Thalamus provides layer 4 of primary visual cortex with orientation- and direction-tuned inputs

Abstract

Understanding the functions of a brain region requires knowing the neural representations of its myriad inputs, local neurons and outputs. Primary visual cortex (V1) has long been thought to compute visual orientation from untuned thalamic inputs, but very few thalamic inputs have been measured in any mammal. We determined the response properties of ∼ 28,000 thalamic boutons and ∼ 4,000 cortical neurons in layers 1-5 of awake mouse V1. Using adaptive optics that allows accurate measurement of bouton activity deep in cortex, we found that around half of the boutons in the main thalamorecipient L4 carried orientation-tuned information and that their orientation and direction biases were also dominant in the L4 neuron population, suggesting that these neurons may inherit their selectivity from tuned thalamic inputs. Cortical neurons in all layers exhibited sharper tuning than thalamic boutons and a greater diversity of preferred orientations. Our results provide data-rich constraints for refining mechanistic models of cortical computation.

Fig. 1. In vivo calcium imaging of thalamic axons in V1

(a) In vivo imaging of GCaMP6s⁺ thalamic axons in V1 of head-fixed awake mouse. (b) GCaMP6s⁺ neurons (green) in dLGN outlined by retinal ganglion cell axons (red). Scale bar: 100 µm. (c) GCaMP6s⁺ axons in V1. Scale bars: 50 µm. (d–f) In vivo images of GCaMP6s⁺ thalamic axons at (d) 40 µm, (e) 200 µm, and (f) 350 µm below pia in V1. Scale bar: 10 µm. (g–i) Varicosities (putative boutons) from d–f color-coded by their preferred orientation. Gray, boutons with visual response but no orientation selectivity (OS). (j–l) Example ΔF/F calcium transients (10 trial average) for boutons from d–f. (m–o) Tuning curves for the bottom six boutons in j–l. Dark gray shadow (j–l) and error bars (m–o): s.e.m.. Representative images from 21 mice.

Figure 2. Adaptive optics is essential for tuning curve characterization

(a) Excitation light aberrated by refractive index differences between water and cranial window/brain. (b) Axial images of a 2-µm bead below a 340-µm window, a 170-µm window, and a 170-µm window with adaptive optics (AO) correction. Images taken without AO have 4× and 2× gain for better visibility. Scale bar: 2 µm. (c) Percentages of non-responsive (NR), not OS (NOS), and OS boutons at 300–350 µm depth under the conditions in b. (d) Images of GCaMP6s+ axons at 170 µm depth measured without and with AO. Images are saturated to improve visibility of dim features. Scale bar: 10 µm. (e) Calcium transients and tuning curves for ROIs labeled in d measured without (red) and with (black) AO. Error bars: s.e.m.. (f) Cumulative distributions of global orientation-selectivity index (gOSI) for boutons at 300–350 µm depth measured without and with AO. (g) Their preferred orientation distributions measured without and with AO. d–g, cranial window thickness: 170 µm. f,g, same data as in c, boutons imaged under 170-µm window without and with AO correction and analyzed with independent ROI selections.

Fig. 3. Characterization of tuning properties of L4, L2/3, and L5 neurons in V1

(a) An example in vivo image of L4 neurons. Numbers label example somata. (b) Calcium transients ΔF/F, and (c) tuning curves of the somata in a; Light gray background in b: 6-sec drifting grating (topmost labels) presentation; Dark gray shade in b and error bars in c: s.e.m.. (d–i) Example in vivo images, calcium transients, and tuning curves of (d–f) L2/3 and (g–i) L5 neurons. Scale bar: 25 µm. Representative results from 3 Scnn1a-Tg3-Cre (L4), 6 Thy1-GCaMP6 GP4.3 (L2/3), and 5 Rbp4-Cre (L5) mice.

Fig. 4. Orientation tuning of thalamic boutons and neurons in V1

(a–o) Orientation tuning of thalamic boutons: histogram distributions of (a–c) preferred orientation, (d–f) global orientation-selective index (gOSI), (g–i) orientation-selective index (OSI), (j–l) tuning width (FWHM), (m–o) direction-selective index (DSI) for OS boutons 0–100 µm, 150–250 µm, and 300–400 µm below pia. (p–dd) Orientation tuning properties of cortical neurons: distributions of preferred orientation, gOSI, OSI, FWHM, and DSI for (p–t) L4, (u–y) L2/3, and (z–dd) L5 neurons. Gray histograms in d–f,q,v,aa: gOSI distributions for non-OS units. Blue dashed lines: distribution medians.

Fig. 5. Direction tuning of thalamic boutons and neurons in V1

(a–q) Direction tuning of thalamic boutons: Example polar-plot tuning curves of (a) axis-selective (AS) and (b) direction-selective (DS) boutons; A, P, S, I in b: anterior, posterior, superior, and inferior directions; Bottom right: DSI & gOSI. (c–e) gOSI, (f–h) OSI, and (i–k) tuning curve FWHM distributions for AS (blue) and DS (red) boutons 0–100 µm, 150–250 µm, and 300–400 µm below pia; (l–n) Circular histograms of preferred motion axis for AS units (bar orientation: preferred motion axis; bar length: # of units); Bars reflected for displaying axis selectivity (faded and full-color bars represent the same data); (o–q) Circular histograms of preferred motion direction for DS units (bar direction: motion direction). (r–ii) Direction tuning of cortical neurons: Example tuning curves, distributions of gOSI, OSI, FWHM, preferred motion axis for AS, and preferred motion direction for DS (r–w) L4, (x–cc) L2/3, and (dd–ii) L5 neurons. Green arrows indicate the anteroinferior-posterosuperior motion axis.