Optogenetic Activation of Cajal-Retzius Cells Reveals Their Glutamatergic Output and a Novel Feedforward Circuit in the Developing Mouse Hippocampus

Optogenetically triggered firing in Cajal-Retzius cells. A, Camera lucida tracing of whole-cell, biocytin-filled, EYFP-expressing neurons of stratum lacunosum-moleculare reveals the typical features of Cajal-Retzius (l-m, stratum lacunosum-moleculare). B, Short flashes of blue light induce action currents in cell-attached configuration (c-a, top traces; five sweeps superimposed) and reveal inward currents after breakthrough into whole-cell voltage-clamp mode (w-c, V_h = −60 mV, bottom traces; five sweeps superimposed). Notice the beginning of the current already during the flash. The empty square indicates latencies measured from the start of the light flash (vertical dotted blue line) to the peak of the action current. The dotted line is for reference. C, Similarly, flashes of blue light trigger firing in the whole-cell current-clamp configuration (five traces superimposed). The empty circle highlights the measurement of the latency from the beginning of the light flash (vertical dotted blue line) to the peak of the action potential. The dotted line indicates the 0 mV level. D, Firing pattern of ChR-expressing cells. Notice the sag in the hyperpolarization response (−25 pA step, 1 s) and the train of action potentials terminated by depolarization block (70 pA, 1 s), which is characteristic of Cajal-Retzius cells. The dotted line indicates −62 mV. E, Summary plot of the latency of the firing measured under cell-attached (c-a, empty squares) and whole-cell configuration (w-c, empty circles). Both mean ± SE and individual data points are shown. F, Unreliable stimulation by trains of flashes. Notice the decreasing probability of firing coupled with increasing latencies during a 10 Hz train of flashes delivered to a Cajal-Retzius cell (recoded in cell-attached mode: c-a, four traces superimposed). Action currents are expanded in the insets, and the blue dotted lines indicate the start of the flash. Same cell after passing in whole-cell mode (w-c); notice the decreasing amplitude of the photocurrents during the repeated stimulation. Black dotted line for reference.

Light stimulation of Cajal-Retzius cells triggers inward currents in stratum lacunosum-moleculare interneurons held in voltage clamp (V_h = −60 mV). A, Anatomical reconstruction of four neurogliaform-like cells with local axonal arborization in stratum lacunosum-moleculare (gray area, l-m). Somatodendritic arborization in red, axon in black. B, Corresponding postsynaptic currents recorded from the interneurons in A after the delivery of light flashes (blue traces) to stratum lacunosum-moleculare. Three overlapping responses are shown for every neuron.

Optogenetic stimulation of Cajal-Retzius cells activates target interneurons via AMPA and NMDA glutamate receptors. A, Different holding potentials (V_h = −60 and +60 mV) reveals the presence of kinetically distinct synaptic responses to flashes of blue light. Three sweeps are shown for each holding potential. B, Summary plots showing the peak amplitude and kinetic properties (rt, 20–80% rise time; hw, half-width) of synaptic responses recorded in interneurons at hyperpolarized (−60 mV) and depolarized (+60 mV) holding potentials. Points joined by lines are data from individual experiments, whereas averages and SE are indicated by empty circles and bars, respectively. C, NBQX (20 μm) blocks responses recorded at −60 mV. Top, Postsynaptic currents before and after application of the drug (four sweeps superimposed for every condition). Bottom, Summary plot of the time course of the effect of NBQX application (black bar) on the normalized amplitude of the light-evoked postsynaptic current (norm peak). D, Similar to C, but for the d-AP5 sensitivity of responses recorded at +60 mV.

