Figure 6. L5a neurons of the medial entorhinal cortex (MEC) provide direct excitatory inputs to interneurons in CA1.
(A) Experimental design showing viral expression, patch pipette, and light stimulation. Interneurons are marked by the expression of venus fluorescent protein driven by the vesicular GABA transporter (Vgat) gene’s promoter. (B) Biocytin filled neurons in the Rbp4-Cre X Vgat-Venus double transgenic mouse line. Fluorescent labelling facilitated patch-clamp recordings made from GABAergic interneurons and distinguishing SR, SL, and SM layers. (C) Representative examples of depolarizing responses recorded at resting membrane potential following 3ms blue light stimulation (blue line) of L5a axons in CA1 (left) (scale bar: 1 mV, 10 ms). Neurons in SO and SM were typically not responsive (<30%). Responses from SR neurons were on average larger than the responses from neurons in other layers (scale bar: 1 mV, 10 ms). Neurons’ spiking response to injecting 200 pA current (right) (scale bar: 10 mV, 100 ms). (D) Proportion of responsive interneurons in all layers of CA1 recorded at resting membrane potential. Green highlighted segment corresponds to the proportion of cells with depolarizing membrane potentials; grey highlighted segment corresponds to neurons with no change in their membrane potential. SO: n = 18 cells, 10 mice; SPFSint: n = 33 cells, 8 Rbp4-Cre X Pvalb-Flp mice, 5 Rbp4-Cre X Vgat-Venus mice, 8 Rbp4-Cre mice; SPNSFint: n = 13 cells, 11 Rbp4-Cre mice, 1 Rbp4-Cre X Vgat-Venus mouse; SR: n = 14 cells, 6 mice; SL: n = 60 cells, 15 mice; SM: n = 21 cells, 10 mice. (E, F) Effects of bath application of Gabazine (orange) and NBQX (green) on postsynaptic potentials (PSPs) recorded from a fast-spiking pyramidal layer interneuron (left) (scale bar: 1 mV, 5 ms). Summary quantification of PSP amplitudes for multiple cells (right). The PSP amplitudes were largely unaffected by application of Gabazine (SPint: n = 6 cells, p = 0.99 two-tailed Student’s t-test) but were largely blocked by NBQX (SPint: n = 5 cells, p = 0.03, two-tailed Student’s t-test) indicating AMPA receptor-mediated glutamatergic synaptic transmission. (G, H) Effects of bath application of tetrodotoxin (TTX; orange) and 4-aminopyridine (4-AP; green) on the response amplitude PSPs recorded from pyramidal layer interneurons. Example traces from a fast-spiking pyramidal layer interneuron (scale bar: 1 mV, 5 ms). Summary plots of response amplitude measurements from multiple recordings (SPint: n = 4 cells). (I, J) An example of ten consecutive PSP responses recorded from a single fast-spiking interneuron in SP illustrates the short and invariant latency of PSPs. Cumulative probability plots of standard deviation of latencies for neurons with PSP responses that were >1 mV in amplitude (mean latency SP: 2.08 ± 0.08 ms, n = 21 cells).