The place of ExNR in the hippocampal excitation chain.

A. z-Projected confocal images of ExNR filled with Alexa 488.

B. Typical voltage responses and firing patterns of ExNR (red) and CA1 pyramidal neuron (black) triggered by hyperpolarizing and depolarizing current injections.

C. Schematic drawing of experimental setup for optogenetic stimulation of CA3 pyramidal cell projections (left panel). Right panel shows blue light evoked individual and averaged responses in CA1Pyr (black) and ExNR (red).

D. Schematic drawing of experimental setup for optogenetic stimulation of EC projections (left panel). Example of blue light triggered individual and averaged IPSCs in ExNR (right panel).

E. Histogram compares latencies of EPSCs induced by optogenetic stimulation in CA1Pyr and ExNR. Averaged values ± SD presented by bars, symbols show data from individual experiments.

F. Schematic diagram of excitatory inputs to major populations of hippocampal principal neurons and ExNR.

Synaptic communication of ExNR with PV+ interneurons.

A. Properties of excitatory projections from ExNR and CA1Pyr to two types of PV+ interneurons. (A1) Diagram shows tested connections. (A2) Example averaged traces recorded from connected pairs between presynaptic CA1Pyr (Pyr) or ExNR and postsynaptic basket cells (BC) or bistratified neurons (Bist). (A3) The plot shows median EPSP values together with the averaged EPSP amplitudes obtained in individual cell pairs formed by presynaptic CA1Pyr or ExNR and postsynaptic BC or Bist. (A4) Comparison of connectivity rates at excitatory projections from CA1Pyr or ExNR to two types of PV+ interneurons.

B. Properties of inhibitory projections from stratum oriens PV+ interneurons to ExNR and CA1Pyr. (B1) Diagram shows tested connections. (B2) Example averaged traces recorded from connected pairs between presynaptic PV+ interneurons and postsynaptic CA1Pyr (Pyr) or ExNR. Note the absence of connections from BC to ExNR. Comparison of connectivity rates (B3) and IPSP amplitudes (B4) for the inhibitory projections.

C. Diagram summarizes data on connectivity within local circuitries formed by CA1Pyr and ExNR with two types of stratum oriens PV+ interneurons. Note, while connectivity rates at reciprocal connections between CA1Pyr and BC or Bist are very similar, excitatory projections from ExNR dominate over recurrent inhibition.

Excitatory drive from a single ExNR is sufficient to trigger polysynaptic inhibition.

A. Diagram shows connections tested for polysynaptic inhibition.

B. Example of connected ExNR → Bist pair where efficiency of synaptic transmission was sufficient to reliably trigger a postsynaptic AP. Subthreshold EPSPs were observed in 10% of ExNR → Bist and 7% 0f ExNR → BC pairs.

C. Polysynaptic IPSPs recorded at ExNR → CA1Pyr. Recruitment of an extra interneuron that receives excitatory drive from the stimulated ExNR and innervates the recorded CA1Pyr was confirmed by the disynaptic delay of IPSPs relative to the peak of AP and the complete occlusion of IPSPs in the presence of an AMPAR antagonist (CNQX; blue trace).

D. Schematic representation of the experimental setup (upper panel): 16-channel electrode array for local field potential (LFP) recording from the pyramidal layer and patch-clamp recording from the ExNR in striatum radiatum. The micrograph (bottom panel) of the hippocampal slice shows the location of the extracellular electrodes and patch pipette.

E. Averaged LFP and current source density profile in the hippocampal pyramidal layer relative to the peak of evoked action potential (AP) of the ExNR (upper panel). Averaged AP of the ExNR (bottom panel; blue trace), LFP for channel #11 (black trace, also marked with an asterisk in the top panel), and the first derivative of Vm of the averaged AP waveform (Vm/Δt, green trace). The LFP trace shows the 4 parameters calculated for all experiments (AP1 to AP2 delay, AP2 amplitude, fIPSP amplitude, AP1 to fIPSP peak time) AP1 is the negative peak on the LFP that coincides in shape with the first derivative of the AP (Vm/Δt) and therefore represents LFP fluctuation caused by evoked AP in ExNR. AP2, following AP1 with a characteristic monosynaptic delay, reflects activity in the second neuron excited by the stimulated ExNR. Finally, the fIPSP following AP2 suggests the GABAergic nature of the second neuron.

(F) Box plots show medians (P25; P75) and corresponding individual values for the following parameters: AP1 to AP2 delay and AP1 to fIPSP delay (upper panel); AP2 and fIPSP amplitudes, (bottom panel).

Possible functional role of ExNR in control of perisomatic inhibition of hippocampal output

A. Schematic drawing depicting excitatory and inhibitory inputs of ExNR and their synaptic targets. The main differences from CA1Pyr are (i) the absence of external excitatory input from EC and (ii) a lack of inhibitory control provided by PV+ backet cells.

B. The left diagram shows the classical view of excitatory and inhibitory circuitries that control the major hippocampal output (CA1 pyramidal cells). Excitation converges from EC via the trisynaptic pathway and directly through the temporoammonal tract and is efficiently controlled by means of feed-back inhibition, primarily via reciprocal connections with BC. The balanced interplay between excitation and inhibition of pyramidal cells was postulated to be the main structural prerequisite for hippocampal rhythmogenesis. However recruitment of ExNR (right diagram), bypassing BC-mediated perisomatic inhibition, can shift the excitation/inhibition balance of CA1Pyr and disrupt the functioning of the CA1Pyr-BC oscillator.

Intrinsic properties of bistratified and basket cells.

A. Voltage responses and spiking to hyperpolarizing and depolarizing 1 second current injections in typical bistratified neuron. Note that minimal amplitude (50 pA) of injected current is sufficient to trigger sustained firing throughout whole period of depolarization.

B. Voltage responses and spiking to hyperpolarizing and depolarizing 1 second current injections in typical basket cell. Note that much higher amplitude (200 pA) of injected current is needed to trigger sustained firing throughout whole period of depolarization.

C. Box plot compares medians (P25; P75) and corresponding individual input resistance values in bistratified and basket cells.

Direct comparison of synaptic efficacy at ExNR → Bist and CA1Pyr → Bist synapses.

Diagram shows experimental design of sequential triple recordings. After finding synoptically connected pair from either ExNR or CA1Pyr to Bist, we searched for the alternative excitatory input (CA1Pyr or ExNR, respectevly) to the same interneuron. Box plot provides pairwise comparison of the avareged EPSP amplitude at two types of excitatory synapses (from presynaptic ExNR and CA1Pyr) converging on the same postsynaptic bistratified cell.