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The neocortex contains a constellation of sensory-motor regions whose functional interactions provide the basis of cognition and behavior. Simultaneously recording neuronal group activity across the cortical hemispheres is essential for understanding the nature of information flow across cerebral networks. Moreover, unbiased and robust methods for measuring functional interactions across the cortex within individual living subjects is critical for substantive tests of genetic and environmental factors that influence brain development and function. To this end, we image pan-neuronally expressed genetically encoded calcium indicators transcranially across the neocortex of unanesthetized, behaving mice throughout development. Recording from behaving mice produces a unique set of challenges, including optical and blood artifacts associated with movement. In addition, areal patterning of the cortex can vary among individuals, ages, and genotypes thus an unbiased, flexible workflow for video acquisition and analysis is necessary for producing high quality segmentations of functional structure across neocortex. To address these challenges, we have developed an eigendecomposition-based workflow that isolates hemodynamic and optical artifacts to recover underlying calcium activity patterns, and segments independent regions of the brain to create maps of functional units across the developing cortex. These unique, data-driven maps provide a reference for understanding developmental, genetic, as well as individual variation between functional units of the brain, and provide a method for extracting optimized time courses from the cortical surface without the need for stimulation-based mapping or anatomical post-processing and alignment. In addition, we quantify the quality of separation of independent sources and use the resulting metrics as feedback to optimize our video acquisition parameters. The open source methods developed here are flexible enough to be utilized with various subject ages and genotypes, as well as numerous experimental configurations and computer architectures. Here we additionally present applications of these methods to anesthesia and development datasets. Overcoming these challenges opens the possibility of using these techniques to help address a number of key objectives in neuroscience, including the quantification of robust inter-areal dynamics between functional motifs across cerebral networks.

Calcium imaging using GCaMP indicators and miniature microscopes has been used to image cellular populations during long timescales and in different task phases, as well as to determine neuronal circuit topology and organization. Because the hippocampus (HPC) is essential for tasks of memory, spatial navigation, and learning, calcium imaging of large populations of HPC neurons can provide new insight on cell changes over time during these tasks. All reported HPCin vivocalcium imaging experiments have been done in mouse. However, rats have many behavioral and physiological experimental advantages over mice. In this paper, we present the first (to our knowledge)in vivocalcium imaging from CA1 HPC in freely moving male rats. Using the UCLA Miniscope, we demonstrate that, in rat, hundreds of cells can be visualized and held across weeks. We show that calcium events in these cells are highly correlated with periods of movement, with few calcium events occurring during periods without movement. We additionally show that an extremely large percent of cells recorded during a navigational task are place cells (77.3 ± 5.0%, surpassing the percent seen during mouse calcium imaging), and that these cells enable accurate decoding of animal position and can be held over days with consistent place fields in a consistent spatial map. A detailed protocol is included, and implications of these advancements onin vivoimaging and place field literature are discussed.SIGNIFICANCE STATEMENTIn vivocalcium imaging in freely moving animals allows the visualization of cellular activity across days. In this paper, we present the firstin vivoCa2+ recording from CA1 hippocampus (HPC) in freely moving rats. We demonstrate that hundreds of cells can be visualized and held across weeks, and that calcium activity corresponds to periods of movement. We show that a high percentage (77.3 ± 5.0%) of imaged cells are place cells, and that these place cells enable accurate decoding and can be held stably over days with little change in field location. Because the HPC is essential for many tasks involving memory, navigation, and learning, imaging of large populations of HPC neurons can shed new insight on cellular activity changes and organization.

Neural progenitor cell/neural stem cell (NPC/NSC) grafts integrate into sites of spinal cord injury (SCI) and show anatomical and electrophysiological evidence of forming neuronal relays across lesions. To determine how signals may be propagated through grafts, we performed ex vivo and in vivo calcium imaging of NPC grafts and host spinal cord neurons in mice. Following optogenetic stimulation of corticospinal tract axon terminals ex vivo, we detected consistent calcium responses throughout grafts. Grafts exhibited spontaneous activity in both independently-firing neurons and emergent neuronal networks, which were also activated by corticospinal stimulation. These patterns of activation resembled those present in intact spinal cord. In turn, optogenetic stimulation of graft axons extending out of lesion sites triggered excitatory post-synaptic responses in caudal host spinal cord neurons. Finally, distinct sensory stimuli elicited responses in the dorsal aspect of grafts in vivo. Thus, NPC/NSC grafts form local networks that are activated by host inputs and can activate host neurons on the distal side of spinal cord lesions.In order to properly assess the potential benefit of neural stem cell grafts in humans, we sought to understand the rate at which human grafts mature in the injury site. We grafted human NSCs into sites of SCI in immunodeficient rats and assessed anatomical and functional outcomes over a 1.5-year period. Although mature neuron markers appeared 3 months after grafting; neurogenesis, neuronal pruning, and neuronal soma enlargement progressed over the next year. Graft size remained stable as axons emerged in large numbers early on, with half of these projections persisting after 1.5 years. Astrocytes expressed mature markers after 6 months, while oligodendrocytes did not display mature markers until 1 year after grafting. A slow migration of astrocytes from graft sites into host tissue was observed. Importantly, functional improvement did not occur until over 1 year after grafting. Thus, human NSCs mature at their intrinsic rate even when grafted into a rodent environment, and they support functional recovery only at this delayed timepoint, a key finding in human clinical trial planning.

Cocaine addiction is one of the leading causes of drug-related deaths and overdose in the United States, and there are currently no cheap and effective treatments for this disease. To combat this problem, it is necessary to study the underlying neuronal circuits and populations that underlie cocaine seeking behavior. Previous studies have demonstrated that the prefrontal cortex (PFC) becomes hypoexcitable following chronic compulsive drug seeking (Chen et. al. 2013), so we decided to see how the inputs and outputs of the prefrontal cortex are modulated following chronic cocaine seeking. To answer both of these questions, we first designed a novel head-fixed cocaine self-administration behavioral setup. This thesis utilizes this model to answer two main questions: 1) how are the inputs to the prefrontal cortex modulated following cocaine addiction? 2) How are the downstream targets of the prefrontal cortex, the neocortex, modulated following cocaine seeking behavior?

Background & Aims Colonic motor patterns have been described by a number of different groups, but the neural connectivity and ganglion architecture supporting patterned motor activity have not been elucidated. Our goals were to describe quantitatively, by region, the structural architecture of the mouse enteric nervous system and use functional calcium imaging, pharmacology, and electrical stimulation to show regional underpinnings of different motor patterns. Methods Excised colon segments from mice expressing the calcium indicator GCaMP6f or GCaMP6s were used to examine spontaneous and evoked (pharmacologic or electrical) changes in GCaMP-mediated fluorescence and coupled with assessment of colonic motor activity, immunohistochemistry, and confocal imaging. Three-dimensional image reconstruction and statistical methods were used to describe quantitatively mouse colon myenteric ganglion structure, neural and vascular network patterning, and neural connectivity. Results In intact colon, regionally specific myenteric ganglion size, architecture, and neural circuit connectivity patterns along with neurotransmitter-receptor expression underlie colonic motor patterns that define functional differences along the colon. Region-specific effects on spontaneous, evoked, and chemically induced neural activity contribute to regional motor patterns, as does intraganglionic functional connectivity. We provide direct evidence of neural circuit structural and functional regional differences that have only been inferred in previous investigations. We include regional comparisons between quantitative measures in mouse and human colon that represent an important advance in showing the usefulness and relevance of the mouse system for translation to the human colon. Conclusions There are several neural mechanisms dependent on myenteric ganglion architecture and functional connectivity that underlie neurogenic control of patterned motor function in the mouse colon., Graphical abstract

The response of cortical neurons to sensory stimuli is shaped both by past events (adaptation) and the expectation of future events (prediction). Here we employed a visual stimulus paradigm with different levels of predictability to characterise how expectation influences orientation selectivity in the primary visual cortex (V1) of mice. We recorded neuronal activity using two-photon calcium imaging (GCaMP6f) while animals viewed sequences of grating stimuli which either varied randomly in their orientations or rotated predictably with occasional transitions to an unexpected orientation. For single neurons and the population, there was significant enhancement in the gain of orientation-selective responses to unexpected gratings. This gain-enhancement for unexpected stimuli was prominent in both awake and anaesthetised mice. We implemented a computational model to demonstrate how trial-to-trial variability in neuronal responses were best characterised when adaptation and expectation effects were combined.

