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Long-range inhibitory intersection of a retrosplenial thalamocortical circuit by apical tuft-targeting CA1 neurons

Nature Neuroscience volume 22, pages 618–626 (2019)Cite this article

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Abstract

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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Fig. 1: CA1 retrosplenial-projecting neurons are a distinct class of GABAergic neurons.
Fig. 2: CA1-RP neurons form inhibitory synapses onto RSCg-L5pyr neurons at L1 apical tuft dendrites.
Fig. 3: ATN-TC axons make excitatory synapses onto RSCg-L5pyr neurons at L1 apical tuft dendrites.
Fig. 4: Opposing actions of CA1-RP inhibition and ATN-TC excitation in contextual fear learning.
Fig. 5: Cellular and subcellular organization of long-range input circuits of the RSCg.

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Code availability

Software routines for morphological analysis are available from ref. 59 or the corresponding authors, and digital reconstructions are available at NeuroMorpho.org.

Data availability

The data are available upon reasonable request.

References

  1. Harris, K. D. & Shepherd, G. M. G. The neocortical circuit: themes and variations. Nat. Neurosci. 18, 170–181 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Lin, C. S., Nicolelis, M. A., Schneider, J. S. & Chapin, J. K. A major direct GABAergic pathway from zona incerta to neocortex. Science 248, 1553–1556 (1990).

    Article  CAS  PubMed  Google Scholar 

  3. Chen, J. & Kriegstein, A. R. A GABAergic projection from the zona incerta to cortex promotes cortical neuron development. Science 350, 554–558 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Saunders, A. et al. A direct GABAergic output from the basal ganglia to frontal cortex. Nature 521, 85–89 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Tomioka, R. et al. Demonstration of long-range GABAergic connections distributed throughout the mouse neocortex. Eur. J. Neurosci. 21, 1587–1600 (2005).

    Article  PubMed  Google Scholar 

  6. He, M. et al. Strategies and tools for combinatorial targeting of GABAergic neurons in mouse cerebral cortex. Neuron 91, 1228–1243 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kimura, F. & Baughman, R. W. GABAergic transcallosal neurons in developing rat neocortex. Eur. J. Neurosci. 9, 1137–1143 (1997).

    Article  CAS  PubMed  Google Scholar 

  8. Rock, C., Zurita, H., Lebby, S., Wilson, C. J. & Apicella, A. J. Cortical Circuits of Callosal GABAergic Neurons. Cereb. Cortex 28, 1154–1167 (2018).

    Article  PubMed  Google Scholar 

  9. Miyashita, T. & Rockland, K. S. GABAergic projections from the hippocampus to the retrosplenial cortex in the rat. Eur. J. Neurosci. 26, 1193–1204 (2007).

    Article  PubMed  Google Scholar 

  10. Jinno, S. et al. Neuronal diversity in GABAergic long-range projections from the hippocampus. J. Neurosci. 27, 8790–8804 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Vann, S. D., Aggleton, J. P. & Maguire, E. A. What does the retrosplenial cortex do? Nat. Rev. Neurosci. 10, 792–802 (2009).

    Article  CAS  PubMed  Google Scholar 

  12. Gabriel, M., Sparenborg, S. P. & Stolar, N. An executive function of the hippocampus: pathway selection for thalamic neuronal significance code. in Hippocampus (eds. Isaacson R. L. & P. K. H.) 1–39 (Springer, Boston, MA, 1986).

  13. Corcoran, K. A., Yamawaki, N., Leaderbrand, K. & Radulovic, J. Role of retrosplenial cortex in processing stress-related context memories. Behav. Neurosci. 132, 388–395 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Sugar, J., Witter, M. P., van Strien, N. M. & Cappaert, N. L. The retrosplenial cortex: intrinsic connectivity and connections with the (para)hippocampal region in the rat. An interactive connectome. Front. Neuroinform. 5, 7 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Yamawaki, N., Corcoran, K. A., Guedea, A. L., Shepherd, G. M. G. & Radulovic, J. Differential contributions of glutamatergic hippocampal∂→retrosplenial cortical projections to the formation and persistence of context memories. Cereb. Cortex https://doi.org/10.1093/cercor/bhy142 (2018).

