Analysis Of Neural Circuits Research Articles

HI-FISH: WHOLE BRAIN IN SITU MAPPING OF NEURONAL ACTIVATION IN DROSOPHILA DURING SOCIAL BEHAVIORS AND OPTOGENETIC STIMULATION.

Monitoring neuronal activity at single-cell resolution in freely moving Drosophila engaged in social behaviors is challenging because of their small size and lack of transparency. Extant methods, such as Flyception, are highly invasive. Whole-brain calcium imaging in head-fixed, walking flies is feasible but the animals cannot perform the consummatory phases of social behaviors like aggression or mating under these conditions. This has left open the fundamental question of whether neurons identified as functionally important for such behaviors using loss- or gain-of-function screens are actually active during the natural performance of such behaviors, and if so during which phase(s). Here we perform brain-wide mapping of active cells expressing the Immediate Early Gene hr38 using a high-sensitivity/low background FISH amplification method called HCR-3.0. Using double-labeling for hr38 mRNA and for GFP, we describe the activity of several classes of aggression-promoting neurons during courtship and aggression, including P1a cells, an intensively studied population of male-specific interneurons. Using HI-FISH in combination with optogenetic activation of aggression-promoting neurons (opto-HI-FISH) we identify candidate downstream functional targets of these cells in a brain-wide, unbiased manner. Finally we compare the activity of P1a neurons during sequential performance of courtship and aggression, using intronic vs. exonic hr38 probes to differentiate newly synthesized nuclear transcripts from cytoplasmic transcripts synthesized at an earlier time. These data provide evidence suggesting that different subsets of P1a neurons may be active during courtship vs. aggression. HI-FISH and associated methods may help to fill an important lacuna in the armamentarium of tools for neural circuit analysis in Drosophila.

EDTP enhances and protects the fluorescent signal of GFP in cleared and expanded tissues

Advanced 3D high-resolution imaging techniques are essential for investigating biological challenges, such as neural circuit analysis and tumor microenvironment in intact tissues. However, the fluorescence signal emitted by endogenous fluorescent proteins in cleared or expanded biological samples gradually diminishes with repeated irradiation and prolonged imaging, compromising its ability to accurately depict the underlying scientific problem. We have developed a strategy to preserve fluorescence in cleared and expanded tissue samples during prolonged high-resolution three-dimensional imaging. We evaluated various compounds at different concentrations to determine their ability to enhance fluorescence intensity and resistance to photobleaching while maintaining the structural integrity of the tissue. Specifically, we investigated the impact of EDTP utilization on GFP, as it has been observed to significantly improve fluorescence intensity, resistance to photobleaching, and maintain fluorescence during extended room temperature storage. This breakthrough will facilitate extended hydrophilic and hydrogel-based clearing and expansion methods for achieving long-term high-resolution 3D imaging of cleared biological tissues by effectively safeguarding fluorescent proteins within the tissue.

Simultaneous removal of noise and correction of motion warping in neuron calcium imaging using a pipeline structure of self-supervised deep learning models.

Calcium imaging is susceptible to motion distortions and background noises, particularly for monitoring active animals under low-dose laser irradiation, and hence unavoidably hinder the critical analysis of neural functions. Current research efforts tend to focus on either denoising or dewarping and do not provide effective methods for videos distorted by both noises and motion artifacts simultaneously. We found that when a self-supervised denoising model of DeepCAD [Nat. Methods18, 1359 (2021)10.1038/s41592-021-01225-0] is used on the calcium imaging contaminated by noise and motion warping, it can remove the motion artifacts effectively but with regenerated noises. To address this issue, we develop a two-level deep-learning (DL) pipeline to dewarp and denoise the calcium imaging video sequentially. The pipeline consists of two 3D self-supervised DL models that do not require warp-free and high signal-to-noise ratio (SNR) observations for network optimization. Specifically, a high-frequency enhancement block is presented in the denoising network to restore more structure information in the denoising process; a hierarchical perception module and a multi-scale attention module are designed in the dewarping network to tackle distortions of various sizes. Experiments conducted on seven videos from two-photon and confocal imaging systems demonstrate that our two-level DL pipeline can restore high-clarity neuron images distorted by both motion warping and background noises. Compared to typical DeepCAD, our denoising model achieves a significant improvement of approximately 30% in image resolution and up to 28% in signal-to-noise ratio; compared to traditional dewarping and denoising methods, our proposed pipeline network recovers more neurons, enhancing signal fidelity and improving data correlation among frames by 35% and 60% respectively. This work may provide an attractive method for long-term neural activity monitoring in awake animals and also facilitate functional analysis of neural circuits.

