and pipette in the auditory cortex of rats. (H) Normalized slopes of fEPSPs before and after HFS with L365,260 (red circle) or vehicle (blue square) injection (two-way RM ANOVA, **P < 0.001). HFS protocol above the curve. Bars at the bottom indicate the periods over which the data are being pooled for the before and after measurements. (I) Representative single fEPSP traces before and after injection of L365,260 (1-2, at the end of each recording period) or vehicle (3-4, at the end of each recording period) and HFS. (J) Positions of recording and stimulating electrodes and pipette in the auditory cortex of rats. (K) Normalized slopes of fEPSPs before and after CCK (red circle) or vehicle (blue square) injection (two-way RM ANOVA, **P < 0.001). The experimental protocol above the curve. (L) Representative single fEPSP traces before and after injection of CCK (1–3) or vehicle (4–6). Data are expressed as mean ± SEM. AC, auditory cortex; Rec., recording electrode; Veh, vehicle.

Fig. 2.

Neocortical LTP and learning an association between two tones were eliminated in CCK^−/− mice. (A) Positions of recording and stimulating electrodes in the mouse auditory cortex. (B) Normalized slopes of fEPSPs before and after HFS in C57 mice (red circle) or CCK^−/− mice (blue square) (two-way RM ANOVA, **P < 0.001). HFS protocol above the curve. (C) Representative single fEPSP traces before and after HFS in C57 mice (1, 2) and CCK^−/− mice (3, 4). (D) Diagram of a training protocol for mice to associate the tones of f1 and f2. (E and F) Bar charts show freezing percentages of the C57 mice (open, E) and CCK^−/− mice (shaded, F) to tones of f1, f2, and f3, before and after the conditioning (two-way RM ANOVA, **P < 0.001). (G) A training protocol for C57 mice to associate the tones of f1 and f2. The sketch shows implanted drug infusion cannulas in both sides of the auditory cortex. (H) Bar charts show freezing percentages of the C57 mice with ACSF infusion (open) and with L365,260 infusion (shaded) to tones of f1 and f2, before and after the conditioning (one-way RM ANOVA, **P < 0.001). Data are expressed as mean ± SEM. N.S., not significant.

Fig. 3.

CCK puffing or stimulation of entorhino-neocortical projections induced LTP in cultured cells, brain slices, and in vivo preparation. (A) Experimental set-up. A cultured neuron was whole-cell–clamped and two pipettes, one for Glu puffing and another for CCK puffing, were placed near the recorded neuron. (Magnification, 20×.) (B) Glutamate-activated current responses are shown before (baseline) and at 5, 10, 30, and 60 min after the pairing. CCK was repeatedly puffed simultaneously with glutamate three times during the pairing. (C) The time course of the change of the group data are shown before (open circle) and after (filled circle) the pairing, together with the control group with no pairing (no CCK puffing, no glutamate puffing, and no depolarization of the neurons, square) (two-way RM ANOVA, **P < 0.001). (D) Positions of the whole-cell recording pipette, electrical stimulation electrode, and the optical fiber in a slice of CCK-Cre mice with AAV-Ef1α-Flex-Chronos-GFP injected in Ent. (E) A photo shows the virus injection site (Ent) and recording site (AC). (F) Voltage-clamp recordings to laser stimulation (blue) (Upper: holding potential, 0 mV; Lower: holding potential, −70 mV; room temperature) under control situation (black) and when incubated with TTX and 4-AP (purple). (G) Immuno-electron microscopy shows the colocalization of vglut1 and the cortical projection terminals of the entorhinal CCK neurons. Gold-particles of 15 nm show the immunoreactivities to mCherry (closed arrowheads), while those of 6 nm show the immunoreactivities to vglut1 (open triangles). AAV-EF1α-DIO-mCherry was injected in the entorhinal cortex of CCK-Cre mouse. (Scale bar, 100 nm.) (H) The ratio of mCherry⁺ terminals that contain vglut1 over all mCherry⁺ terminals. (I) HF/ES and LF/ES pairing protocol in which 60 Hz (HF) or 1 Hz (LF) laser stimulation at the auditory cortex was followed by 1 Hz ES. (J) Normalized slopes of EPSCs in response to the electrical stimulation before and after the HF/ES (red circle) or LF/ES (blue square) stimulation protocols (two-way RM ANOVA, *P < 0.001). (K) Positions of the recording electrode in the auditory cortex and laser fiber in the entorhinal cortex of Thy1-Chr2-eYFP mice. (L) Ent HF/AS and Ent LF/AS protocols in which high frequency or low-frequency laser stimulation of the entorhinal cortex was followed by presentations of an auditory stimulus. (M) Normalized slopes of fEPSPs in response to the auditory stimulus before and after the Ent HF/AS (red circle) or Ent LF/AS (blue square) stimulation protocols (two-way RM ANOVA, **P < 0.001). Data are expressed as mean ± SEM. Ent, entorhinal cortex; Ent HF/AS, high-frequency laser stimulation at entorhinal cortex paired with auditory stimuli; Ent LF/AS, low-frequency laser stimulation at entorhinal cortex paired with auditory stimuli; Glu, glutamate; HF/ES, high-frequency laser stimulation paired with electrical stimulation; LF/ES, low-frequency laser stimulation paired with electrical stimulation; TTX/4-AP, tetrodotoxin/4-Aminopyridine.

