Differential connectivity and response dynamics of excitatory and inhibitory neurons in visual cortex

Abstract

Neuronal responses during sensory processing are influenced by both the organization of intracortical connections and the statistical features of sensory stimuli. How these intrinsic and extrinsic factors govern the activity of excitatory and inhibitory populations is unclear. Using two-photon calcium imaging in vivo and intracellular recordings in vitro, we investigated the dependencies between synaptic connectivity, feature selectivity and network activity in pyramidal cells and fast-spiking parvalbumin-expressing (PV) interneurons in mouse visual cortex. In pyramidal cell populations, patterns of neuronal correlations were largely stimulus-dependent, indicating that their responses were not strongly dominated by functionally biased recurrent connectivity. By contrast, visual stimulation only weakly modified co-activation patterns of fast-spiking PV cells, consistent with the observation that these broadly tuned interneurons received very dense and strong synaptic input from nearby pyramidal cells with diverse feature selectivities. Therefore, feedforward and recurrent network influences determine the activity of excitatory and inhibitory ensembles in fundamentally different ways.

Figure 1. Calcium imaging and electrophysiological recordings of visually evoked responses in Parvalbumin (PV)-expressing neurons.

a. Image of an OGB-1 labeled PV-positive (PV) neuron in a Cre-PV-lsl-tdTomato mouse from which a cell-attached recording was made. Scale bar, 20 μm b. Average calcium signal (ΔF/F, top) and action potential (AP) rate per imaging frame (bottom) from simultaneous calcium imaging and electrophysiological recording from a PV neuron during stimulation with drifting gratings. Scale bars, 5% ΔF/F, 4 spikes per bin, bin size 131 ms. The directions of drifting gratings are indicated on top, dashed lines show drift onset. c. Polar plot of normalized responses to different grating directions from neuron in b, calculated from APs (black solid line) and calcium signal (red dashed line). d, e. Average peak calcium signal plotted against absolute AP number (d) or against normalized AP number (normalized to maximum number of APs) (e) for each of 12 PV neurons from 6 mice, calculated for bins of 393 ms. Error bars are omitted for clarity. f, g. Correspondence of preferred grating orientation (f) and orientation selectivity index (OSI, see methods) (g) calculated from calcium signal and from APs for 9 PV neurons (red circles) and 7 PCs (green diamonds) that were visually stimulated and responsive to moving gratings. h. Image of OGB-1 AM labeled tissue, including four PV neurons (red) in layer 2/3. Scale bar, 20 μm i. Average calcium traces (ΔF/F) from three PV (red, left) and two PV-negative neurons (putative PCs, green, right) during stimulation with episodically presented drifting gratings (8 directions, 6 repetitions). The directions of drifting gratings are indicated on top, dashed lines show drift onset. j. Orientation selectivity index (OSI) for PCs (green) and PV interneurons (red), which significantly responded to grating stimuli (ANOVA, P < 0.0001). OSI for highly-selective, sharply-tuned neurons approaches 1, whereas OSI for broadly-tuned, non-selective neurons approaches zero. Black lines indicate median OSI. PC: median OSI = 0.60, PV: median OSI = 0.26; 15 regions, 7 animals, P < 10^-6).

Figure 2. Assessing synaptic connectivity in vitro between neurons functionally characterized in vivo.

