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Neocortical Excitation/Inhibition Balance in Information Processing and Social Dysfunction

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Neocortical Excitation/Inhibition Balance in Information Processing and Social Dysfunction

Ofer Yizhar et al. Nature.

Abstract

Severe behavioural deficits in psychiatric diseases such as autism and schizophrenia have been hypothesized to arise from elevations in the cellular balance of excitation and inhibition (E/I balance) within neural microcircuitry. This hypothesis could unify diverse streams of pathophysiological and genetic evidence, but has not been susceptible to direct testing. Here we design and use several novel optogenetic tools to causally investigate the cellular E/I balance hypothesis in freely moving mammals, and explore the associated circuit physiology. Elevation, but not reduction, of cellular E/I balance within the mouse medial prefrontal cortex was found to elicit a profound impairment in cellular information processing, associated with specific behavioural impairments and increased high-frequency power in the 30-80 Hz range, which have both been observed in clinical conditions in humans. Consistent with the E/I balance hypothesis, compensatory elevation of inhibitory cell excitability partially rescued social deficits caused by E/I balance elevation. These results provide support for the elevated cellular E/I balance hypothesis of severe neuropsychiatric disease-related symptoms.

Figures

Figure 1
Figure 1. Kinetic and absorbance properties of a stabilized SFO
a–c, Absorption spectra recorded after illumination with 450 nm light for 30 s. Absorption difference spectra (Δ Abs.) taken from the corresponding absorption spectra are shown in the insets. Spectra were collected at the indicated times after the end of illumination; note prominent recovery after 3 min in the single mutants, in contrast to the double mutant. d, Representative whole-cell patch-clamp recording of photocurrent in a cultured hippocampal neuron expressing ChR2(C128S/D156A) SSFO. Blue and orange bars indicate activation and deactivation light pulses. e, Monoexponential fits of photocurrent decay in cells expressing ChR2(C128S/D156A) (dark blue; τ = 29 min) or ChR2(D156A) (light blue; τ = 6.9 min). f, Activation (blue) and deactivation (orange) spectra recorded from cultured neurons expressing ChR2(C128S/D156A). g, Simplified photocycle scheme; in C128/D156 mutants the transition P520 to P480 is probably slowed down or blocked, avoiding the desensitized state Des480 which cannot be reactivated with 470 nm light. Both yellow and violet light (yellow and violet arrows) shuttle the channels to an inactive state (see Supplementary Fig. 1). h,Whole-cell photocurrent responses of a cultured neuron expressing SSFO to 470 nm light pulses of indicated power (left). Pulse lengths were 2 s (grey traces) or 5 s (black traces). Dashed lines mark light pulse termination. Apparent time constants for activation (τon) are shown on a log–log plot versus light power (n = 27 recordings from 5 cells; middle). Within this broad range of light power, the calculated number of incident photons arriving at each cell for photocurrents to reach the exponential curve constant (63% of Imax) for that cell was constant (right). Each point represents a photon number from a single recording at a given light power.
Figure 2
Figure 2. Elevated, but not reduced, prefrontal E/I balance leads to behavioural impairment
a, Experimental setup for behavioural experiments. Green region marks viral injection area; fibre-optic connector was attached to the light guide only transiently before testing. Cg, cingulate cortex; IL, infralimbic cortex; M2, secondary motor cortex; PL, prelimbic cortex. b, Confocal image from a mouse injected with CaMKIIα::SSFO–EYFP virus shows expression in prelimbic and infralimbic cortex. D, dorsal; L, lateral; M, medial; V, ventral. Contra, contralateral; Ipsi, ipsilateral. c, Representative images of prefrontal slices from PV::SSFO and CaMKIIα::SSFO mice stained for c-Fos 90 min after a 2 s 473 nm light pulse. Scale bar, 25 µm. Graph shows average c-Fos-positive cell counts in the mPFC of control, CaMKIIα::SSFO and PV::SSFO mice. d, Social exploration in control, CaMKIIα::SSFO and PV::SSFO mice of a juvenile intruder in the home cage. CaMKIIα::SSFO mice showed a significant reduction in social exploration. e, Mice administered one 2 s 473 nm pulse of light before fear conditioning were tested the next day for freezing in response to the conditioned context or auditory cue; CaMKIIα::SSFO mice were significantly impaired in freezing responses to both conditioned stimuli. On the following day, mice were reconditioned without optical stimulation and freezing was evaluated 24 h later. All mice showed similar freezing behaviour in the absence of light. f, Open-field exploration in CaMKIIα::SSFO (blue) and CaMKIIα::EYFP (grey) mice, before (Test 1) and after (Test 2) light activation. Example tracks from a CaMKIIα::SSFO mouse are shown. g, Novel object exploration over a 10 min period is similar in mice expressing CaMKIIα::SSFO (blue) and CaMKIIα::EYFP (grey). h, Phase contrast and fluorescence images of coronal sections from wild-type mice injected with CaMKIIα::SSFO in PFC (top) or V1 (bottom). i, Social behaviour in the three-chamber test is impaired following a 2 s 473 nm light pulse in mice expressing CaMKIIα::SSFO in the PFC (n = 5), but not in control mice (n = 6) or in mice expressing CaMKIIα::SSFO in V1 (n = 8). Time spent in a given chamber after stimulation is only significantly altered in mice expressing CaMKIIα::SSFO. All bar graphs depict mean ± s.e.m. (*P<0.05, **P < 0.005).
