In Vivo Optophysiology Reveals Lateral Inhibition among Layer 1 Interneurons
Authors: Pritish Patil, Ofer Yizhar
Journal: Neuron (2020)

Neurons in neocortical layer 1 (L1) are thought to regulate attentional processes through integration of long-range inputs and disinhibitory effects on the underlying cortex. A new study combines genetically targeted voltage imaging and optogenetics to elucidate the input-output transformations of the L1 network in the mouse somatosensory cortex, revealing unique features of sensory-evoked dynamics in L1 neurons. Neurons in neocortical layer 1 (L1) are thought to regulate attentional processes through integration of long-range inputs and disinhibitory effects on the underlying cortex. A new study combines genetically targeted voltage imaging and optogenetics to elucidate the input-output transformations of the L1 network in the mouse somatosensory cortex, revealing unique features of sensory-evoked dynamics in L1 neurons. The outermost layer of neocortex, cortical L1, possesses unique cytoarchitecture as it is composed mostly of axons from distant brain regions and dendritic arbors of underlying pyramidal neurons. It also contains a small population of neurons that are predominantly inhibitory. What is the impact of these interneurons on the activity of the cortical circuit in which they are embedded? How do they contribute to sensory processing and cortical dynamics? These questions are recently being addressed thanks to the availability of recombinase-expressing mouse lines targeting these cells and advanced technology for large-scale recordings of neural activity in the living brain. Most reports of population activity in awake behaving mice have relied either on extracellular electrophysiology, recently boosted by the advent of high-density silicon probes, or on improved imaging technology combining genetically encoded calcium indicators (GECIs) and fluorescence microscopy. Although both techniques permit simultaneous recording of hundreds of individual neurons, both are mostly restricted to supra-threshold activity: silicon probes record extracellular spiking activity whereas calcium imaging relies on an indirect signal, the correlation of which with action potential firing can vary between neuronal subtypes. It is widely accepted that subthreshold neural activity contains valuable information about the synaptic input to neurons and the computations they perform. Patch-clamp recordings measure the sub-threshold dynamics of neurons with high fidelity, even in vivo, and provide a glimpse into the internal state of single neurons. Patch-clamp recordings can further disentangle the excitatory and inhibitory components of a neuron’s input by directly controlling the membrane potential to isolate specific ionic conductances. However, despite their ability to reveal detailed information about a neuron’s “inner world,” patch-clamp techniques are inherently low throughput and do not allow simultaneous population-level recording of subthreshold dynamics. To circumvent these limitations, several research groups have been developing a complementary technology that enables fluorescence-based recording of the neuronal membrane potential. Genetically encoded voltage indicators (GEVIs) are proteins whose change in fluorescence correlates with the voltage across the membrane in which they reside. One major advantage of GEVI imaging is that it directly reports the spiking activity of neurons. Another is that under optimal imaging conditions, voltage imaging can also capture the sub-threshold dynamics of targeted neurons. Combined with optogenetic manipulation (Hochbaum et al., 2014Hochbaum D.R. Zhao Y. Farhi S.L. Klapoetke N. Werley C.A. Kapoor V. Zou P. Kralj J.M. Maclaurin D. Smedemark-Margulies N. et al.All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.Nat. Methods. 2014; 11: 825-833Crossref PubMed Google Scholar), GEVI imaging allows a fine dissection of excitatory and inhibitory synaptic events. Although voltage imaging is a fledgling technology, GEVIs are being rapidly optimized and further enhanced to increase signal-to-noise ratios (SNRs) and biocompatibility. Early attempts to develop GEVIs focused on combining fluorescent proteins with voltage-sensitive domains from ion channels or enzymes (Jin et al., 2012Jin L. Han Z. Platisa J. Wooltorton J.R.A. Cohen L.B. Pieribone V.A. Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe.Neuron. 2012; 75: 779-785Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). More recent work led to the development of GEVIs based on the naturally occurring type-I rhodopsin family (Kralj et al., 2011Kralj J.M. Hochbaum D.R. Douglass A.D. Cohen A.E. Electrical spiking in Escherichia coli probed with a fluorescent voltage-indicating protein.Science. 2011; 333: 345-348Crossref PubMed Scopus (251) Google Scholar). These 7-transmembrane retinal proteins have been shown to emit a fluorescence signal that is strongly modulated by membrane potential, and they have given rise to a range of GEVIs with diverse functional properties. Currently available GEVIs vary widely in their functional properties, their spectral attributes, their temporal kinetics, and their fluorescence SNR. Rhodopsin-based GEVIs show excellent SNR and a red-shifted excitation spectrum, making them compatible with optogenetic stimulation. However, these GEVIs have poor voltage sensitivity under two-photon excitation, and their application is therefore restricted to superficial neurons in vivo. Conversely, the GFP-based sensors retain their voltage sensitivity under two-photon imaging and allow access to deeper-lying neurons in cortex and other brain structures (Wu et al., 2020Wu J. Liang Y. Chen S. Hsu C.-L. Chavarha M. Evans S.W. Shi D. Lin M.Z. Tsia K.K. Ji N. Kilohertz two-photon fluorescence microscopy imaging of neural activity in vivo.Nat. Methods. 2020; 17: 287-290Crossref PubMed Scopus (87) Google Scholar). One of the major advantages of rhodopsin-based sensors is their near-infrared absorption spectrum. SomArchon1, a GEVI based on the archaeal proton pump archaerhodopsin (Piatkevich et al., 2019Piatkevich K.D. Bensussen S. Tseng H.-A. Shroff S.N. Lopez-Huerta V.G. Park D. Jung E.E. Shemesh O.A. Straub C. Gritton H.J. et al.Population imaging of neural activity in awake behaving mice.Nature. 2019; 574: 413-417Crossref PubMed Scopus (125) Google Scholar) can be excited with red light to record changes in membrane voltage, while its fluorescence response is minimally affected by co-administration of blue light. This permits the integration of optogenetic tools in experiments utilizing this GEVI. In their recent study appearing in Cell, Fan et al., 2020Fan L.Z. Kheifets S. Böhm U.L. Wu H. Piatkevich K.D. Xie M.E. Parot V. Ha Y. Evans K.E. Boyden E.S. et al.All-Optical Electrophysiology Reveals the Role of Lateral Inhibition in Sensory Processing in Cortical Layer 1.Cell. 2020; 180: 521-535.e18Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar capitalize on this multiplexed approach and combine SomArchon1 with the blue-shifted channelrhodopsin SomCheRiff (together called Optopatch4) or with the anion-conducting channelrhodopsin stGtACR2 (together dubbed i-Optopatch) to record from a genetically targeted subset of layer 1 (L1) interneurons in the mouse somatosensory cortex (Figure 1A). To precisely stimulate or inhibit L1 interneurons, they utilize a digital micromirror device (DMD) and provide targeted illumination that allows them to address L1 neurons individually or in small groups. The fluorescence excitation light is also focused using a spatial light modulator (SLM), which allows holographically targeted GEVI recording at 1 kHz. Combining these two approaches, the authors manipulate and record arbitrary subsets of L1 neurons while providing sensory input through whisker stimulation. Through the use of voltage imaging in L1 neurons, Fan et al., 2020Fan L.Z. Kheifets S. Böhm U.L. Wu H. Piatkevich K.D. Xie M.E. Parot V. Ha Y. Evans K.E. Boyden E.S. et al.All-Optical Electrophysiology Reveals the Role of Lateral Inhibition in Sensory Processing in Cortical Layer 1.Cell. 2020; 180: 521-535.e18Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar could precisely detect single spikes from L1 interneurons in response to whisker stimulation (Figure 1B). Averaging the voltage traces around detected spikes revealed a hyperpolarization