The store-operated calcium (Ca2+) entry (SOCE) is the Ca2+ entry mechanism used by cells to replenish depleted Ca2+ store. The dysregulation of SOCE has been reported in metastatic cancer. It is believed that SOCE promotes migration and invasion by remodeling the actin cytoskeleton and cell adhesion dynamics. There is recent evidence supporting that SOCE is critical for the spatial and the temporal coding of Ca2+ signals in the cell. In this review, we critically examined the spatiotemporal control of SOCE signaling and its implication in the specificity and robustness of signaling events downstream of SOCE, with a focus on the spatiotemporal SOCE signaling during cancer cell migration, invasion and metastasis. We further discuss the limitation of our current understanding of SOCE in cancer metastasis and potential approaches to overcome such limitation.

Summary The immature characteristics and metabolic phenotypes of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) restrict their applications for disease modeling, drug discovery, and cell-based therapy. Leveraging on the metabolic shifts from glycolysis to fatty acid oxidation as CMs mature, a human hexokinase1-GFP metabolic reporter cell line (H7 HK1-GFP) was generated to facilitate the isolation of fetal or more matured hPSC-CMs. RNA sequencing of fetal versus more matured CMs uncovered a potential role of interferon-signaling pathway in regulating CM maturation. Indeed, IFN-γ-treated CMs resulted in an upregulation of the JAK-STAT pathway, which was found to be associated with increased expression of CM maturation genes, shift from MYH6 to MYH7 expression, and improved sarcomeric structure. Functionally, IFN-γ-treated CMs exhibited a more matured electrophysiological profile, such as increased calcium dynamics and action potential upstroke velocity, demonstrated through calcium imaging and MEA. Expectedly, the functional improvements were nullified with a JAK-STAT inhibitor, ruxolitinib., Highlights • RNA-seq revealed upregulation of IFN-signaling pathways during CM maturation • IFN-γ-treated PSC-derived fetal CMs display increased MYH7:MYH6 ratio • IFN-γ-treated PSC-derived fetal CMs exhibited improved electrophysiological profile, In this article, Soh and colleagues utilize a metabolic reporter to identify a novel role of JAK-STAT signaling in hPSC-CM maturation. IFN-γ treatment activates the JAK-STAT pathway and upregulates STAT1 expression in hPSC-derived fetal CMs, thereby enhancing the expression of CM maturation genes including MYH7, which subsequently resulted in improved sarcomeric structure, and a relatively more matured electrophysiological profile in vitro.

Although calcium waves have been widely observed in glial cells, their occurrence in vivo during behavior remains less understood. Here, we investigated the recruitment of glial cells in the hindbrain and spinal cord after acousto-vestibular (AV) stimuli triggering escape responses using in vivo population calcium imaging in larval zebrafish. We observed that gap-junction-coupled spinal glial network exhibits large and homogenous calcium increases that rose in the rostral spinal cord and propagated bi-directionally toward the spinal cord and toward the hindbrain. Spinal glial calcium waves were driven by the recruitment of neurons and in particular, of noradrenergic signaling acting through α-adrenergic receptors. Noradrenergic neurons of the medulla-oblongata (NE-MO) were revealed in the vicinity of where the calcium wave started. NE-MO were recruited upon AV stimulation and sent dense axonal projections in the rostro-lateral spinal cord, suggesting these cells could trigger the glial wave to propagate down the spinal cord. Altogether, our results revealed that a simple AV stimulation is sufficient to recruit noradrenergic neurons in the brainstem that trigger in the rostral spinal cord two massive glial calcium waves, one traveling caudally in the spinal cord and another rostrally into the hindbrain.

AgRP neurons trigger one of the most potent orexigenic responses and are both necessary and sufficient for feeding. Recent technical advances for monitoring in vivo neuronal activity have revisited a previously well-established model of AgRP neurons' feeding regulatory effects. Our current understanding of AgRP neurons has increased in complexity and revealed a fine-tuned regulation of their activity dynamics across the whole sequence of feeding-related behaviours. This review focuses on recent studies that refined and re-evaluated our understanding of the regulatory principles and behavioural effects of AgRP circuits. We aim to cover major discoveries on the dynamic regulation of AgRP neuronal activity by exteroceptive and interoceptive food-related cues, their pleiotropic effects in feeding and whole-body homeostasis, and the associated AgRP circuits. The function and regulation of AgRP neuron will be sequentially discussed across the temporal series of behavioural and physiological changes occurring during the appetitive (food craving and foraging), the anticipatory (discovery of food-predicting cues), and the consummatory/post-ingestive phase of feeding (calorie ingestion).

Postnatal neurogenesis provides an opportunity to understand how newborn neurons functionally integrate into circuits to restore lost function. Newborn olfactory sensory neurons (OSNs) wire into highly organized olfactory bulb (OB) circuits throughout life, enabling lifelong plasticity and regeneration. Immature OSNs can form functional synapses capable of evoking firing in OB projection neurons. However, what contribution, if any, immature OSNs make to odor processing is unknown. Indeed, because immature OSNs can express multiple odorant receptors, any input that they do provide could degrade the odorant selectivity of input to OB glomeruli. Here, we used a combination of in vivo 2-photon calcium imaging, optogenetics, electrophysiology and behavioral assays to show that immature OSNs provide odor input to the OB, where they form monosynaptic connections with excitatory neurons. Importantly, immature OSNs responded as selectively to odorants as mature OSNs. Furthermore, mice successfully performed odor detection tasks using sensory input from immature OSNs alone. Immature OSNs responded more strongly to low odorant concentrations but their responses were less concentration dependent than those of mature OSNs, suggesting that immature and mature OSNs provide distinct odor input streams to each glomerulus. Together, our findings suggest that sensory input mediated by immature OSNs plays a previously unappreciated role in olfactory-guided behavior.

Objective: the switch between nonseizure and seizure states involves profound alterations in network excitability and synchrony. In this study, we aimed to identify and compare features of neural excitability and dynamics across multiple zebrafish seizure and epilepsy models. Methods: inspired by video-electroencephalographic recordings in patients, we developed a framework to study spontaneous and photically evoked neural and locomotor activity in zebrafish larvae, by combining high-throughput behavioral tracking and whole-brain in vivo two-photon calcium imaging. Results: our setup allowed us to dissect behavioral and physiological features that are divergent or convergent across multiple models. We observed that spontaneous locomotor and neural activity exhibit great diversity across models. Nonetheless, during photic stimulation, hyperexcitability and rapid response dynamics were well conserved across multiple models, highlighting the reliability of photically evoked activity for high-throughput assays. Intriguingly, in several models, we observed that the initial elevated photic response is often followed by rapid decay of neural activity and a prominent depressed state. Elevated photic response and following depressed state in seizure-prone networks are significantly reduced by the antiseizure medication valproic acid. Finally, rapid decay and depression of neural activity following photic stimulation temporally overlap with slow recruitment of astroglial calcium signals that are enhanced in seizure-prone networks. Significance We argue that fast decay of neural activity and depressed states following photic response are likely due to homeostatic mechanisms triggered by excessive neural activity. An improved understanding of the interplay between elevated and depressed excitability states might suggest tailored epilepsy therapies., European Research Council (ERC); European Union (EU); Horizon 2020; Helse Midt--Norge; Norges Forskningsrad

Cross-sectional studies have established a variety of structural, synaptic, and cell physiological changes corresponding to critical periods in cortical development. However, the emergence of functional connectivity (FC) in development has not been fully characterized, and hemodynamic-based measures are vulnerable to any neurovascular coupling changes occurring in parallel. We therefore used optical fluorescence imaging to trace longitudinal calcium FC in the awake, resting-state mouse cortex at 5 developmental timepoints beginning at postnatal day 15 (P15) and ending in early adulthood at P60. Calcium FC displayed coherent functional maps as early as P15, and FC significantly varied in connections between many regions across development, with the developmental trajectory’s shape specific to the functional region. Evaluating 325 seed–seed connections, we found that there was a significant increase in FC between P15 and P22 over the majority of the cortex as well as bilateral connectivity and node degree differences in frontal, motor, and retrosplenial cortices after P22. A rebalancing of inter- and intrahemispheric FC and local-distal FC dominance was also observed during development. This longitudinal developmental calcium FC study therefore provides a resource dataset to the field and identifies periods of dynamic change which cross-sectional studies may target for examination of disease states.

