Hippocampus, granular retrosplenial cortex (RSCg), and anterior thalamic nuclei (ATN) interact to mediate diverse cognitive functions. To identify cellular mechanisms underlying hippocampo–thalamo–retrosplenial interactions, we investigated the potential circuit suggested by projections to RSCg layer 1 (L1) from GABAergic CA1 neurons and ATN. We find that CA1→RSCg projections stem from GABAergic neurons with a distinct morphology, electrophysiology, and molecular profile. Their long-range axons inhibit L5 pyramidal neurons in RSCg via potent synapses onto apical tuft dendrites in L1. These inhibitory inputs intercept L1-targeting thalamocortical excitatory inputs from ATN to coregulate RSCg activity. Subicular axons, in contrast, excite proximal dendrites in deeper layers. Short-term plasticity differs at each connection. Chemogenetically abrogating CA1→RSCg or ATN→RSCg connections oppositely affects the encoding of contextual fear memory. Our findings establish retrosplenial-projecting CA1 neurons as a distinct class of long-range dendrite-targeting GABAergic neuron and delineate an unusual cortical circuit specialized for integrating long-range inhibition and thalamocortical excitation.
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We thank N. Bernstein, A. Guedea, and D. Wokosin for a technical assistance and advice. We thank A. Apicella, J. Barrett, K. Guo, K. Harris, G. Maccaferri, and A. Tanimura for comments and suggestions. This research was supported by NIH grants NS061963 (G.M.G.S.), EB017695 (G.M.G.S.), and MH108837 (J.R.).
The authors declare no competing interests.
Journal peer review information: Nature Neuroscience thanks John Aggleton, Leopoldo Petreanu, and Peter Somogyi for their contribution to the peer review of this work.
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Integrated supplementary information
a, Top: Schematic depicting injection of AAVretro-FLEX-tdTomato into L1 of the RSCg in a Gad2-Cre mouse, for retrograde labeling of CA1-RP neurons. Bottom: Merged epifluorescence and bright-field image showing the injection site in RSCg. Right: 2-photon maximum-intensity projection image of dorsal CA1, showing several retrogradely transfected neurons labeled with tdTomato around the stratum radiatum/lacunosum-moleculare (SR-SLM) border. Repeated on 2 different mice. b, Immunostaining characterization of retrogradely labeled CA1-RP neurons. The scale bar applies to all panels. Arrows mark retrogradely labeled neurons. Anti-parvalbumin (PV) staining labeled 0 out of 47 retrogradely labeled neurons (6 dorsal hippocampal fields of view, 2 mice). Anti-calbindin (CB) staining labeled 0 out of 32 retrogradely labeled neurons (3 dorsal hippocampal fields of view, 2 mice). Anti-calretinin (CR) staining labeled 1 out of 24 retrogradely labeled neurons (4 dorsal hippocampal fields of view, 2 mice). Anti-somatostatin (SOM) staining labeled 0 out of 29 retrogradely labeled neurons (5 dorsal hippocampal fields of view, 2 mice). See main text for description of anti-reelin (Reln) and anti-NOS immunolabeling results.
Supplementary Figure 2 Additional characterizations of the pattern of CA1-RP labeling by viral injections in Gad2-Cre mice.
a, Top row: Eight sections (0.15 mm thick, 0.3 mm between sections) through the dorsal hippocampal region, after injection of Cre-dependent AAV5-Ef1a-DIO-hChR2-EYFP into CA1 of a Gad2-Cre mouse. Distances from bregma (in mm) are indicated at top right. Boxes mark regions of interest that were imaged at higher magnification (shown in the bottom row). All images were captured using the same light intensity, and displayed with identical brightness and contrast settings. Bottom row: Confocal images of the 5 regions of interest in CA1 indicated above. Yellow arrows indicate labeled neurons at the border of stratum radiatum (SR) and lacunosum-moleculare (SLM). Pyr: pyramidal layer. All images were captured using the same light intensity, and displayed with identical brightness and contrast settings. Repeated on 3 different mice. b, For anterograde labeling of GABAergic projections, AAV5-Ef1a-DIO-hChR2-EYFP was injected into dorsal CA1. Right: Merged image of a 0.6 mm-thick sagittal slice immediately adjacent to the midline, through L1 of RSCg, showing the anteroposterior extent of the CA1 axons projection (red), which is focused onto the RSCg. Brain slice recordings in this study were made from RSCg neurons located in coronal sections taken through the region indicated (white dashed box).
