A bistable inhibitory optoGPCR for multiplexed optogenetic control of neural circuits.

Information is transmitted between brain regions through the release of neurotransmitters from long-range projecting axons. Understanding how the activity of such long-range connections contributes to behavior requires efficient methods for reversibly manipulating their function. Chemogenetic and optogenetic tools, acting through endogenous G-protein-coupled receptor pathways, can be used to modulate synaptic transmission, but existing tools are limited in sensitivity, spatiotemporal precision or spectral multiplexing capabilities. Here we systematically evaluated multiple bistable opsins for optogenetic applications and found that the Platynereis dumerilii ciliary opsin (PdCO) is an efficient, versatile, light-activated bistable G-protein-coupled receptor that can suppress synaptic transmission in mammalian neurons with high temporal precision in vivo. PdCO has useful biophysical properties that enable spectral multiplexing with other optogenetic actuators and reporters. We demonstrate that PdCO can be used to conduct reversible loss-of-function experiments in long-range projections of behaving animals, thereby enabling detailed synapse-specific functional circuit mapping.

Studying dominance and aggression requires ethologically relevant paradigms.

Although aggression is associated with several psychiatric disorders, there is no effective treatment nor a rigorous definition for "pathological aggression". Mice make a valuable model for studying aggression. They have a dynamic social structure that depends on the habitat and includes reciprocal interactions between the mice's aggression levels, social dominance hierarchy (SDH), and resource allocation. Nevertheless, the classical behavioral tests for territorial aggression and SDH in mice are reductive and have limited ethological and translational relevance. Recent work has explored the use of semi-natural environments to simultaneously study dominance-related behaviors, resource allocation, and aggressive behavior. Semi-natural setups allow experimental control of the environment combined with manipulations of neural activity. We argue that these setups can help bridge the translational gap in aggression research toward discovering neuronal mechanisms underlying maladaptive aggression.

A non-canonical striatopallidal Go pathway that supports motor control.

In the classical model of the basal ganglia, direct pathway striatal projection neurons (dSPNs) send projections to the substantia nigra (SNr) and entopeduncular nucleus to regulate motor function. Recent studies have re-established that dSPNs also possess axon collaterals within the globus pallidus (GPe) (bridging collaterals), yet the significance of these collaterals for behavior is unknown. Here we use in vivo optical and chemogenetic tools combined with deep learning approaches in mice to dissect the roles of dSPN GPe collaterals in motor function. We find that dSPNs projecting to the SNr send synchronous motor-related information to the GPe via axon collaterals. Inhibition of native activity in dSPN GPe terminals impairs motor activity and function via regulation of Npas1 neurons. We propose a model by which dSPN GPe axon collaterals (striatopallidal Go pathway) act in concert with the canonical terminals in the SNr to support motor control by inhibiting Npas1 neurons.

A bistable inhibitory OptoGPCR for multiplexed optogenetic control of neural circuits.

Information is transmitted between brain regions through the release of neurotransmitters from long-range projecting axons. Understanding how the activity of such long-range connections contributes to behavior requires efficient methods for reversibly manipulating their function. Chemogenetic and optogenetic tools, acting through endogenous G-protein coupled receptor (GPCRs) pathways, can be used to modulate synaptic transmission, but existing tools are limited in sensitivity, spatiotemporal precision, or spectral multiplexing capabilities. Here we systematically evaluated multiple bistable opsins for optogenetic applications and found that the Platynereis dumerilii ciliary opsin (PdCO) is an efficient, versatile, light-activated bistable GPCR that can suppress synaptic transmission in mammalian neurons with high temporal precision in-vivo. PdCO has superior biophysical properties that enable spectral multiplexing with other optogenetic actuators and reporters. We demonstrate that PdCO can be used to conduct reversible loss-of-function experiments in long-range projections of behaving animals, thereby enabling detailed synapse-specific functional circuit mapping.

Neural basis for fasting activation of the hypothalamic-pituitary-adrenal axis.