Intrinsic neuronal variability significantly limits information encoding in the primary visual cortex (V1). However, under certain conditions, neurons can respond reliably with highly precise responses to the same visual stimuli from trial to trial. This suggests that there exists intrinsic neural circuit mechanisms that dynamically modulate the intertrial variability of visual cortical neurons. Here, we sought to elucidate the role of different inhibitory interneurons (INs) in reliable coding in mouse V1. To study the interactions between somatostatin-expressing interneurons (SST-INs) and parvalbumin-expressing interneurons (PV-INs), we used a dual-color calcium imaging technique that allowed us to simultaneously monitor these two neural ensembles while awake mice, of both sexes, passively viewed natural movies. SST neurons were more active during epochs of reliable pyramidal neuron firing, whereas PV neurons were more active during epochs of unreliable firing. SST neuron activity lagged that of PV neurons, consistent with a feedback inhibitory SST→PV circuit. To dissect the role of this circuit in pyramidal neuron activity, we used temporally limited optogenetic activation and inactivation of SST and PV interneurons during periods of reliable and unreliable pyramidal cell firing. Transient firing of SST neurons increased pyramidal neuron reliability by actively suppressing PV neurons, a proposal that was supported by a rate-based model of V1 neurons. These results identify a cooperative functional role for the SST→PV circuit in modulating the reliability of pyramidal neuron activity.SIGNIFICANCE STATEMENTCortical neurons often respond to identical sensory stimuli with large variability. However, under certain conditions, the same neurons can also respond highly reliably. The circuit mechanisms that contribute to this modulation remain unknown. Here, we used novel dual-wavelength calcium imaging and temporally selective optical perturbation to identify an inhibitory neural circuit in visual cortex that can modulate the reliability of pyramidal neurons to naturalistic visual stimuli. Our results, supported by computational models, suggest that somatostatin interneurons increase pyramidal neuron reliability by suppressing parvalbumin interneurons via the inhibitory SST→PV circuit. These findings reveal a novel role of the SST→PV circuit in modulating the fidelity of neural coding critical for visual perception.

In all animals the optic tectum (OT) (or superior colliculi in mammals) provides guidance for external attention; this is not the only function of the tectum but is critically important for the development of behavioral visual reactions. In fish, the OT is the main primary visual center. It receives signals from most ganglion cells (GC) of different (known) types in the retina. Knowledge of the properties – both structural and physiological – of the neurons in the OT is important for understanding the mechanisms organizing behavior. We recorded extracellular electrical activity in the OT in living adult fish (Carassius auratus gibelio). Simultaneous recordings were made of the responses of retinal GC (from their axon terminals) and tectal neurons (TN), probably from cell bodies. Four types of TN are described with directional selectivity (DS) (henceforth these neurons are termed DS TN) at different (defined) depths of the OT. In addition to these, rare sporadic spikes lacking DS and arising on stimulation at any locus in a large area were consistently recorded simultaneously (superficially) with the responses of caudorostral DS GC with the electrode in one position. These are presumptively the responses of superficial tectal neurons (superficial inhibitory neurons, SIN). Various different types of stimulation were applied with the aim of obtaining clear SIN responses. Comparison of the results of our electrophysiological studies with published data (most studies in this direction have used calcium imaging in transparent Danio rerio fry) showed that DS TN were identical to glutamatergic periventricular interneurons in the OT, while SIN were identical to GABAergic inhibitory interneurons (SIN). These latter presumptively mediate detection of the main object (pop-out) in the field of vision.

Seizure initiation is the least understood and most disabling element of epilepsy. Studies of ictogenesis require high speed recordings at cellular resolution in the area of seizure onset. However, in vivo seizure onset areas cannot be determined at the level of resolution necessary to enable such studies. To circumvent these challenges, we used novel GCaMP7-based calcium imaging in the organotypic hippocampal slice culture model of post-traumatic epilepsy in mice. Organotypic hippocampal slice cultures generate spontaneous, recurrent seizures in a preparation in which it is feasible to image the activity of the entire network (with no unseen inputs existing). Chronic calcium imaging of the entire hippocampal network, with paired electrophysiology, revealed three patterns of seizure onset: (i) low amplitude fast activity; (ii) sentinel spike; and (iii) spike burst and low amplitude fast activity onset. These patterns recapitulate common features of human seizure onset, including low voltage fast activity and spike discharges. Weeks-long imaging of seizure activity showed a characteristic evolution in onset type and a refinement of the seizure onset zone. Longitudinal tracking of individual neurons revealed that seizure onset is stochastic at the single neuron level, suggesting that seizure initiation activates neurons in non-stereotyped sequences seizure to seizure. This study demonstrates for the first time that transitions to seizure are not initiated by a small number of neuronal ‘bad actors’ (such as overly connected hub cells), but rather by network changes which enable the onset of pathology among large populations of neurons.

Caenorhabditis elegans (C. elegans) is an ideal model organism for studying neuronal functions at the system level. This article develops a customized system for whole-body motor neuron calcium imaging of freely moving C. elegans without the coverslip pressed. Firstly, we proposed a fast centerline localization algorithm that could deal with most topology-variant cases costing only 6 ms for one frame, not only benefits for real-time localization but also for post-analysis. Secondly, we implemented a full-time two-axis synchronized motion strategy by adaptively adjusting the motion parameters of two motors in every short-term motion step (~50 ms). Following the above motion tracking configuration, the tracking performance of our system has been demonstrated to completely support the high spatiotemporal resolution calcium imaging on whole-body motor neurons of wild-type (N2) worms as well as two mutants (unc-2, unc-9), even the instantaneous speed of worm moving without coverslip pressed was extremely up to 400 μm/s.

Summary Stem cell transplantation shows enormous potential for treatment of incurable retinal degeneration (RD). To determine if and how grafts connect with the neural circuits of the advanced degenerative retina (ADR) and improve vision, we perform calcium imaging of GCaMP5-positive grafts in retinal slices. The organoid-derived C-Kit+/SSEA1− (C-Kit+) retinal progenitor cells (RPCs) become synaptically organized and build spontaneously active synaptic networks in three major layers of ADR. Light stimulation of the host photoreceptors elicits distinct neuronal responses throughout the graft RPCs. The graft RPCs and their differentiated offspring cells in inner nuclear layer synchronize their activities with the host cells and exhibit presynaptic calcium flux patterns that resemble intact retinal neurons. Once graft-to-host network is established, progressive vision loss is stabilized while control eyes continually lose vision. Therefore, transplantation of organoid-derived C-Kit+ RPCs can form functional synaptic networks within ADR and it holds promising avenue for advanced RD treatment., Highlights •C-Kit+ RPCs migrate and integrate in 3 major layers of advanced degenerative retina • Host inputs evoked by light activates graft neural response • 2-photon imaging visually defines graft-to-host synaptic Ca2+ transients in retina • Grafts connecting with the host retinal circuits preserves vision, In this article Liu, Yin, and colleagues show C-Kit+ retinal progenitor cells can migrate and integrate in three major layers of advanced degenerative retina, form functional synapses with the hosts, and connect with the neural circuits. They provide state-of-the-art techniques to visually demonstrate exceptional synaptic repair and vision restoration, opening up a feasible avenue for advanced RD treatment.

Auditory-cued goal-oriented behaviors requires the participation of cortical and subcortical brain areas, but how neural circuits associate sensory-based decisions with goal locations through learning remains poorly understood. The hippocampus is critical for spatial coding, suggesting its possible involvement in transforming sensory inputs to the goal-oriented decisions. Here, we developed an auditory discrimination task in which rats learned to navigate to goal locations based on the frequencies of auditory stimuli. Using in vivo calcium imaging in freely behaving rats over the course of learning, we found that dentate granule cells became more active, spatially tuned, and responsive to task-related variables as learning progressed. Furthermore, only after task learning, the activity of dentate granule cell ensembles represented the navigation path and predicts auditory decisions as early as when rats began to approach the goals. Finally, chemogenetic silencing of dentate gyrus suppressed task learning. Our results demonstrate that dentate granule cells gain task-relevant firing pattern through reinforcement learning and could be a potential link of sensory decisions to spatial navigation.