Supplementary Figure 3 Pharmacological assessment of GABAB receptors in CA1-RP inhibitory synaptic transmission to L5pyr neurons.
a, Laser scanning photostimulation was used to activate CA1-RP axons in L1 of RSCg. b, Example traces of photo-evoked IPSCs after sequential application of GABAB receptor antagonist CGP55845 (red, 5 μM) and GABAA receptor antagonist SR-95531 (blue, 10 μM). c, Group comparison of drug effects, showing data from individual neurons (gray) and overall mean (black). CGP55845 reduced IPSC mean (± s.e.m.) amplitude by 16% (control: 32.7 ± 4.4 pA, CGP55845: 27.6 ± 3.8 pA, signed-rank test, n = 7 neurons). The remaining responses were eliminated after addition of SR-95531 (0.5 ± 0.4 pA).
Supplementary Figure 4 Short-term synaptic plasticity of CA1, thalamic and subicular inputs to RSCg-L5pyr neurons.
a, Schematics showing approach used to repetitively stimulate CA1 and ATN axons (laser aimed at sites in L1; top) or subicular axons (laser aimed at sites in deeper layers; bottom). b, Example traces showing responses recorded in L5pyr neurons to 20 Hz repetitive stimulation of CA1, thalamic (ATN), and subicular (SUB) inputs. c, Group data (mean ± s.e.m. shown as colored line and gray shade). Top: analysis of CA1 inputs, showing pattern of strong synaptic depression (p = 0.006 or less for all responses when compared against 1st response, signed-rank test, Bonferroni-corrected, n = 26 L5pyr neurons). Middle, analysis of ATN inputs, showing initially non-depressing followed by depressing responses (p = 0.006 or less for 3rd to 10th response when compared against 1st response, signed-rank test, Bonferroni-corrected, n = 18 L5pyr neurons). Bottom, analysis of subicular inputs, showing facilitating responses (p = 0.006 or less for 2nd and 3rd response vs 1st response, signed-rank test, Bonferroni-corrected, n = 23 L5pyr neurons). d, Schematic depicting injection performed to labeled CA1-RP neurons with a tracer and ChR2, for control experiment testing reliability of photo-evoking action potentials in CA1-RP axons. e, Recording configuration. In the presence of synaptic blockers (NBQX, CPP, and SR-95531), a cell-attached voltage recording was made from a CA1-RP neuron to monitor (antidromically propagated) action potentials that were evoked by repetitive laser stimulation (10 pulses, 1 ms duration each, 20 Hz) targeted to a site near the SLM border. f, Representative example showing 5 consecutive sweeps (inter-sweep interval of 30 s) recorded in one CA1-RP neuron. As shown in this example, there was 100% reliability of photo-evoked action potentials (same results observed in 7 of 7 neurons tested).
Supplementary Figure 5 Effect of CA1 inhibitory input on interspike intervals in active L5pyr neurons.
a, Schematic of the experimental strategy, involving whole-cell recording from an RSCg-L5pyr neuron while briefly flashing the focused beam of the laser to activate CA1 axons at a location slightly away from the dendrites of the recorded neuron. b, Example trace, showing a train of spikes evoked in a L5pyr neuron by a step of current injected via the somatic patch pipette (1 s, +200 pA), and the timing of the laser pulse (1-ms duration) used to photostimulate CA1 axons. Subsequent analysis of inter-spike intervals (ISI) focused on the peristimulus region indicated by red. c, Top: Zoomed-in view of the peristimulus spiking shown in the previous panel. Bottom: The spiking patterns recorded in multiple sweeps are shown as a raster plot; the red arrow indicates the sweep of the example trace. d, Plot of peristimulus ISIs for one neuron, showing the mean (black) and individual sweeps (gray). The zeroeth ISI is defined as the one during which the laser stimulus was delivered. e, Plot of the mean peristimulus ISIs for multiple (n = 11) neurons (gray), and the overall mean across all neurons (black). f, Plot of the change in ISI, calculated by subtracting the preceding ISI, for multiple (n = 11) neurons (gray) and the overall mean across all neurons (black). The mean (± s.e.m.) change in ISI did not differ significantly for the two ISIs immediately before the L1 stimulation (2.4 ± 1.0 ms vs 1.3 ± 0.7 ms, p = 0.12, signed-rank test), but did for the ISI before vs during stimulation (1.3 ± 0.7 ms vs 6.9 ± 2.2 ms, p = 0.003, signed-rank test).
