Cortico-cortical projections in mouse visual cortex are functionally target specific

Abstract

Neurons in primary sensory cortex have diverse response properties, whereas higher cortical areas are specialized. Specific connectivity may be important for areal specialization, particularly in the mouse, where neighboring neurons are functionally diverse. To examine whether higher visual areas receive functionally specific input from primary visual cortex (V1), we used two-photon calcium imaging to measure responses of axons from V1 arborizing in three areas with distinct spatial and temporal frequency preferences. We found that visual preferences of presynaptic boutons in each area were distinct and matched the average preferences of recipient neurons. This specificity could not be explained by organization within V1 and instead was due to both a greater density and greater response amplitude of functionally matched boutons. Projections from a single layer (layer 5) and from secondary visual cortex were also matched to their target areas. Thus, transmission of specific information to downstream targets may be a general feature of cortico-cortical communication.

Figure 1

Functional two-photon calcium imaging from the axons of V1 projection neurons. (a) Two models of mouse visual cortex. Higher visual areas may receive functionally nonspecific (top) or specific (bottom) inputs from V1. Specificity may arise through diverse mechanisms, including a bias in projection probability, arborization size or neural excitability. (b) Labeling of V1 axonal projections with GCaMP3.3. Top, tangential section of visual cortex; A, anterior; P, posterior; L, lateral; M, medial. Bottom, infected somata in layer 2/3 (L2/3) of V1 (left) and V1 axonal arborizations in LM (right). Scale bars, 500 μm (top) and 30 μm (bottom). (c) In vivo calcium imaging. Left, in vivo image of visual cortex. V1 was covered to prevent saturation and the inset (gray box) taken with lower illumination. Middle and right, example average two-photon fluorescence responses (dF/F; 24 trials) of axons in PM (white outlined region in left panel). Stimuli (insets) are sinusoidal drifting gratings of different spatial and temporal frequencies. Scale bars, 500 μm (left) and 50 μm (middle and right). (d) Identification of visually responsive boutons. Left, maximum (max) response projection across stimuli. Middle, magnification of the boxed region at left. Right, gray and colored circles indicate locations of boutons whose tunings are well fit by a two-dimensional Gaussian. Black circles indicate poor fits (excluded from further analysis). Scale bars, 50 μm (left) and 15 μm (middle and right). (e) Visual responses of boutons in colored circles from d. Top, average response of each bouton (for the indicated directions of motion; 12 trials per stimulus) and the fit of the average response (right). The area of each circle is proportional to dF/F. SF, spatial frequency; TF, temporal frequency. Bottom, average dF/F time course for each stimulus. Blue lines (in left panel) represent duration of stimulus (5 s). Shaded regions are ± s.e.m.

Figure 2

V1 axons projecting to LM, AL and PM are functionally distinct. (a) Example responses from six boutons from the same field of view in LM (top), PM (middle) or AL (bottom) of the same mouse. Boutons were ordered from lowest to highest speed and chosen as evenly spaced percentiles (14th, 29th, 43rd, 57th, 71st and 86th). Amplitude of peak dF/F is noted above each response. SF, spatial frequency; TF, temporal frequency. (b) Average response of all boutons (n represents number of boutons) in LM (left), PM (middle) and AL (right) for three example mice. (c) Top, average response in each area for all boutons imaged. Bottom, average response in each area for all mice imaged (n represents number of mice). (d) Average tuning curves for SF (left), TF (middle) and speed (right) in each area for all boutons imaged. Number of boutons is given in parentheses. Error bars, ± s.e.m.

Figure 3

Lack of functional organization for speed in V1. (a) Coronal section of V1 stained with DAPI to show nuclei (left) and expressing GCaMP3 (second from left). Third from left, projection of a z-stack taken through the injection site in vivo (2-μm steps; same mouse as on left). Right, relative positions of four volumes imaged in this mouse. Layers are indicated at left. (b) Example responses from six neurons in L2/3 (top), L4 (middle) or L5 (bottom) from the same mouse as in a. Neurons were ordered from lowest to highest speed and chosen as evenly spaced percentiles. Amplitude of peak dF/F is noted above each response. (c) Scatter plot of peak speed by cortical depth for all neurons (n = 302) imaged in the mouse in a. Dashed lines delineate the borders of L4. (d) x–z (left) and x–y (right) projection of all neurons imaged in the mouse in a, colored according to peak speed. Note the lack of clustering in any dimension. (e) Average tuning distance (see Online Methods) for all pairs of neurons binned according to their distance in z (left) and x–y (right) (n = 542 neurons, two mice; the number of pairs for each bin is given below; bins with <10 pairs were excluded). (f) Average tuning for speed for neurons in L2/3 (n = 166 neurons, four mice) and L5 (n = 312 neurons, two mice). (g) Cumulative distribution of peak speeds for neurons in L2/3 and L5 (same population of neurons as in f). All error bars, ± s.e.m.

