A dedicated circuit links direction-selective retinal ganglion cells to the primary visual cortex

Journal name:
Nature
Volume:
507,
Pages:
358–361
Date published:
DOI:
doi:10.1038/nature12989
Received
Accepted
Published online
Corrected online

How specific features in the environment are represented within the brain is an important unanswered question in neuroscience. A subset of retinal neurons, called direction-selective ganglion cells (DSGCs), are specialized for detecting motion along specific axes of the visual field1. Despite extensive study of the retinal circuitry that endows DSGCs with their unique tuning properties2, 3, their downstream circuitry in the brain and thus their contribution to visual processing has remained unclear. In mice, several different types of DSGCs connect to the dorsal lateral geniculate nucleus (dLGN)4, 5, 6, the visual thalamic structure that harbours cortical relay neurons. Whether direction-selective information computed at the level of the retina is routed to cortical circuits and integrated with other visual channels, however, is unknown. Here we show that there is a di-synaptic circuit linking DSGCs with the superficial layers of the primary visual cortex (V1) by using viral trans-synaptic circuit mapping7, 8 and functional imaging of visually driven calcium signals in thalamocortical axons. This circuit pools information from several types of DSGCs, converges in a specialized subdivision of the dLGN, and delivers direction-tuned and orientation-tuned signals to superficial V1. Notably, this circuit is anatomically segregated from the retino-geniculo-cortical pathway carrying non-direction-tuned visual information to deeper layers of V1, such as layer 4. Thus, the mouse harbours several functionally specialized, parallel retino-geniculo-cortical pathways, one of which originates with retinal DSGCs and delivers direction- and orientation-tuned information specifically to the superficial layers of the primary visual cortex. These data provide evidence that direction and orientation selectivity of some V1 neurons may be influenced by the activation of DSGCs.

At a glance

Figures

  1. The layer of the dLGN that receives input from DSGCs projects to V1.
    Figure 1: The layer of the dLGN that receives input from DSGCs projects to V1.

    a, GFP+ DSGC axons in dLGN. OT, optic tract. Inset, GFP+ boutons. d, dorsal; m, medial. b, Merged GFP/CTβ−594 (all RGC axons) and c, with DAPI (4′,6-diamidino-2-phenylindole; cell nuclei). ac, Scale bar, 125μm. d, Summary. e, AAV2-tdTomato injection to dLGN. Asterisk, tdTomato+ neurons in core. Scale bar, 100μm. f, tdTomato+ thalamocortical axons in V1. L1–L6, layers 1–6; wm, white matter. Scale bar, 150μm. gi, Higher magnification of L4 (g), L5 and L6 (h) and L1 and L2 (i). Arrows, axons in L1. Scale bars, g, h, 50μm; i, 100μm. Sixteen mice were used for these experiments.

  2. Parallel, layer-specific thalamocortical circuits in the mouse.
    Figure 2: Parallel, layer-specific thalamocortical circuits in the mouse.

    ac, Tracer injections to all V1 layers (a), layer 4 (b) and superficial V1 (c). df, dLGN neurons labelled after full depth injection (d; asterisk, axons of L6 neurons; yellow box, neurons in shell; green box, middle; blue box, core) deep V1 injection (e) and superficial V1 injection (f; arrows, labelled cells; arrowhead, labelled cell outside shell). gi, Position of retrogradely labelled cells along dLGN width. g, Full depth, 35.54±1.62% (4 mice; n = 232 cells); h, Deep, 68.53±1.22% (4 mice; n = 69 cells); i, Superficial, 14.68±1.57% (4 mice; n = 78 cells). Superficial versus deep = ***P<0.0001; Tukey’s multiple comparisons. c, Scale bar, 200μm. f, Scale bar, 100μm.