The present study focused on lithocholic acid (LCA), a secondary bile acid that contributes to cholestatic pruritus. Although recent studies have found that LCA acts on MAS-related G protein-coupled receptor family member X4 (MRGPRX4) in humans, it is unclear which subtypes of MRGPRs are activated by LCA in mice since there is no precise ortholog of human MRGPRX4 in the mouse genome. Using calcium imaging, we found that LCA could activate mouse Mrgpra1 when transiently expressed in HEK293T cells. Moreover, LCA similarly activates mouse Mrgprb2. Importantly, LCA-induced responses showed dose-dependent effects through Mrgpra1 and Mrgprb2. Moreover, treatment with QWF (an antagonist of Mrgpra1 and Mrgprb2), YM254890 (Gαq inhibitor), and U73122 (an inhibitor of phospholipase C) significantly suppressed the LCA-induced responses, implying that the LCA-induced responses are indeed mediated by Mrgpra1 and Mrgprb2. Furthermore, LCA activated primary cultures of mouse sensory neurons and peritoneal mast cells, suggesting that Mrgpra1 and Mrgprb2 contribute to LCA-induced pruritus. However, acute injection of LCA did not induce noticeable differences in scratching behavior, implying that the pruritogenic role of LCA may be marginal in non-cholestatic conditions. In summary, the present study identified for the first time that LCA can activate Mrgpra1 and Mrgprb2. The current findings provide further insight into the similarities and differences between human and mouse MRGPR families, paving a way to understand the complex roles of these pruriceptors.

Neuro-immune interaction during development is strongly implicated in the pathogenesis of neurodevelopmental disorders, but the mechanisms that cause neuronal circuit dysregulation are not well understood. We performed in vivo imaging of the developing retinotectal system in the larval zebrafish to characterize the effects of immune system activation on refinement of an archetypal sensory processing circuit. Acute inflammatory insult induced hyper-dynamic remodeling of developing retinal axons in larval fish and increased axon arbor elaboration over days. Using calcium imaging in GCaMP6s transgenic fish we showed that these morphological changes were accompanied by a shift toward decreased visual acuity in tectal cells. This finding was supported by poorer performance in a visually guided behavioral task. We further found that the pro-inflammatory cytokine, interleukin-1β (IL-1β) is upregulated by the inflammatory insult, and that down-regulation of IL-1β abrogated the effects of inflammation on axonal dynamics and growth. Moreover, baseline branching of the RGC arbors in IL-1β morphant animals was significantly different from that in control larvae, and their performance in a predation assay was impaired, indicating a role for this cytokine in normal neuronal development. This work establishes a simple and powerful non-mammalian model of developmental immune activation and demonstrates a role for IL-1β in mediating the pathological effects of inflammation on neuronal circuit development.SIGNIFICANCE STATEMENTMaternal immune activation (MIA) can increase the risk of neurodevelopmental disorders in offspring, however the mechanisms involved are not fully understood. Using a non-mammalian vertebrate model of developmental immune activation, we show that even brief activation of inflammatory pathways has immediate and long-term effects on the arborization of axons, and that these morphological changes have functional and behavioral consequences. Finally, we show that the pro-inflammatory cytokine IL-1β plays an essential role in both the effects of inflammation on circuit formation and normal axonal development. Our data add to a growing body of evidence supporting epidemiological studies linking immune activation to neurodevelopmental disorders, and help shed light on the molecular and cellular processes that contribute to the etiology of these disorders.

Adaptive behaviour necessitates the formation of memories for fearful events, but also that these memories can be extinguished. Effective extinction prevents excessive and persistent reactions to perceived threat, as can occur in anxiety and ‘trauma- and stressor-related’ disorders1. However, although there is evidence that fear learning and extinction are mediated by distinct neural circuits, the nature of the interaction between these circuits remains poorly understood2–6. Here, through a combination of in vivo calcium imaging, functional manipulations, and slice physiology, we show that distinct inhibitory clusters of intercalated neurons (ITCs) in the mouse amygdala exert diametrically opposed roles during the acquisition and retrieval of fear extinction memory. Furthermore, we find that the ITC clusters antagonize one another through mutual synaptic inhibition and differentially access functionally distinct cortical- and midbrain-projecting amygdala output pathways. Our findings show that the balance of activity between ITC clusters represents a unique regulatory motif that orchestrates a distributed neural circuitry, which in turn regulates the switch between high- and low-fear states. These findings suggest that the ITCs have a broader role in a range of amygdala functions and associated brain states that underpins the capacity to adapt to salient environmental demands. Distinct clusters of inhibitory neurons in the mouse amygdala perform opposing roles in fear extinction.

Impairment of episodic memory, a class of memory for spatiotemporal context of an event, is an early symptom of Alzheimer's disease. Both spatial and temporal information are encoded and represented in the hippocampal neurons, but how these representations are impaired under amyloid β (Aβ) pathology remains elusive. We performed chronic imaging of the hippocampus in awake male amyloid precursor protein (App) knock-in mice behaving in a virtual reality environment to simultaneously monitor spatiotemporal representations and the progression of Aβ depositions. We found that temporal representation is preserved, while spatial representation is significantly impaired in the App knock-in mice. This is due to the overall reduction of active place cells but not time cells, and compensatory hyperactivation of remaining place cells near Aβ aggregates. These results indicate the differential impact of Aβ aggregates on two major modalities of episodic memory, suggesting different mechanisms for forming and maintaining these two representations in hippocampus.SIGNIFICANCE STATEMENT:Spatiotemporal memory impairments are common at the early stage of Alzheimer's disease patients. We demonstrate the different impairment patterns of place and time cells in the dorsal hippocampus of head-fixed App knock-in mouse by in vivo two-photon calcium imaging over months under the virtual reality spatiotemporal tasks. These results highlight that place cells were preferentially and gradually damaged nearby Aβ aggregates, while time cells were less vulnerable. We further show these impairments were due to neuronal hyperactivity that occurs near the Aβ deposition. We suggest the differential and gradual impairment in two major modalities of episodic memory under Aβ pathology.

Background: Thyrotropin (TSH) is well known as the hormone of the anterior pituitary thyrotrophs responsible for acting in the thyroid gland, where it stimulates synthesis and release of thyroid hormones through G(s) and G(q/11) protein coupled TSH receptors (TSHRs). Methods: In this study, we examined whether the functional TSHRs are also expressed in cultured rat pituitary cells, using double immunocytochemistry, quantitative reverse transcription-polymerase chain reaction analysis, cAMP and hormone measurements, and single-cell calcium imaging. Results: Double immunocytochemistry revealed the expression of TSHRs in cultured corticotrophs and melanotrophs, in addition to previously identified receptors in folliculostellate cells. The functional coupling of these receptors to the G(q/11) signaling pathway was not observed, as demonstrated by the lack of TSH activation of IP(3)-dependent calcium mobilization in these cells when bathed in calcium-deficient medium. However, TSH increased cAMP production in a time- and concentration-dependent manner and facilitated calcium influx in single corticotrophs and melanotrophs, indicating their coupling to the G(s) signaling pathway. Consistent with these findings, TSH stimulated adrenocorticotropin and β-endorphin release in male and female pituitary cells in a time- and concentration-dependent manner without affecting the expression of proopiomelanocortin gene. Conclusions: These results indicate that TSH is a potential paracrine modulator of anterior pituitary corticotrophs and melanotrophs, controlling the exocytotic but not the transcriptional pathway in a cAMP/calcium influx-dependent manner.