a, Characterization of thalamic labeling in Grp_KH288-Cre mice. CTB647 was injected into L1 RSCg of Grp_KH288-Cre mice crossed with a fluorescent reporter line (Ai14). b, Bright-field (left) and epifluorescence (right) images of a coronal brain slice (1.0 mm posterior to bregma) containing anterior thalamic nuclei, including the AV, anterodorsal (AD), and anteromedial (AM) nuclei. Retrograde labeling is also shown (green). c, Higher-magnification view. Cre-positive neurons are present in AV, but not in AD. d, Higher-magnification view of the labeling in AV in the previous image. A subset of RSCg-projecting neurons is Cre-positive. e, Coronal brain slice (1.1 mm posterior to bregma, immediately posterior to that shown in panel b), containing anterior thalamic nuclei, including AV and AD (dashed circle), along with the lateral dorsal (LD) and AM nuclei. f, Higher-magnification view. Retrograde labeling is also shown (green). g, Higher-magnification view of the labeling in AV. A subset of RSCg-projecting neurons is Cre-positive. b-g is repeated on 2 mice. h, Left: Quantification of double-labeled neurons relative to the CTB-labeled neurons in AV (analysis of 4 slices from 2 mice, 2 slices per mice). Right: Quantification of double-labeled neurons relative to the Cre+ neurons. i-m, Additional characterizations of thalamic labeling, showing the spread of labeling at the injection site. Repeated on 6 mice. i, Bright-field images of 4 consecutive brain sections (0.25 mm thick) of through the injection site, collected from Grp_KH288-Cre mouse injected with Cre-dependent AAV5-Ef1a-DIO-hChR2-EYFP targeted to the AV nucleus of the thalamus. Distances from bregma are indicated at top right. Two regions of interest (cortex for projection pattern and thalamus for spread of AAV) are further imaged at high magnification. j, Epifluorescence images of the same sections. k, Higher-magnification bright-field images of the thalamic regions shown in the preceding panels. l, Corresponding epifluorescence images of the same sections. Labeled thalamocortical neurons are mostly confined in AV. m, Images of cortical labeling resulting from the injection, showing intense labeling of axons in L1 of RSCg. Axons were absent more anteriorly in the anterior cingulate (AC).
a, Thalamocortical excitation and feed-forward (disynaptic) inhibition. Example traces from a L5pyr neuron in an RSCg brain slice, showing responses to photostimulation of ATN-TC axons while the voltage was held at −70 mV (blue traces) to sample inward current (EPSC), or at +10 mV (red traces) to sample outward current (IPSC). Addition of TTX and 4-AP eliminated the IPSC but not the EPSC, indicating that the IPSC is mediated by feed-forward (disynaptic) mechanism (n = 7). b, Comparison of EPSC and IPSC amplitudes in the same neurons. ATN-TC axons were stimulated by wide-field LED (black circles, n = 7) or focal stimulation of L1 using a laser (red circles, n = 4). c, Example traces, recorded from a RSCg-projecting TC neuron in AV, showing regular spiking vs bursting firing patterns evoked by somatic injection of a current step stimulus from depolarized (−50 mV) versus hyperpolarized (−70 mV) membrane potentials (n = 9). Trace on the right shows the middle trace’s burst on a faster time base.
