Functional specificity of local synaptic connections in neocortical networks.

Neuronal connectivity is fundamental to information processing in the brain. Therefore, understanding the mechanisms of sensory processing requires uncovering how connection patterns between neurons relate to their function. On a coarse scale, long-range projections can preferentially link cortical regions with similar responses to sensory stimuli. But on the local scale, where dendrites and axons overlap substantially, the functional specificity of connections remains unknown. Here we determine synaptic connectivity between nearby layer 2/3 pyramidal neurons in vitro, the response properties of which were first characterized in mouse visual cortex in vivo. We found that connection probability was related to the similarity of visually driven neuronal activity. Neurons with the same preference for oriented stimuli connected at twice the rate of neurons with orthogonal orientation preferences. Neurons responding similarly to naturalistic stimuli formed connections at much higher rates than those with uncorrelated responses. Bidirectional synaptic connections were found more frequently between neuronal pairs with strongly correlated visual responses. Our results reveal the degree of functional specificity of local synaptic connections in the visual cortex, and point to the existence of fine-scale subnetworks dedicated to processing related sensory information. ©2011 Macmillan Publishers Limited. All rights reserved

Paired intracellular recordings in cortical slices indicate that synaptic connectivity between neighboring neurons is heterogeneous and depends on factors such as cell type and electrophysiological properties5-10. In fact, even within relatively homogenous groups of neurons, connectivity is not uniformly distributed5,6. While this non-random connectivity raises the possibility that functionally similar neurons form synaptically coupled subnetworks6,7, the relationship between a neuron’s synaptic partners and their functional properties in local cortical circuits has not been determined.

To elucidate this relationship, we developed an approach to relate connectivity to function in identified neurons of the layer 2/3 (L2/3) network in mouse visual cortex (V1), where neurons with diverse preferences for sensory stimuli are locally intermixed11,12. In anaesthetized mice, the monocular region of V1 was bulk labeled with injections of the calcium indicator dye OGB□1 AM and the astrocyte marker SR10113 (see Methods). We first used in vivo two-photon imaging14,15 to sample spike□related somatic calcium signals from L2/3 neurons during presentation of drifting gratings and natural movie sequences (see Methods). We repeated this mapping at consecutive depths beneath the cortical surface in order to characterize visually evoked responses of all neurons within a cortical volume of approximately 285×285×90 μm3, starting at the upper border of L2/3 (Fig. 1a, b, depth range covered 60-120 μm). In this way, we obtained information about orientation/direction tuning and response correlation from a complete sample of L2/3 neurons (Fig. 1c, d).

Figure 1

Imaging functional properties of neurons in vivo and indentifying the same neurons in vitro

a, Two-photon imaging was used to sample somatic calcium signals from a complete population of L2/3 neurons within a 285×285×119 μm3 volume. Imaging was carried out at 7 μm depth increments. Neurons were labeled with the calcium indicator dye OGB-1 AM (green) and the astrocyte marker SR101 (red). b, Example traces of calcium signals from four different cells in the imaged volume while presenting six trials of grating stimuli drifting in eight different directions. c, All orientation selective cells in the volume were color-coded according to preferred orientation and plotted as spheres. d, Signal correlations were computed from average responses to natural movies. Red lines represent strongly correlated neuronal pairs (signal correlation > 0.2). e and f, After imaging visually evoked calcium signals, a detailed image stack was obtained in vivo. The brain was sliced coronally and another stack of the same tissue was obtained in vitro (a single optical plane is shown in f). Affine transformation was used to align the in vivo to the in vitro stack, allowing precise matching of OGB-1-filled cells in the two stacks. g and h, Close-ups of the regions outlined with dashed lines in e and f, respectively.

