Improved green and red GRAB sensors for monitoring spatiotemporal serotonin release in vivo.

The serotonergic system plays important roles in both physiological and pathological processes, and is a therapeutic target for many psychiatric disorders. Although several genetically encoded GFP-based serotonin (5-HT) sensors were recently developed, their sensitivities and spectral profiles are relatively limited. To overcome these limitations, we optimized green fluorescent G-protein-coupled receptor (GPCR)-activation-based 5-HT (GRAB5-HT) sensors and developed a red fluorescent GRAB5-HT sensor. These sensors exhibit excellent cell surface trafficking and high specificity, sensitivity and spatiotemporal resolution, making them suitable for monitoring 5-HT dynamics in vivo. Besides recording subcortical 5-HT release in freely moving mice, we observed both uniform and gradient 5-HT release in the mouse dorsal cortex with mesoscopic imaging. Finally, we performed dual-color imaging and observed seizure-induced waves of 5-HT release throughout the cortex following calcium and endocannabinoid waves. In summary, these 5-HT sensors can offer valuable insights regarding the serotonergic system in both health and disease.

Genetically encoded sensors for in vivo detection of neurochemicals relevant to depression.

Depressive disorders are a common and debilitating form of mental illness with significant impacts on individuals and society. Despite the high prevalence, the underlying causes and mechanisms of depressive disorders are still poorly understood. Neurochemical systems, including serotonin, norepinephrine, and dopamine, have been implicated in the development and perpetuation of depressive symptoms. Current treatments for depression target these neuromodulator systems, but there is a need for a better understanding of their role in order to develop more effective treatments. Monitoring neurochemical dynamics during depressive symptoms is crucial for gaining a better a understanding of their involvement in depressive disorders. Genetically encoded sensors have emerged recently that offer high spatial-temporal resolution and the ability to monitor neurochemical dynamics in real time. This review explores the neurochemical systems involved in depression and discusses the applications and limitations of current monitoring tools for neurochemical dynamics. It also highlights the potential of genetically encoded sensors for better characterizing neurochemical dynamics in depression-related behaviors. Furthermore, potential improvements to current sensors are discussed in order to meet the requirements of depression research.

Improved green and red GRAB sensors for monitoring dopaminergic activity in vivo.

Dopamine (DA) plays multiple roles in a wide range of physiological and pathological processes via a large network of dopaminergic projections. To dissect the spatiotemporal dynamics of DA release in both dense and sparsely innervated brain regions, we developed a series of green and red fluorescent G-protein-coupled receptor activation-based DA (GRABDA) sensors using a variety of DA receptor subtypes. These sensors have high sensitivity, selectivity and signal-to-noise ratio with subsecond response kinetics and the ability to detect a wide range of DA concentrations. We then used these sensors in mice to measure both optogenetically evoked and behaviorally relevant DA release while measuring neurochemical signaling in the nucleus accumbens, amygdala and cortex. Using these sensors, we also detected spatially resolved heterogeneous cortical DA release in mice performing various behaviors. These next-generation GRABDA sensors provide a robust set of tools for imaging dopaminergic activity under a variety of physiological and pathological conditions.

Improved dual-color GRAB sensors for monitoring dopaminergic activity in vivo.

Dopamine (DA) plays multiple roles in a wide range of physiological and pathological processes via a vast network of dopaminergic projections. To fully dissect the spatiotemporal dynamics of DA release in both dense and sparsely innervated brain regions, we developed a series of green and red fluorescent GPCR activation-based DA (GRABDA) sensors using a variety of DA receptor subtypes. These sensors have high sensitivity, selectivity, and signal-to-noise properties with subsecond response kinetics and the ability to detect a wide range of DA concentrations. We then used these sensors in freely moving mice to measure both optogenetically evoked and behaviorally relevant DA release while measuring neurochemical signaling in the nucleus accumbens, amygdala, and cortex. Using these sensors, we also detected spatially resolved heterogeneous cortical DA release in mice performing various behaviors. These next-generation GRABDA sensors provide a robust set of tools for imaging dopaminergic activity under a variety of physiological and pathological conditions.

Serotonergic neurons control cortical neuronal intracellular energy dynamics by modulating astrocyte-neuron lactate shuttle.

The central serotonergic system has multiple roles in animal physiology and behavior, including sleep-wake control. However, its function in controlling brain energy metabolism according to the state of animals remains undetermined. Through in vivo monitoring of energy metabolites and signaling, we demonstrated that optogenetic activation of raphe serotonergic neurons increased cortical neuronal intracellular concentration of ATP, an indispensable cellular energy molecule, which was suppressed by inhibiting neuronal uptake of lactate derived from astrocytes. Raphe serotonergic neuronal activation induced cortical astrocytic Ca2+ and cAMP surges and increased extracellular lactate concentrations, suggesting the facilitation of lactate release from astrocytes. Furthermore, chemogenetic inhibition of raphe serotonergic neurons partly attenuated the increase in cortical neuronal intracellular ATP levels as arousal increased in mice. Serotonergic neuronal activation promoted an increase in cortical neuronal intracellular ATP levels, partly mediated by the facilitation of the astrocyte-neuron lactate shuttle, contributing to state-dependent optimization of neuronal intracellular energy levels.