  16. Odagiri, S., Meguro, R., Asano, Y., Tani, T. & Ichinohe, N. Single axon branching analysis in rat thalamocortical projection from the anteroventral thalamus to the granular retrosplenial cortex. Front. Neuroanat. 5, 63 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Van Groen, T., Vogt, B. A. & Wyss, J. M. Interconnections between the thalamus and retrosplenial cortex in the rodent brain. in Neurobiol. Cingulate Cortex Limbic Thalamus (eds. Vogt, B. A. & Gabriel, M.) 123–150 (Birkhäuser, Boston, MA, 1993).

  18. Price, C. J. et al. Neurogliaform neurons form a novel inhibitory network in the hippocampal CA1 area. J. Neurosci. 25, 6775–6786 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Overstreet-Wadiche, L. & McBain, C. J. Neurogliaform cells in cortical circuits. Nat. Rev. Neurosci. 16, 458–468 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Price, C. J., Scott, R., Rusakov, D. A. & Capogna, M. GABA(B) receptor modulation of feedforward inhibition through hippocampal neurogliaform cells. J. Neurosci. 28, 6974–6982 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Fuentealba, P. et al. Expression of COUP-TFII nuclear receptor in restricted GABAergic neuronal populations in the adult rat hippocampus. J. Neurosci. 30, 1595–1609 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Tasic, B. et al. Adult mouse cortical cell taxonomy revealed by single cell transcriptomics. Nat. Neurosci. 19, 335–346 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Harris, K. D. et al. Classes and continua of hippocampal CA1 inhibitory neurons revealed by single-cell transcriptomics. PLoS Biol. 16, e2006387 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Gabriel, M., Vogt, B. A., Kubota, Y., Poremba, A. & Kang, E. Training-stage related neuronal plasticity in limbic thalamus and cingulate cortex during learning: a possible key to mnemonic retrieval. Behav. Brain Res. 46, 175–185 (1991).

    Article  CAS  PubMed  Google Scholar 

  25. Frank, A. C. et al. Hotspots of dendritic spine turnover facilitate clustered spine addition and learning and memory. Nat. Commun. 9, 422 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Yamawaki, N., Radulovic, J. & Shepherd, G. M. G. A Corticocortical circuit directly links retrosplenial cortex to M2 in the mouse. J. Neurosci. 36, 9365–9374 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wyss, J. M., Van Groen, T. & Sripanidkulchai, K. Dendritic bundling in layer I of granular retrosplenial cortex: intracellular labeling and selectivity of innervation. J. Comp. Neurol. 295, 33–42 (1990).

    Article  CAS  PubMed  Google Scholar 

  28. Kurotani, T. et al. Pyramidal neurons in the superficial layers of rat retrosplenial cortex exhibit a late-spiking firing property. Brain Struct. Funct. 218, 239–254 (2013).

    Article  PubMed  Google Scholar 

  29. Ichinohe, N., Fujiyama, F., Kaneko, T. & Rockland, K. S. Honeycomb-like mosaic at the border of layers 1 and 2 in the cerebral cortex. J. Neurosci. 23, 1372–1382 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Stepanyants, A. & Chklovskii, D. B. Neurogeometry and potential synaptic connectivity. Trends Neurosci. 28, 387–394 (2005).

    Article  CAS  PubMed  Google Scholar 

  31. Rees, C. L., Moradi, K. & Ascoli, G. A. Weighing the evidence in Peters’ Rule: does neuronal morphology predict connectivity? Trends Neurosci. 40, (63–71 (2017).

    Google Scholar 

  32. Petreanu, L., Mao, T., Sternson, S. M. & Svoboda, K. The subcellular organization of neocortical excitatory connections. Nature 457, 1142–1145 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Larkum, M. A cellular mechanism for cortical associations: an organizing principle for the cerebral cortex. Trends Neurosci. 36, 141–151 (2013).

    Article  CAS  PubMed  Google Scholar 

  34. Spruston, N. Pyramidal neurons: dendritic structure and synaptic integration. Nat. Rev. Neurosci. 9, 206–221 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Anwar, H., Li, X., Bucher, D. & Nadim, F. Functional roles of short-term synaptic plasticity with an emphasis on inhibition. Curr. Opin. Neurobiol. 43, 71–78 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Regehr, W. G. Short-term presynaptic plasticity. Cold Spring Harb. Perspect. Biol. 4, a005702 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Aggleton, J. P. & Brown, M. W. Episodic memory, amnesia, and the hippocampal-anterior thalamic axis. Behav. Brain Sci. 22, 425–444 (1999). discussion 444–489.

