Tuned thalamic excitation is amplified by visual cortical circuits

Abstract

Cortical neurons in thalamic recipient layers receive excitation from the thalamus and the cortex. The relative contribution of these two sources of excitation to sensory tuning is poorly understood. We optogenetically silenced the visual cortex of mice to isolate thalamic excitation onto layer 4 neurons during visual stimulation. Thalamic excitation contributed to a third of the total excitation and was organized in spatially offset, yet overlapping, ON and OFF receptive fields. This receptive field structure predicted the orientation tuning of thalamic excitation. Finally, both thalamic and total excitation were similarly tuned to orientation and direction and had the same temporal phase relationship to the visual stimulus. Our results indicate that tuning of thalamic excitation is unlikely to be imparted by direction- or orientation-selective thalamic neurons and that a principal role of cortical circuits is to amplify tuned thalamic excitation.

Figure 1. Isolating thalamic excitation

a, Schematic of experimental approach for isolating thalamic excitation recorded in response to visual stimulation in layer 4 neurons voltage clamped at the reversal potential for inhibition. Thalamic excitation is isolated by silencing cortical activity via photostimulation of ChR2-expressing PV cells. b, Optogenetic silencing of visually evoked cortical activity. Top: The spiking responses of two example cortical neurons recorded in the loose patch configuration with (LED, blue) and without (control, red) PV cell photostimulation are shown as raster plots. Gray rectangle, visual stimulus (1.5 s). Blue bar, LED illumination (2.6 s). Control and LED trials were interleaved, but separated here for clarity. Bottom, population peristimulus time histogram (PSTH) averaged across all cells and all stimulus directions (n = 14 cells, 2 mice). Note complete suppression of spiking during LED illumination. The high spontaneous activity and its stimulus-evoked reduction in cell 2 were rare. c, Total and thalamic excitation in response to flashed squares. Top, visually evoked EPSCs recorded in two example cortical neurons with (blue) and without (red) cortical silencing in response to a flashed square (5 degrees) at optimal location and luminance. Average of 8 (cell 1) and 4 (cell 2) trials. Gray rectangle, visual stimulus. Blue bar, LED illumination. Bottom, distribution of the fraction of excitatory charge contributed by the thalamus (n = 18 cells, 16 mice). d, Same as c for drifting gratings. The response to the preferred direction is shown. Average of 8 (cell 1) and 5 (cell 2) trials. Bottom, distribution of the fraction of excitatory charge contributed by the thalamus (n = 42 cells, 33 mice).

Figure 2. Receptive field structure of thalamic excitation

a, Example cell. Isolated thalamic excitation (EPSC_Thal) in response to black (“OFF”, magenta traces, left) or white (“ON”, green traces, right) squares at each of 64 locations on an 8×8 grid. Stimuli appear at the beginning of each trace and last for 100 ms. The raw receptive field heat maps calculated from the thalamic excitatory charge (Q_Thal) evoked at each grid location and normalized to the peak ON response are depicted by the background gray-level of each trace. Average of 4 trials per location. b, Contour plot of the OFF and ON subfields for the cell in a. Each contour represents 2 z-scores. Filled magenta and green circles mark the peaks of the OFF and ON receptive fields, respectively. c, Profile plot of OFF and ON receptive fields in c along the axis connecting their peaks. d, Same as b and c for 3 additional neurons. e, Population average of OFF and ON receptive fields. Before averaging, the receptive fields for each cell were centered on the peak of the OFF subfield and rotated so that the peak of the ON subfield was directly to the right of the OFF. OFF and ON subfields were separately normalized. Top, heat maps of the population average OFF and ON receptive fields. Bottom, contour and profile plots of the population average receptive fields as in b and c. The outermost contour represents 10% of the peak and each additional contour is an increment of 20% of the peak. f, Quantification of subfield area (n = 26 subfields) and overlap between ON and OFF subfields (n = 13 cells). g, Distance between the peaks of ON and OFF subfields (n = 13 cells). h, Comparison of subfield width along the axis connecting the ON and OFF peaks (ON/OFF axis) or the orthogonal axis (n = 26 subfields). Black line, unity. Data in e–h are from 13 cells each exhibiting 1 OFF and 1 ON subfield from 12 mice. Error bars, mean ± s.e.m.