Figure 4

Layer 5 axons projecting to LM, AL and PM are functionally distinct. (a) Coronal section of visual cortex stained for DAPI (left) from an Rbp4-cre × tdTomato reporter (right) mouse. Note the dense expression in layer 5 (L5). (b) Left, x–z projection of an in vivo z-stack of an Rbp4-cre mouse after infection with GCaMP3 (2-μm steps). Right, scatter plot of peak speed by cortical depth for all neurons imaged (n = 121) in the mouse on left. (c) Average tuning for speed for neurons in L5 of WT (n = 312 neurons, two mice) and Rbp4-cre mice (n = 159 neurons, two mice). (d) Cumulative distribution of peak speeds for neurons in L5 of WT and Rbp4-cre (same population of neurons as in c). (e) Tangential section of visual cortex of an Rbp4-cre mouse (same mouse as in b) after infection with GCaMP3. Scale bar, 500 μm; abbreviations as in Figure 1. (f) Average response of all boutons (n represents number of boutons) in LM (left), AL (middle) and PM (right) for two example mice (top and middle) and for all boutons imaged in each area (bottom). (g) Average tuning curves for spatial frequency (SF; left), temporal frequency (TF; middle) and speed (right) for all boutons imaged in each area. All error bars, ± s.e.m.

Figure 5

Bias in the number of boutons with target-specific preferences. (a) Two models for the different average tuning of all boutons in AL and PM (see Fig. 2c). These differences may be due to the number of inputs, either axons or axonal branches, with matched properties (left; schematized by number of arrows) or area-specific biases in response amplitudes (right; schematized by the thickness of the arrows). (b) Cumulative distribution of preferred spatial frequency (SF; left), preferred temporal frequency (TF; middle) and peak speed (right) for all boutons imaged in each area in WT mice. (c) Same as in b, for boutons in Rbp4-cre mice. Dotted lines include boutons selected in AL with relaxed criteria (P < 0.01 for responsivity and 75% confidence intervals; n = 143). (d) Left, median speed of all boutons imaged in each WT (open circles) or Rbp4-cre (open triangles) mouse and for all boutons (filled circles). Areas imaged in the same mouse are connected with a gray line. Numbers of mice with each area imaged (black) and with multiple areas imaged (gray) are indicated below. Filled squares are the median speed of cell bodies imaged in AL (red) and PM (blue; from ref. 4). Right, cumulative distribution of peak speed for boutons (thick lines) and cell bodies (thin lines) imaged in AL and PM. Arrows at 10% and 90% highlight the difference in distributions at the low and high speeds.

Figure 6

Areal bias in the amplitude of responses at different speeds is explained by the activity of a small fraction of boutons. (a) Two models for the different average tuning of all boutons in AL and PM (see Fig. 2c). These differences may be solely due to the number of inputs with matched properties (left; schematized by number of arrows) or extra, area-specific biases in response amplitudes (right; schematized by the thickness of the arrows). (b) Distributions of peak speeds for all boutons in AL and PM. (c) Average response amplitude (dF/F) for all boutons in AL and PM binned according to their peak speed. Boutons in AL with high peak speeds have a larger average response than boutons with low peak speeds; the opposite is true for boutons in PM. (d) Distributions of peak speeds for all boutons in each area, for different ranges of peak dF/F. Each bin notes the range of dF/F and the percentage of boutons from each area contained in that bin. The response preferences in AL and PM are segregated at all ranges of dF/F, but they become increasingly segregated for boutons with larger peak responses. (e) Average peak speed (top) and preferred spatial frequency (SF; middle) and temporal frequency (TF; bottom) for boutons grouped according to their peak dF/F (same bins as in b). All error bars, ± s.e.m.

Figure 7

Axons projecting from LM to AL and PM are functionally distinct. (a) Tangential section of visual cortex after infection with GCaMP3 in LM. Scale bar, 500 μm; abbreviations as in Figure 1. (b) Example responses from six boutons from the same field of view in PM (top row) or AL (bottom row) of the same mouse. Boutons were ordered from lowest to highest speed and chosen as evenly spaced percentiles. Amplitude of dF/F is noted above each response. (c) Average response of all boutons (n represents number of boutons) in AL and PM for two example mice. (d) Average response for all boutons (top; n represents number of boutons) and for all mice (bottom; n represents number of mice) imaged in each area. (e) Average tuning curves for spatial frequency (SF; left), temporal frequency (TF; middle) and speed (right) for all boutons imaged in each area. Error bars, ± s.e.m. (f) Cumulative distribution of preferred SF (left), preferred TF (middle) and peak speed (right) for all boutons in AL and PM from LM (thick lines) and V1 (thin lines). (g) Median speed of all boutons from LM within each mouse (open circles) and for all boutons (filled circles). Areas imaged in the same mouse are connected with a gray line. Numbers of mice imaged in each area (black) and in both areas (gray) are given below. Filled squares are the median speed of boutons from V1 imaged in AL and PM (same data as in Fig. 5).