Activation of the CXCR4 receptor has parallel effects on optogenetically triggered excitability of Cajal-Retzius cells and optogenetically evoked postsynaptic responses in stratum lacunosum-moleculare interneurons. A, Bath application of CXCL12 (50 nm) either reduces the number of light-triggered action potentials (r1, example recording 1) or delays (r2, example recording 2) firing of Cajal-Retzius cells. Top, Insets, The distinct effects are shown by four overlapped sweeps in control and in the presence of CXCL12. The vertical dashed blue lines are for temporal reference. Notice that in the presence of CXCL12 (black bar) the latency of the firing increases in r2. Bottom, Summary graph (empty circles, mean; bars, SE) of the time course of the effect of CXCL12 application on light-evoked spikes in Cajal-Retzius cells (norm APs, normalized number of action potentials). B, Similar to A, but the analysis is performed on synaptic responses recorded from stratum lacunosum-moleculare interneurons. Notice in the top panels either the abolishment of the synaptic response (r1, example recording 1) or the delay of its onset (r2, example recording 2). The vertical dashed blue lines are for temporal reference. Bottom, Summary plot of the time course of the normalized peak amplitude (norm peak) of the postsynaptic response during CXCL12 application (black bar).

Cajal-Retzius cells contact CA1 pyramidal cells via AMPA and NMDA glutamate receptors. A, Anatomical reconstruction of a CA1 pyramidal cell in which a postsynaptic response with a d-AP5-sensitive component was recorded following light stimulation [gray, stratum lacunosum-moleculare (l-m)]. B, Top, Postsynaptic responses recorded at V_h = −60 mV in control conditions and after the addition of NBQX (20 μm). Three sweeps are superimposed for each condition. Bottom, Summary plot (empty circles, mean; bars, SE) of the time course of the effect of NBQX application (black bar) on the postsynaptic responses (norm peak, normalized peak). C, Top, Postsynaptic responses (three sweeps superimposed) recorded at V_h = −30 mV before and after the application of d-AP5 (50 μm). Averaged traces in the two conditions (black trace, control; gray trace, d-AP5) are superimposed after scaling. Bottom, Summary graphs of the effect of d-AP5 on the normalized amplitude (left, norm peak, empty circles are individual experiments) and the half-width (right, empty squares are individual experiments) of the postsynaptic responses.

Comparison of the basic electrophysiological properties of postsynaptic currents recorded in interneurons and pyramidal cells. A, Normalized averaged traces of synaptic responses recorded in an interneuron of stratum lacunosum-moleculare (black line) and in CA1 pyramidal cells (gray line). Notice the different kinetics of the two responses. B, Cumulative probability distribution plot of the latencies of the responses from the beginning of the light flash (black, interneurons; gray, pyramidal cells). Notice the striking similarity of the two curves. C–E, Cumulative probability distribution plots of the peak amplitude, rise time, and half-width of the light-evoked responses. Notice the largely different kinetics, with interneurons showing much faster rise times and half-widths.

Developmental profile of the probability of finding light-evoked responses in interneurons (black empty circles) versus pyramidal cells (gray empty circles). A, Summary plot of the probability of obtaining a response (p response) and linear fits, weighted according to the number of cells tested at each postnatal age. B, Bar plot of the number of interneurons/pyramidal cells (black empty bars/gray empty bars) tested to estimate the probability of obtaining a response.

Optogenetically evoked postsynaptic currents in interneurons and pyramidal cells are action potential-dependent. A, Response recorded in interneurons before [control (ctrl)] and after the addition of TTX (500 nm). Four sweeps were superimposed in both conditions. B, As in A, but data are from a pyramidal cell recording.

Effects of combined application of TTX (1 μm) and 4-AP (1 mm) on light-evoked action potential in Cajal-Retzius cells, and light-evoked synaptic currents in interneurons and pyramidal cells. A, Top, Light-evoked action potentials in Cajal-Retzius cells are prevented by adding TTX and 4-AP (black bar) to the external solution (four sweeps superimposed in both conditions). Bottom, Summary plot showing the time course of the effect (norm APs, normalized number of action potentials; empty circles and bars, mean and SE). B, Top, Light-evoked synaptic currents in interneurons are not abolished by the combined application of TTX and 4-AP. Notice however, the increased latency of the light-evoked events (dotted blue line indicates the beginning of the light flash for reference). Four responses superimposed in both condition. Bottom, Summary graph form several experiments (norm peak, normalized peak of the synaptic response). C, As in B; however, notice that the application of TTX and 4-AP completely abolishes responses in pyramidal neurons.