Fast three-dimensional imaging of freely-swimming zebrafish is essential to understand the link between neuronal activity and behavioral changes during epileptic seizures. Studying the complex spatiotemporal patterns of neuronal activity at the whole-brain or -body level typically requires physical restraint, thus hindering the observation of unperturbed behavior. Here we report on real-time volumetric optoacoustic imaging of aberrant circular swimming activity and calcium transients in freely behaving zebrafish larvae, continuously covering their motion across an entire three-dimensional region. The high spatiotemporal resolution of the technique enables capturing ictal-like epileptic seizure events and quantifying their propagation speed, independently validated with simultaneous widefield fluorescence recordings. The work sets the stage for discerning functional interconnections between zebrafish behavior and neuronal activity for studying fundamental mechanisms of epilepsy and in vivo validation of treatment strategies., Frontiers in Molecular Neuroscience, 15, ISSN:1662-5099

Chordotonal neurons are responsible for sound sensation in Drosophila. However, little is known about how they respond to sound with high sensitivity. Using genetic labeling, we found one of the Drosophila axonemal dynein heavy chains, CG9492 (DNAH5), was specifically expressed in larval chordotonal neurons and showed a distribution restricted to proximal cilia. While DNAH5 mutation did not affect the cilium morphology or the trafficking of Inactive, a candidate auditory transduction channel, larvae with DNAH5 mutation had reduced startle responses to sound at low and medium intensities. Calcium imaging confirmed that DNAH5 functioned autonomously in chordotonal neurons for larval sound sensation. Furthermore, disrupting DNAH5 resulted in a decrease of spike firing responses to low-level sound in chordotonal neurons. Intriguingly, DNAH5 mutant larvae displayed an altered frequency tuning curve of the auditory organs. All together, our findings support a critical role of DNAH5 in tuning the frequency selectivity and the sound sensitivity of larval auditory neurons. SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12264-021-00631-w) contains supplementary material, which is available to authorized users.

Microglia proliferation is critical for proper development and function of the central nervous system (CNS), while dysregulation of proliferation contributes to pathology. We recently reported that male inducible nitric oxide synthase knockout (iNOS(−/−)) mice displayed significantly more proliferating microglia in their postnatal cortex than age-matched wildtype (WT) male mice. Moreover, nitric oxide (NO) signaling in mouse microglia greatly upregulates calcium entry through transient receptor potential vanilloid type 2 (TRPV2) channels. Considering that TRPV2 activity restricts astrocytic proliferation within glioma tissues, we investigated the roles of iNOS/NO signaling and TRPV2 expression in the regulation of microglial proliferation in vitro using assays of calcium imaging, immunocytochemistry, western blot, and polymerase chain reaction. Results showed that non-dividing microglia exhibited substantially higher expression of TRPV2 on the plasma membrane and significantly larger calcium influx through TRPV2 channels in comparison to dividing microglia. Additionally, non-dividing WT microglia exhibited significantly more NO production than dividing WT microglia. Furthermore, the NO-donor NOC18 increased the nuclear translocation of nuclear factor of activated T-cells cytoplasmic 2 (NFATC2) and the mRNA of the cyclin-dependent kinase inhibitor p21 and decreased the percentage of dividing WT and iNOS(–/–) microglia in culture. Importantly, the presence of the TRPV2 inhibitor tranilast abolished these effects of NOC18. Together, results from this study indicated that iNOS/NO signaling inhibits microglial proliferation through TRPV2-mediated calcium influx, nuclear translocation of the transcription factor NFATC2, and p21 expression. [Figure: see text]

Genetically encoded calcium indicators have proven useful for characterizing dorsal root ganglion neuron excitability in vivo. Challenges persist in achieving high spatial–temporal resolutions in vivo, however, due to deep tissue imaging and motion artifacts that may be limiting technical factors in obtaining measurements. Here we report an ex vivo imaging method, using a peripheral neuron-specific Advillin-GCaMP mouse line and electric field stimulation of dorsal root ganglion tissues, to assess the sensitivity of neurons en bloc. The described method rapidly characterizes Ca(2+) activity in hundreds of dorsal root ganglion neurons (221 ± 64 per dorsal root ganglion) with minimal perturbation to the in situ soma environment. We further validate the method for use as a drug screening platform with the voltage-gated sodium channel inhibitor, tetrodotoxin. Drug treatment led to decreased evoked Ca(2+) activity; half-maximal response voltage (EV(50)) increased from 13.4 V in untreated tissues to 21.2, 23.3, 51.5 (p < 0.05), and 60.6 V (p < 0.05) at 0.01, 0.1, 1, and 10 μM doses, respectively. This technique may help improve an understanding of neural signaling while retaining tissue structural organization and serves as a tool for the rapid ex vivo recording and assessment of neural activity.

Olfactory dysfunctions, including hyposmia and anosmia, affect ~100 million people around the world and the underlying causes are not fully understood. Degeneration of olfactory sensory neurons and incapacity of globose basal cells to generate olfactory sensory neurons are found in elder people and patients with smell disorders. Thus, olfactory stem cell may function as a promising tool to replace inactivated globose basal cells and to generate sensory neurons. Methods: We established clonal expansion of cells from the murine olfactory epithelium as well as colony growth from human olfactory mucosa using Matrigel-based three-dimensional system. These colonies were characterized by immunostaining against olfactory epithelium cellular markers and by calcium imaging of responses to odors. Chemical addition was optimized to promote Lgr5 expression, colony growth and sensory neuron generation, tested by quantitative PCR and immunostaining against progenitor and neuronal markers. The differential transcriptomes in multiple signaling pathways between colonies under different base media and chemical cocktails were determined by RNA-Seq. Results: In defined culture media, we found that VPA and CHIR99021 induced the highest Lgr5 expression level, while LY411575 resulted in the most abundant yield of OMP+ mature sensory neurons in murine colonies. Different base culture media with drug cocktails led to apparent morphological alteration from filled to cystic appearance, accompanied with massive transcriptional changes in multiple signaling pathways. Generation of sensory neurons in human colonies was affected through TGF-β signaling, while Lgr5 expression and cell proliferation was regulated by VPA. Conclusion: Our findings suggest that targeting expansion of olfactory epithelium/mucosa colonies in vitro potentially results in discovery of new source to cell replacement-based therapy against smell loss.

Microglia have crucial roles in sculpting synapses and maintaining neural circuits during development. To test the hypothesis that microglia continue to regulate neural circuit connectivity in adult brain, we have investigated the effects of chronic microglial depletion, via CSF1R inhibition, on synaptic connectivity in the visual cortex in adult mice of both sexes. We find that the absence of microglia dramatically increases both excitatory and inhibitory synaptic connections to excitatory cortical neurons assessed with functional circuit mapping experiments in acutely prepared adult brain slices. Microglia depletion leads to increased densities and intensities of perineuronal nets. Furthermore,in vivocalcium imaging across large populations of visual cortical neurons reveals enhanced neural activities of both excitatory neurons and parvalbumin-expressing interneurons in the visual cortex following microglia depletion. These changes recover following adult microglia repopulation. In summary, our new results demonstrate a prominent role of microglia in sculpting neuronal circuit connectivity and regulating subsequent functional activity in adult cortex.SIGNIFICANCE STATEMENTMicroglia are the primary immune cell of the brain, but recent evidence supports that microglia play an important role in synaptic sculpting during development. However, it remains unknown whether and how microglia regulate synaptic connectivity in adult brain. Our present work shows chronic microglia depletion in adult visual cortex induces robust increases in perineuronal nets, and enhances local excitatory and inhibitory circuit connectivity to excitatory neurons. Microglia depletion increasesin vivoneural activities of both excitatory neurons and parvalbumin inhibitory neurons. Our new results reveal new potential avenues to modulate adult neural plasticity by microglia manipulation to better treat brain disorders, such as Alzheimer's disease.

Mural cells (smooth muscle cells and pericytes) are integral components of brain blood vessels that play important roles in vascular formation, blood-brain barrier maintenance, and regulation of regional cerebral blood flow (rCBF). These cells are implicated in conditions ranging from developmental vascular disorders to age-related neurodegenerative diseases. Here we present complementary tools for cell labeling with transgenic mice and organic dyes that allow high-resolution intravital imaging of the different mural cell subtypes. We also provide detailed methodologies for imaging of spontaneous and neural activity-evoked calcium transients in mural cells. In addition, we describe strategies for single- and two-photon optogenetics that allow manipulation of the activity of individual and small clusters of mural cells. Together with measurements of diameter and flow in individual brain microvessels, calcium imaging and optogenetics allow the investigation of pericyte and smooth muscle cell physiology and their role in regulating rCBF. We also demonstrate the utility of these tools to investigate mural cells in the context of Alzheimer's disease and cerebral ischemia mouse models. Thus, these methods can be used to reveal the functional and structural heterogeneity of mural cells in vivo, and allow detailed cellular studies of the normal function and pathophysiology of mural cells in a variety of disease models. The implementation of this protocol can take from several hours to days depending on the intended applications.