Fasting initiates a multitude of adaptations to allow survival. Activation of the hypothalamic-pituitary-adrenal (HPA) axis and subsequent release of glucocorticoid hormones is a key response that mobilizes fuel stores to meet energy demands1-5. Despite the importance of the HPA axis response, the neural mechanisms that drive its activation during energy deficit are unknown. Here, we show that fasting-activated hypothalamic agouti-related peptide (AgRP)-expressing neurons trigger and are essential for fasting-induced HPA axis activation. AgRP neurons do so through projections to the paraventricular hypothalamus (PVH), where, in a mechanism not previously described for AgRP neurons, they presynaptically inhibit the terminals of tonically active GABAergic afferents from the bed nucleus of the stria terminalis (BNST) that otherwise restrain activity of corticotrophin-releasing hormone (CRH)-expressing neurons. This disinhibition of PVHCrh neurons requires γ-aminobutyric acid (GABA)/GABA-B receptor signalling and potently activates the HPA axis. Notably, stimulation of the HPA axis by AgRP neurons is independent of their induction of hunger, showing that these canonical 'hunger neurons' drive many distinctly different adaptations to the fasted state. Together, our findings identify the neural basis for fasting-induced HPA axis activation and uncover a unique means by which AgRP neurons activate downstream neurons: through presynaptic inhibition of GABAergic afferents. Given the potency of this disinhibition of tonically active BNST afferents, other activators of the HPA axis, such as psychological stress, may also work by reducing BNST inhibitory tone onto PVHCrh neurons.

Determinants of functional synaptic connectivity among amygdala-projecting prefrontal cortical neurons in male mice.

The medial prefrontal cortex (mPFC) mediates a variety of complex cognitive functions via its vast and diverse connections with cortical and subcortical structures. Understanding the patterns of synaptic connectivity that comprise the mPFC local network is crucial for deciphering how this circuit processes information and relays it to downstream structures. To elucidate the synaptic organization of the mPFC, we developed a high-throughput optogenetic method for mapping large-scale functional synaptic connectivity in acute brain slices. We show that in male mice, mPFC neurons that project to the basolateral amygdala (BLA) display unique spatial patterns of local-circuit synaptic connectivity, which distinguish them from the general mPFC cell population. When considering synaptic connections between pairs of mPFC neurons, the intrinsic properties of the postsynaptic cell and the anatomical positions of both cells jointly account for ~7.5% of the variation in the probability of connection. Moreover, anatomical distance and laminar position explain most of this fraction in variation. Our findings reveal the factors determining connectivity in the mPFC and delineate the architecture of synaptic connections in the BLA-projecting subnetwork.

A non-canonical striatopallidal "Go" pathway that supports motor control.

In the classical model of the basal ganglia, direct pathway striatal projection neurons (dSPNs) send projections to the substantia nigra (SNr) and entopeduncular nucleus to regulate motor function. Recent studies have re-established that dSPNs also possess "bridging" collaterals within the globus pallidus (GPe), yet the significance of these collaterals for behavior is unknown. Here we use in vivo optical and chemogenetic tools combined with deep learning approaches to dissect the roles of bridging collaterals in motor function. We find that dSPNs projecting to the SNr send synchronous motor-related information to the GPe via axon collaterals. Inhibition of native activity in dSPN GPe terminals impairs motor activity and function via regulation of pallidostriatal Npas1 neurons. We propose a model by which dSPN GPe collaterals ("striatopallidal Go pathway") act in concert with the canonical terminals in the SNr to support motor control by inhibiting Npas1 signals going back to the striatum.

Localized chemogenetic silencing of inhibitory neurons: a novel mouse model of focal cortical epileptic activity.