Effects of suppressing fast GABAergic inhibition on light-evoked responses in pyramidal cells. A, Top, Traces from a nonresponsive pyramidal cell in control conditions and in the presence of gabazine (gbz; 12.5 μm). Notice that gabazine application does not reveal latent polysynaptic pathway. The gray line shows the time window used to measure the peak current following the light flash. Four sweeps were superimposed in both condition. Bottom, Summary plot from 9 of 10 pyramidal neurons that did not produce light-evoked currents either in the control condition or after the addition of gabazine (empty circles, mean; bars, SE). B, Sweeps from the single experiment where gabazine application unmasked latent polysynaptic pathways. Top, Four traces superimposed in control conditions. Bottom, Four records in the presence of gabazine. Notice the long latency from the beginning of the light flash (dotted blue lines for reference), which is incompatible with the much shorter latencies usually recorded in light-responsive pyramidal cells (compare Fig. 7). C, Top, Light-evoked responses in pyramidal cells in control conditions and after the addition of gabazine. Four sweeps were superimposed in both condition. Notice that responses are unchanged by the addition of the drug. Bottom, Summary graph for all the responsive cells tested (norm peak, normalized peak of the synaptic response; empty circles and bars, mean and SE, respectively). The mice used for these experiments (n = 3) were selected at a postnatal stage associated with GABA_A receptor-mediated inhibition in hippocampal pyramidal neurons (P15, P17, and P18; see Banke and McBain, 2006).

Lack of direct light-evoked firing and of ChR expression in entorhinal cortex neurons. A, Immunocytochemistry showing EYFP labeling of layer I in the entorhinal cortex, but no clearly labeled neurons in the deeper layers. B, C, Enlargements of the areas marked by an asterisk in A for the lateral and medial entorhinal cortices, respectively. Notice the typical morphology of Cajal-Retzius cells and the staining associated with layer I. D, Comparison of the response to light in hippocampal Cajal-Retzius neurons (left) versus layer II/III entorhinal cortex cells (middle) in cell-attached conditions. Notice the reliable firing reflected by action currents in Cajal-Retzius cells and the lack of light-induced effects in entorhinal cortex neurons. Four sweeps superimposed in both cases. Right, The percentage of cells responding to light with firing in the two different samples. CR, Cajal-Retzius cells; EC, entorhinal cortex neurons. Notice that entorhinal cortex neurons do not fire under these experimental conditions. E, ChR-mediated currents in hippocampal Cajal-Retzius cells (left), and layer II/III entorhinal cortex neurons (middle and right). A short flash of light (1 ms) produces significant currents in Cajal-Retzius cells, as shown by the recording (four traces superimposed, top) and by the cumulative distribution plot for several experiments (bottom). In contrast, light flashes either of the same (1 ms; middle) or of longer duration (5 ms; right) do not produce significant currents in entorhinal neurons. Top, Four traces superimposed showing the response of entorhinal cortex neurons to 1 and 5 ms flashes. Bottom, Summary plots for the two conditions (the distribution of the photocurrents in Cajal-Retzius cells is also shown in gray for comparison).

Cajal-Retzius cells drive feedforward GABAergic input to pyramidal cells. A, Example of an interneuron firing following blue light stimulation. Anatomical reconstruction (left); gray area labels stratum lacunosum-moleculare. Red, Somatodendritic arborization; black, axon. Right, Action potentials triggered by light stimulation (five sweeps superimposed). B, Light-evoked long-latency currents recorded in pyramidal cells with high (135 mm; left, V_h = −60 mV) or low intracellular [Cl⁻] (10 mm; right, V_h = 0 mV) pipette solutions (four sweeps superimposed). Notice that these long-latency responses are fully abolished by gabazine (gbz; 12.5 μm). C, GABAergic long-latency currents evoked by light flashes are the result of a feedforward circuit and are sensitive to NBQX (20 μm; V_h = 0 mV). Two examples are shown in the left and right panels, respectively (four sweeps superimposed).