Figure 3. Orientation tuning of thalamic excitation

a, Example cell: Top, Isolated thalamic excitation (EPSC_Thal) in response to drifting gratings of various orientations (average of 8 trials per direction). Gray rectangle, visual stimulus (1.7 s). Blue bar, LED illumination (2.6 s). Bottom, F1 modulation of EPSC_Thal. Cycle average (blue) and best-fitting sinusoid (green) at the grating temporal frequency (2 Hz). The y-offset was removed to aid comparison of F1 amplitude across different orientations. Right, EPSC_Thal and cycle average in response to 330 degree grating at expanded time scale showing how Q_Thal and F1_Thal are determined. b, Orientation tuning curves of Q_Thal (dashed line) and F1_Thal (solid line) for the neuron in (a). c, Population tuning curves of Q_Thal (dashed line) and F1_Thal (solid line). Left, Population tuning curves in which Q_Thal and F1_Thal tuning curves for each cell were equally shifted so that the preferred direction of F1_Thal occurred at 0 degrees (F1_Thal reference). Right, Population tuning curves in which Q_Thal and F1_Thal tuning curves for each cell were independently shifted so that preferred direction of Q_Thal and F1_Thal both occurred at 0 degrees (self reference). d, Orientation selectivity index (OSI) of Q_Thal plotted against OSI of F1_Thal for each cell. e, Direction selectivity index (DSI) of Q_Thal plotted against DSI of F1_Thal for each cell. Filled red markers in d and e denote the OSI and DSI values of the example cell. Data in c–e are from n = 42 cells from 33 mice. Error bars, mean ± s.e.m.

Figure 4. Separation of ON and OFF thalamic subfields predicts preferred orientation of thalamic excitation

a–d, Example recording of isolated thalamic excitation (EPSC_Thal) where both the ON and OFF receptive fields and the responses to drifting gratings at various orientations were obtained in the same cell. a, EPSC_Thal in response to black and white squares. Average of 5 trials per location. b, Contour plot of the OFF and ON receptive field maps for the cell in a. Each contour represents 2 z-scores. Filled magenta and green circles mark the peaks of the OFF and ON receptive fields, respectively. Dashed black line connects the OFF and ON peaks to define the ON-OFF axis. The preferred orientation predicted from the ON-OFF axis, RF_Pref, is indicated by the small grating. c, EPSC_Thal in response to drifting gratings of various orientations (average of 3 trials per direction). Gray rectangle, visual stimulus (1.7 s). Blue bar, LED illumination (2.6 s). d, Orientation tuning curves of F1_Thal (blue) and Q_Thal (gray) in polar coordinates for the responses in c. The blue line indicates the preferred orientation of F1_Thal (Grating_Pref) and the black dashed line corresponds to RF_Pref. e, Same as b and d for three additional cells. f, RF_Pref plotted against Grating_Pref (n = 8 cells, 7 mice). Black line, unity. The dashed lines denote the region in which cells the difference between RF_Pref and Grating_Pref is less than 30 degrees. The distributions of Grating_Pref (n = 42, 33 mice) and RF_Pref (n = 13 cells, 12 mice) across the population of cells in which either value was measured are shown along the top and right, respectively. g, Absolute difference in RF_Pref and Grating_Pref (ΔPref Ori) (n = 8 cells, 7 mice). Error bars, mean ± s.e.m. h, Diagram of how orientation tuning of F1_Thal arises from spatially offset OFF and ON thalamic excitatory input. The area of the blue shaded region corresponds to Q_Thal. The difference between the peak and the trough of EPSC_Thal corresponds to F1_Thal.