We then identified the same OGB-1-filled neurons in acute slices (Fig. 1e-h, 2a) by registering image stacks obtained in vivo and in vitro using affine transformation (see Methods and Supplementary Fig. 1), and carried out simultaneous whole-cell patch clamp recordings from up to four neighboring L2/3 pyramidal neurons (mean distance ± s.d. = 25 ± 9 μm). Synaptic connectivity between cells was assessed by evoking action potentials in each neuron in turn while simultaneously recording membrane potential in the other neurons. Monosynaptic connections appeared as spike□locked postsynaptic potentials with millisecond latency (mean latency ± SEM = 1.69 ± 0.11 ms; see Fig. 2c, 3b for sample traces). This approach allowed us to determine connectivity rates and patterns (i.e. unidirectional, bidirectional), and to relate these to cell functionality in the intact brain (Fig. 1c, d, 2b, 3a).

Figure 2

Relating orientation and direction preference to connection probability among L2/3 pyramidal neurons

a White circles denote the locations of in vivo to in vitro matched cells that were targeted for whole-cell recording and filled with Alexa 594. b. Left: average calcium responses of the four cells to oriented drifting gratings. Right: corresponding polar plots of inferred spike rate responses, normalized to the maximum response of cell 4. Three of the cells (cells 2, 3 and 4) were reliably responsive and orientation selective. Arrow shows a connection detected from cell 3 to cell 2. c, Membrane potential recordings from the four cells. Currents were injected into each cell in sequence, and from average traces of postsynaptic potentials an excitatory connection was found from cell 3 to cell 2. No other connections were found. Vertical dashed lines indicate timing of presynaptic spikes. In some traces, stimulation artefacts are visible that coincided exactly with presynaptic spikes and therefore could be clearly distinguished from EPSPs. d, Relationship between connection probability and difference in preferred orientation (ΔOri) among pairs in which both neurons were responsive to grating stimuli and were orientation selective (OSI > 0.4). There was a significant decreasing trend in connection probability as ΔOri increased (P = 0.040, Cochran-Armitage test). Dotted line indicates connection probability for all pairs included in this analysis (25/94, 0.27). The bins include difference in orientation values of 0 to 22.5° (zero degree bin), 22.5° to 67.5° (45 degree bin), and 67.5° to 90° (90 degree bin). e, Relationship between connection probability and difference in preferred direction (ΔDir) in the subset of neurons which were direction-selective (DSI > 0.3). The same decreasing trend with respect to ΔOri was detected (P = 0.034, Cochran-Armitage test). Neurons connected with specificity to preferred orientation but not to preferred direction. Dotted line indicates connection probability for all directionally selective pairs (19/72, 0.26). The bins include difference in orientation values of 0 to 22.5° (zero degree bin), 22.5° to 67.5° (45 degree bin), and so on.

Figure 3

Relationship between response correlation to natural movies and connection probability

a, An example of a triplet of neurons targeted for whole cell recording in vitro, with associated in vivo calcium responses to the natural movie (average of 6 repetitions) and spike rate correlation values. Neuron 1 and 2 showed correlated firing (signal correlation = 0.31), whereas other pairs did not. b, Triple recordings from the same neurons reveals the pattern of connections: neurons 1 and 2 were bidirectionally connected, while neuron 3 provided input to neuron 1. Dashed lines indicate timing of presynaptic spikes. c, There was a significant increase in connection probability with increasing signal correlation to natural movies (P = 0.0002, Cochran-Armitage test). Dotted line indicates connection probability for all pairs included in this analysis (30/108, 0.28). d, Connection probability increased significantly with increase in noise correlation (P = 0.011, Cochran-Armitage test). Correlation values were binned, with ranges from −0.15 to −0.05, from −0.05 to 0.05, and so on.

The dataset contained imaging experiments performed on 16 mice and whole-cell recordings from 126 L2/3 pyramidal cells, 116 of which could be matched to neurons functionally characterized in vivo (see Methods). The rate of connectivity was 0.19 (43 connections out of 222 potential connections assayed), in keeping with previous reports6,8,10. Connection probability, synaptic strength and electrophysiological properties of OGB-1 labeled neurons were not significantly different to those recorded in slices from naive age-matched visual cortex which was not injected with OGB-1 AM (connectivity rate 0.18; 25 connected of 143 tested; Supplementary Fig. 2), indicating that dye loading, anesthesia and prolonged exposure to infra-red laser light during imaging in vivo did not alter these parameters.

