As reported by others (Xu et al, 2010), 15 min administration of 5 μM WIN, a synthetic cannabinoid agonist that activates both CB1Rs and CB2Rs, induced a persistent depression of synaptic responses in the CA1 region (EPSP slope: 47.7±7.9% of control EPSPs 60 min after WIN washout, N=4, data not shown). WIN-induced LTD was not altered by co-administration of 30 μM D,L-APV, a competitive NMDAR antagonist (51.0±3.5%, N=3, Figure 1a), although the same concentration of APV administered during the delivery of 900 pulse 1 Hz LFS completely blocked homosynaptic LTD induction (98.5±0.7% of control, N=3, data not shown). WIN-induced LTD was blocked completely by 5 μM AM251, a selective CB1R inhibitor (EPSPs 60 min after washout; 103.0±5.1%, N=5, P<0.001, Figure 1b). Continuous administration of AM251 after washout of WIN failed to prevent the depression induced by WIN (47.1±9.1%, N=3, data not shown), indicating that WIN-LTD does not result from ongoing CB1R activation. At 5 μM, AM630, a CB2R antagonist, had no significant effect on WIN-LTD (74.5±9.5%, N=6, P=0.305, Figure 1c). These observations suggest that activation of CB1Rs, but not CB2Rs, drives a form of chemical LTD in the CA1 region. Consistent with this, 15 min administration of 5 μM ACEA, another synthetic cannabinoid with relative selectivity for CB1Rs that induces LTD in corticostriatal synapses (Sergeeva et al, 2007), also induced persistent synaptic depression (Figure 1d).

Figure 1 Chemical LTD induced by synthetic EC analogs. (a) Administration of 5 μM WIN55,212-2 for 15 min (black bar) induced persistent depression (LTD) in the presence of 30 μM APV. (b, c) The ability of WIN to induce LTD was blocked by co-administration of 5 μM AM251, a CB1R antagonist (panel b, hatched bar), but not by 5 μM AM630, a CB2R antagonist (panel c, hatched bar). (d). Administration of 5 μM ACEA for 15 min (black bar) also induced persistent depression. Traces depict EPSPs before (dashed lines) and 60 min after administration of synthetic EC analogs. Scale; 1 mV and 5 ms. PowerPoint slide Full size image

These observations suggest that central nervous system (CNS)-generated ECs may also induce or contribute to persistent synaptic depression of glutamate transmission. This prompted us to examine several ECs and other endogenous cannabinoid receptor ligands for their ability to mimic the effects of WIN and ACEA. Administration of 5 μM anandamide (92.3±4.9% change, data, N=5, Figure 2a) or 30 μM oleamide (94.5±7.7% change, data, N=3, not shown) for 15 min had no significant effect on EPSPs. Anandamide also failed to induce robust LTD in slices pretreated with 0.3 μM URB-597, a fatty-acid amide hydrolase inhibitor (85.8±8.1%, N=7, P=0.343 vs anandamide alone by Mann–Whitney test). Although 5 μM 2AG depressed EPSPs, the degree of persistent LTD was marginal (90.1±12.7%, N=5, Figure 2b). A higher concentration of 2AG (20 μM) induced only slightly greater synaptic depression (82.4±4.7%, N=3) that was less than the depression observed with WIN or ACEA. In contrast, administration of 5 μM NLDE slowly but persistently depressed EPSPs (69.7±5.2%, N=3, Figure 2c). Owing to the more robust and reliable effects of NLDE, we explored its actions in greater detail and found that a similar degree of LTD could be induced at concentrations as low as 0.1 μM. We thus used 0.1 μM NLDE for all subsequent studies. LTD induced by NLDE seemed to be saturated by a single administration because a second administration of NLDE did not induce further LTD (EPSP slope: 54.0±7.3% 60 min after the first NLDE administration and 60.2±7.5% 60 min after the second NLDE administration, N=5).