Focal cortical epilepsies are frequently refractory to available anticonvulsant drug therapies. One key factor contributing to this state is the limited availability of animal models that allow to reliably study focal cortical seizures and how they recruit surrounding brain areas in vivo. In this study, we selectively expressed the inhibitory chemogenetic receptor, hM4D, in GABAergic neurons in focal cortical areas using viral gene transfer. GABAergic silencing using Clozapine-N-Oxide (CNO) demonstrated reliable induction of local epileptiform events in the electroencephalogram signal of awake freely moving mice. Anesthetized mice experiments showed consistent induction of focal epileptiform-events in both the barrel cortex (BC) and the medial prefrontal cortex (mPFC), accompanied by high-frequency oscillations, a known characteristic of human seizures. Epileptiform-events showed propagation indication with favored propagation pathways: from the BC on 1 hemisphere to its counterpart and from the BC to the mPFC, but not vice-versa. Lastly, sensory whisker-pad stimulation evoked BC epileptiform events post-CNO, highlighting the potential use of this model in studying sensory-evoked seizures. Combined, our results show that targeted chemogenetic inhibition of GABAergic neurons using hM4D can serve as a novel, versatile, and reliable model of focal cortical epileptic activity suitable for systematically studying cortical ictogenesis in different cortical areas.

Exercise increases information content and affects long-term stability of hippocampal place codes.

Physical exercise is known to augment brain functioning, improving memory and cognition. However, while some of the physiological effects of physical activity on the brain are known, little is known about its effects on the neural code. Using calcium imaging in freely behaving mice, we study how voluntary exercise affects the quality and long-term stability of hippocampal place codes. We find that running accelerates the emergence of a more informative spatial code in novel environments and increases code stability over days and weeks. Paradoxically, although runners demonstrated an overall more stable place code than their sedentary peers, their place code changed faster when controlling for code quality level. A model-based simulation shows that the combination of improved code quality and faster representational drift in runners, but neither of these effects alone, could account for our results. Thus, exercise may enhance hippocampal function via a more informative and dynamic place code.

Optogenetics at the presynapse.

Optogenetic actuators enable highly precise spatiotemporal interrogation of biological processes at levels ranging from the subcellular to cells, circuits and behaving organisms. Although their application in neuroscience has traditionally focused on the control of spiking activity at the somatodendritic level, the scope of optogenetic modulators for direct manipulation of presynaptic functions is growing. Presynaptically localized opsins combined with light stimulation at the terminals allow light-mediated neurotransmitter release, presynaptic inhibition, induction of synaptic plasticity and specific manipulation of individual components of the presynaptic machinery. Here, we describe presynaptic applications of optogenetic tools in the context of the unique cell biology of axonal terminals, discuss their potential shortcomings and outline future directions for this rapidly developing research area.

Molecular Optimization of Rhodopsin-Based Tools for Neuroscience Applications.

There is no question that genetically encoded tools have revolutionized neuroscience. These include optically modulated tools for writing-in (optogenetics) and reading-out (calcium, voltage, and neurotransmitter indicators) neural activity as well as precision expression of these reagents using virally mediated delivery. With few exceptions, these powerful approaches are derived from naturally occurring molecules that are sourced from diverse organisms that span all kingdoms of life. Successful expression of genetic tools in standard neuroscience model organisms requires optimizing gene structure, taking into account differences in both protein translation and trafficking. Myriad approaches have resolved these two challenges, resulting in order-of-magnitude increases in functional expression. In this chapter, we focus on synthesizing prior experience in successfully enabling the transition of genes across kingdoms with a goal of facilitating the production of the next generation of molecular tools for neuroscience. We then provide a detailed protocol that allows expression and testing of novel genetically encoded tools in mammalian cell lines and primary cultured neurons.

Rhodopsin-bestrophin fusion proteins from unicellular algae form gigantic pentameric ion channels.

Many organisms sense light using rhodopsins, photoreceptive proteins containing a retinal chromophore. Here we report the discovery, structure and biophysical characterization of bestrhodopsins, a microbial rhodopsin subfamily from marine unicellular algae, in which one rhodopsin domain of eight transmembrane helices or, more often, two such domains in tandem, are C-terminally fused to a bestrophin channel. Cryo-EM analysis of a rhodopsin-rhodopsin-bestrophin fusion revealed that it forms a pentameric megacomplex (~700 kDa) with five rhodopsin pseudodimers surrounding the channel in the center. Bestrhodopsins are metastable and undergo photoconversion between red- and green-absorbing or green- and UVA-absorbing forms in the different variants. The retinal chromophore, in a unique binding pocket, photoisomerizes from all-trans to 11-cis form. Heterologously expressed bestrhodopsin behaves as a light-modulated anion channel.

