Criteria for isolating synaptic responses from the medial and lateral perforant paths onto granule cells of the dentate gyrus
The DG granule cells receive glutamatergic inputs via the axons of stellate cells and pyramidal neurons of the lateral and medial EC. These axons, collectively named perforant paths (PP), exhibit unique pharmacological and synaptic properties depending on whether they originate from the lateral or medial EC (LEC and MEC, respectively). We first isolated and characterized their synaptic responses before exploring whether neonatal treatment with MK-801 alters the LPP and MPP synapses. A stimulation electrode was positioned in the outer one-third section of the molecular layer (≈200-220 µm above the granule cell layer) or the middle one-third section (≈100-120 µm above) of the supra-pyramidal blade of the DG (Petersen et al., 2013), and recording pipettes were positioned 200 – 300 µM onto the stimulation electrode to record a fEPSPs from the LPP or MPP, respectively (Figure 1a). Presynaptic terminals of LEC-contacting granule cells selectively express group III metabotropic glutamate receptors (mGluRs), while those of MEC-contacting granule cells express group II mGluRs (Macek et al., 1996; Shigemoto et al., 1997). We used these pharmacological criteria to verify the origin of the evoked responses. Bath perfusion of DCG-IV (5 µM), a group II mGluRs agonist, did not depress the LPP fEPSP (92.9 ± 3.77% of baseline response); however, the subsequent perfusion of L-AP4 (20 µM), a group III mGluRs agonist, abolished the synaptic response (17.18 ± 2.8% of baseline response; n = 7 slices / 6 animals, upper traces in Figure 1b). On the other hand, bath perfusion of L-AP4 did not depress the MPP fEPSP (94.8 ± 3.28% of baseline response). However, the synaptic response was abolished during the subsequent application of DCG-IV (20.35 ± 3.2% of baseline response; n = 7 slices / 6 animals; lower traces in Figure 1b). The pharmacological activation of mGluRs shows that responses evoked from the LPP and the MPP converging on DG granule cells can be reliably isolated. Therefore, we systematically used these criteria to corroborate the synaptic origins of the evoked responses included in this study.