Figure 2 Effects of exogenously applied ECs on synaptic transmission. (a) Administration of 5 μM anandamide for 15 min (black bar) had marginal effects on EPSPs. (b) Administration of 5 μM 2AG for 15 min (hatched bar) only partially depressed EPSPs. (c) Administration of 5 μM NLDE for 15 min (black bar) slowly depressed EPSPs with depression becoming even more prominent after washout. Traces depict EPSPs before (dashed lines) and 60 min after administration of each EC. Scale; 1 mV and 5 ms. PowerPoint slide Full size image

These results indicate that certain cannabinoids, acting through CB1Rs induce a form of chemical LTD that does not involve NMDARs, and NLDE is an endogenous candidate to participate in LTD resulting from stimulation of the Schaffer collateral pathway. As Schaffer collateral LTD can also involve mGluR5, we examined whether cannabinoid-induced LTD shares mechanisms with mGluR-induced LTD. For these studies, we used CHPG, a selective mGluR5 agonist that has previously been shown to induce chemical LTD (Lanté et al, 2006). At 250 μM, CHPG acutely suppressed EPSPs and resulted in synaptic depression that persisted after washout (75.6±3.6%, 60min after CHPG, N=11, Figure 3a and c). Repeated administrations of CHPG did not result in further LTD (71.5±6.4% 60 min after the first CHPG administration and 71.2±6.3% 60 min after the second CHPG administration, N=6). Administration of NLDE after induction of stable LTD with CHPG also failed to produce further LTD (Figure 3a). Similarly, administration of CHPG after establishing NLDE-LTD resulted in no further lasting change in synaptic responses (Figure 3b), suggesting that these two forms of LTD share common mechanisms.

Figure 3 Non-additivity of LFS-induced LTD and two forms of chemically induced LTD. (a) Administration of 250 μM CHPG for 15 min (gray bar) acutely depressed EPSPs and resulted in sustained LTD. After induction of LTD with CHPG, 0.1 μM NLDE (black bar) failed to induce further LTD. (b) Administration of 0.1 μM NLDE for 15 min (black bar) slowly depressed EPSPs and resulted in LTD. After induction of NLDE-LTD, 250 μM CHPG (gray bar) failed to induce further LTD. (c) After induction of CHPG-LTD, LFS (1 Hz for 15 min, arrows) failed to induce further LTD, although short-term depression was observed during LFS. (d) After induction of NLDE-LTD, LFS (arrows) failed to induce further LTD, although short-term depression was observed during delivery of LFS. Traces depict EPSPs before (dashed lines) and 60 min after each treatment. Scale; 1 mV and 5 ms. PowerPoint slide Full size image

Delivery of Schaffer collateral LFS after induction of CHPG-LTD also failed to produce further LTD (69.9±5.8% after CHPG and 69.6±5.2% after LFS, N=5, Figure 3c), although LFS reliably induced LTD in naive slices (70.5±9.0%, N=5, see Figure 7). Moreover, LFS failed to induce further depression when administered after NLDE-LTD (70.6±2.8% after NLDE and 71.3±2.4% after LFS, N=5, Figure 3d). Taken together, these data support the idea that these three forms of LTD share common induction mechanisms. We also examined whether these three forms of LTD have similar effects on paired-pulse plasticity. After stable LTD induction, all three forms of LTD resulted in a statistically significant increase in the paired-pulse ratio (Figure 4).

Figure 4 Effects of LTD on paired-pulse plasticity. The bar graphs show the effects of LFS (a, N=11), CHPG (b, N=10), and NLDE (c, N=10) on paired-pulse plasticity using an interpulse interval of 21 ms. Under baseline conditions, a 50% maximal stimulus resulted in a small degree of paired-pulse facilitation (PPF) (white bars). Sixty minutes after LTD induction, all three treatments resulted in significantly increased PPF using this same stimulation (gray bars). Black bars show enhanced PPF after LTD even when the stimulus intensity was increased to activate EPSPs of the same size as in the initial baseline recordings. The traces to the right of the graphs (d) show raw EPSPs under baseline conditions (dashed traces) and after LFS induced LTD (solid traces). Upper traces use the baseline 50% maximal stimulus, whereas bottom traces show PPF after increasing the stimulus intensity to generate a conditioning EPSP similar to that observed in the initial baseline recordings (solid traces). Scale bar: 1 mV, 5 ms. *P<0.05, **P<0.01, compared with baseline PPF. PowerPoint slide Full size image

As LFS-LTD is known to require NMDAR activation (Dudek and Bear, 1992; Fujii et al, 1991), we examined whether other forms of LTD also involve NMDARs. Neither NLDE-LTD nor CHPG-LTD was altered by 30 μM D,L-APV (70.3±6.9%, 74.9±2.5%, N=5, respectively. Figure 5a and b). Given the interactions among these forms of LTD and the fact that LFS-induced LTD is blocked completely by this concentration of APV, this suggests that activation of mGluR5 and CB1Rs likely occurs downstream of NMDARs in the cascade leading to LFS-LTD. To determine the role of mGluR5 in these forms of LTD, we examined MPEP, a relatively selective mGluR5 antagonist. In the presence of 10 μM MPEP, LFS failed to induce LTD (97.8±3.1%, N=5, Figure 6a). MPEP also blocked CHPG-induced LTD (96.6±3.3%, N=5, Figure 6b). In contrast, NLDE administered in the presence of MPEP resulted in a typical slowly developing form of LTD (71.1±9.8%, N=5, Figure 6c). These results suggest that CB1R activation occurs downstream of mGluR5 in the LTD pathway.