  3. DSGC axons contact thalamic relay neurons projecting to superficial V1.
    Figure 3: DSGC axons contact thalamic relay neurons projecting to superficial V1.

    a, ΔG-RABV-mCherry injection to superficial V1, to infect axons of DSGC-RZ neurons. b, V1 injection. Asterisk, infected L5/6 neurons. Scale bar, 200μm. c, d, mCherry+ dLGN neurons. c, Arrowhead, axon. Scale bars, 75μm. d, Arrow, proximal dendrites; arrowheads, distal dendrites. Scale bars, 25μm. e, Percentage of mCherry+ somas within GFP+ DSGC-RZ (74.77±11.62%; 8 mice, n = 83 cells) (Extended Data Fig. 4). fh, DSGC axons and dLGN somas and dendrites. g, Magnified view of frame in f. Arrowheads, putative contact sites13. Scale bars; f, 125μm; g, 20μm. hk, GFP, mCherry, VGLUT2 (per cent mCherry signal contacted by GFP+/VGLUT2+ profiles = 5.23±1.39%; 4 mice; n = 4 cells). k, Scale bar, 2μm.

  4. Synaptic circuit linking DSGCs to superficial V1, and non-DSGCs to L4.
    Figure 4: Synaptic circuit linking DSGCs to superficial V1, and non-DSGCs to L4.

    a, Trans-synaptic tracing. b, Infected dLGN neurons. Arrow and arrowhead: double-infected cells; arrow is same cell as in c, d. Scale bar, 100μm. c, d, Cell from b. Scale bar, 15μm. Dashed line, lateral border. e, Distribution of double-infected dLGN cells = 9.29±1.82% (8 mice, n = 21 cells). f, On-Off DSGC trans-synaptically labelled from superficial V1. g, On (red) and Off (black) dendrites. Arrowhead, axon. Scale bar, 50μm. hj, Cell (f) is GFP+ On-Off DSGC6. Scale bar, 10μm. km, Trans-synaptically labelled GFP+ and Cart+ DSGC5 shown at low (left) and high (right) magnifications. m, Scale bar left, 75μm; right, 10μm. n, Trans-synaptically labelled J-RGC4. o, Off dendrites (black). Scale, 50μm. p, Same as a, but layer 4 injection. qs, Infected neurons in core; q, arrow, arrowhead: double-infected cells. Scale, 100μm. r, s, Cell from q (arrow). s, Scale bar, 15μm. t, Distribution of double-infected dLGN cells = 70±2.65% (7 mice, n = 53 cells) (P<0.0001 versus e; two-tailed t-test). u, v, Alpha RGC labelled from V1 layer 4. u, Scale bar, 100μm; sideview, 50μm. wy, mCherry+ RGC same as from u, v, is SMI-32+. Scale bar, 20μm. zbb, SMI-32 and Cart. Scale bar, 25μm. ccee, ΔG-RABV-mCherry+ alpha RGC; lacks GFP6 and Cart. Scale bar, 150μm.

  5. In vivo imaging of visually evoked Ca2+ signals in thalamocortical axons.
    Figure 5: In vivo imaging of visually evoked Ca2+ signals in thalamocortical axons.

    a, AAV2-GCaMP6 injection to dLGN shell. b, GCaMP6+ neurons (arrows). Scale bar, 50μm; inset, 10μm. c, GCaMP6+ dLGN axons, superficial V1. Arrows: varicosities. Scale, 50μm. d, GCaMP6+ axons, superficial V1. Circles, square in d correspond to polar plots l, o, p. Scale bar (d), 5μm. e, In vivo imaging/visual stimulation. f, Visually evoked Ca2+ signal in thalamocortical axon (top trace: photodiode signal; bottom trace: ΔF/F). g, Directional stimuli (0°, 45°, 90°, 135°, 180°, 225°, 270°, 315°). hj, Direction- (h, i) and orientation-tuned (j) varicosities. 5–8 trial average. ks, Polar plots of F1 (red) or F2 (black) magnitude responses (Methods). Inner solid ring, average response to mean grey stimulus. Shaded, 3 standard deviations greater than the mean response to grey stimuli. Lower right of each plot, OSI/DSI. Upper right, Fourier amplitudes. t, DSI/OSI, all varicosities (5 mice, n = 58 varicosities). Mean±s.e.m. u, Cumulative distributions: OSI (circles), DSI (squares).