Top row: Eight sections (0.15 mm thick, 0.3 mm between sections) through the dorsal hippocampal region, from a wild-type mouse injected with Cre-independent AAV1-CamKIIa-hChR2-mCherry in the subiculum (SUB). Distances from bregma (in mm) are indicated at top right. Boxes: Regions of interest in CA1 and subiculum were imaged at higher magnification (shown in the bottom row). All images were captured using the same light intensity, and displayed with identical brightness and contrast settings. Bottom row: Confocal images of the 2 regions of interest in CA1 and the 3 in subiculum (indicated above). All images were captured using the same light intensity, and displayed with identical brightness and contrast settings. Although AAV injected into subiculum transfected some neurons in CA1, these were exclusively in the pyramidal (Pyr) layer (arrows); no labeling was found at the stratum radiatum/lacunosum-moleculare border or in other layers, reflecting the specificity of the CaMKIIa promotor for driving expression in excitatory neurons. Repeated on 3 different mice.
a, Schematic of the injection strategy for assessing effect of CNO on synaptic transmission from presynaptic hM4D(Gi)-expressing CA1-RP axons to postsynaptic RSCg pyramidal neurons. The optogenetic virus, AAV5-Ef1a-DIO-hChR2-EYFP, was injected with or without co-injection of the chemogenetic virus, AAV8-hSyn-DIO-hM4D(Gi)-mCherry, into dorsal CA1 of Gad2-Cre mice. b, Example traces showing photo-evoked responses at baseline (left) and increasing concentrations of CNO (right), for axons that either did (top row, +hM4D) or did not (bottom row, -hM4D) co-express the chemogenetic construct. c, Time course of the amplitude (mean ± s.e.m., normalized to baseline) of the optogenetically evoked response, for axons that either did (red, n = 7) or did not (black, n = 7) co-express the chemogenetic construct. d, Group comparison of photo-evoked IPSC amplitudes recorded with different concentrations of CNO. Left: Synaptic transmission from terminals with hM4D(Gi). The mean (± s.e.m.) IPSC amplitude 2.5 mins immediately before application of 0.1 μM CNO was 70.7 ± 21.9 pA. The mean (± s.e.m.) IPSC amplitudes 2.5 mins before application of 1 μM CNO and 2.5 mins before the end of recording session were 53.4 ± 17.7 pA and 39.2 ± 13.6 pA, respectively (baseline vs 0.1 μM or 1 μM CNO: n = 7, p = 0.016 for both, signed-rank test; 0.1 μM CNO vs 1 μM CNO, p = 0.03, signed-rank test). Right: Synaptic transmission from terminals without hM4D(Gi), measured at same time window as plot on the left. The mean (± s.e.m.) IPSC amplitude at 0, 0.1 and 1 uM CNO was 47.2 ± 3.2, 46.8 ± 4.7, and 42.1 ± 6.0 pA, respectively (baseline vs 0.1 μM or 1 μM CNO: n = 7, p = 0.94 or 0.16, signed-rank test; 0.1 μM CNO vs 1 μM CNO, p = 0.11, signed-rank test). e-g, Same, for assessing effect of CNO on synaptic transmission from presynaptic hM4D(Gi)-expressing ATN-TC axons to postsynaptic RSCg pyramidal neurons (n = 7). Cre-independent AAV1-CamKIIa-hChR2-mCherry was injected, with or without co-injection of AAV8-hSyn-HA-hM4D(Gi)-mCherry, into AV of wild-type mice. h, Left: Synaptic transmission from terminals with hM4D(Gi). The mean (± s.e.m.) EPSC amplitude 2.5 mins immediately before application of 0.1 μM CNO was 42.1 ± 10.7 pA. The mean EPSC amplitudes 2.5 mins before application of 1 μM CNO and 2.5 mins before the end of recording session were −36.9 ± 9.2 pA and 21.5 ± 6.3 pA, respectively (baseline vs 0.1 μM or 1 μM CNO: n = 7, p = 0.031 or 0.016, signed-rank test; 0.1 μM CNO vs 1 μM CNO, p = 0.031, signed-rank test). Right: Synaptic transmission from terminals without hM4D(Gi). The mean (± s.e.m.) EPSC amplitude at 0, 0.1 and 1 uM CNO was −20.5 ± 3.8, −21.5 ± 3.7, and −18.8 ± 3.2 pA, respectively (baseline vs 0.1 μM