Figure 5 CHPG-LTD and NLDE-LTD do not require NMDAR activation. (a) Administration of 250 μM CHPG (black bar) in the presence of 30 μM APV (hatched bar) successfully induced LTD. (b) Similarly, administration of 0.1 μM NLDE (black bar) in the presence of APV induced LTD. Traces depict EPSPs before (dashed lines) and 60 min after administration of CHPG and NLDE. Scale; 1 mV and 5 ms. PowerPoint slide Full size image

Figure 6 An mGluR5 antagonist blocks LFS-LTD and CHPG-LTD but not NLDE-LTD. (a) LFS (arrows) in the presence of 10 μM MPEP (hatched bar) acutely depressed EPSPs but did not induce LTD. (b) Administration of 250 μM CHPG (gray bar) in the presence of MPEP failed to induce LTD. (c) In contrast, administration of 0.1 μM NLDE (black bar) in the presence of MPEP resulted in a slowly developing form of LTD. Traces depict EPSPs before (dashed lines) and 60 min after administration of LFS, CHPG, and NLDE. Scale; 1 mV and 5 ms. PowerPoint slide Full size image

To determine whether ECs act downstream of mGluR5, we examined the effects of AM251 against LFS, CHPG, and NLDE. All three forms of LTD were blocked by AM251 (90.0±5.0%, 105.0±4.7%, 94.8±1.2%, respectively, N=5 each, Figure 7a–c). In contrast, 5 μM AM630, a specific CB2R antagonist, failed to block LTD induced by any of these treatments (66.2±11.4%, 64.4±1.6%, 65.5±10.1%, respectively, N=3 each, data not shown). Continuous administration of AM251 after washout of NLDE also failed to prevent NLDE-LTD (77.1±4.1%, N=5, Figure 7d), again indicating that the slowly developing LTD does not result from ongoing CB1R activation.

Figure 7 Effects of a CB1R antagonist on LTD. (a) LFS (arrows) in the presence of 5 μM AM251 (hatched bar) acutely depressed EPSPs but did not induce LTD. (b) Administration of 250 μM CHPG (gray bar) in the presence of AM251 acutely depressed EPSPs but also failed to induce LTD. (c) AM251 also blocked the ability of 0.1 μM NLDE (black bar) to induce LTD. (d) AM251 administered after washout of NLDE did not prevent the depression. Traces depict EPSPs before (dashed lines) and 60 min after administration of LFS, CHPG, and NLDE. Scale; 1 mV and 5 ms. PowerPoint slide Full size image

As DHPG, a less selective group I mGluR agonist, has been reported to induce a unique form of LTD (Palmer et al, 1997), we also examined the sensitivity of DHPG-induced LTD to MPEP and AM251. In contrast to CHPG, we found that DHPG induced robust LTD in the presence of MPEP (72.7±5.9%, N=5, Figure 8a), suggesting that this form of LTD is not mediated solely by activation of mGluR5. DHPG-LTD was also induced in the presence of AM251 (59.0±7.8%, N=6, Figure 8b). LTD in the presence of MPEP and AM251 did not differ from DHPG alone (62.6±8.7%, N=5). To examine interactions of DHPG-LTD with other forms of LTD, CHPG and DHPG were administered sequentially after stable induction of LFS-LTD. Although administration of CHPG did not induce further LTD after LFS-LTD, administration of DHPG induced additional and persistent synaptic depression (EPSP change 70.5±9.0% 60 min after LFS, 84.8±4.6% 60 min after CHPG administration, and 27.7±4.1% 60 min after DHPG administration, N=5, Figure 8c). These observations again suggest that LFS-LTD and CHPG-LTD share common mechanisms, but differ from DHPG-induced LTD. Moreover, DHPG induced further depression after establishing stable NDLE-LTD (57.1±10.4% by NLDE and 32.8±7.2% by DHPG after NLDE, P<0.05, N=4, data not shown).