of the cells’ membrane potential following evoked spikes, but not after spontaneous ones (Figure 1C). They conjectured that this precisely timed inhibitory input was a result of feedforward lateral inhibition from other L1 cells. By increasing the driving force for synaptic inhibition using optogenetic excitation (Figure 1D), their recordings revealed that this hyperpolarization emanated from increased inhibition rather than decreased excitation. To test whether neighboring L1 interneurons mediate this hyperpolarization, they briefly optogenetically stimulated nearby L1 interneurons while simultaneously depolarizing the recorded cell to increase inhibitory drive (Figure 1E). The results (Figure 1F) were remarkably similar to those observed after whisker stimulation: selective activation of L1 interneurons led to a hyperpolarizing response in neighboring neurons, suggesting that L1 interneurons are linked through lateral inhibitory connections. However, the presence of such an inhibitory connection does not provide sufficient evidence to conclude that these connections are responsible for the inhibitory responses associated with sensory stimulation. The authors thus combined their GEVI imaging with an optogenetic silencing approach (i-Optopatch) to ask whether nearby L1 neuron activity is required for the observed inhibitory responses during sensory stimulation. By expressing the blue-light-activated stGtACR2 in L1 cells, they could silence these neurons with blue light during whisker stimulation. Their results (Figure 1G) showed that the sensory-evoked hyperpolarization disappears when neighboring cells are silenced, supporting the role of lateral inhibition in this process. These experiments together suggested that the stimulus-evoked inhibition of L1 interneurons results from lateral inhibition from similar neighboring cells. Cortical L1 interneurons are known to be modulated by cholinergic input (Letzkus et al., 2011Letzkus J.J. Wolff S.B.E. Meyer E.M.M. Tovote P. Courtin J. Herry C. Lüthi A. A disinhibitory microcircuit for associative fear learning in the auditory cortex.Nature. 2011; 480: 331-335Crossref PubMed Scopus (570) Google Scholar). The authors confirmed that air puffs delivered to awake animals during the recording triggered an excitatory response in L1 neurons that was attenuated by a cholinergic antagonist. They further demonstrated that during cholinergic activation, L1 neurons respond with substantial diversity: some neurons fire single spikes while others display sustained firing. This “winner-take-all” dynamics in the L1 network can be reproduced in a spiking neural network model containing lateral inhibition between L1 interneurons. The model further predicts that excitation of a single L1 neuron would lead to sustained spiking while global excitation of several L1 neurons would result in winner-take-all dynamics. Patterned stimulation of single or multiple neurons allowed a direct experimental test of these predictions. Although many possible mechanisms could explain this finding, the authors use simulations to show that these population dynamics can arise from spontaneous spatial symmetry breaking in a nearly homogeneous network. This type of symmetry breaking relies on amplification of small inhomogeneities in population activity and is reminiscent of Turing patterns, a mechanism first hypothesized to explain morphogenesis in living organism. A similar mechanism relying on lateral connections has been theorized to be present in cortical networks and was proposed to explain the shapes of drug-induced geometric visual hallucinations based on connectivity in the primary visual cortex (Bressloff et al., 2002Bressloff P.C. Cowan J.D. Golubitsky M. Thomas P.J. Wiener M.C. What geometric visual hallucinations tell us about the visual cortex.Neural Comput. 