Local 5-HT signaling bi-directionally regulates the coincidence time window for associative learning.

The coincidence between conditioned stimulus (CS) and unconditioned stimulus (US) is essential for associative learning; however, the mechanism regulating the duration of this temporal window remains unclear. Here, we found that serotonin (5-HT) bi-directionally regulates the coincidence time window of olfactory learning in Drosophila and affects synaptic plasticity of Kenyon cells (KCs) in the mushroom body (MB). Utilizing GPCR-activation-based (GRAB) neurotransmitter sensors, we found that KC-released acetylcholine (ACh) activates a serotonergic dorsal paired medial (DPM) neuron, which in turn provides inhibitory feedback to KCs. Physiological stimuli induce spatially heterogeneous 5-HT signals, which proportionally gate the intrinsic coincidence time windows of different MB compartments. Artificially reducing or increasing the DPM neuron-released 5-HT shortens or prolongs the coincidence window, respectively. In a sequential trace conditioning paradigm, this serotonergic neuromodulation helps to bridge the CS-US temporal gap. Altogether, we report a model circuitry for perceiving the temporal coincidence and determining the causal relationship between environmental events.

Brainstem serotonin neurons selectively gate retinal information flow to thalamus.

Retinal ganglion cell (RGC) types relay parallel streams of visual feature information. We hypothesized that neuromodulators might efficiently control which visual information streams reach the cortex by selectively gating transmission from specific RGC axons in the thalamus. Using fiber photometry recordings, we found that optogenetic stimulation of serotonergic axons in primary visual thalamus of awake mice suppressed ongoing and visually evoked calcium activity and glutamate release from RGC boutons. Two-photon calcium imaging revealed that serotonin axon stimulation suppressed RGC boutons that responded strongly to global changes in luminance more than those responding only to local visual stimuli, while the converse was true for suppression induced by increases in arousal. Converging evidence suggests that differential expression of the 5-HT1B receptor on RGC presynaptic terminals, but not differential density of nearby serotonin axons, may contribute to the selective serotonergic gating of specific visual information streams before they can activate thalamocortical neurons.

Serotonin-releasing agents with reduced off-target effects.

Increasing extracellular levels of serotonin (5-HT) in the brain ameliorates symptoms of depression and anxiety-related disorders, e.g., social phobias and post-traumatic stress disorder. Recent evidence from preclinical and clinical studies established the therapeutic potential of drugs inducing the release of 5-HT via the 5-HT-transporter. Nevertheless, current 5-HT releasing compounds under clinical investigation carry the risk for abuse and deleterious side effects. Here, we demonstrate that S-enantiomers of certain ring-substituted cathinones show preference for the release of 5-HT ex vivo and in vivo, and exert 5-HT-associated effects in preclinical behavioral models. Importantly, the lead cathinone compounds (1) do not induce substantial dopamine release and (2) display reduced off-target activity at vesicular monoamine transporters and 5-HT2B-receptors, indicative of low abuse-liability and low potential for adverse events. Taken together, our findings identify these agents as lead compounds that may prove useful for the treatment of disorders where elevation of 5-HT has proven beneficial.

Distinct serotonergic pathways to the amygdala underlie separate behavioral features of anxiety.

Anxiety-like behaviors in mice include social avoidance and avoidance of bright spaces. Whether these features are distinctly regulated is unclear. We demonstrate that in mice, social and anxiogenic stimuli, respectively, increase and decrease serotonin (5-HT) levels in basal amygdala (BA). In dorsal raphe nucleus (DRN), 5-HT∩vGluT3 neurons projecting to BA parvalbumin (DRN5-HT∩vGluT3-BAPV) and pyramidal (DRN5-HT∩vGluT3-BAPyr) neurons have distinct intrinsic properties and gene expression and respond to anxiogenic and social stimuli, respectively. Activation of DRN5-HT∩vGluT3→BAPV inhibits 5-HT release via GABAB receptors on serotonergic terminals in BA, inducing social avoidance and avoidance of bright spaces. Activation of DRN5-HT∩vGluT3→BA neurons inhibits two subsets of BAPyr neurons via 5-HT1A receptors (HTR1A) and 5-HT1B receptors (HTR1B). Pharmacological inhibition of HTR1A and HTR1B in BA induces avoidance of bright spaces and social avoidance, respectively. These findings highlight the functional significance of heterogenic inputs from DRN to BA subpopulations in the regulation of separate anxiety-related behaviors.