  6. The retino-geniculo-cortical pathway links retinal cells and circuits to the brain.
    Extended Data Fig. 1: The retino-geniculo-cortical pathway links retinal cells and circuits to the brain.

    a, Diagram of retina, dorsal lateral geniculate nucleus (dLGN) and primary visual cortex (V1). The optic tract which carries retinal ganglion cell (RGC) axons and thalamocortical (dLGN to V1) pathway also shown. b, Diagram of retinal layers: PRL, photoreceptor layer; opl, outer plexiform layer; INL, inner nuclear layer; ipl, inner plexiform layer; GCL, ganglion cell layer; nfl, nerve fibre layer. c, Retina diagram with cells shown (labels same as in b).

  7. Approach for assessing laminar specificity of mouse geniculocortical projections.
    Extended Data Fig. 2: Approach for assessing laminar specificity of mouse geniculocortical projections.

    a, Focal retrograde tracer injection to V1. Scale bar, 3mm. b, Diagram of the three different injection depths used to generate data in Fig. 2. c, Percentage of fluorescence in V1 from superficial (black line) versus deep (grey line) injections. Superficial, peak intensity occurs at 25μm from pial surface (4 mice). Deep, peak intensity occurs at 350μm from pial surface. Gray shaded regions, s.e.m. (superficial vs deep = ***P<0.0001; two-way ANOVA). d, Assessment of retrogradely labelled cells across the width of the dLGN. 0% is at optic tract, 100% is at medial border (see Fig. 2g–i).

  8. Retrograde tracers to superficial V1 label cells in the DSGC-RZ.
    Extended Data Fig. 3: Retrograde tracers to superficial V1 label cells in the DSGC-RZ.

    ac, Same dLGN as in main Fig. 2f but with GFP+ On-Off DSGC6 axons shown. a, most of the retrogradely labelled cells (magenta/dashed circles) reside in the DSGC-RZ (green terminals). Asterisk, labelled cell outside the DSGC-RZ. Scale bar, 200μm. b, c, High magnification views of retrogradely labelled dLGN neuron cell bodies with potential contact from GFP+ DSGC axons (arrow in b); c, this cell is in vicinity of DSGC axonal boutons (arrowheads). b, c, Scale, 15μm. d, Diagram of laminar-specific connections between DSGC-RZ and superficial V1 and dLGN core and deeper V1 layers 4 and 6.

  9. Analysis of dLGN neurons retrogradely infected from superficial V1.
    Extended Data Fig. 4: Analysis of dLGN neurons retrogradely infected from superficial V1.

    af, Example serial sections of anterior, middle and posterior portions of dLGN in a mouse with GFP expressing On-Off DSGC axons that was injected with ΔG-RABV-mCherry in superficial layers of V1. a, DAPI to show cytoarchitectural landmarks and dLGN borders. b, GFP+ DSGC axons and AAV2-Glyco-hGFP-infected cell bodies (see main Fig. 4 and text). c, Mask of GFP+ DSGC axons (Methods). d, ΔG-RABV-mCherry+ dLGN relay neurons. e, GFP+ DSGC axon mask superimposed with mCherry signal; this was used to determine colocalization. f, mCherry and GFP signals merged. Scale bar, 200μm.