We first examined how connectivity depended on orientation selectivity and on responsiveness to natural movies. Out of the 116 neurons, 77 were responsive to the natural movie, and 79 were orientation selective for grating stimuli (see Methods). Connection probability between orientation-tuned neurons was more than two-fold higher than among non-selective and/or non-responsive cells (0.27; 25/94 vs 0.10; 3/31; P = 0.050, Chi-square test). Connectivity rate between neurons responsive to the natural movie was significantly higher than among cells non-responsive to the movie (0.28; 30/108 vs 0.04; 2/48; P = 0.001, Chi-square test). Taken together, these data suggest that reliably responsive and feature selective neurons belong to more densely interconnected neocortical subnetworks.

We then related connection probability to neuronal preference for the angle and direction of drifting gratings (Fig. 2). For this analysis, we only included pairs in which both neurons were responsive (74/113), orientation selective (OSI > 0.4; 53/74), or direction selective (DSI > 0.3; 41/53; see Methods and Supplementary Fig. 3a-c). Connectivity rate decreased with increasing difference in orientation preference (P = 0.040, Cochran-Armitage test for trend, Figure 2d). For similarly tuned cells, connection probability was high (0.38; 10/26; ΔORI < 22.5°), more than two-fold higher than for cells with a large difference in orientation preference (0.17; 4/24; ΔORI > 67.5°). Thus, neuronal pairs similarly tuned for orientation were more likely to connect to each other, although a considerable connectivity rate was still observed between neurons tuned to dissimilar or orthogonal orientations. These results are consistent with the narrow suprathreshold yet broader subthreshold tuning for orientation and direction in mouse V1 neurons16. The same decrease of connection probability with increase in ΔOri was found for direction selective pairs (P = 0.034, Cochran-Armitage test, Fig. 2e), but these neurons only connected specifically with respect to orientation not preferred direction (Fig. 2e). These data suggest that directional preference is not conferred by biased local excitatory input, so other cell intrinsic or network mechanisms (e.g., biased long range input, specific inhibition) may be needed to explain the emergence of direction selectivity. Varying the criteria for orientation or direction selectivity (OSI/DSI from 0.2 to 0.6) did not change the dependence of connectivity on difference in orientation/direction preference (Supplementary Fig. 3d, e), suggesting that neurons which are broadly or sharply tuned both tend to connect preferentially to others with similar functional preference. In our dataset we did not find evidence suggesting that neurons with similar preferred orientation or direction are connected by stronger (EPSP amplitude) or more facilitating (paired-pulse ratio, PPR) connections than neurons with different preferred orientation or direction (Supplementary Fig. 4a, b, d and e; also see Supplementary Fig. 5 for a sample pair with strong connections), although the sample size may not be adequate for ruling out any subtle trends.

The visual cortical circuit is constantly engaged in processing natural scenes, so statistical dependencies between neuronal activities in the presence of such stimuli may reflect connectivity. We therefore tested how network connectivity relates to the similarity of neuronal responses during presentation of stimuli with natural spatiotemporal statistics (Figure 3, see Methods). For each neuronal pair in which both neurons responded reliably to natural movies (56/113), we computed the time-varying firing-rate correlation of average responses (signal correlation) to repeated presentations of a 30 to 40-second-long natural movie sequence (Fig. 3a). On average, signal correlations were low (mean ± s.d. = 0.08 ± 0.10). The probability of finding a connection between two neurons significantly increased with signal correlation to natural movies (P = 0.0002, Cochran-Armitage test, Fig. 3c). For pairs with close to zero or weakly negative signal correlation (<0.05), the connection probability was low (0.11, 5/44). In contrast, for neuronal pairs with stronger signal correlation (>0.15), the connection probability was more than four-fold higher (0.5; 13/26). Therefore, connectivity in mouse visual cortex is highly selective with respect to neuronal responses to natural movies. EPSP amplitude and paired-pulse ratio, however, were not found to change significantly with increase in signal correlation (Supplementary Fig. 4c, f; also see Supplementary Fig. 5), although the sample size may not be large enough to rule out subtle trends.