Fig. 4.

HFS of CCK-containing entorhino-neocortical projections enables the association between an auditory stimulus and electrical stimulation of the auditory cortex, leading to behavioral changes. (A) AAV-EF1α-DIO-hChR2(E123T/T159C)-eYFP (for B1, 2, 4, and 5) or AAV-EF1α-DIO-hChR2(E123T/T159C)-mCherry (for B3) was injected into the entorhinal cortex of CCK-Cre mice. (B) Images of virus expression in the entorhinal cortex (1–3) and the auditory cortex (4, 5). (Scale bars: 500 μm for 1 and 4; 100 µm for 2, 3, and 5.) In 3, mCherry (CCK), CamKII, and DAPI were overlapped (Arrowhead: neurons express both CamkII and CCK; arrow: a neuron only expresses CCK). (C) Positions of the laser fiber and stimulating/recording electrodes in the auditory cortex of CCK-Cre mice injected with AAV-EF1α-DIO-hChR2(E123T/T159C)-eYFP in the entorhinal cortex. (D) fEPSP responses to laser stimulation (Upper: 1 Hz; Lower: 80 Hz) in the auditory cortex. (E) HF/AS/ES and LF/AS/ES pairing protocols. (F) Normalized slopes of fEPSPs after the HF/AS/ES (red circle) or LF/AS/ES (blue square) pairing protocols (two-way RM ANOVA, **P < 0.001). (G) Representative single fEPSP traces before and after the HF/AS/ES (1–3) and LF/AS/ES (4–6) protocols. (H) Unit responses to the auditory stimulus before and after the pairings of HF/AS/ES (1–3) and LF/AS/ES (4–6). (I) Cued fear conditioning and pairing protocols. (J) Freezing percentages in response to the paired auditory stimulus before and after the HF/AS/ES (Red) or LF/AS/ES (Blue) pairing (two-way RM ANOVA, *P < 0.05). Data are expressed as mean ± SEM. HF/ES/AS, high-frequency laser stimulation paired with electrical stimulation and auditory stimuli; LF/ES/AS, low-frequency laser stimulation paired with electrical stimulation and auditory stimuli.

Fig. 5.

NMDARs control the release of CCK, which in turn enables CCK-dependent neocortical LTP. (A) Either an NMDAR antagonist or CCKBR antagonist blocked HFS-induced LTP in C57 cortical slices. APV was applied throughout all of the recording period, while L365,260 was applied after the baseline recording, but before the HFS. Inset shows positions of recording and electrical stimulating sites. (B) Coupled with LFS, both NMDA and CCK induced LTP in C57 cortical slices. (C) Blocking of NMDARs did not block LTP in the presence of CCK in C57 cortical slices with HFS. (D) Blocking of NMDARs did not block LTP in the presence of CCK in C57 cortical slices with LFS. (E) NMDA induced LTP was blocked by application of CCKBR antagonist L365,260 in the C57 slices. (F) NMDA induced LTP was blocked by application of CCKBR antagonist, L365,260, in the auditory cortex of anesthetized C57 mice. L365,260 or control (DMSO+ACSF) was infused during the whole experiment. (G) No LTP was induced by NMDA application, but a marked induction of LTP was generated by CCK application in the cortical slices of CCK^−/− mice. Inset shows representative single fEPSP traces before and after the CCK+LFS (1, 2). (H) Diagram illustrating injecting cannula for NMDA (100 μM, 1 μL) application and microdialysis with a schedule showing time points of ACSF collection and NMDA application. (I) Local application of NMDA triggered CCK release in the auditory cortex of C57 mice. (J–M) A new paradigm for LTP induction: a 25 PPS protocol. (J) Twenty-five pairs of paired-pulses with varied IPuI between 5 ms and 200 ms and fixed IPaI of 1 s. (K) The induction of LTP with different IPuIs. (L) LTP was induced by the PPS protocol when IPuI was 20 ms, but not 200 ms in the auditory cortex of anesthetized C57 mice (two-way RM ANOVA, **P < 0.001). (M) LTP induced with the PPS protocol was blocked by infusion of either APV or L365,260 (two-way RM ANOVA, **P < 0.001). Data are expressed as mean ± SEM.

Fig. 6.

A schematic illustration shows that CCK release triggered by either/both pre- or/and postsynaptic NMDA receptors produces LTP and sound–sound associative memory. CCK released from the entorhinal CCK neuron (Ent, neuron A) that projects to the auditory cortex enhances the connectivity of two neurons in the auditory cortex (neuron B to neuron C) (Left part). When the first action potential comes to neuron A, glutamate is released from A and activates its own NMDAR on its terminal (Lower Center, Left). The NMDAR will enable the release of CCK from A, if the second action potential comes to the terminal of neuron A within a period of 10–100 ms (Lower Center, Right). Together with other conditions, such as activation of the B → C synapse and action potentials of neuron C, the released CCK from A that activates the CCKBR on neuron C enables neuroplasticity of B → C pathway, inducing potentiated EPSC for the B → C synapse (Upper Center).