  10. Putative sites of contact between DSGC axons and a dLGN neuron retrogradely infected from superficial V1.
    Extended Data Fig. 5: Putative sites of contact between DSGC axons and a dLGN neuron retrogradely infected from superficial V1.

    ai, GFP+ On-Off DSGC axons (green in all panels except black in b) and mCherry+ dLGN relay neuron (magenta in all panels except white in c) infected by injection to superficial V1. Framed region in a is shown at higher magnification in bd. Arrowhead (a), thalamocortical axon of mCherry+ dLGN cell. Scale bar in a, 50μm. Yellow boxed region in c, d, is shown at higher magnification in ei. Scale bar in d, 15μm. ei, Some DSGC axon–dendrite contacts contain VGLUT2 (blue). fi, Arrowhead, site of GFP/mCherry co-localization that does not contain VGLUT2; arrow, GFP/mCherry/VGLUT2+ contact.

  11. The axons of GFP+ On-Off DSGCs and dLGN neurons infected with AAV2-Glyco-hGFP can be distinguished on the basis of their cellular localization.
    Extended Data Fig. 6: The axons of GFP+ On-Off DSGCs and dLGN neurons infected with AAV2-Glyco-hGFP can be distinguished on the basis of their cellular localization.

    High magnification view of DSGC-RZ in mouse with GFP+ posterior-tuned On-Off DSGCs that was injected 14 days earlier with AAV2-Glyco-hGFP. Glyco-hGFP+ neurons have nuclear GFP labelling (arrows), whereas DSGCs have GFP in axon terminals (arrowheads). Dashed line, lateral border of dLGN. OT, optic tract. Scale bar, 50μm.

  12. Signature anatomical and physiological characteristics of GFP-tagged On-Off DSGCs.
    Extended Data Fig. 7: Signature anatomical and physiological characteristics of GFP-tagged On-Off DSGCs.

    a, b, Flat-mount retina with GFP+ On-Off DSGCs (a) and co-stained with DAPI (b). c, Positions of GFP+ RGCs. Scale bar in c, 150μm. df, High magnification views. Scale bar, 12μm. g, Targeted fill of a GFP+ DSGC. Scale bar, 50μm. h, Schematic of On-Off DSGC stratification and starburst amacrine cells (magenta). Labelling as in Extended Data Fig. 1. i, j, Higher magnification of framed region in g stained for VAChT (starburst amacrine processes). Asterisk, ‘looping arborizations’; dashed line, GFP arborization, which matches VAChT plexus. Scale bar, 10μm. k, l, Side (xz plane) views of cell in g. GFP+ dendrites co-stratify with both the On and Off sublayers. Scale bar, 5μm. m, Direction-tuned response of a GFP+ On-Off DSGC targeted for recording and receptive field characterization. The spike count is highest for bars moving towards ~270° in the cardinal axes.

  13. Injections of [Dgr]G-RABV-mCherry into both superficial and deep V1 combined with AAV2-Glyco-hGFP infection of dLGN core.
    Extended Data Fig. 8: Injections of ΔG-RABV-mCherry into both superficial and deep V1 combined with AAV2-Glyco-hGFP infection of dLGN core.

    a, mCherry+ neurons in the DSGC-RZ and the core of the dLGN. b, AAV2-Glyco-hGFP: many neurons throughout the dLGN, but mostly along the medial border and not in the shell/DSGC-RZ express Glyco-hGFP. DSGC-RZ marked by axons of GFP+ On-Off DSGCs. c, Merged of a, b. Scale in a, 100μm. Boxed regions with arrows: two dLGN neurons; both RABV-mCherry+ and AAV2-Glyco-hGFP+. One or both of these cells infected their presynaptic partner, the RGC shown in Fig. 4 (panels cc-ee) of the main text. Scale bar, 15μm.

Change history

Corrected online 19 March 2014
Minor edits were made to the numbering of the affiliations list, and minor typographical edits were made to the legends of Figs 3 and 4.

