Decorrelating action of inhibition in neocortical networks

Abstract

Inhibitory GABAergic interneurons have been extensively studied but their contribution to circuit dynamics remain poorly understood. Although it has been suggested that interneurons, especially those belonging to the same subclass, synchronize their activity and impart this synchrony onto their local network, recent theoretical and experimental work have challenged this view. To better understand the activity of interneurons during cortical activity, we combined molecular identification, two-photon imaging, and electrophysiological recordings in thalamocortical slices from mouse somatosensory cortex. Using calcium imaging to monitor cortical activity, we found low spiking correlations among parvalbumin or somatostatin interneurons during cortical UP states, indicating that interneurons do not synchronize their firing. Intracellular recordings confirmed that nearby interneurons do not display more synchronous spiking than excitatory cells. The lack of interneuron synchrony was also evident during slow oscillations, even among interneurons that were electrically coupled via gap junctions, suggesting that their coupling does not function to synchronize their activity. Using voltage-clamp recordings from nearby pyramidal cells, we found that inhibitory currents (IPSCs) are more correlated than excitatory ones, but that correlated IPSCs arise from the activation of common presynaptic inhibitory cells, rather than from synchronization of interneuron activity. Finally, we demonstrate that pharmacologically reducing inhibitory currents increases correlated excitatory activity. We conclude that inhibitory interneurons do not have synchronous activity during UP states, and that their function may be to decorrelate rather than to synchronize the firing of neurons within the local network.

Figure 1.

Layer-specific colocalization of GFP and PV in G42 animals. A1–A3, Cortical S1 was double labeled for GFP and PV. Left panel (green) shows antibody labeling for GFP, middle column shows PV immunolabeling (red), and far right column is the overlay. Top row is L2/3, middle row is L4, and bottom row is L5. Scale bar, 30 μm. B, The percentage of GFP-positive cells that were also positive for PV was significantly higher in layer 2/3 than either layer 4 or 5 (*p < 0.01, one-way ANOVA). C, The percentage of PV cells that were labeled for GFP was significantly lower in L4 than either layer 2/3 or 5 (**p < 0.001, one-way ANOVA).

Figure 2.

Two-photon fast calcium imaging with a single-spike deconvolution algorithm. A, Light micrograph of a S1 thalamocortical slice preparation with intact thalamic input nucleus VB, thalamocortical axons, and the somatosensory cortex. A stimulating electrode is placed in VB. Superimposed yellow box indicates location, over layers 2/3 and 4, of illustrated two photon z stack to right. Neurons pictured in this field are loaded with fura-2 AM dye, and targeted with an SLM (far right). B, Two-photon image of a single frame showing neuronal cell bodies targeted with two photon illumination with the SLM. Cell outlined in red was targeted in cell-attached mode (D). Scale bar, 30 μm. C, Examples of fluorescence signals showing changes in fluorescence, normalized to baseline (ΔF/F), from four cells imaged at 66.6 Hz with an EMCCD. D, Three examples from calibration experiments in which a PC (left), a PV cell (middle), and SOM cell (right) were targeted for cell attached recording during simultaneous stimulation of the thalamus. Top trace shows raw fluorescence signal from that cell imaged at 66.6 Hz. Middle trace is the deconvolution of the calcium signals using parameters obtained from electrophysiology to obtain estimated spike times. Red dots above both traces indicate the time of the actual spikes. Bottom trace shows the associated electrophysiological trace. E, Sensitivity (true-positive rate) and specificity (1-false-positive rate) of the deconvolution algorithm. These rates were calculated while allowing for either no window around each spike to search for a signal (15 ms), or for a window of ± 1 frame around each spike (±15 ms).

Figure 3.

