Glutamatergic neurons and SST+ GABAergic neurons project from MEC to LEC.

(A) Horizontal brain section through MEC of a GAD67eGFP mouse showing GFP+ neurons (green) together with retrogradelly labelled neurons (light cyan) from LEC. Inset: Schematic of the retrograde tracer injection in LEC. Scale bar, 500 µm. See Supplementary Fig. 1 for images showing the dorsoventral extent of the injection site. (B) SST+ GABAergic neurons in MEC project to LEC. Arrowheads indicate examples of triple-labelled neurons (SST+, GAD67eCFP, FG) from the experiment in A. Scale bar, 100 µm. (C) Proportions of GABAergic neurons (SST+ and non-SST+) and glutamatergic neurons in the MEC-LEC pathway (neuron counts from 3 mice). (D) Delivery of an equal mix of AAV1-CAG-FLEX-tdTomato and AAV1-CamKII-eGFP-WPRE-rBG into MEC of SSTCre mice (upper left schematic) selectively labels SST+ neurons with tdTomato and glutamatergic neurons with GFP. Sub, subiculum. Scale bar, 500 µm. See Supplementary Fig. 2 for images showing the dorsoventral extent of the injection site. (E) Different laminar innervation patterns in LEC of glutamatergic axons and SST+ axons from the experiment in D. Wm, white matter. Scale bar, 100 µm.

Target preference of medial entorhinal axons in LEC.

(A) Left: Virus injection in MEC of a wild-type mouse. Right: High magnification image showing YFP-labelled photosensitive (ChR2) axons in LEC. MML, DG middle molecular layer. Scale bars, 500 µm (left) and 100 µm (right). (B) Membrane current recordings of a representative PNIIA to the indicated pharmacological treatments during optogenetic stimulation (blue bars). The average membrane current traces (red/black) are superimposed on the individual traces (grey). (C) Laser-evoked EPSCs (black) and IPSCs (red) in representative PNIIA, PNIIB and PNIII recorded without (top, control ACSF) or with glutamatergic synaptic blockers (bottom, DNQX/APV). (D) Normalized IPSC amplitudes plotted against the location of photostimulation. Boxes indicate 25th-75th percentiles, red line is median, whiskers extend to all data points not considered outliers. All data points are superimposed on the box plots. Each value represents data from one cell resulting from photostimulation of the given layer. (E) Patch-clamp recorded neurons together with photosensitive MEC SST+ axons in a semicoronal slice of LEC from an SSTCre mouse. PER, perirhinal cortex. Schematic: Injection to virally label MEC SST+ neurons with ChrimsonR-tdTomato. Scale bars, 200 µm. (F) Membrane current recordings of the numbered neurons in E during photostimulation (orange bars). (G) Membrane current amplitudes recorded from all neurons in the experiment in E. (H) Charge transfers in response to optogenetic activation when clamping neurons at 0 mV. Non-responding neurons have charge transfer values of 0 pC. (I) Same as in H, but for data obtained when clamping neurons to −50 mV. The virus used in each experiment is indicated below each individual panel. The color code for PNIIA, PNIIB and PNIII in (H) and (I) follow the color code as defined in G.

Target-specific control of postsynaptic neuron activity by medial entorhinal axons in layer I.

