Substance P induces plasticity and synaptic tagging/capture in rat hippocampal area CA2

Abstract

The hippocampal area Cornu Ammonis (CA) CA2 is important for social interaction and is innervated by Substance P (SP)-expressing supramammillary (SuM) nucleus neurons. SP exerts neuromodulatory effects on pain processing and central synaptic transmission. Here we provide evidence that SP can induce a slowly developing NMDA receptor- and protein synthesis-dependent potentiation of synaptic transmission that can be induced not only at entorhinal cortical (EC)-CA2 synapses but also at long-term potentiation (LTP)-resistant Schaffer collateral (SC)-CA2 synapses. In addition, SP-induced potentiation of SC-CA2 synapses transforms a short-term potentiation of EC-CA2 synaptic transmission into LTP, consistent with the synaptic tagging and capture hypothesis. Interestingly, this SP-induced potentiation and associative interaction between the EC and SC inputs of CA2 neurons is independent of the GABAergic system. In addition, CaMKIV and PKMζ play a critical role in the SP-induced effects on SC-CA2 and EC-CA2 synapses. Thus, afferents from SuM neurons are ideally situated to prime CA2 synapses for the formation of long-lasting plasticity and associativity.

Keywords: CA2 region; Substance P; long-term potentiation; social memory; synaptic tagging.

Fig. 1.

SP induces long-lasting potentiation of synaptic transmission in CA2 neurons. (A) The schema shows the location of electrodes for stimulation of synaptic inputs MF-CA3 (green), SC-CA2 (blue), SC-CA1 (pink), and the recording sites of fEPSPs within the hippocampal CA3, CA2, and CA1 areas. (B) Bath application of 5 µM SP induced a slow-onset, long-lasting potentiation in SC-CA2, but not in MF-CA3 and SC-CA1 synaptic inputs (n = 5). (C) The schema shows the location of electrodes for the stimulation of SC-CA2 (blue) and EC-CA2 (red) synaptic inputs and the recording sites of fEPSPs within the hippocampal CA2 area. (D) Bath application of SP for 15 min after a stable baseline of 30 min induced a synaptic potentiation in SC-CA2 and EC-CA2 synaptic inputs (n = 10). (Insets) Representative fEPSPs 15 min before (closed line), 60 min after (dotted line), and 180 min after (hatched line) SP application. (E) Whole-cell voltage-clamp recording of EPSCs with the application of SP (5 µM) induced a slow-onset potentiation in SC-CA2 (n = 10). (F) Control experiments indicated the stability of the recordings (n = 6). (Insets) Representative EPSC 5 min before (closed line), 30 min after (dotted line), and 50 min after (hatched line) SP application. Calibration bars are 2 mV/3 ms for fEPSP and 50 pA/40 ms for EPSC traces.

Fig. S1.

NK1 mRNA expression levels in CA1, CA2, and CA3 of hippocampus: qRT-PCR analysis shows that NK1 mRNA expression is significantly greater in CA2 and CA3 regions compared with the CA1 region (approximately 100 slices from five rats). Significant differences between groups (CA1 vs. CA2 and CA1 vs. CA3) are indicated by ***P < 0.001. Individual data points of fold change are represented within the bar graphs.

Fig. S2.

(A) The relative location of the electrodes to stimulate independent SC-CA2 (blue) and EC-CA2 synaptic inputs (red) to the CA2 neuronal population and the location of additional electrodes to stimulate SC synaptic inputs to CA1-pyramidal neurons (pink) are depicted in the schema. Two recording electrodes were used to record fEPSPs from the CA2 or CA1 area simultaneously. (B) Baseline test stimulation of CA2 and CA1 synapses yielded stable responses throughout the recording period (n = 7). (C) Bath application of a potent and selective antagonist for the adenosine A₁ receptor, DPCPX (10 nM), for 15 min induced potentiation in SC-CA2 (blue circles) and EC-CA2 (red circles) synapses but not in the CA1 area (pink circles; n = 8). (D) To test that the stimulation of EC-CA2 and SC-CA2 synapses within individual experiments activated independent synaptic inputs, a paired-pulse stimulation protocol (50-ms interstimulus interval) was used. The synaptic inputs were sequentially activated with SC; then paired EC-SC, EC; then paired SC-EC, paired EC-EC, and paired SC-SC. The bar graph summarizes the ratios for the different pairings. The synaptic inputs were independent because the ratio of EC-SC and SC-EC equals 1 and differed significantly from the facilitation values obtained by the same fiber pairings. Representative fEPSP traces at 15 min (closed line), 60 min (dotted line), and 180 min (hatched line) are depicted. Calibration bars for fEPSP traces are 2 mV/3 ms.

Fig. S3.