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Author information

Affiliations

  1. Department of Neurosciences, University of California, San Diego, California 92093, USA

    • Alberto Cruz-Martín,
    • Rana N. El-Danaf,
    • Onkar S. Dhande,
    • Phong L. Nguyen &
    • Andrew D. Huberman
  2. Neurobiology Section in the Division of Biological Sciences, University of California, San Diego, California 92093, USA

    • Alberto Cruz-Martín,
    • Rana N. El-Danaf,
    • Balaji Sriram,
    • Onkar S. Dhande,
    • Phong L. Nguyen &
    • Andrew D. Huberman
  3. Salk Institute for Biological Studies, La Jolla, California 92097, USA

    • Fumitaka Osakada,
    • Edward M. Callaway &
    • Andrew D. Huberman
  4. Neuroscience Discovery, F. Hoffman La Roche, 4070 Basel, Switzerland

    • Anirvan Ghosh
  5. Department of Ophthalmology, University of California, San Diego, California 92093, USA

    • Andrew D. Huberman

Contributions

A.D.H., A.C.-M., A.G. and R.N.E. designed the experiments. A.D.H., A.C.-M., R.N.E. and P.L.N. carried out and analysed the circuit connectivity experiments. A.C.-M. carried out the in vivo imaging experiments. B.S. and A.C.-M. analysed imaging data. O.S.D. collected data on molecular markers of cell types. E.M.C. and F.O. designed and made the rabies viruses. A.D.H. and A.C.-M. wrote the paper in collaboration with the other authors. A.D.H. and A.C.-M. prepared the figures. A.D.H. oversaw the project.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

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Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: The retino-geniculo-cortical pathway links retinal cells and circuits to the brain. (100 KB)

    a, Diagram of retina, dorsal lateral geniculate nucleus (dLGN) and primary visual cortex (V1). The optic tract which carries retinal ganglion cell (RGC) axons and thalamocortical (dLGN to V1) pathway also shown. b, Diagram of retinal layers: PRL, photoreceptor layer; opl, outer plexiform layer; INL, inner nuclear layer; ipl, inner plexiform layer; GCL, ganglion cell layer; nfl, nerve fibre layer. c, Retina diagram with cells shown (labels same as in b).

  2. Extended Data Figure 2: Approach for assessing laminar specificity of mouse geniculocortical projections. (108 KB)

    a, Focal retrograde tracer injection to V1. Scale bar, 3mm. b, Diagram of the three different injection depths used to generate data in Fig. 2. c, Percentage of fluorescence in V1 from superficial (black line) versus deep (grey line) injections. Superficial, peak intensity occurs at 25μm from pial surface (4 mice). Deep, peak intensity occurs at 350μm from pial surface. Gray shaded regions, s.e.m. (superficial vs deep = ***P<0.0001; two-way ANOVA). d, Assessment of retrogradely labelled cells across the width of the dLGN. 0% is at optic tract, 100% is at medial border (see Fig. 2g–i).

  3. Extended Data Figure 3: Retrograde tracers to superficial V1 label cells in the DSGC-RZ. (410 KB)

    ac, Same dLGN as in main Fig. 2f but with GFP+ On-Off DSGC6 axons shown. a, most of the retrogradely labelled cells (magenta/dashed circles) reside in the DSGC-RZ (green terminals). Asterisk, labelled cell outside the DSGC-RZ. Scale bar, 200μm. b, c, High magnification views of retrogradely labelled dLGN neuron cell bodies with potential contact from GFP+ DSGC axons (arrow in b); c, this cell is in vicinity of DSGC axonal boutons (arrowheads). b, c, Scale, 15μm. d, Diagram of laminar-specific connections between DSGC-RZ and superficial V1 and dLGN core and deeper V1 layers 4 and 6.