Interneurons are weakly correlated during thalamically triggered activity. A, Two-photon image of a loaded slice with labeled interneurons (pvGFP) pseudocolored in green. Scale bar, 30 μm. B, Quantification of the average percentage active pvGFP (red), sGFP (green), and GFPneg (blue) neurons as determined by SLM imaging and deconvolution. C, Example fluorescence traces from simultaneously imaged pvGFP and GFPneg cells (top) or sGFP cells and GFPneg cells (bottom) during a thalamically triggered UP state. Spike inference for each trace is shown below each example. D, Normalized distribution of correlation coefficients of spike inference for pvGFP interneurons and GFPneg cells (top) and sGFP interneurons and GFPneg cells (bottom). These distributions were not significantly different. E, Distance versus correlation for all cell pairs imaged. No significant relationship was found between distance and strength of correlation for pvGFP cells (left, p = 0.27), sGFP cells (middle, p = 0.78), or GFPneg cells (right, p = 0.27).

Figure 4.

Morphological and physiological characterization of interneurons. A, Reconstruction of a pvGFP-positive basket cell (top), with typical fast spiking physiology (below). Axons in red, dendrites in black. B, Reconstruction of a sGFP-positive Martinotti cell, with typical accommodating firing pattern shown below. Axons in green, dendrites in black. C, Reconstruction of a layer 2/3 PC with characteristic regular firing pattern shown below. Axons in blue, dendrites in black.

Figure 5.

Spiking of interneuron subtypes is not more synchronous than pyramidal cells. A, Diagram depicting a layer 4 small recurrent network of cells consisting of interneurons (green and blue) and principal cells (gray). Two nearby cells, either pvGFP, sGFP, or unlabeled PCs were patched in whole-cells current-clamp mode within 200 μm of one another. B, Left, electrophysiological traces from two nearby sGFP cells. Right, a schematic depicting how the minimum intercell spike interval was calculated for each spike in the top trace to the right. C, Representative traces from pairs of simultaneously patched pvGFP (top), sGFP (middle), and PC (bottom) pairs during thalamic stimulation during trials in which both cells fired action potentials. D, Probability distributions of minimum intercell spike time intervals for pvGFP cells, sGFP cells and PCs during thalamic stimulation were not significantly different from one another. E, Probability distributions of minimum intercell spike time intervals during spontaneously occurring activations were not significantly different from one another.

Figure 6.

Electrical coupling does not influence synchrony of interneurons. A, Anatomical reconstruction of two pvGFP cells patched within 60 μm of one another that were gap junction coupled (axons of cell 1 are in green, dendrites in red, axons cell 2 in blue, dendrites orange). B, Intracellular current injections in a PV neuron and a SOM neuron at 2× rheobase (blue traces top), with a nearby electrically coupled neuron patched within 100 μm (green traces, bottom). C, Representative traces from a pair of PV interneurons, and SOM interneurons, respectively, in response to thalamic stimulation. D, Distribution of minimum spike times in PV (left) and SOM (right) interneurons in juvenile (D1) and adult animals (D2). Mean minimum intercell spike time distributions did not differ between uncoupled and coupled neurons in either juvenile or adult slices.

Figure 7.

Interneurons are not synchronous during low-frequency oscillations. A, Thalamic activation of cortical neurons in two different UP states regimes. Recordings were made from pvGFP interneurons and PCs in ACSF with either normal K⁺ (top trace, black) or high K⁺ (bottom trace, purple). The thalamus was stimulated in both of these conditions, and the response to this stimulation is shown in the expanded traces to the right. B, The frequency of UP states in high K⁺ was significantly higher than normal K⁺. C, the duration of the UP state was significantly shorter in high K⁺ versus normal K⁺ for both spont and thalamically stim UP states. No significant difference was found when comparing spont versus stim in either high K⁺ or normal K⁺. D, the number of action potentials fired by PV cells and PCs during the UP state was significantly lower in high K⁺ versus normal K⁺ for both spont and stim UP states. No significant difference was found when comparing spont versus stim in either high K⁺ or normal K⁺. **p < 0.001, *p < 0.01. E, Distributions of minimum intercell spike intervals for pvGFP interneurons in high K⁺ for both spont (left) and stim (right) conditions. F, Distributions of minimum intercell spike intervals for PCs in high K⁺ for both spont (left) and stim (right) conditions. No significant difference was found when comparing pvGFP versus PC distributions in spont and stim conditions.