Orientation tuning is a fundamental response property of V1 neurons and has been extensively studied with single-/multiunit recording and intrinsic signal optical imaging. Long-term 2-photon calcium imaging allows simultaneous recording of hundreds of neurons at single neuron resolution over an extended time in awake macaques, which may help elucidate V1 orientation tuning properties in greater detail. We used this new technology to study the microstructures of orientation functional maps, as well as population tuning properties, in V1 superficial layers of 5 awake macaques. Cellular orientation maps displayed horizontal and vertical clustering of neurons according to orientation preferences, but not tuning bandwidths, as well as less frequent pinwheels than previous estimates. The orientation tuning bandwidths were narrower than previous layer-specific single-unit estimates, suggesting more precise orientation selectivity. Moreover, neurons tuned to cardinal and oblique orientations did not differ in quantities and bandwidths, likely indicating minimal V1 representation of the oblique effect. Our experimental design also permitted rough estimates of length tuning. The results revealed significantly more end-stopped cells at a more superficial 150 μm depth (vs. 300 μm), but unchanged orientation tuning bandwidth with different length tuning. These results will help construct more precise models of V1 orientation processing.

Neurons in the visual system can be spatially organized according to their response properties such as receptive field location and feature selectivity. For example, the visual cortex of many mammalian species contains orientation and direction columns where neurons with similar preferences are clustered. Here, we examine whether such a columnar structure exists in the mouse superior colliculus (SC), a prominent visual center for motion processing. By performing large-scale physiological recording and two-photon calcium imaging in adult male and female mice, we show that direction-selective neurons in the mouse SC are not organized into stereotypical columns as a function of their preferred directions, although clusters of similarly tuned neurons are seen in a minority of mice. Nearby neurons can prefer similar or opposite directions in a largely position-independent manner. This finding holds true regardless of animal state (anesthetized vs awake, running vs stationary), SC depth (most superficial lamina vs deeper in the SC), research technique (calcium imaging vs electrophysiology), and stimulus type (drifting gratings vs moving dots, full field vs small patch). Together, these results challenge recent reports of region-specific organizations in the mouse SC and reveal how motion direction is represented in this important visual center.

There has been considerable research showing populations of neurons encoding for different aspects of space in the brain. Recently, several studies using two-photon calcium imaging and virtual navigation have identified “spatially” modulated neurons in the posterior cortex. We enquire here whether the presence of such spatial representations may be a cortex-wide phenomenon and, if so, whether these representations can be organized in the absence of the hippocampus. To this end, we imaged the dorsal cortex of mice running on a treadmill populated with tactile cues. A high percentage (40–80%) of the detected neurons exhibited sparse, spatially localized activity, with activity fields uniformly localized over the track. The development of this location specificity was impaired by hippocampal damage. Thus, there is a substantial population of neurons distributed widely over the cortex that collectively form a continuous representation of the explored environment, and hippocampal outflow is necessary to organize this phenomenon.SIGNIFICANCE STATEMENTIncreasing evidence points to the role of the neocortex in encoding spatial information. Whether this feature is linked to hippocampal functions is largely unknown. Here, we systematically surveyed multiple regions in the dorsal cortex of the same animal for the presence of signals encoding for spatial position. We described populations of cortical neurons expressing sequential patterns of activity localized in space in primary, secondary, and associational areas. Furthermore, we showed that the formation of these spatial representations was impacted by hippocampal lesion. Our results indicate that hippocampal inputs are necessary to maintain a precise cortical representation of space.

To obtain insights into striatal neural processes underlying reward-based learning and movement control, we examined spatial organizations of striatal neurons related to movement and reward-based learning. For this, we recorded the activity of direct- and indirect-pathway neurons (D1 and A2a receptor-expressing neurons, respectively) in mice engaged in probabilistic classical conditioning and open-field free exploration. We found broadly organized functional clusters of striatal neurons in the direct as well as indirect pathways for both movement- and reward-related variables. Functional clusters for different variables were partially overlapping in both pathways, but the overlap between outcome- and value-related functional clusters was greater in the indirect than direct pathway. Also, value-related spatial clusters were progressively refined during classical conditioning. Our study shows the broad and learning-dependent spatial organization of functional clusters of dorsal striatal neurons in the direct and indirect pathways. These findings further argue against the classic model of the basal ganglia and support the importance of spatiotemporal patterns of striatal neuronal ensemble activity in the control of behavior.

Background: Dysregulated signaling in neurons and astrocytes participates in pathophysiological alterations seen in the Alzheimer’s disease brain, including increases in amyloid-β, hyperphosphorylated tau, inflammation, calcium dysregulation, and oxidative stress. These are often noted prior to the development of behavioral, cognitive, and non-cognitive deficits. However, the extent to which these pathological changes function together or independently is unclear. Objective: Little is known about the temporal relationship between calcium dysregulation and oxidative stress, as some reports suggest that dysregulated calcium promotes increased formation of reactive oxygen species, while others support the opposite. Prior work has quantified several key outcome measures associated with oxidative stress in aldehyde dehydrogenase 2 knockout (Aldh2–/–) mice, a non-transgenic model of sporadic Alzheimer’s disease. Methods: Here, we tested the hypothesis that early oxidative stress can promote calcium dysregulation across aging by measuring calcium-dependent processes using electrophysiological and imaging methods and focusing on the afterhyperpolarization (AHP), synaptic activation, somatic calcium, and long-term potentiation in the Aldh2–/– mouse. Results: Our results show a significant age-related decrease in the AHP along with an increase in the slow AHP amplitude in Aldh2–/– animals. Measures of synaptic excitability were unaltered, although significant reductions in long-term potentiation maintenance were noted in the Aldh2–/– animals compared to wild-type. Conclusion: With so few changes in calcium and calcium-dependent processes in an animal model that shows significant increases in HNE adducts, Aβ, p-tau, and activated caspases across age, the current findings do not support a direct link between neuronal calcium dysregulation and uncontrolled oxidative stress.

Introduction Cannabidiol (CBD) is reported to produce pain relief, but the clinically relevant cellular and molecular mechanisms remain uncertain. The TRPV1 receptor integrates noxious stimuli and plays a key role in pain signaling. Hence, we conducted in vitro studies, to elucidate the efficacy and mechanisms of CBD for inhibiting neuronal hypersensitivity in cultured rat sensory neurons, following activation of TRPV1. Methods Adult rat dorsal root ganglion (DRG) neurons were cultured and supplemented with the neurotrophic factors NGF and GDNF, in an established model of neuronal hypersensitivity. Neurons were stimulated with CBD (Adven 150, EMMAC Life Sciences) at 1, 10, 100 nMol/L and 1, 10 and 50 µMol/L, 48 h after plating. In separate experiments, DRG neurons were also stimulated with capsaicin with or without CBD (1 nMol/L to10 µMol/L), in a functional calcium imaging assay. The effects of the adenylyl cyclase activator forskolin and the calcineurin inhibitor cyclosporin were determined. We also measured forskolin-stimulated cAMP levels, without and after treatment with CBD, using a homogenous time-resolved fluorescence (HTRF) assay. The results were analysed using Mann-Whitney test. Results DRG neurons treated with 10 and 50 µMol/L CBD showed calcium influx, but not at lower doses. Neurons treated with capsaicin demonstrated robust calcium influx, which was dose-dependently reduced in the presence of low dose CBD (IC50 = 100 nMol/L). The inhibition or desensitization by CBD was reversed in the presence of forskolin and cyclosporin. Forskolin-stimulated cAMP levels were significantly reduced in CBD treated neurons. Conclusion CBD at low doses corresponding to plasma concentrations observed physiologically inhibits or desensitizes neuronal TRPV1 signalling by inhibiting the adenylyl cyclase - cAMP pathway, which is essential for maintaining TRPV1 phosphorylation and sensitization. CBD also facilitated calcineurin-mediated TRPV1 inhibition. These mechanisms may underlie nociceptor desensitization and the therapeutic effect of CBD in animal models and patients with acute and chronic pain.

