Low-frequency stimulation induces the MPP long-term depression dependent on the cannabinoid receptor type 1.
Thus far, our data show decreased synaptic strength and altered control of glutamate release of the PP terminals synapsing DG granule cells. Previous studies demonstrated that those MK-801-driven changes negatively impact the induction of long-term synaptic plasticity in the hippocampus (Griego et al., 2022; Márquez et al., 2023). Therefore, it is reasonable to assume that neonatal treatment with MK-801 will negatively impact the induction of long-term plasticity at the PP synapses on DG granule cells of the hippocampus.
However, since long-term depression has been scarcely explored in the PP – DG synapses, we first determined a reliable stimulation protocol to induce LTD. Our exploratory experiments show that low-frequency stimulation (LFS, 900 pulses, 1 Hz), a protocol commonly used to induce glutamatergic LTD in the hippocampus (Dudek and Bear, 1992), fails to induce stable synaptic depression in both the LPP – DG and the MPP – DG synapse. In the LPP – DG synapse, LFS did not alter the baseline response (fEPSP at 50 min post-LFS: 108 ± 7.73% of baseline, n = 4 slices / 4 animals; white symbols and upper traces in Figures 1a and 1b). In the MPP – DG synapse, LFS caused transitory synaptic depression, and the fEPSP returned to baseline values within 50 min (fEPSP at 50 min post-LFS: 93.15 ± 5, n = 5 slices / 4 animals; white symbols and upper traces in supplementary Figure 1d and 1e).
Stellar cells and pyramidal neurons of EC layer II discharge volleys in the delta and theta range (0.4 – 10 Hz) to DG granule cells (Gloveli et al., 1997; Deshmukh et al., 2010). Therefore, we explored whether a stimulation paradigm within this physiological range induces LTD. Contrary to our prediction, we found that 900 pulses delivered at 3 Hz triggered synaptic potentiation in the LPP – DG synapse (fEPSP at 50 min post-LFS: 121.2 ± 9.32% of baseline, n = 3 slices / 3 animals; black circles and lower traces in supplementary figures 1a and 1b). This form of synaptic potentiation requires additional investigation beyond the scope of the present study.
In sharp contrast, the delivery of 900 pulses at 3 Hz in the MPP – DG synapse induced stable glutamatergic LTD that lasted up to 50 min (fEPSP at 50 min post-LFS: 69.64 ± 6.65% of baseline, n = 4 slices / 4 animals; Mann-Whitney test, P < 0.05 vs. LFS at 1 Hz; black circles and lower traces in Figures 1d and 1e). Because the delivery of 900 pulses at 3 Hz induced a reliable LTD in the MPP but not in the LPP synapse, subsequent experiments were restricted to the MPP – DG synapse.
Figure 3a-b summarizes the induction of LTD in the MPP – DG synapse and its sensitivity to perfusion DCG-IV (fEPSP at 90 min post-LFS: 69 ± 6.25% of baseline response,n = 6 slices / 6 animals; fEPSP in the presence of DGC-IV: 15.66 ± 2.55% of baseline response, Wilcoxon test, P < 0.05 vs. baseline response). Moreover, the induction of MPP LTD was accompanied by an increment in the PPF (MPP PPF in control condition: 1.18 ± 0.08; at 90 min post-LFS: 1.54 ± 0.17; Wilcoxon test, P< 0.05; black bars in Figure 3f), indicating presynaptic locus for expression of LTD.
According to a previous study, induction of LTD in the MPP synapse requires the delivery of 6,000 pulses at 10 Hz while simultaneously blocking GABAA receptors (Peñasco et al., 2019). Under these experimental conditions, the authors reported that this form of LTD requires activation of the cannabinoid receptor type 1 (CB1R). Therefore, our next experiment aimed to determine if CB1R is required for LTD induced with 3 Hz while maintaining active GABAergic transmission. Consistent with Peñasco et al., we found that perfusion of the CB1R antagonist, AM 251 (5 µM), blocks the induction of LTD (MPP fEPSP at 90 min post-LFS: 119.7 ± 10.94% of baseline response, n = 4 slices / 4 animals; lower traces and white symbols in Figure 3a-b and white bars in Figure 3e). The subsequent perfusion of the DCG-IV (5 µM) at the end of experiments corroborated the MPP origin of the synaptic responses (fEPSP in the presence of DCG-IV: 15.7 ± 5.22% of baseline; white bar in Figure 3e). Likewise, perfusion of AM 251 prevented the changes in the MPP PPF observed after the induction of LTD (MPP PPF in AM 251 treated slices in control condition: 1.22 ± 0.1; at 90 min post-LFS: 1.2 ± 0.08; Wilcoxon test, P > 0.05; white bars in Figure 3f). Together, these results confirm that the MPP – DG synapse exhibits a stable LTD with a presynaptic locus of expression and requires CB1R activation.