Optogenetics for light control of biological systems.

Optogenetic techniques have been developed to allow control over the activity of selected cells within a highly heterogeneous tissue, using a combination of genetic engineering and light. Optogenetics employs natural and engineered photoreceptors, mostly of microbial origin, to be genetically introduced into the cells of interest. As a result, cells that are naturally light-insensitive can be made photosensitive and addressable by illumination and precisely controllable in time and space. The selectivity of expression and subcellular targeting in the host is enabled by applying control elements such as promoters, enhancers and specific targeting sequences to the employed photoreceptor-encoding DNA. This powerful approach allows precise characterization and manipulation of cellular functions and has motivated the development of advanced optical methods for patterned photostimulation. Optogenetics has revolutionized neuroscience during the past 15 years and is primed to have a similar impact in other fields, including cardiology, cell biology and plant sciences. In this Primer, we describe the principles of optogenetics, review the most commonly used optogenetic tools, illumination approaches and scientific applications and discuss the possibilities and limitations associated with optogenetic manipulations across a wide variety of optical techniques, cells, circuits and organisms.

Optogenetic strategies for high-efficiency all-optical interrogation using blue-light-sensitive opsins.

All-optical methods for imaging and manipulating brain networks with high spatial resolution are fundamental to study how neuronal ensembles drive behavior. Stimulation of neuronal ensembles using two-photon holographic techniques requires high-sensitivity actuators to avoid photodamage and heating. Moreover, two-photon-excitable opsins should be insensitive to light at wavelengths used for imaging. To achieve this goal, we developed a novel soma-targeted variant of the large-conductance blue-light-sensitive opsin CoChR (stCoChR). In the mouse cortex in vivo, we combined holographic two-photon stimulation of stCoChR with an amplified laser tuned at the opsin absorption peak and two-photon imaging of the red-shifted indicator jRCaMP1a. Compared to previously characterized blue-light-sensitive soma-targeted opsins in vivo, stCoChR allowed neuronal stimulation with more than 10-fold lower average power and no spectral crosstalk. The combination of stCoChR, tuned amplified laser stimulation, and red-shifted functional indicators promises to be a powerful tool for large-scale interrogation of neural networks in the intact brain.

Efficient optogenetic silencing of neurotransmitter release with a mosquito rhodopsin.

Information is carried between brain regions through neurotransmitter release from axonal presynaptic terminals. Understanding the functional roles of defined neuronal projection pathways requires temporally precise manipulation of their activity. However, existing inhibitory optogenetic tools have low efficacy and off-target effects when applied to presynaptic terminals, while chemogenetic tools are difficult to control in space and time. Here, we show that a targeting-enhanced mosquito homolog of the vertebrate encephalopsin (eOPN3) can effectively suppress synaptic transmission through the Gi/o signaling pathway. Brief illumination of presynaptic terminals expressing eOPN3 triggers a lasting suppression of synaptic output that recovers spontaneously within minutes in vitro and in vivo. In freely moving mice, eOPN3-mediated suppression of dopaminergic nigrostriatal afferents induces a reversible ipsiversive rotational bias. We conclude that eOPN3 can be used to selectively suppress neurotransmitter release at presynaptic terminals with high spatiotemporal precision, opening new avenues for functional interrogation of long-range neuronal circuits in vivo.

The social dilemma: prefrontal control of mammalian sociability.

Mammalian social interactions are orchestrated by a wide array of neural circuits. While some aspects of social behaviors are driven by subcortical circuits, and are considered to be highly conserved and hard-wired, others require dynamic and context-dependent modulation that integrates current state, past experience and goal-driven action selection. These cognitive social processes are known to be dependent on the integrity of the prefrontal cortex. However, the circuit mechanisms through which the prefrontal cortex supports complex social functions are still largely unknown, and it is unclear if and how they diverge from prefrontal control of behavior outside of the social domain. Here we review recent studies exploring the role of prefrontal circuits in mammalian social functions, and attempt to synthesize these findings to a holistic view of prefrontal control of sociability.