a. Images of OGB-1 labeled V1 tissue in a slice (top, left) and of the same cells in vivo before slicing (bottom, left, after registration of the image stacks, see Methods), white circles denote matched neurons in vivo and in vitro, which were targeted for whole-cell recording and filled with Alexa 594 (top, right). Three pyramidal cells (PCs) and one fast-spiking interneuron (FS) were patched. Bottom, right: Polar plots of normalized responses to gratings drifting in 8 different directions, illustrating their orientation/direction preference and tuning. b. Action potential firing pattern in response to depolarizing current injection for the cells from a. Scale bars: 20 mV, 50 ms. c. Average traces of postsynaptic potentials in the FS interneuron in response to spike-evoking current injections in each of the three PCs from a, showing that all three were providing synaptic input onto the FS neuron. Scale bars: left panel: 40 mV, 50 ms; right panel: 2 mV for upper two traces, 0.2 mV for bottom trace; 50 ms. d. Probability of finding synaptic connections between pairs of PCs and from PC to PV/FS neurons. e. Amplitudes of excitatory postsynaptic potentials (EPSPs) between PCs and from PCs to PV/FS cells. Black lines depict median amplitudes. f. Another example of connectivity between six PCs and one PV/FS interneuron and their orientation preferences. Five out of the six PCs provided input onto the PV/FS neuron, which was held in whole-cell mode continuously while two sets of three PCs were patched and their connectivity assayed sequentially. g. Polar plots with normalized responses to drifting grating stimuli (8 directions) of 15 additional visually-responsive PV/FS interneurons (red lines) overplotted with normalized responses of the PCs that were found to provide synaptic input onto them (green lines). PCs which provided stronger connections (> 2mV EPSP amplitude) are indicated by darker and thicker green lines. h. Relationship between connection probability and difference of preferred orientation (ΔOri) for pairs of orientation-tuned (OSI > 0.4) PCs (green), from PCs to PV/FS interneurons (black, filled bars) and from PCs to PV/FS interneurons with OSI > 0.25 (black, open bars). Two PCs were more likely to be connected if they preferred similar grating orientations. Connection probability from PC onto PV/FS cells was not dependent on response similarity, irrespective of response selectivity. i. Connection strength (EPSP amplitude) from PCs to PV/FS cells plotted against difference in preferred orientation of each cell pair (ΔOri). Closed circles, pairs with OSI ≤ 0.25; open circles, pairs with OSI > 0.25. Strength of input was not dependent of orientation preference similarity: all cell pairs, P = 0.59, only cell pairs with OSI > 0.25, P = 0.94, Kruskal-Wallis test. j. Relationship between paired-pulse ratio of synaptic connections from PCs to PV/FS cells and ΔOri. Degree of facilitation (PPR>1) or depression (PPR<1) of synapses was not related to response similarity to gratings: all cell pairs, P = 0.54, only cell pairs with OSI > 0.25, P = 0.11, Kruskal-Wallis test. Black lines depict median amplitudes, dotted lines median amplitudes for pairs with OSI > 0.25. Bins include difference in preferred 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).

Figure 3. Relationship between response similarity and pair-wise correlations during spontaneous activity.

a,b. Spontaneous pair-wise correlation coefficients plotted against pair-wise signal correlation coefficients (obtained from averaged responses to gratings drifting in eight different directions) from two different imaging regions, for pairs of PV-negative neurons (PC, green), mixed pairs of one PC and one PV-positive (PV) neuron (black), and pairs of PV neurons (red). c. Boxplots of the correlation coefficients (R) and slopes of the relationship between spontaneous correlations and signal correlations from all imaged regions. d. Pooled data from all PC pairs (green), mixed pairs (black) and PV cell pairs (red) normalized for comparison across animals and imaged regions by computing z-scores (see Methods). PC-pairs: R = 0.10, slope = 0.11; PC/PV-pairs: R = 0.22, slope = 0.37; PV-pairs: R = 0.61, slope = 1.08. 15 regions, 7 animals, 7285 PC cell pairs, 2562 PC/PV cell pairs, 187 PV cell pairs.

Figure 4. Comparison of population activity patterns with and without visual stimulation.

a,c,e. Calcium signals of 30 PC (top) and 6 PV (bottom) neurons simultaneously imaged in darkness with the monitor switched off (a), and during stimulation with episodically drifting gratings (c), or with natural movie sequences (e). Schematic stimulus sequence is shown above each plot. b,d,f. Strength of pair-wise time-varying (total) correlations from calcium signals for PC pairs (left), PV pairs (right) and mixed PC/PV pairs (middle) during spontaneous activity (b), and during visual stimulation with gratings (d), or natural movies (f). Circles depict median values of each imaged region, colored lines indicate group median. Grey lines connect values obtained from the same imaged region. g. Matrices of pair-wise response rate correlation coefficients between significantly responsive PV and PC neurons of one imaged region. Cells were ordered such that the strongest correlations during spontaneous activity were close to the diagonal in the spontaneous condition, and the same order was applied to correlation matrices of the other conditions. Positions on the diagonal were set to the lowest value. h. The similarity between two matrices is the correlation coefficient of their off-diagonal elements (pattern correlation). Comparisons were made between correlation matrices of spontaneous and each of the evoked conditions, and between different visually evoked conditions for PC cells (green), PV cells (red) and for matrices from mixed PC/PV pairs (black). Boxplots represent median values of all imaged regions that included three or more responsive PV cells (vertical lines are group medians, 6 animals, 13 regions).

Figure 5. Comparison of spontaneous and noise correlation patterns during visual stimulation.

a,b. Noise correlation coefficients from calcium signals during stimulation with drifting gratings (a) or with natural movie sequences (b) for PC cell pairs (left, green) and PV cell pairs (right, red). Circles depict median values of each imaged region, colored lines indicate group median. c. Similarity of matrices of noise correlations during visually-evoked conditions (see Methods) and correlations during spontaneous activity (left and middle) and similarity of noise correlation matrices during grating and natural movie stimulation (right) for PC (green) and PV (red) cell populations. Pattern correlation values are correlation coefficients of off-diagonal matrix elements for each imaged regions with three or more responsive PV cells, 6 animals, 13 regions.