Kalium channelrhodopsins effectively inhibit neurons

The analysis of neural circuits has been revolutionized by optogenetic methods. Light-gated chloride-conducting anion channelrhodopsins (ACRs)—recently emerged as powerful neuron inhibitors. For cells or sub-neuronal compartments with high intracellular chloride concentrations, however, a chloride conductance can have instead an activating effect. The recently discovered light-gated, potassium-conducting, kalium channelrhodopsins (KCRs) might serve as an alternative in these situations, with potentially broad application. As yet, KCRs have not been shown to confer potent inhibitory effects in small genetically tractable animals. Here, we evaluated the utility of KCRs to suppress behavior and inhibit neural activity in Drosophila, Caenorhabditiselegans, and zebrafish. In direct comparisons with ACR1, a KCR1 variant with enhanced plasma-membrane trafficking displayed comparable potency, but with improved properties that include reduced toxicity and superior efficacy in putative high-chloride cells. This comparative analysis of behavioral inhibition between chloride- and potassium-selective silencing tools establishes KCRs as next-generation optogenetic inhibitors for in vivo circuit analysis in behaving animals.

Development of wafer-scale multifunctional nanophotonic neural probes for brain activity mapping.

Optical techniques, such as optogenetic stimulation and functional fluorescence imaging, have been revolutionary for neuroscience by enabling neural circuit analysis with cell-type specificity. To probe deep brain regions, implantable light sources are crucial. Silicon photonics, commonly used for data communications, shows great promise in creating implantable devices with complex optical systems in a compact form factor compatible with high volume manufacturing practices. This article reviews recent developments of wafer-scale multifunctional nanophotonic neural probes. The probes can be realized on 200 or 300 mm wafers in commercial foundries and integrate light emitters for photostimulation, microelectrodes for electrophysiological recording, and microfluidic channels for chemical delivery and sampling. By integrating active optical devices to the probes, denser emitter arrays, enhanced on-chip biosensing, and increased ease of use may be realized. Silicon photonics technology makes possible highly versatile implantable neural probes that can transform neuroscience experiments.

Cell assembly analysis of neural circuits for innate behavior in Drosophila melanogaster using an immediate early gene stripe/egr-1

Innate behavior, such as courtship behavior, is controlled by a genetically defined set of neurons. To date, it remains challenging to visualize and artificially control the neural population that is active during innate behavior in a whole-brain scale. Immediate early genes (IEGs), whose expression is induced by neural activity, can serve as powerful tools to map neural activity in the animal brain. We screened for IEGs in vinegar fly Drosophila melanogaster and identified stripe/egr-1 as a potent neural activity marker. Focusing on male courtship as a model of innate behavior, we demonstrate that stripe-GAL4-mediated reporter expression can label fruitless (fru)-expressing neurons involved in courtship in an activity (experience)-dependent manner. Optogenetic reactivation of the labeled neurons elicited sexual behavior in males, whereas silencing of the labeled neurons suppressed courtship and copulation. Further, by combining stripe-GAL4-mediated reporter expression and detection of endogenous Stripe expression, we established methods that can label neurons activated under different contexts in separate time windows in the same animal. The cell assembly analysis of fru neural population in males revealed that distinct groups of neurons are activated during interactions with a female or another male. These methods will contribute to building a deeper understanding of neural circuit mechanisms underlying innate insect behavior.

GluCl.Cre ON enables selective inhibition of molecularly defined pain circuits.

Insight into nociceptive circuits will ultimately build our understanding of pain processing and aid the development of analgesic strategies. Neural circuit analysis has been advanced greatly by the development of optogenetic and chemogenetic tools, which have allowed function to be ascribed to discrete neuronal populations. Neurons of the dorsal root ganglion, which include nociceptors, have proved challenging targets for chemogenetic manipulation given specific confounds with commonly used DREADD technology. We have developed a cre/lox dependant version of the engineered glutamate-gated chloride channel (GluCl) to restrict and direct its expression to molecularly defined neuronal populations. We have generated GluCl.Cre ON that selectively renders neurons expressing cre-recombinase susceptible to agonist-induced silencing. We have functionally validated our tool in multiple systems in vitro, and subsequently generated viral vectors and tested its applicability in vivo. Using Nav1.8 Cre mice to restrict AAV-GluCl.Cre ON to nociceptors, we demonstrate effective silencing of electrical activity in vivo and concomitant hyposensitivity to noxious thermal and noxious mechanical pain, whereas light touch and motor function remained intact. We also demonstrated that our strategy can effectively silence inflammatory-like pain in a chemical pain model. Collectively, we have generated a novel tool that can be used to selectively silence defined neuronal circuits in vitro and in vivo. We believe that this addition to the chemogenetic tool box will facilitate further understanding of pain circuits and guide future therapeutic development.