or 1 μM CNO: n = 7, p = 0.81 or 0.16, signed-rank test; 0.1 μM CNO vs 1 μM CNO, p = 0.16, signed-rank test). i, Group comparison of holding current recorded with different concentrations of CNO. Left: Mean (± s.e.m.) holding current during recording of CA1 input in absence of CNO was 180.3 ± 20.1 pA. The mean (± s.e.m.) holding current during recording with 0.1 and 1 μM CNO was 183.0 ± 16.1 and189.4 ± 14.6 pA (baseline vs 0.1 μM or 1 μM CNO: n = 14, p = 0.855 and 0.808, signed-rank test; 0.1 μM CNO vs 1 μM CNO, p = 0.626, signed-rank test). Right: Mean holding (± s.e.m.) current during recording of ATN input in absence of CNO was −96.0 ± 30.4 pA. The mean (± s.e.m.) holding current during recording with 0.1 and 1 μM CNO was −100.9 ± 35.5 and −89.7 ± 35.9 pA (baseline vs 0.1 μM or 1 μM CNO: n = 14, p = 0.903 and 0.153, signed-rank test; 0.1 μM CNO vs 1 μM CNO, p = 0.104, signed-rank test). j, mCherry expression at the injection site in CA1, and in RSCg (axonal labeling). Arrow indicates region containing labeled neurons. Repeated on 9 mice. k, mCherry expression at the injection site in AV, and in RSCg (axonal labeling). Arrow indicates region containing labeled neurons. Repeated on 10 mice. l, Merged bright-field and epifluorescence image of a coronal slice at the level of the RSGg showing restriction to the RSCg of a fluorescent dye (fluorescent muscimol) that was cannula-infused into the RSCg using the same parameters used for the behavioral experiments. No labeling of dye was observed in dorsal subiculum. Repeated on 2 mice. m, Schematic of experimental procedure to assess the extent of spread of the infusate. One week after cannula implantation into RSC, an infusate containing fluorescent muscimol was delivered using the same parameters used for infusing CNO (0.5 μL/min until 0.2 μL per side was reached). Approximately 30 mins after infusion, sagittal sections were prepared for imaging. n, Consecutive sagittal sections of right hemisphere (merged image of epifluorescence and bright-field; the fluorescent signal is pseudo-colored red). Dorsal hippocampus typically appeared ~0.3 mm lateral from midline. All fluorescent images were acquired with the same light intensity, and the same gain and contrast setting were used for display. o, Schematic depicting the regions of interest for fluorescent signal analysis. Cortex (major source of fluorescent signal) was isolated by dissection from other regions to avoid light scattering artifacts. Hippo: hippocampus. Right: Images of isolated cortex and hippocampal/subcortical regions that were separately acquired, with regions of interest marked by rectangles. n-o was repeated on 3 mice. p, Group data comparing fluorescent signals in RSC, hippocampus, and subcortex in each hemisphere Fluorescent signals were normalized to the strongest signal across all slices (~0.3 to ~0.9 mm from midline). Mean signal (± s.e.m.) for RSC, CA1, and subcortex was 0.90 ± 0.06, 0.00 ± 0.02, and 0.00 ± 0.01 for 0.3 mm lateral section, 0.53 ± 0.17, 0.00 ± 0.02, and 0.00 ± 0.01 for 0.6 mm lateral section, and 0.18 ± 0.12, 0.01 ± 0.00, and 0.01 ± 0.01 for 0.9 mm lateral section (RSC vs CA1 or subcortex, and CA1 vs subcortex in 0.3 mm lateral section: n = 6 hemisphere from 3 mice, p = 0.03 and 0.03, and p = 0.56, signed-rank test; in 0.6 mm lateral section, p = 0.03 and 0.03, and p = 0.69, signed-rank test; in 0.9 mm lateral section, p = 0.06 and 0.06, and p = 0.63, signed-rank test).
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Yamawaki, N., Li, X., Lambot, L. et al. Long-range inhibitory intersection of a retrosplenial thalamocortical circuit by apical tuft-targeting CA1 neurons. Nat Neurosci 22, 618–626 (2019). https://doi.org/10.1038/s41593-019-0355-x
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