(A) STET did not induce LTP in the SC-CA2 inputs (blue filled circles; n = 7). The fEPSPs of the EC-CA2 input remained stable. (B) STET induced long-lasting input-specific LTP in EC-CA2 inputs (red circles) with stable control potentials in SC-CA2 (blue circles; n = 8). Representative fEPSP traces 15 min before (closed line), 60 min after (dotted line), and 180 min after (hatched line) STET are depicted. (C) Application of WTET in the EC-CA2 input (red circles; n = 7) resulted in an early LTP that decayed to baseline level within 120 min. The baseline responses from the SC-CA2 input were unaffected throughout the recording period. Representative fEPSPs 15 min before (closed line), 5 min after (dotted line), and 180 min after (hatched line) WTET are depicted. Calibration bars are 2 mV/3 ms.

Fig. S4.

SP-induced potentiation requires NMDA receptor activation and protein synthesis. (A) SP had no effect when NMDA receptors were inhibited by bath application of APV (50 μM, n = 8). The drug was initially applied for 15 min before coapplication with SP for the next 15 min. APV application was continued for an additional 15 min (i.e., total of 45 min APV application; hatched bar). (B and C) Coapplication of the protein synthesis inhibitors ANI (25 μM, n = 7; B) or EME (20 μM, n = 7; C) together with SP abolished the slow-onset potentiation of fEPSPs in both studied synaptic inputs. (D) Application of the NK1 receptor antagonist L-733060 (5 μM) 15 min before and after coapplication with SP similarly impaired the initiation of a slow-onset potentiation in both synaptic inputs: SC-CA2 (blue circles) and EC-CA2 (red circles; n = 7). Representative fEPSP traces 15 min before (closed line), 60 min after (dotted line), and 180 min after (hatched line) SP application are depicted. Calibration bars are 2 mV/3 ms.

Fig. 2.

Expression of SP-mediated potentiation requires test stimulation. (A) The potentiation by SP requires test stimulations and is input-specific. Only SC-CA2 synapses, but not the EC-CA2 synapses, showed potentiation when the EC-CA2 test stimulation was suspended at the time of SP application and for a subsequent 1 h (n = 8). (B) In a reverse scenario, i.e., suspension of SC-CA2 test stimulation during and 45 min after drug application, no potentiation was observed in SC-CA2, unlike the case for EC-CA2 synapses, in which potentiation was expressed (n = 7). (C) Suspended baseline stimulation of SC-CA2 and EC-CA2 synaptic inputs at the time of SP application and for a subsequent 1 h prevented potentiation in either synaptic input (n = 8). (D) A control experiment showing no potentiation in SC-CA2 and EC-CA2 synaptic inputs in response to the suspension of baseline stimulation and in the absence of drug application (n = 6). Representative fEPSP traces 15 min before (closed line), 95 min after (dotted line), and 180 min after (hatched line) SP application are depicted. Calibration bars for fEPSP traces in all panels are 2 mV/3 ms.

Fig. 3.

SP initiates STC in SC-CA2 and EC-CA2 synapses. (A) Experiment showing a WTET-induced early LTP in EC-CA2 synaptic inputs. After recording a stable baseline of 30 min, SP was bath-applied for 15 min, during which time the baseline stimulation was suspended for 1 h (n = 7). After that, a baseline was recorded for another 30 min followed by WTET protocol. The potentiation decayed to baseline level within 180 min. (B) STC initiated by SP. Stimulation of EC-CA2 inputs was suspended during the application of SP and, at the 90th minute, WTET was delivered and the SC-CA2 fEPSPs were further recorded. Unlike in A, SP transformed the EC-CA2 fEPSP potentiation into an L-LTP (n = 8). (C and E) Experiments similar to A but with STET protocol was applied at the 90th minute in the presence of protein synthesis inhibitors ANI (25 µM; n = 8; C) or EME (20 µM; n = 8; E). The inhibitors were applied 20 min before STET (i.e., 10 min after resuming the baseline recording) and washed out 40 min after STET of the EC-CA2 input. (D and F) STC experiments in which L-LTP in the EC-CA2 inputs (red circles) was expressed in the presence of protein synthesis inhibitors ANI (25 µM; n = 8; D) or EME (20 µM; n = 9; F) as a result of the capture of previously SP-induced plasticity-relevant proteins. The experimental design was similar to that in B with the exception that, in this case, STET was applied to EC-CA2 inputs at the 90th minute. Representative fEPSP traces 15 min before (closed line), 95 min after (dotted line), and 180 min after (hatched line) SP application or WTET/STET are depicted. Calibration bars for fEPSP traces are 2 mV/3 ms. Arrows indicate the time points of WTET or STET.

Fig. S5.