Figure 8.

IPSCs are more highly correlated than EPSCs during triggered and spontaneous activations. A, Recordings from two PCs with cell bodies ∼68 μm apart. Top trace shows EPSCs during thalamic stimulation, bottom shows IPSCs recorded in the same cells on an alternate trial. B, Cross-correlations between currents at 0 mV (blue) and −70 mV (red). C, Box plots of all correlation coefficients calculated for EPSCs recorded at −70 mV and IPSCs recorded at 0 mV during evoked activity (left) and spontaneous activity (right). D, Half-width of the cross-correlations for EPSCs and IPSCs. **p < 0.001.

Figure 9.

Correlation of unitary EPSCs and IPSCs confirms IPSCs are more synchronous that EPSCs. Individual IPSCs and EPSCs were detected and binary vectors of the event times were correlated among simultaneously patched cells pairs. These vectors were binned at either A, 1 ms; B, 10 ms; or C, 100 ms. For all time bins, the correlation coefficient for IPSCs was significantly higher than EPSCs for both evoked and spontaneous activity. **p < 0.001.

Figure 10.

High correlation of IPSCs is due to common input, rather than synchronous firing of interneurons. A, Schematic depicting two possible mechanisms underlying correlated IPSCs. In the first scenario, depicted to the left, synchronous firing, correlated IPSCs would be caused by two or more interneurons firing simultaneously. In this case, each IPSC would be the sum of the spiking of several interneurons. In the second scenario, shared input, in a system where every interneuron has highly divergent axons and contacts many postsynaptic PCs, each time an interneuron fires a spike, an IPSC would be recorded from all of its downstream postsynaptic targets nearly simultaneously. B, Connection probabilities for PC→PC pairs pvGFP→PC pairs and sGFP→PC pairs showing significantly higher connection probability for interneuron→PC than for PC→PC (p = 0.001 Kruskal–Wallis; p < 0.001 for PC vs pvGFP and PC vs sGFP; p > 0.05 for pvGFP vs sGFP, Dunn's multiple-comparison test; **p < 0.01). C, Distance versus correlations coefficients of IPSCs (blue circles) and EPSCs (red squares) were plotted for all cell pairs. Both EPSC and IPSC correlations drop off with distance with slopes that were not significantly different from one another. D, Normalized distribution of conductances for IPSCs recorded during thalamically triggered activations (top, red), and synaptic conductances measured from pvGFP→ PC pairs (blue) or sGFP→ PC pairs (green). The mean of these distributions did not differ from one another.

Figure 11.

Pharmacologically decreasing inhibition decorrelates excitation. A, Schematic showing experimental configuration. Two excitatory cells were patched within 100 μm of one another while a thalamically triggered UP state was simultaneously recorded in the two cells at different holding potentials ranging from 0 mV (mostly IPSCs) to −70 mV (mostly EPSCs), with a mixture of IPSCs and EPSCs recorded at intermediate potentials. B, The correlation coefficient was calculated for EPSCs at these different holding potentials. Starting from −70 mV, correlation values decreased, hitting their lowest value at intermediate potentials, and increasing again as holding potential approached 0 mV. C, Example traces from two PCs recorded 55 μm from one another. These two cells were voltage-clamped at −70 mV so EPSCs could be recorded. Above traces are control, and below in 200 nm GZ. Red lines below each set of traces indicated EPSCs in both cells that occurred within 10 ms of one another. D, Nanomolar concentrations of GZ significantly increased correlations in EPSCs (*p < 0.05, Mann–Whitney, n = 5 pairs). Dashed line shows correlation of shuffled data, which did not differ significantly between control and either 100 or 200 nm GZ.