Editorial: Cutting-edge technologies for the comprehensive analysis of neural circuits

Editorial: Cutting-edge technologies for the comprehensive analysis of neural circuits

Recent technological advances in correlative light and electron microscopy for the comprehensive analysis of neural circuits.

Light microscopy (LM) covers a relatively wide area and is suitable for observing the entire neuronal network. However, resolution of LM is insufficient to identify synapses and determine whether neighboring neurons are connected via synapses. In contrast, the resolution of electron microscopy (EM) is sufficiently high to detect synapses and is useful for identifying neuronal connectivity; however, serial images cannot easily show the entire morphology of neurons, as EM covers a relatively narrow region. Thus, covering a large area requires a large dataset. Furthermore, the three-dimensional (3D) reconstruction of neurons by EM requires considerable time and effort, and the segmentation of neurons is laborious. Correlative light and electron microscopy (CLEM) is an approach for correlating images obtained via LM and EM. Because LM and EM are complementary in terms of compensating for their shortcomings, CLEM is a powerful technique for the comprehensive analysis of neural circuits. This review provides an overview of recent advances in CLEM tools and methods, particularly the fluorescent probes available for CLEM and near-infrared branding technique to match LM and EM images. We also discuss the challenges and limitations associated with contemporary CLEM technologies.

Microfluidics for Neuronal Cell and Circuit Engineering.

The widespread adoption of microfluidic devices among the neuroscience and neurobiology communities has enabled addressing a broad range of questions at the molecular, cellular, circuit, and system levels. Here, we review biomedical engineering approaches that harness the power of microfluidics for bottom-up generation of neuronal cell types and for the assembly and analysis of neural circuits. Microfluidics-based approaches are instrumental to generate the knowledge necessary for the derivation of diverse neuronal cell types from human pluripotent stem cells, as they enable the isolation and subsequent examination of individual neurons of interest. Moreover, microfluidic devices allow to engineer neural circuits with specific orientations and directionality by providing control over neuronal cell polarity and permitting the isolation of axons in individual microchannels. Similarly, the use of microfluidic chips enables the construction not only of 2D but also of 3D brain, retinal, and peripheral nervous system model circuits. Such brain-on-a-chip and organoid-on-a-chip technologies are promising platforms for studying these organs as they closely recapitulate some aspects of in vivo biological processes. Microfluidic 3D neuronal models, together with 2D in vitro systems, are widely used in many applications ranging from drug development and toxicology studies to neurological disease modeling and personalized medicine. Altogether, microfluidics provide researchers with powerful systems that complement and partially replace animal models.

Brain-wide TVA compensation allows rabies virus to retrograde target cell-type-specific projection neurons

Retrograde tracers based on viral vectors are powerful tools for the imaging and manipulation of upstream neural networks projecting to a specific brain region, and they play important roles in structural and functional studies of neural circuits. However, currently reported retrograde viral tracers have many limitations, such as brain area selectivity or the inability to retrograde label genetically defined brain-wide projection neurons. To overcome these limitations, a new retrograde tracing method, AAV-PHP.eB assisted retrograde tracing systems (PARTS) based on rabies virus, was established through brain-wide TVA-dependent targeting using an AAV-PHP.eB that efficiently crosses the blood–brain barrier in C57BL/6 J mice, and complementation of EnvA-pseudotyped defective rabies virus that specifically recognizes the TVA receptor. Furthermore, combined with Cre transgenic mice, cell-type-specific PARTS (cPARTS) was developed, which can retrograde label genetically defined brain-wide projection neurons. Our research provides new tools and technical support for the analysis of neural circuits.

Environmental Enrichment Differentially Activates Neural Circuits in FVB/N Mice, Inducing Social Interaction in Females but Agonistic Behavior in Males.