    Article  CAS  PubMed  Google Scholar 

  38. Wright, N. F., Vann, S. D., Erichsen, J. T., O’Mara, S. M. & Aggleton, J. P. Segregation of parallel inputs to the anteromedial and anteroventral thalamic nuclei of the rat. J. Comp. Neurol. 521, 2966–2986 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Shibata, H. Efferent projections from the anterior thalamic nuclei to the cingulate cortex in the rat. J. Comp. Neurol. 330, 533–542 (1993).

    Article  CAS  PubMed  Google Scholar 

  40. Llinás, R. & Jahnsen, H. Electrophysiology of mammalian thalamic neurones in vitro. Nature 297, 406–408 (1982).

    Article  PubMed  Google Scholar 

  41. Jiang, X., Wang, G., Lee, A. J., Stornetta, R. L. & Zhu, J. J. The organization of two new cortical interneuronal circuits. Nat. Neurosci. 16, 210–218 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Llinás, R. & Ribary, U. Coherent 40-Hz oscillation characterizes dream state in humans. Proc. Natl Acad. Sci. USA 90, 2078–2081 (1993).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Takahashi, H. & Magee, J. C. Pathway interactions and synaptic plasticity in the dendritic tuft regions of CA1 pyramidal neurons. Neuron 62, 102–111 (2009).

    Article  CAS  PubMed  Google Scholar 

  44. Higley, M. J. Localized GABAergic inhibition of dendritic Ca2+ signalling. Nat. Rev. Neurosci. 15, 567–572 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Murayama, M. et al. Dendritic encoding of sensory stimuli controlled by deep cortical interneurons. Nature 457, 1137–1141 (2009).

    Article  CAS  PubMed  Google Scholar 

  46. Egger, R. et al. Robustness of sensory-evoked excitation is increased by inhibitory inputs to distal apical tuft dendrites. Proc. Natl Acad. Sci. USA 112, 14072–14077 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Ranganath, C. & Ritchey, M. Two cortical systems for memory-guided behaviour. Nat. Rev. Neurosci. 13, 713–726 (2012).

    Article  CAS  PubMed  Google Scholar 

  48. Viney, T. J. et al. Shared rhythmic subcortical GABAergic input to the entorhinal cortex and presubiculum. eLife 7, 7 (2018).

    Article  Google Scholar 

  49. Llinás, R. R., Ribary, U., Jeanmonod, D., Kronberg, E. & Mitra, P. P. Thalamocortical dysrhythmia: A neurological and neuropsychiatric syndrome characterized by magnetoencephalography. Proc. Natl Acad. Sci. USA 96, 15222–15227 (1999).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Ohashi, K. et al. Neuron-derived neurotrophic factor functions as a novel modulator that enhances endothelial cell function and revascularization processes. J. Biol. Chem. 289, 14132–14144 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Taniguchi, H. et al. A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex. Neuron 71, 995–1013 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Peron, S. P., Freeman, J., Iyer, V., Guo, C. & Svoboda, K. A cellular resolution map of barrel cortex activity during tactile behavior. Neuron 86, 783–799 (2015).

    Article  CAS  PubMed  Google Scholar 

  53. Gerfen, C. R., Paletzki, R. & Heintz, N. GENSAT BAC cre-recombinase driver lines to study the functional organization of cerebral cortical and basal ganglia circuits. Neuron 80, 1368–1383 (2013).

    Article  CAS  PubMed  Google Scholar 

  54. Tervo, D. G. et al. A designer AAV variant permits efficient retrograde access to projection neurons. Neuron 92, 372–382 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Suter, B. A. et al. Ephus: multipurpose data acquisition software for neuroscience experiments. Front. Neural Circuit 4, 100 (2010).

    Article  Google Scholar 

  56. Yamawaki, N., Suter, B. A., Wickersham, I. R. & Shepherd, G. M. G. Combining optogenetics and electrophysiology to analyze projection neuron circuits. Cold Spring Harb. Protoc. 2016, prot090084 (2016).