(A) Biocytin-filled principal neurons recorded from layers IIa, IIb and III. Scale bar, 100 µm. (B) Different laser-evoked responses of PNIIA versus PNIIB and PNIII (PNIIA, hyperpolarizing; PNIIB, PNIII; depolarizing). Neurons were depolarized by somatic current injections (yellow) while MEC axons were photostimulated with light (blue bar). Schematic upper left: A 100 ms laser stimulation (blue) of MEC axons (ChR2, green) in LEC layer I during current-clamp recordings. (C) Action potential firing across multiple sweeps of the photostimulation (blue bar) protocol in representative neurons. Top: Voltage recordings. Middle: Raster plots. Bottom: Histograms. Note the transient suppression of spiking activity in PNIIA and the increase in spiking activity in PNIIB and PNIII during photostimulation. (D) Mean firing rate (mean number of action potentials per bin) of all tested PNIIA, PNIIB and PNIII. Plots are mean (black) ± standard error of the mean (grey). (E) Box plots showing the deviation in action potential firing relative to the average firing activity without laser stimulation. Box plots as in Fig. 2. Outliers are shown as grey points. (F) Donut plots showing the proportion of laser-evoked responses. (G) Laser-evoked hyperpolarizing responses are resistant to application of glutamatergic synaptic blockers (DNQX/APV). The recording is from a representative PNIIA. (H) Left: Laser-evoked depolarizing responses recorded from a representative PNIII. Right: The responses disappear when blocking glutamatergic synaptic transmission with APV and DNQX. RMP, resting membrane potential. In the rightmost panels the average voltage trace (black) is overlaid the individual voltage traces (grey).

Cortical glutamatergic inputs to principal neurons in layer IIa.

(A) Labelled axons in superficial layers of LEC following anterograde tracer injections into PER (left), cLEC (middle) and PIR (right). BDA, biotinylated dextran amine. See Supplementary Fig.11 for images of the injection sites. (B) Confocal images of neurons retrogradely labelled from LEC in a GAD67eGFP mouse (same case as the experiment in Fig. 1A). FG+ cell bodies were found in MEC, PER, cLEC and PIR. In this brain, co-localization of GFP and FG was evident in PER and MEC (white square insets). Bar graphs show the proportions of neurons retrogradely labelled with FG or AAV2-CAG-tdTomato that also expressed GFP (FG/tdTomato and GFP; mean ± standard deviation, n = 3 mice). (C) Electrophysiological interrogation of afferent pathways in wild-type mice. Optogenetic activation (blue bars) of photosensitive axons from PIR (top) or cLEC (middle) in LEC elicited monosynaptic EPSPs in PNIIA. Insets show average membrane potential traces in control ACSF (black), and ACSF added with TTX (blue) or TTX + 4-AP (orange). Activation of PER neurons (bottom) through UV-photostimulation (purple bars) of caged glutamate evoked EPSPs in PNIIA in LEC. All membrane potential traces show the average trace (black) superimposed on individual trials (grey). Note that the recordings in the three panels are from different mice. Scale bars are 100 µm in (A) and (B).

Synaptic inputs from MEC diminish EPSPs of principal neurons in layer IIa.

(A) Experimental design of dual photostimulation within layer I of LEC while recording from PNIIA. Depicted is an example of a biocytin-filled PNIIA showing its typical dendritic arborizations in layers I/IIa. (B) Example PNIIA recording showing EPSPs evoked by glutamate uncaging alone (left, right) or together with MEC input (middle). The average membrane potential trace (black) is superimposed on the individual traces (grey). The dotted blue line indicates the peak of the average EPSP in response to photolysis of glutamate alone, showing a decrease in EPSP amplitude when pairing glutamate release with MEC activation. (C) Quantification of EPSP amplitude for all individual trials recorded from the neuron in B. (D) Average voltage traces from the recording in B. (E) Scatter plots of EPSP amplitude (left) and halfwidth (right) for individual sweeps of the experimental protocol from all recorded PNIIA (n = 38). (F) Relative deviations in EPSP amplitude between responses elicited when glutamate was released alone (purple) and when glutamate release was paired with photostimulation of MEC axons (purple and red). Data show relative deviations for all recorded PNIIA (n = 38). (G) Same as in F, but for EPSP halfwidth.

Anatomical organization of the circuit connecting MEC to LEC.

Inhibitory and excitatory pathways differentially target principal neurons in the superficial layers of LEC. The main pathways (solid lines) described in this study are shown together with the well-established connectivity between LEC and the hippocampus (dotted lines; Nilssen et al., 2019). For the MEC–LEC pathway, the thickness of the GABAergic/glutamatergic arrows indicate the relative strength of these inputs onto postsynaptic neurons. HC, hippocampus

Primary antibodies used in the study

Secondary antibodies used in the study