Correlated variability in neuronal firing independent of a sensory stimulus is assumed to reflect neuronal connectivity in the network17-19. Correlated fluctuations in neuronal firing may either be driven by common input or by recurrent synaptic connections, or both. For a subset of visually responsive neuronal pairs (12/56) that were imaged simultaneously in vivo (i.e. on the same optical planes), we computed noise correlations (see Methods), which provide an indication of correlated response variability. Noise correlations were low (mean ± s.d. = 0.02 ± 0.04). Despite the small sample size, connection probability was found to increase significantly with increase in noise correlation (Fig. 3d, P = 0.011, Cochran-Armitage test), indicating that recurrent connectivity may contribute to correlated fluctuations of neuronal firing.

We next compared how visual response similarity relates to connectivity motifs in the local network. Previous work indicates that bidirectional connections are over-represented in a network of sparsely connected pyramidal neurons5. We found that the connectivity bias between neurons responding similarly to drifting gratings or to natural movies was further accentuated when investigating the distribution of unidirectionally or bidirectionally connected pairs (Fig. 4). We found a decreasing trend relating probability of bidirectional connections and difference in orientation preference (Fig. 4a, b; P = 0.070 for all orientation selective pairs; P = 0.036 for direction selective pairs, Cochran-Armitage test). Importantly, the monotonic fall-off in the incidence of bidirectional motifs was steeper than the overall decrease in probability of finding connected pairs as ΔOri increased (Fig. 4a, b). Similarly, the incidence of bidirectional connections increased sharply as signal correlation to natural movies increased (P = 0.003, Cochran-Armitage test, Fig. 4c), such that signal correlation was almost three-fold higher for recurrently connected pairs than unconnected pairs (mean signal correlation of bidirectionally connected pairs ± s.d. = 0.16 ± 0.07 vs 0.06 ± 0.10 for unconnected pairs; P = 0.01, rank sum test). Since probability of unidirectionally connected pairs did not show a monotonic trend with increase in response similarity (Fig. 4a-c, P > 0.4 for all conditions, Cochran-Armitage test), reciprocal connectivity reflects functional similarity better than does unidirectional connectivity.

Figure 4

Relationship between similarity of visual responses and probability of finding unidirectionally and bidirectionally connected pairs

a, Among orientation selective neurons, probability of finding connected pairs decreased as ΔOri increased. The fall-off in probability of finding bidirectionally connected pairs was steeper than the decrease in overall probability of finding connected pairs. A trend of decrease in probability of finding bidirectionally connected pairs was found (P = 0.070, Cochran-Armitage test). b, The same observation holds in the subset of directionally selective pairs, and probability of finding bidirectionally connected pairs decreased as ΔOri increased (P = 0.036, Cochran-Armitage test). c, The probability of finding bidirectionally connected pairs increased sharply as signal correlation to natural movies increased (P = 0.003, Cochran-Armitage test). Dotted lines indicate probability of finding connected pairs from all pairs included in analysis (panel a: 15/41, 0.37; panel b: 11/31, 0.35; panel c: 20/52, 0.38).

In this study, we have characterized the functional specificity of local connections in mouse V1. Our results demonstrate that connectivity between neighboring neurons (<50 μm apart) is not random, but specifically structured; visually driven neurons were more likely to connect to each other, and this probability increased with the degree of their response similarity. This relationship between connectivity and function was stronger when comparing responses to natural sensory input than for relatively artificial grating stimuli.

