Figure 5. L5a neurons of the medial entorhinal cortex (MEC) provide direct excitatory and indirect inhibitory inputs to pyramidal cells in CA1.

(A) Experimental design showing viral expression, placement of patch-clamp electrode, and light delivery over CA1. (B) Examples of biocytin filled pyramidal neurons recorded in CA1. (C) An example electrical recording of a pyramidal neuron at rest (−67 mV) showing depolarizing responses upon 3-ms light stimulation (top) (blue line, scale bar: 1 mV, 10 ms). Train of action potentials upon 200 pA step-current injection (bottom; scale bar: 20 mV, 100 ms). (D) Proportion of responsive pyramidal neurons located in the distal and proximal halves of CA1 (top). The difference in proportions was not significant (X-squared = 1.9071, df = 1, p value = 0.167291, chi-squared test, n = 12 cells in proximal and n = 39 cells in distal CA1). Proportion of responsive pyramidal neurons located in the deep versus superficial locations of CA1 pyramidal cell layer (bottom) (X-squared = 0.2969, df = 1, p value = 0.585804, chi-squared test, n = 8 cells in deep SP and n = 13 cells in superficial SP). (E) Effects of bath application of Gabazine (orange, n = 10 cells, 9 mice) and NBQX (green, n = 5 cells) on postsynaptic potentials (PSPs) recorded from a pyramidal neuron (scale bar: 0.5 mV, 10 ms) and a summary plot of PSP amplitude measurements for all tested pyramidal neurons. Note that some neurons were only treated with Gabazine which did not cause a significant change in amplitudes (p = 0.97, two-tailed Student’s t-test, n = 10 cells, 9 mice). (F) An example of ten consecutive PSP responses recorded from a single pyramidal neuron illustrates the short and invariant latency of PSPs (scale bar: 0.5 mV, 10 ms) and a cumulative probability plot of standard deviation of latencies for neurons with PSP responses that were >1 mV in amplitude (n = 20 cells). (G) Effects of bath application of tetrodotoxin (TTX; orange) and 4-aminopyridine (4-AP; green) on PSPs recorded from a pyramidal neuron (scale bar: 0.5 mV, 10 ms) and a summary plot of changes in PSP amplitudes for all tested pyramidal neurons. TTX application abolished responses (n = 5 cells, 5 mice, p = 0.01, two-tailed Student’s t-test). (H, I) An example inhibitory PSP response recorded from a pyramidal neuron upon 10 Hz light stimulation (blue bars). Response polarity reversed when the neuron’s membrane potential was adjusted to –50 mV and was abolished after application of Gabazine (scale bars: 0.2 mV, 100 ms). Inset shows the long latency (>10 ms) of PSP onset indicating polysynaptic connectivity.

Figure 5—figure supplement 1. Experimental design and properties of responses to optogenetic stimulation of axons originating from L5a of the medial entorhinal cortex (MEC).

(A) Summary table for biophysical properties of neurons recorded in the study. (B) An example of a biphasic response recorded from a pyramidal neuron upon 3-ms stimulation of axons of MEC L5a neurons in CA1. A late hyperpolarizing (red) response was abolished by application of Gabazine to reveal a larger depolarizing postsynaptic potential (PSP; black) which was abolished by application of NBQX (grey) (scale bar: 10 ms, 0.5 mV). (C) Experimental strategy for fluorescence guided patch-clamp recordings made from parvalbumin-expressing neurons. Left: a FlpX-dependent green fluorescent protein (GFP)-expressing virus was injected in the hippocampus in Rbp4-Cre X Pvalb-Flp mice. Right: confocal image of horizontal slice showing labelled interneurons in the hippocampus. Bottom: an example fast-spiking interneuron marked as described in (C), that showed depolarizing PSP upon light stimulation. During recording, the neuron was filled with biocytin and later stained with streptavidin and anti-parvalbumin antibody. (D) (i) Traces of responses to a train of 10 light stimulation (blue lines), showing relatively stable EPSP amplitude across simulations (scale bar: 30 ms, 1 mV). (ii) Magnified images of 1st and 10th pulse illustrate the invariant latency in one example cell (scale bars: 0.5 ms, 0.5 mV). (iii) Quantification of latency across the train in all cells. Error bars represent standard error of the mean (SEM). (E) Plots showing the frequency distribution of PSP amplitudes in pyramidal neurons and interneurons. PSPs over 12.5 mV were detected in interneurons and often resulted in spiking responses (SP_Pyr: n = 19, SP_int: n = 19, SR: n = 9, SL and SM: n = 22 cells). (F) Effects of bath application of Gabazine and NBQX on PSPs recorded from interneurons in SR and SL. The PSP amplitudes were largely unaffected by application of Gabazine (SR: n = 2 cells and SL: n = 2 cells, p = 0.28, two-tailed Student’s t-test) but were largely blocked by NBQX (SR: n = 2 cells and SL: n = 2 cells, p = 0.04, two-tailed Student’s t-test) indicating AMPA receptor-mediated glutamatergic synaptic transmission. (G) Effects of bath application of tetrodotoxin (TTX) and 4-aminopyridine (4-AP) on the response amplitude PSPs recorded from interneurons in SR, SL, and SM (SR: n = 2, SL: n = 3, SM: n = 1 cells). (H) Cumulative probability plots of standard deviation of latencies for neurons with PSP responses that were >1 mV in amplitude (SR: n = 6 cells, SL: n = 15 cells, SM: n = 4 cells). EPSPs of interneurons in all layers except SO had a short latency after light stimulation (mean latency SR: 2.00 ± 0.08 ms, n = 6 cells; SL and SM: 2.12 ± 0.21 ms, n = 23 cells). In SO, two out of three of the evoked PSPs had a larger latency of onset (mean latency = 8.5 ± 0.63 ms, n = 2 cells, 2 mice, data not shown).