Stress is a physiological response that promotes maintenance of balance against harmful stimuli. Unfortunately, chronic activation of stress systems facilitates the development of psychiatric disorders. A stress-mediated hypercholinergic state could underlie this facilitation, as cholinergic mechanisms have been suggested to play a role in anxiety, depression, and substance use disorder (SUD). Stimulation by stress hormones, urocortin (Ucn1) or corticotropin-releasing factor (CRF), of the CRF receptor type 1 (CRFR1) of acetylcholine-containing neurons of the laterodorsal tegmental nucleus (LDT) could be involved in modulation of cholinergic transmission during periods of stress hormone activation, which could play a role in psychiatric disorders as cholinergic LDT neurons project to, and control activity in, mood-, arousal- and SUD-controlling regions. The present study investigated for the first time the membrane effects and intracellular outcomes of CRFR1 activation by endogenous stress hormones on LDT neurons. Patch clamp recordings of immunohistochemically-identified cholinergic and non-cholinergic LDT neurons with concurrent calcium imaging were used to monitor cellular responses to CRFR1 stimulation with Ucn1 and CRF. Postsynaptically-mediated excitatory currents were elicited in LDT cholinergic neurons, accompanied by an enhancement in synaptic events. In addition, CRFR1 activation resulted in rises in intracellular calcium levels. CRFR1 stimulation recruited MAPK/ERK and SERCA-ATPase involved pathways. The data presented here provide the first evidence that Ucn1 and CRF exert pre and postsynaptic excitatory membrane actions on LDT cholinergic neurons that could underlie the hypercholinergic state associated with stress which could play a role in the heightened risk of psychiatric disorders associated with a chronic stress state.

We used a novel microendoscope system to record simultaneously optical activity (fluorescence of a calcium indicator dye) and electrical activity (multi-unit activity and local field potentials) from the dorsal inferior colliculus of the echolocating bat, Carollia perspicillata. Optically recorded calcium responses to wide-band noise and to frequency-modulated bursts were recorded at probe depths down to 1300 µm, with the majority of active sites encountered at more shallow depths down to 800 µm. Calcium activity exhibited long latencies, within the time span of 50-100 ms after stimulus onset, significantly longer than onset latencies of either multi-unit activity or local field potentials. Latencies and amplitude/latency trading of these electrical responses were consistent with those seen in standard electrophysiological recordings, confirming that the microendoscope was able to record both neural and optical activity successfully. Optically recorded calcium responses rose and decayed slowly and were correlated in time with long-latency negative deflections in local field potentials. These data suggest that calcium-evoked responses may reflect known, sustained inhibitory interactions in the inferior colliculus.

To identify odors, the mammalian nose deploys hundreds of olfactory receptors (ORs) from the rhodopsin-like class of the G protein-coupled receptor superfamily. Odorants having multiple rotatable bonds present a problem for the stereochemical shape-based matching process assumed to govern the sense of smell through OR-odorant recognition. We conformationally restricted the carbon chain of the odorant octanal to ask whether an OR can respond differently to different odorant conformations. By using calcium imaging to monitor signal transduction in sensory neurons expressing the mouse aldehyde OR, Olfr2, we found that the spatial position of the C7 and C8 carbon atoms of octanal, in relation to its -CHO group, determines whether an aliphatic aldehyde functions as an agonist, partial agonist or antagonist. Our experiments provide evidence that an odorant can manipulate an OR through its intrinsic conformational repertoire, in unexpected analogy to the photon-controlled aldehyde manipulation observed in rhodopsin.

Perception of visual motion is important for a range of ethological behaviors in mammals. In primates, specific visual cortical regions are specialized for processing of coherent visual motion. However, whether mouse visual cortex has a similar organization remains unclear, despite powerful genetic tools available for measuring population neural activity. Here, we use widefield and 2-photon calcium imaging of transgenic mice to measure mesoscale and cellular responses to coherent motion. Imaging of primary visual cortex (V1) and higher visual areas (HVAs) during presentation of natural movies and random dot kinematograms (RDKs) reveals varied responsiveness to coherent motion, with stronger responses in dorsal stream areas compared to ventral stream areas. Moreover, there is considerable anisotropy within visual areas, such that neurons representing the lower visual field are more responsive to coherent motion. These results indicate that processing of visual motion in mouse cortex is distributed heterogeneously both across and within visual areas., Processing of coherent motion has been extensively studied in the primate visual system, but has not been well characterized in mice. Here, the authors use widefield calcium imaging to reveal that coherent motion responses are organized anisotropically both across and within visual areas in mice.

Duplications of MECP2-containing genomic segments led to severe autistic symptoms in male. Transgenic mice overexpressing the human MECP2 gene exhibit autistic-like behaviors. Neural circuits underlying social defects in MECP2 transgenic (MECP2-TG) mice remain unknown. To observe neural activity of MECP2-TG mice in vivo, we performed calcium imaging by implantation of microendoscope in the hippocampal CA1 regions of MECP2-TG and wild type (WT) mice. We identified neurons whose activities were tightly associated with social interaction, which activity patterns were compromised in MECP2-TG mice. Strikingly, we rescued the social-related neural activity in CA1 and social defects in MECP2-TG mice by deleting the human MECP2 transgene using the CRISPR/Cas9 method during adulthood. Our data points to the neural circuitry responsible for social interactions and provides potential therapeutic targets for autism in adulthood.

The bed nucleus of the stria terminalis (BNST) plays a critical role in regulating anxiety, yet the involved specific cell types and their connections functioning in anxiety-related behaviors remains elusive. Here we identified two cell subpopulations—corticotropin-releasing hormone-positive (CRH+) and protein kinase C-δ-positive (PKC-δ+) neurons—each displayed discrete emotionally valenced behaviors in the anterior-dorsal BNST (adBNST). Using whole-cell patch-clamp recordings and virus-assisted circuit tracing techniques, we delineated the local and long-range connectivity networks in a cell-type-specific manner. The results show that the CRH+ and PKC-δ+ neurons received inputs from similar brain regions and exhibited significant differences in the downstream projection density. In addition, in vivo calcium imaging as well as gain- and loss-of-function studies characterized the physiological response properties and the functional heterogeneities in modulating anxiety, further suggesting the similarity and individuality between the two adBNST cell types. These results provide novel insights into the circuit architecture of adBNST neurons underlying the functionally specific neural pathways that relate to anxiety disorders.

Hippocampus receives dense serotonergic input specifically from raphe nuclei. However, what information is carried by this input and its impact on behavior has not been fully elucidated. Here we used in vivo two-photon imaging of activity of hippocampal median raphe projection fibers in behaving male and female mice and identified two distinct populations: one linked to reward delivery and the other to locomotion. Local optogenetic manipulation of these fibers confirmed a functional role for these projections in the modulation of reward-induced behavior. The diverse function of serotonergic inputs suggests a key role in integrating locomotion and reward information into the hippocampal CA1.SIGNIFICANCE STATEMENT Information constantly flows in the hippocampus, but only some of it is captured as a memory. One potential process that discriminates which information should be remembered is concomitance with reward. In this work, we report a neuromodulatory pathway, which delivers reward signal as well as locomotion signal to the hippocampal CA1. We found that the serotonergic system delivers heterogeneous input that may be integrated by the hippocampus to support its mnemonic functions. It is dynamically involved in regulating behavior through interaction with the hippocampus. Our results suggest that the serotonergic system interacts with the hippocampus in a dynamic and behaviorally specific manner to regulate reward-related information processing. Ministry of Education (MOE) National Medical Research Council (NMRC) Published version A.L. received a Japan Society for the Promotion of Science Postdoctoral Fellowship for Foreign Researchers and Japan Society for the Promotion of Science Grant-in-Aid for Japan Society for the Promotion of ScienceFellows (15F14409). This work was supported by RIKEN, RIKEN Presidents Fund, Grant-in-Aid for Scientific Research (A, 20240032, 16H02455) and for Scientific Research on Innovative Area “Foundation of Synapse and Neurocircuit Pathology” (22110006) and “Constructive understanding of multi-scale dynamism of neuropsychiatric disorders” (18H05434) from the MEXT, Japan, Human Frontier Science Program, Uehara Memorial Foundation, Naito Foundation, Taked Science Foundation, Japan Foundation for Applied Enzymology, Novartis Foundation (Japan) for the Promotion of Science, Research Foundation for Opto-Scienceand Technology, and Brain Science Foundation to Y.H. This work was also supported by Ministry of Education, Singapore, AcRF Tier 2 (MOE2015-T2-2-035), and National Medical Research Council, Singapore, OF-IRG(NMRC/OFIRG/0046/2017-00). We thank Drs. Kota Tamada and Toru Takumi for sharing unpublished data and materials.