A serotonergic axon-cilium synapse drives nuclear signaling to alter chromatin accessibility.

Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, the 5-hydroxytryptamine receptor 6 (5-HTR6). Using a cilia-targeted serotonin sensor, we show that opto- and chemogenetic stimulation of serotonergic axons releases serotonin onto cilia. Ciliary 5-HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility. Ablation of this pathway reduces chromatin accessibility in CA1 pyramidal neurons. As a signaling apparatus with proximity to the nucleus, axo-ciliary synapses short circuit neurotransmission to alter the postsynaptic neuron's epigenetic state.

Synaptic mechanism underlying serotonin modulation of transition to cocaine addiction.

Compulsive drug use despite adverse consequences defines addiction. While mesolimbic dopamine signaling is sufficient to drive compulsion, psychostimulants such as cocaine also boost extracellular serotonin (5-HT) by inhibiting reuptake. We used SERT Met172 knockin (SertKI) mice carrying a transporter that no longer binds cocaine to abolish 5-HT transients during drug self-administration. SertKI mice showed an enhanced transition to compulsion. Conversely, pharmacologically elevating 5-HT reversed the inherently high rate of compulsion transition with optogenetic dopamine self-stimulation. The bidirectional effect on behavior is explained by presynaptic depression of orbitofrontal cortex­to­dorsal striatum synapses induced by 5-HT via 5-HT1B receptors. Consequently, in projection-specific 5-HT1B receptor knockout mice, the fraction of individuals compulsively self-administering cocaine was elevated.

A genetically encoded sensor for measuring serotonin dynamics.

Serotonin (5-HT) is a phylogenetically conserved monoamine neurotransmitter modulating important processes in the brain. To directly visualize the release of 5-HT, we developed a genetically encoded G-protein-coupled receptor (GPCR)-activation-based 5-HT (GRAB5-HT) sensor with high sensitivity, high selectivity, subsecond kinetics and subcellular resolution. GRAB5-HT detects 5-HT release in multiple physiological and pathological conditions in both flies and mice and provides new insights into the dynamics and mechanisms of 5-HT signaling.

New frontiers in translational research: Touchscreens, open science, and the mouse translational research accelerator platform.

Many neurodegenerative and neuropsychiatric diseases and other brain disorders are accompanied by impairments in high-level cognitive functions including memory, attention, motivation, and decision-making. Despite several decades of extensive research, neuroscience is little closer to discovering new treatments. Key impediments include the absence of validated and robust cognitive assessment tools for facilitating translation from animal models to humans. In this review, we describe a state-of-the-art platform poised to overcome these impediments and improve the success of translational research, the Mouse Translational Research Accelerator Platform (MouseTRAP), which is centered on the touchscreen cognitive testing system for rodents. It integrates touchscreen-based tests of high-level cognitive assessment with state-of-the art neurotechnology to record and manipulate molecular and circuit level activity in vivo in animal models during human-relevant cognitive performance. The platform also is integrated with two Open Science platforms designed to facilitate knowledge and data-sharing practices within the rodent touchscreen community, touchscreencognition.org and mousebytes.ca. Touchscreencognition.org includes the Wall, showcasing touchscreen news and publications, the Forum, for community discussion, and Training, which includes courses, videos, SOPs, and symposia. To get started, interested researchers simply create user accounts. We describe the origins of the touchscreen testing system, the novel lines of research it has facilitated, and its increasingly widespread use in translational research, which is attributable in part to knowledge-sharing efforts over the past decade. We then identify the unique features of MouseTRAP that stand to potentially revolutionize translational research, and describe new initiatives to partner with similar platforms such as McGill's M3 platform (m3platform.org).

Nanoscopic Visualization of Restricted Nonvolume Cholinergic and Monoaminergic Transmission with Genetically Encoded Sensors.

How neuromodulatory transmitters diffuse into the extracellular space remains an unsolved fundamental biological question, despite wide acceptance of the volume transmission model. Here, we report development of a method combining genetically encoded fluorescent sensors with high-resolution imaging and analysis algorithms which permits the first direct visualization of neuromodulatory transmitter diffusion at various neuronal and non-neuronal cells. Our analysis reveals that acetylcholine and monoamines diffuse at individual release sites with a spread length constant of ∼0.75 µm. These transmitters employ varied numbers of release sites, and when spatially close-packed release sites coactivate they can spillover into larger subcellular areas. Our data indicate spatially restricted (i.e., nonvolume) neuromodulatory transmission to be a prominent intercellular communication mode, reshaping current thinking of control and precision of neuromodulation crucial for understanding behaviors and diseases.