2002; 14: 473-491Crossref PubMed Scopus (132) Google Scholar). Since neuromodulatory receptors mediate both drug-induced geometric hallucinations and the winner-take-all phenomenon described in this study, it is tempting to speculate that neuromodulatory systems act on neural processing through formation of Turing-like patterns to amplify small underlying inhomogeneities. Fan et al., 2020Fan L.Z. Kheifets S. Böhm U.L. Wu H. Piatkevich K.D. Xie M.E. Parot V. Ha Y. Evans K.E. Boyden E.S. et al.All-Optical Electrophysiology Reveals the Role of Lateral Inhibition in Sensory Processing in Cortical Layer 1.Cell. 2020; 180: 521-535.e18Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar further propose that such Turing-like patterns could be of behavioral relevance and might provide a mechanism for the Yerkes-Dodson law, which describes the non-monotonic effects of arousal on cognitive performance. Recent work has suggested that patterned manipulation might be necessary for revealing specific inhibition-driven dynamics in neural networks (Sadeh and Clopath, 2020Sadeh S. Clopath C. Patterned perturbation of inhibition can reveal the dynamical structure of neural processing.eLife. 2020; 9: 9Crossref Scopus (17) Google Scholar). The Optopatch technology utilized in this study allows neuroscientists to pursue such theoretical ideas at a greater level of detail than ever before. As better GEVIs are developed along with advanced microscopy techniques (Wu et al., 2020Wu J. Liang Y. Chen S. Hsu C.-L. Chavarha M. Evans S.W. Shi D. Lin M.Z. Tsia K.K. Ji N. Kilohertz two-photon fluorescence microscopy imaging of neural activity in vivo.Nat. Methods. 2020; 17: 287-290Crossref PubMed Scopus (87) Google Scholar), we might be able to record voltage from behaving and even freely moving animals. Although the use of Optopatch is restricted to superficial layers of cortex due to light scattering, the use of GRIN lenses, prisms, or adaptive optics can facilitate recording in deeper cortical layers and subcortical structures. Improvements in GEVIs with two-photon capabilities (Villette et al., 2019Villette V. Chavarha M. Dimov I.K. Bradley J. Pradhan L. Mathieu B. Evans S.W. Chamberland S. Shi D. Yang R. et al.Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice.Cell. 2019; 179: 1590-1608.e23Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) will further remove the restriction to superficial layers and reduce crosstalk. Beyond the recoding of somatic voltage, GEVI-based imaging has the potential to improve our understanding of the dynamics in distal cellular compartments within a neuron, such as dendrites and axons. Until recently, dendritic processing has been studied primarily using GECIs or dendritic patch clamp. Dendritic patch-clamp recordings are, however, difficult to obtain and nearly impossible to perform longitudinally. Further, due to space clamping issues, neither of these techniques allow for dissection of local conductances. Two-photon GEVIs could reveal local voltage changes in thinner dendrites (Wu et al., 2020Wu J. Liang Y. Chen S. Hsu C.-L. Chavarha M. Evans S.W. Shi D. Lin M.Z. Tsia K.K. Ji N. Kilohertz two-photon fluorescence microscopy imaging of neural activity in vivo.Nat. Methods. 2020; 17: 287-290Crossref PubMed Scopus (87) Google Scholar) and perhaps even spines and presynaptic boutons. Coupled with optogenetics, such optopatching of spines and presynaptic terminals would greatly enhance our understanding of neural processing and synaptic physiology. As the study by Fan et al., 2020Fan L.Z. Kheifets S. Böhm U.L. Wu H. Piatkevich K.D. Xie M.E. Parot V. Ha Y. Evans K.E. Boyden E.S. et al.All-Optical Electrophysiology Reveals the Role of Lateral Inhibition in Sensory Processing in Cortical Layer 1.Cell. 2020; 180: 521-535.e18Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar demonstrates, the combination of optogenetics with GEVI imaging could allow interrogation of in vivo “functional connectomics,” the quantification of the effective connection strength between neurons and its relationship to ongoing and sensory-driven population activity. All-Optical Electrophysiology Reveals the Role of Lateral Inhibition in Sensory Processing in Cortical Layer 1Fan et al.CellJanuary 23, 2020In BriefBy simultaneously combining genetically targeted voltage imaging with optogenetic modulation of neuronal activity, Fan et al. demonstrate that all-optical electrophysiology in awake animals can be a powerful tool for revealing hidden principles of neural circuit function. Full-Text PDF Open Archive