Pair bond formation depends vitally on neuromodulatory signaling within the nucleus accumbens, but the neuronal dynamics underlying this behavior remain unclear. Using in vivo Ca2+ imaging in monogamous prairie voles, we found that pair bonding does not elicit differences in overall nucleus accumbens Ca2+ activity. Instead, we identified distinct neuronal ensembles in this region recruited during approach to either a partner or novel vole. The partner-approach neuronal ensemble increased in size following bond formation and differences in the size of approach ensembles for partner and novel voles predicts bond strength. In contrast, neurons comprising departure ensembles do not change over time and are not correlated with bond strength indicating that ensemble plasticity is specific to partner approach. Further, the neurons comprising partner and novel approach ensembles are non-overlapping while departure ensembles are more overlapping than chance, which may reflect another key feature of approach ensembles. We posit that the features of the partner approach ensemble and its expansion upon bond formation make it a potential key substrate underlying bond formation and maturation.HighlightsWe performed in vivo Ca2+ in the nucleus accumbens of pair bonded prairie volesOverall nucleus accumbens activity did not differ during partner versus stranger interactionDistinct approach neurons exist for the partner and for the strangerPartner-approach ensemble increases as partner preference emergesWe identify a putative neuronal substrate underlying bond formation and maturation

G protein–coupled receptor signaling is required for the navigation of immune cells along chemoattractant gradients. However, chemoattractant receptors may couple to more than one type of heterotrimeric G protein, each of which consists of a Gα, Gβ, and Gγ subunit, making it difficult to delineate the critical signaling pathways. Here, we used knockout mouse models and time-lapse microscopy to elucidate Gα and Gβ subunits contributing to complement C5a receptor-mediated chemotaxis. Complement C5a-mediated chemokinesis and chemotaxis were almost completely abolished in macrophages lacking Gnai2 (encoding Gα(i2)), consistent with a reduced leukocyte recruitment previously observed in Gnai2(−/−) mice, whereas cells lacking Gnai3 (Gα(i3)) exhibited only a slight decrease in cell velocity. Surprisingly, C5a-induced Ca(2+) transients and lamellipodial membrane spreading were persistent in Gnai2(−/−) macrophages. Macrophages lacking both Gnaq (Gα(q)) and Gna11 (Gα(11)) or both Gna12 (Gα(12)) and Gna13 (Gα(13)) had essentially normal chemotaxis, Ca(2+) signaling, and cell spreading, except Gna12/Gna13-deficient macrophages had increased cell velocity and elongated trailing ends. Moreover, Gnaq/Gna11-deficient cells did not respond to purinergic receptor P2Y(2) stimulation. Genetic deletion of Gna15 (Gα(15)) virtually abolished C5a-induced Ca(2+) transients, but chemotaxis and cell spreading were preserved. Homozygous Gnb1 (Gβ(1)) deletion was lethal, but mice lacking Gnb2 (Gβ(2)) were viable. Gnb2(−/−) macrophages exhibited robust Ca(2+) transients and cell spreading, albeit decreased cell velocity and impaired chemotaxis. In summary, complement C5a-mediated chemotaxis requires Gα(i2) and Gβ(2), but not Ca(2+) signaling, and membrane protrusive activity is promoted by G proteins that deplete phosphatidylinositol 4,5-bisphosphate.

Trajectory-dependent splitter neurons in the hippocampus encode information about a rodent’s prior trajectory during performance of a continuous alternation task. As such, they provide valuable information for supporting memory-guided behavior. Here, we employed single-photon calcium imaging in freely moving mice to investigate the emergence and fate of trajectory-dependent activity through learning and mastery of a continuous spatial alternation task. In agreement with others, the quality of trajectory-dependent information in hippocampal neurons correlated with task performance. We thus hypothesized that, due to their utility, splitter neurons would exhibit heightened stability. We find that splitter neurons were more likely to remain active and retained more consistent spatial information across multiple days than other neurons. Furthermore, we find that both splitter neurons and place cells emerged rapidly and maintained stable trajectory-dependent/spatial activity thereafter. Our results suggest that neurons with useful functional coding exhibit heightened stability to support memory guided behavior., Trajectory-coding neurons in the hippocampus convey important information for performing memory tasks. Here, Kinsky et al. track long-term neural activity in the hippocampus to find that trajectory-coding emerges rapidly and remains stable across long time-scales.

Smoking during pregnancy is associated with deleterious physiological and cognitive effects on the offspring, which are likely due to nicotine-induced alteration in the development of neurotransmitter systems. Prenatal nicotine exposure (PNE) in rodents is associated with changes in behaviors controlled in part by the pontine laterodorsal tegmentum (LDT), and LDT excitatory signaling is altered in a sex and age-dependent manner by PNE. As effects on GABAergic LDT signaling are unknown, we used calcium imaging to evaluate GABAA receptor- (GABAA R as well as GABAA -ρ R) and GABAB receptor (GABAB R)-mediated calcium responses in LDT brain slices from female and male PNE mice in two different age groups. Overall, in older PNE females, changes in calcium induced by stimulation of GABAA R and GABAB R, including GABAA -ρ R were shifted toward calcium rises. In both young and old males, PNE was associated with alterations in calcium mediated by all three receptors; however, the GABAA R was the most affected. These results show for the first time that PNE is associated with alterations in GABAergic transmission in the LDT in a sex- and age-dependent manner, and these data are the first to show PNE-associated alterations in functionality of GABA receptors in any nucleus. PNE-associated alterations in LDT GABAergic transmission within the LDT would be expected to alter output to target regions and could play a role in LDT-implicated, negative behavioral outcomes following gestational exposure to smoking. Accordingly, our data provide further supportive evidence of the importance of eliminating the consumption of nicotine during pregnancy.

Human pluripotent stem cells (hPSCs) are a powerful tool for modelling human development. In recent years, hPSCs have become central in cell-based therapies for neurodegenerative diseases given their potential to replace affected neurons. However, directing hPSCs into specific neuronal types is complex and requires an accurate protocol that mimics endogenous neuronal development. Here we describe step-by-step a fast feeder-free neuronal differentiation protocol to direct hPSCs to mature forebrain neurons in 37 days in vitro (DIV). The protocol is based upon a combination of specific morphogens, trophic and growth factors, ions, neurotransmitters and extracellular matrix elements. A human-induced PSC line (Ctr-Q33) and a human embryonic stem cell line (GEN-Q18) were used to reinforce the potential of the protocol. Neuronal activity was analysed by single-cell calcium imaging. At 8 DIV, we obtained a homogeneous population of hPSC-derived neuroectodermal progenitors which self-arranged in bi-dimensional neural tube-like structures. At 16 DIV, we generated hPSC-derived neural progenitor cells (NPCs) with mostly a subpallial identity along with a subpopulation of pallial NPCs. Terminal in vitro neuronal differentiation was confirmed by the expression of microtubule associated protein 2b (Map 2b) by almost 100% of hPSC-derived neurons and the expression of specific-striatal neuronal markers including GABA, CTIP2 and DARPP-32. HPSC-derived neurons showed mature and functional phenotypes as they expressed synaptic markers, voltage-gated ion channels and neurotransmitter receptors. Neurons displayed diverse spontaneous activity patterns that were classified into three major groups, namely “high”, “intermediate” and “low” firing neurons. Finally, transplantation experiments showed that the NPCs survived and differentiated within mouse striatum for at least 3 months. NPCs integrated host environmental cues and differentiated into striatal medium-sized spiny neurons (MSNs), which successfully integrated into the endogenous circuitry without teratoma formation. Altogether, these findings demonstrate the potential of this robust human neuronal differentiation protocol, which will bring new opportunities for the study of human neurodevelopment and neurodegeneration, and will open new avenues in cell-based therapies, pharmacological studies and alternative in vitro toxicology. Electronic supplementary material The online version of this article (10.1007/s12035-020-01907-4) contains supplementary material, which is available to authorized users.