Requirement of test stimulation for SP-induced STC. (A) SP was bath-applied for 15 min, during which time the baseline stimulation was suspended for 1 h in EC-CA2 but not in SC-CA2. After 1 h, EC-CA2 synapses recordings were resumed and continued for another 30 min, followed by WTET at the 90th minute. At that time, the SC-CA2 stimulation input was suspended until the end of the experiment (n = 8). In this case, SC-CA2 synapses that expressed potentiation within the first 1 h was sufficient to transform the fEPSP potentiation in EC-CA2 to an L-LTP. (B) In a similar control experiment, SC- and EC-CA2 inputs were silenced for 1 h after a stable baseline recording for 30 min. A WTET at the 90th minute in EC-CA2 failed to transform the fEPSP potentiation to an L-LTP in this case (n = 7). (C) In another set of experiment, the EC-CA2 input received normal test stimulation and displayed significant potentiation, whereas the SC-CA2 input was silenced during SP application and then for as long as 1 h. At 30 min after resuming the test stimulation, STET was applied, which failed to induce potentiation in this input (n = 6). Representative fEPSP traces 15 min before (closed line), 95 min after (dotted line), and 180 min after (hatched line) SP application or WTET/STET are depicted. In A, traces are shown up to 60 min after SP application (dotted line). Calibration bars are 2 mV/3 ms.

Fig. S6.

CaMKIV and PRKCZ mRNA expression levels in the CA2 region of hippocampus after SP treatment: qRT PCR analysis shows that CaMKIV and PRKCZ mRNA expression were significantly increased in the CA2 region of hippocampus after SP treatment compared with untreated control slices and snap-frozen rat hippocampus. Significant difference between groups (control vs. SP-treated, naïve rat hippocampus vs. SP-treated) are indicated by *P < 0.05 or **P < 0.01 (slices from three biological replicates). Individual data points of fold change are represented within the bar graphs.

Fig. 4.

PKMζ and CaMKIV are required for SP-induced plasticity and associativity in CA2 neurons. (A) The CaMKIV inhibitor KN-93 (10 µM) and SP were coapplied as indicated by horizontal bars in the graph (total of 45 min). The coapplication prevented the induction of SP-mediated potentiation in both synaptic inputs (n = 8). (B) Experimental design and drug application similar to that in A except that the nonactive drug KN-92 was used (n = 8). (C, a and b) Western blot analysis showed a significant increase of CaMKIV protein phosphorylation in the CA2 region after SP treatment compared with the respective control. The significant difference between the groups (CA2 control vs. CA2 SP-treated) is indicated by **P < 0.01 (from three biological replicates). Individual data points of fold change are represented within the bar graphs. (D) Preincubation (1.5 h) and continuous application of PKMζ antisense oligodeoxynucleotides (20 μM) for as long as 240 min prevented SP-induced fEPSP potentiation (n = 7). (E) Experimental design similar to that in D except that a scrambled version of PKMζ antisense oligodeoxynucleotides was applied (n = 7). Representative fEPSP traces 15 min before (closed line), 60 min after (dotted line), and 180 min after (hatched line) SP application are depicted. Calibration bars for fEPSP traces are 2 mV/3 ms. (F, a and b) Western blot analysis of PKMζ protein expression also showed a significant up-regulation in CA2 region after SP treatment compared with control. The significant difference between the groups (CA2 control vs. CA2 SP-treated) is indicated by **P < 0.01 (from three biological replicates). Individual data points of fold change are represented within the bar graphs.

Fig. S7.

(A) Western blot analysis of PKMζ protein expression in acute hippocampal slices using anti-PKCZ antibody detected a major band at ∼55 kDa (for PKMζ) in the three lanes loaded with control (untreated), treated with antisense oligodeoxynucleotides plus SP, and scrambled plus SP (Upper). (Lower) Equal loading of samples in all three lanes using anti-tubulin antibody (50 kDa). (B) A significant decrease in the protein expression level was observed in the slices treated with PKMζ antisense oligodeoxynucleotides compared with control slices. In contrast, the protein expression was higher (equivalent to untreated control slices) when they were treated with the corresponding scrambled version of the oligodeoxynucleotides. Significant differences between groups (control vs. antisense and antisense vs. scrambled) are indicated by **P < 0.01 (slices from three biological replicates).

Fig. 5.

SP-induced potentiation does not require GABA_A or GABA_B receptors. (A) Inhibitors of GABA_A receptors, PTX (100 μM), or of GABA_B receptors, CGP55845 (2 μM), were applied together during the entire incubation and recording period. SP was applied for 15 min. Potentiation was intact in SC-EC-CA2 synaptic inputs (n = 6). (B) Similar to the experiments in Fig. 3, STC experiments were carried out in the presence of the same GABA_A and GABA_B receptor inhibitors during the entire incubation and recording period. Even in the absence of GABAergic transmission, SP-mediated potentiation in SC-CA2 could transform the EC-CA2 fEPSP potentiation into an L-LTP (n = 7). (C) Experimental design was similar to that in Fig. 3F, but the experiment was carried out in the presence of GABA_A and GABA_B receptor inhibitors during the entire incubation and recording period (n = 6). STC was still observed even without GABAergic transmission. Representative fEPSP traces 15 min before (closed line), 95 min after (dotted line), and 180 min after (hatched line) SP application or WTET/STET are depicted. Calibration bars for fEPSP traces are 2 mV/3 ms.