In situ electrochemical generation of nitric oxide for neuronal modulation.

Understanding the function of nitric oxide, a lipophilic messenger in physiological processes across nervous, cardiovascular and immune systems, is currently impeded by the dearth of tools to deliver this gaseous molecule in situ to specific cells. To address this need, we have developed iron sulfide nanoclusters that catalyse nitric oxide generation from benign sodium nitrite in the presence of modest electric fields. Locally generated nitric oxide activates the nitric oxide-sensitive cation channel, transient receptor potential vanilloid family member 1 (TRPV1), and the latency of TRPV1-mediated Ca2+ responses can be controlled by varying the applied voltage. Integrating these electrocatalytic nanoclusters with multimaterial fibres allows nitric oxide-mediated neuronal interrogation in vivo. The in situ generation of nitric oxide in the ventral tegmental area with the electrocatalytic fibres evoked neuronal excitation in the targeted brain region and its excitatory projections. This nitric oxide generation platform may advance mechanistic studies of the role of nitric oxide in the nervous system and other organs.

Anatomically and functionally distinct thalamocortical inputs to primary and secondary mouse whisker somatosensory cortices.

Subdivisions of mouse whisker somatosensory thalamus project to cortex in a region-specific and layer-specific manner. However, a clear anatomical dissection of these pathways and their functional properties during whisker sensation is lacking. Here, we use anterograde trans-synaptic viral vectors to identify three specific thalamic subpopulations based on their connectivity with brainstem. The principal trigeminal nucleus innervates ventral posterior medial thalamus, which conveys whisker-selective tactile information to layer 4 primary somatosensory cortex that is highly sensitive to self-initiated movements. The spinal trigeminal nucleus innervates a rostral part of the posterior medial (POm) thalamus, signaling whisker-selective sensory information, as well as decision-related information during a goal-directed behavior, to layer 4 secondary somatosensory cortex. A caudal part of the POm, which apparently does not receive brainstem input, innervates layer 1 and 5A, responding with little whisker selectivity, but showing decision-related modulation. Our results suggest the existence of complementary segregated information streams to somatosensory cortices.

Wireless Optogenetic Stimulation of Oxytocin Neurons in a Semi-natural Setup Dynamically Elevates Both Pro-social and Agonistic Behaviors.

Complex behavioral phenotyping techniques are becoming more prevalent in the field of behavioral neuroscience, and thus methods for manipulating neuronal activity must be adapted to fit into such paradigms. Here, we present a head-mounted, magnetically activated device for wireless optogenetic manipulation that is compact, simple to construct, and suitable for use in group-living mice in an enriched semi-natural arena over several days. Using this device, we demonstrate that repeated activation of oxytocin neurons in male mice can have different effects on pro-social and agonistic behaviors, depending on the social context. Our findings support the social salience hypothesis of oxytocin and emphasize the importance of the environment in the study of social neuromodulators. Our wireless optogenetic device can be easily adapted for use in a variety of behavioral paradigms, which are normally hindered by tethered light delivery or a limited environment.

Associative and plastic thalamic signaling to the lateral amygdala controls fear behavior.

Decades of research support the idea that associations between a conditioned stimulus (CS) and an unconditioned stimulus (US) are encoded in the lateral amygdala (LA) during fear learning. However, direct proof for the sources of CS and US information is lacking. Definitive evidence of the LA as the primary site for cue association is also missing. Here, we show that calretinin (Calr)-expressing neurons of the lateral thalamus (Calr+LT neurons) convey the association of fast CS (tone) and US (foot shock) signals upstream from the LA in mice. Calr+LT input shapes a short-latency sensory-evoked activation pattern of the amygdala via both feedforward excitation and inhibition. Optogenetic silencing of Calr+LT input to the LA prevents auditory fear conditioning. Notably, fear conditioning drives plasticity in Calr+LT neurons, which is required for appropriate cue and contextual fear memory retrieval. Collectively, our results demonstrate that Calr+LT neurons provide integrated CS-US representations to the LA that support the formation of aversive memories.