Figure 3
Figure 3. Elevated cellular E/I balance in the mPFC drives baseline high frequency rhythmicity in freely moving, socially impaired mice
a, Implantable CMO for awake, behaving recordings in mouse M2 and PFC. Arrowheads indicate wire termination sites; arrow shows cleaved end of fibre-optic connector. b, Electolytic lesions mark the sites from which recordings were taken in a mouse expressing CaMKIIα::SSFO. c, Social (left) and novel object exploration (right) before (grey) and after (blue) activation with 473 nm light in mice used for CMO recordings (n = 3 mice). d, e, Local-field-potential wavelet spectrogram from a representative unmodulated channel (d) and a representative modulated channel (e). Example traces are shown for the baseline, activation and deactivation periods. Average wavelet spectra for the three indicated periods (n = 5 trials in 1 mouse) and population data (insets; n = 3 mice) are shown, demonstrating a specific increase in gamma rhythmicity on the modulated channel after SSFO activation in PFC pyramidal neurons. All bar graphs depict mean ± s.e.m. (*P < 0.05).
Figure 4
Figure 4. Multistep engineering of a potent redshifted ChR
a, Confocal images of cultured hippocampal neurons transfected with VChR1–EYFP or C1V1-ts–EYFP under the control of the CaMKIIα promoter. Yellow box denotes region expanded in the last panel, showing dendritic membrane localization of C1V1-ts–EYFP. Scale bars: 20 µm (left, middle), 4 µm (right). b, Model of the C1V1 chromophore binding pocket, showing ChR1 helices in blue, VChR1 helices in orange, the retinal Schiff base (RSB) in purple, and key amino acid residues (with corresponding ChR2 numbering in parentheses and the modelled location of the SSFO mutations C128 and D156 shown for context). c, Representative traces and summary plot of channel closure time constant (τoff) in cultured neurons expressing the indicated channelrhodopsins; traces are normalized to peak current. d, Action spectra collected for the indicated channelrhodopsins (colour code as in c). Photocurrents were collected with 2 ms light pulses in HEK293 cells. e, Mean peak photocurrents recorded in cultured neurons expressing the indicated channelrhodopsins in response to a 2 ms 542 nm light pulse. Colours are as indicated in c; numbers in brackets indicate n. f, Fluorescence–photocurrent relationship in ChR2(H134R) (blue) and C1V1(E122T/E162T) (red). a.u., arbitrary units. g, Acute slice recordings in prefrontal pyramidal neurons (PYR) expressing C1V1(E122T/E162T) and stimulated with 560 nm light pulse trains or current injections at the indicated frequencies. Summary graphs show light and current-evoked spike probability versus stimulation frequency (n = 6; P > 0.4 at all frequencies). All error bars indicate s.e.m.
Figure 5
Figure 5. Combinatorial optogenetics enables partial reversal of elevated E/I-balance social behaviour disruption
a, mPFC optrode recording in an anaesthetized PV::Cre mouse injected with CaMKIIα::C1V1(E162T)-ts–EYFP and Ef1a-DIO::ChR2–EYFP (diagram illustrates experimental setup). Violet (405 nm) light pulses are presented with variable delay (Δt) relative to green light pulses (example traces). b, Summary graph shows probability of green-light-evoked spikes with violet pulses preceding the green light pulses by the indicated delays. Individual points are from single recordings. Black line shows average for all recordings (>3 recording sites per bin). c, Experimental paradigm for SSFO activation in pyramidal neurons and C1V1 activation in PV neurons. d, Action spectra of SSFO (blue) and C1V1(E122T/E162T) (C1V1, red). Orange and blue vertical lines indicate stimulation wavelengths used in the experiments. e, Experiment design and pulse patterns; no-light control was used for baseline behaviour; 2 s 473 nm light for prolonged SSFO activation; 10 Hz 473 nm for co-activation of SSFO and C1V1; 10 Hz 590 nm for C1V1 activation. f, Mice expressing CaMKIIα::SSFO (n = 7) showed significant social preference at baseline, but exhibited social dysfunction after either 2 s 473 nm activation or during 10 Hz 473 nm activation. g, Mice expressing both CaMKIIα::SSFO and DIO-PV::C1V1 (n = 7) showed impaired social behaviour after a 2 s 473 nm pulse, but showed partially restored social behaviour during the 10 Hz 473 nm light stimulation. Activation of C1V1 alone with 10 Hz 590 nm pulses did not impair social behaviour. NS, not significant. Supplementary Fig. 16 shows normal social behaviour under all illumination conditions in YFP-expressing control cohorts. All error bars indicate s.e.m. (*P < 0.05; **P < 0.005).

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