Figure 5. Tuning of non-thalamic excitatory charge

a, Example cell: Top, EPSC_Thal (blue) and EPSC_Tot (red) in response to drifting gratings of various orientations. Bottom, EPSC_Sub derived from point-by-point subtraction of EPSC_Thal from EPSC_Tot. Gray rectangle, visual stimulus (1.7 s). Blue bar, LED illumination (2.6 s). b, Orientation tuning curves of Q_Tot (red) and Q_Thal (blue) for the example cell in (a). c, Population tuning curves of Q_Tot (red) and Q_Thal (blue). Tuning curves are aligned to the preferred direction of Q_Tot (Q_tot reference) and normalized by the value of Q_Tot at its preferred direction. d, OSI of Q_Tot plotted against OSI of Q_Thal for all neurons. e, DSI of Q_Tot plotted against DSI of Q_Thal for all neurons. f–i, Same as (b–e) for Q_Sub (black) and Q_Thal (blue). Filled green markers in d, e, h, and i denote the OSI and DSI values of the example cell. Data in c–e and g–i are from n = 42 cells from 33 mice. Error bars, mean ± s.e.m.

Figure 6. Tuning of non-thalamic excitatory F1 modulation

a, Example cell: Top, EPSC_Sub in response to drifting gratings of various orientations. Gray rectangle, visual stimulus (1.5 s). Blue bar, LED illumination (2.6 s). Bottom, F1 modulation of EPSC_Sub. Cycle average (black) and best-fitting sinusoid (green) at the grating temporal frequency (2 Hz). b, Orientation tuning curves of Q_Sub (dashed curve) and F1_Sub (solid curve) for the example cell in a. c, Population tuning curve of Q_Sub (dashed curve) and F1_Sub (solid curve). Left, Population tuning curves in which Q_Sub and F1_Sub tuning curves for each cell were equally shifted so that the preferred direction of Q_sub occurred at 0 degrees (Q_sub reference). Right, Population tuning curves in which Q_Sub and F1_Sub tuning curves for each cell were independently shifted so that preferred direction of Q_Sub and F1_Sub both occurred at 0 degrees (self reference). d, OSI of F1_Sub is plotted against OSI of Q_Sub for all neurons. e, Distribution of absolute differences in preferred orientation (ΔPref Ori) between Q_Sub and F1_Sub. Dark curve, all cells (n = 42). Gray curve, cells in the top 50^th percentile of F1_Sub OSI (n = 21). f–g, Same as (d–e) for DSI and absolute differences in preferred direction (ΔPref Dir). Filled green markers in d and f denote the OSI and DSI values of the example cell. Data in c–g are from n = 42 cells from 33 mice. Error bars, mean ± s.e.m.

Figure 7. Co-tuning and phase relationship between thalamic and non-thalamic excitation

a, Cycle average of EPSC_Thal (blue) and EPSC_Sub (black) for an example cell. Green curves are the best-fitting sinusoids at the grating temporal frequency (2 Hz). b, Orientation tuning curves of F1_Thal (blue) and F1_Sub (black) for cell in a. c, Population tuning curves of F1_Thal (blue) and F1_Sub (black). F1_Thal and F1_Sub tuning curves were aligned to the preferred direction of F1_Thal (F1_thal reference) d, Top, distribution of absolute difference in preferred orientation (ΔPref Ori) between F1_Thal and F1_Sub. Dark curve, all cells (n = 42). Gray curve, cells in the top 50th percentile of F1_Thal OSI (n = 21). Bottom, OSI of F1_Thal is plotted against OSI of F1_Sub for all neurons. e, Same as d for absolute differences in preferred direction (ΔPref Dir) and DSI. f, Population average of EPSC_Thal (blue) and EPSC_Sub (black) over two grating cycles at the preferred direction of F1_Thal and aligned to the F1 phase of EPSC_Thal. g, Distribution of F1 phase difference (ΔPhase) between EPSC_Thal and EPSC_Sub for responses from f. F1 phase of EPSC_Thal is set to 0 degrees. Data in c–g are from n = 42 cells from 33 mice. Error bars, mean ± s.e.m.