We have shown in mouse V1 that—although a given neuron receives input from nearby neurons preferring a wide range of stimulus orientations—more than twice as many connections are made between similarly tuned neurons as between disparately tuned cells. In keeping, subthreshold tuning in L2/3 pyramidal neurons in mouse V1 is broad but nonetheless biased towards the preferred orientation16. This is similar to the tuning of neurons in pinwheel centers of orientation maps in visual cortex of other species20. In carnivores and primates, long-range horizontal projections in L2/3 (>500 μm) are biased towards cortical columns with similar orientation preference1-4. Our results indicate that similar principles of connectivity apply at the level of local neocortical networks in the mouse—a species without columnar architecture—suggesting that functionally biased connectivity may be a general feature of organization in the visual cortex. In the visual cortex this selective connection scheme may serve as mechanism for amplification of thalamic input and sharpening of tuning21,22 or for local contour integration23.

Analysis of connectivity rate with respect to similarity of responses to natural movies revealed a striking degree of specificity of local connections (Fig. 3c, d). Connection probability increased sharply with increase in both signal and noise correlation to natural movies. Neurons with higher signal correlation to natural movies likely share similar receptive field structure, and may therefore be driven by common feed-forward input24. Our results are therefore consistent with the finding that L2/3 pyramidal neurons form highly interconnected subnetworks sharing common input from layer 4 in slices of rat visual cortex6. Developmentally, this organization of lateral connections based on receptive field similarity may arise through activity-dependent synaptic plasticity, whereby neurons driven by common input develop stable bidirectional connections25. Indeed, our data show that the majority of bidirectionally connected neurons had stronger signal correlations to natural movies and shared similar orientation preference. Since individual neurons exhibit variability in their responses to the same visual stimulus26, recurrent excitation between similarly tuned neurons may reduce response variance, while introducing redundancy into the population code for robustness against errors27.

Our results do not preclude the possibility that other factors—including gap junction coupling, inhibitory connections or synaptic strength—also contribute to functional specificity in the circuit. Since inhibition, in particular, may be important in determining the receptive field properties of neurons in V128, it will be important to examine the extent to which inhibitory connections are functionally specific7.

In conclusion, by using a novel and relatively straightforward approach for in vitro mapping of synaptic connectivity among neurons that had been identified functionally in vivo, we found that neighboring neurons with similar feature selectivity preferentially but not exclusively connected to each other in L2/3 of mouse V1. Together with other powerful approaches29,30, our method can be used to uncover functional biases of connectivity between different cell types and cortical layers, and in other brain areas. This information will be critical for understanding the functional wiring of circuits mediating perception and behavior.

Methods Summary

Anaesthetized C57Bl/6 mice between postnatal day 22 and 26 were injected with the calcium-sensitive dye Oregon Green Bapta-1 AM into monocular V1 as described previously11 and in vivo two-photon calcium imaging14,15 was used to record responses of layer 2/3 neurons to 8 different drifting square-wave gratings (0.035 cycles/degree, 2 cycles/s, 100% contrast) and natural movie sequences. Spike trains were inferred from calcium signals using a non-negative deconvolution method. Preferred orientation and direction, as well as orientation selectivity index (OSI) and direction selectivity index (DSI) were calculated using Fourier-interpolated tuning curves. Pearson’s correlation coefficient was used to obtain pair-wise response correlations, either from average responses to the stimulus (signal correlation) or from mean-subtracted responses (noise correlation). Small volumes of fluorescent microspheres were injected into the imaged region to facilitate identification of the region in the sliced brain. Coronal slices were cut after dissection of the brain, and whole-cell recordings from up to four cells simultaneously were carried out in the vicinity of the microsphere tract (identified by two-photon microscopy). The presence of synaptic connections was tested by evoking five spikes at 30-Hz in each cell, repeated for 30-90 times. Connection probability is the number of detected connections over the total number of potential connections assayed. Probability of finding uni- or bidirectionally connected pairs was calculated as the number of uni- or bidirectionally connected pairs over the total number of pairs. To register in vivo and in vitro image stacks and to match the same neurons imaged in vivo and recorded from in vitro, three□dimensional image registration by affine transformation using custom-written MATLAB software was performed subsequent to the experiment. To relate connectivity to functional properties, the asymptotic Cochran-Armitage test for trend was used to test for significance.