  4. Extended Data Figure 4: Analysis of dLGN neurons retrogradely infected from superficial V1. (320 KB)

    af, Example serial sections of anterior, middle and posterior portions of dLGN in a mouse with GFP expressing On-Off DSGC axons that was injected with ΔG-RABV-mCherry in superficial layers of V1. a, DAPI to show cytoarchitectural landmarks and dLGN borders. b, GFP+ DSGC axons and AAV2-Glyco-hGFP-infected cell bodies (see main Fig. 4 and text). c, Mask of GFP+ DSGC axons (Methods). d, ΔG-RABV-mCherry+ dLGN relay neurons. e, GFP+ DSGC axon mask superimposed with mCherry signal; this was used to determine colocalization. f, mCherry and GFP signals merged. Scale bar, 200μm.

  5. Extended Data Figure 5: Putative sites of contact between DSGC axons and a dLGN neuron retrogradely infected from superficial V1. (526 KB)

    ai, GFP+ On-Off DSGC axons (green in all panels except black in b) and mCherry+ dLGN relay neuron (magenta in all panels except white in c) infected by injection to superficial V1. Framed region in a is shown at higher magnification in bd. Arrowhead (a), thalamocortical axon of mCherry+ dLGN cell. Scale bar in a, 50μm. Yellow boxed region in c, d, is shown at higher magnification in ei. Scale bar in d, 15μm. ei, Some DSGC axon–dendrite contacts contain VGLUT2 (blue). fi, Arrowhead, site of GFP/mCherry co-localization that does not contain VGLUT2; arrow, GFP/mCherry/VGLUT2+ contact.

  6. Extended Data Figure 6: The axons of GFP+ On-Off DSGCs and dLGN neurons infected with AAV2-Glyco-hGFP can be distinguished on the basis of their cellular localization. (563 KB)

    High magnification view of DSGC-RZ in mouse with GFP+ posterior-tuned On-Off DSGCs that was injected 14 days earlier with AAV2-Glyco-hGFP. Glyco-hGFP+ neurons have nuclear GFP labelling (arrows), whereas DSGCs have GFP in axon terminals (arrowheads). Dashed line, lateral border of dLGN. OT, optic tract. Scale bar, 50μm.

  7. Extended Data Figure 7: Signature anatomical and physiological characteristics of GFP-tagged On-Off DSGCs. (277 KB)

    a, b, Flat-mount retina with GFP+ On-Off DSGCs (a) and co-stained with DAPI (b). c, Positions of GFP+ RGCs. Scale bar in c, 150μm. df, High magnification views. Scale bar, 12μm. g, Targeted fill of a GFP+ DSGC. Scale bar, 50μm. h, Schematic of On-Off DSGC stratification and starburst amacrine cells (magenta). Labelling as in Extended Data Fig. 1. i, j, Higher magnification of framed region in g stained for VAChT (starburst amacrine processes). Asterisk, ‘looping arborizations’; dashed line, GFP arborization, which matches VAChT plexus. Scale bar, 10μm. k, l, Side (xz plane) views of cell in g. GFP+ dendrites co-stratify with both the On and Off sublayers. Scale bar, 5μm. m, Direction-tuned response of a GFP+ On-Off DSGC targeted for recording and receptive field characterization. The spike count is highest for bars moving towards ~270° in the cardinal axes.

  8. Extended Data Figure 8: Injections of ΔG-RABV-mCherry into both superficial and deep V1 combined with AAV2-Glyco-hGFP infection of dLGN core. (484 KB)

    a, mCherry+ neurons in the DSGC-RZ and the core of the dLGN. b, AAV2-Glyco-hGFP: many neurons throughout the dLGN, but mostly along the medial border and not in the shell/DSGC-RZ express Glyco-hGFP. DSGC-RZ marked by axons of GFP+ On-Off DSGCs. c, Merged of a, b. Scale in a, 100μm. Boxed regions with arrows: two dLGN neurons; both RABV-mCherry+ and AAV2-Glyco-hGFP+. One or both of these cells infected their presynaptic partner, the RGC shown in Fig. 4 (panels cc-ee) of the main text. Scale bar, 15μm.

Additional data