    Article  Google Scholar 

  57. Suter, B. A. & Shepherd, G. M. G. Reciprocal interareal connections to corticospinal neurons in mouse M1 and S2. J. Neurosci. 35, 2959–2974 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Jackman, S. L., Beneduce, B. M., Drew, I. R. & Regehr, W. G. Achieving high-frequency optical control of synaptic transmission. J. Neurosci. 34, 7704–7714 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Yamawaki, N., Borges, K., Suter, B. A., Harris, K. D. & Shepherd, G. M. G. A genuine layer 4 in motor cortex with prototypical synaptic circuit connectivity. eLife 3, e05422 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Li, X., Yamawaki, N., Barrett, J. M., Körding, K. P. & Shepherd, G. M. G. Scaling of optogenetically evoked signaling in a higher-order corticocortical pathway in the anesthetized mouse. Front. Syst. Neurosci. 12, 16 (2018).

    PubMed  PubMed Central  Google Scholar 

  61. Corcoran, K. A. et al. NMDA receptors in retrosplenial cortex are necessary for retrieval of recent and remote context fear memory. J. Neurosci. 31, 11655–11659 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Nelson, A. J., Powell, A. L., Holmes, J. D., Vann, S. D. & Aggleton, J. P. What does spatial alternation tell us about retrosplenial cortex function? Front. Behav. Neurosci. 9, 126 (2015).

    PubMed  PubMed Central  Google Scholar 

  63. Cowansage, K. K. et al. Direct reactivation of a coherent neocortical memory of context. Neuron 84, 432–441 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Tanaka, K. Z. et al. Cortical representations are reinstated by the hippocampus during memory retrieval. Neuron 84, 347–354 (2014).

    Article  CAS  PubMed  Google Scholar 

  65. Kwapis, J. L., Jarome, T. J., Lee, J. L. & Helmstetter, F. J. The retrosplenial cortex is involved in the formation of memory for context and trace fear conditioning. Neurobiol. Learn. Mem. 123, 110–116 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  66. Jovasevic, V. et al. GABAergic mechanisms regulated by miR-33 encode state-dependent fear. Nat. Neurosci. 18, 1265–1271 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Keene, C. S. & Bucci, D. J. Contributions of the retrosplenial and posterior parietal cortices to cue-specific and contextual fear conditioning. Behav. Neurosci. 122, 89–97 (2008).

    Article  PubMed  Google Scholar 

  68. Stachniak, T. J., Ghosh, A. & Sternson, S. M. Chemogenetic synaptic silencing of neural circuits localizes a hypothalamus→midbrain pathway for feeding behavior. Neuron 82, 797–808 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Mahler, S. V. et al. Designer receptors show role for ventral pallidum input to ventral tegmental area in cocaine seeking. Nat. Neurosci. 17, 577–585 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Van Groen, T. & Wyss, J. M. Projections from the anterodorsal and anteroventral nucleus of the thalamus to the limbic cortex in the rat. J. Comp. Neurol. 358, 584–604 (1995).

    Article  PubMed  Google Scholar 

  71. Yamawaki, N. & Shepherd, G. M. G. Synaptic circuit organization of motor corticothalamic neurons. J. Neurosci. 35, 2293–2307 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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.).

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Authors and Affiliations

  1. Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Naoki Yamawaki, Xiaojian Li, Laurie Lambot, Jelena Radulovic & Gordon M. G. Shepherd

  2. Department of Psychiatry and Behavioral Sciences, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Lynn Y. Ren & Jelena Radulovic

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Contributions

N.Y., J.R., and G.M.G.S. designed the research; N.Y. performed injections, ex vivo recording, imaging, immunostaining, and reconstruction; X.L. performed in vivo recording; L.L performed immunostaining and reconstruction. L.Y.R. and J.R. performed behavioral studies; N.Y., J.R, and G.S. analyzed data and wrote the paper.

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Correspondence to Naoki Yamawaki or Gordon M. G. Shepherd.

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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

Supplementary Figure 1 Additional characterizations of CA1-RP neurons and axons.

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).

Supplementary Figure 6 Labeling of ATN→RSCg projections.

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).

Supplementary Figure 7 Additional electrophysiological characterizations of ATN inputs to RSCg.

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.

Supplementary Figure 8 Spread of AAV in dorsal hippocampus after injection into subiculum.

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.

Supplementary Figure 9 Chemogenetic control experiments.

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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