Environmental enrichment induces behavioral and structural modifications in rodents and influences the capability of mice to cope with stress. However, little is understood about hippocampal neurogenesis and the appearance of social/agonistic (aggressive) behavior upon activation of different neuronal circuits in FVB/N mice. Thus, in this study we hypothesized that environmental enrichment differentially regulates neurogenesis, neural circuit activation and social/agonistic behavior in male and female FVB/N mice. We explored the (1) neurogenic process as an indicative of neuroplasticity, (2) neuronal activation in the limbic system, and (3) social behavior using the resident-intruder test. On postnatal day 23 (PD23), mice were assigned to one of two groups: Standard Housing or Environmental Enrichment. At PD53, rodents underwent the resident-intruder test to evaluate social behaviors. Results revealed that environmental enrichment increased neurogenesis and social interaction in females. In males, environmental enrichment increased neurogenesis and agonistic behavior. Enriched male mice expressed higher levels of agonistic-related behavior than female mice housed under the same conditions. Neural circuit analysis showed lower activation in the amygdala of enriched males and higher activation in enriched females than their respective controls. Enriched females also showed higher activation in the frontal cortex without differences in male groups. Moreover, the insular cortex was less activated in females than in males. Thus, our results indicate that environmental enrichment has different effects on neuroplasticity and social/agonistic behavior in FVB/N mice, suggesting the relevance of sexual dimorphism in response to environmental stimuli.

Emotional Well-Being Animal Models

Clinical studies suggest an association between EWB and the risk or progression of AD. However, the mechanistic link and causal relationship between EWB and AD remain unknown, due to limited experimental access and control of the underlying human brain processes. Animal models offer genetic control of AD mutations and neural circuit analysis tools, but subjective feelings of EWB cannot be assessed through self-report. To study EWB across species, we adopt a theoretical framework that views emotions as central brain states that respond to exteroceptive or interoceptive stimuli and cause multiple cognitive, somatic and behavioral changes. Recent neuroanatomical and functional imaging studies have identified evolutionarily related brain circuits in the encoding and regulation of central emotional states in animals. Dr. Wang will review progress in elucidating the functional activities of these circuits and discuss the challenges and opportunities to link these neural representations to EWB and AD related pathological progression.

A Multimodal Multi-Shank Fluorescence Neural Probe for Cell-Type-Specific Electrophysiology in Multiple Regions across a Neural Circuit.

Cell‐type‐specific, activity‐dependent electrophysiology can allow in‐depth analysis of functional connectivity inside complex neural circuits composed of various cell types. To date, optics‐based fluorescence recording devices enable monitoring cell‐type‐specific activities. However, the monitoring is typically limited to a single brain region, and the temporal resolution is significantly low. Herein, a multimodal multi‐shank fluorescence neural probe that allows cell‐type‐specific electrophysiology from multiple deep‐brain regions at a high spatiotemporal resolution is presented. A photodiode and an electrode‐array pair are monolithically integrated on each tip of a minimal‐form‐factor silicon device. Both fluorescence and electrical signals are successfully measured simultaneously in GCaMP6f expressing mice, and the cell type from sorted neural spikes is identified. The probe's capability of combined electro‐optical recordings for cell‐type‐specific electrophysiology at multiple brain regions within a neural circuit is demonstrated. The new experimental paradigm to enable the precise investigation of functional connectivity inside and across complex neural circuits composed of various cell types is expected.

Analysis of Brain Functional Connectivity Neural Circuits in Children With Autism Based on Persistent Homology

Autism spectrum disorder (ASD) is a complex neuropsychiatric disorder with a complex and unknown etiology. Statistics demonstrate that the number of people diagnosed with ASD is increasing in countries around the world. Currently, although many neuroimaging studies indicate that ASD is characterized by abnormal functional connectivity (FC) patterns within brain networks rather than local functional or structural abnormalities, the FC characteristics of ASD are still poorly understood. In this study, a Vietoris-Rips (VR) complex filtration model of the brain functional network was established by using resting-state functional magnetic resonance imaging (fMRI) data of children aged 6–13 years old [including 54 ASD patients and 52 typical development (TD) controls] from the Autism Brain Imaging Data Exchange (ABIDE) public database. VR complex filtration barcodes are calculated by using persistent homology to describe the changes in the FC neural circuits of brain networks. The number of FC neural circuits with different length ranges at different threshold values is calculated by using the barcodes, the different brain regions participating in FC neural circuits are discussed, and the connectivity characteristics of brain FC neural circuits in the two groups are compared and analyzed. Our results show that the number of FC neural circuits with lengths of 8–12 is significantly decreased in the ASD group compared with the TD control group at threshold values of 0.7, 0.8 and 0.9, and there is no significant difference in the number of FC neural circuits with lengths of 4–7 and 13–16 and lengths 16. When the thresholds are 0.7, 0.8, and 0.9, the number of FC neural circuits in some brain regions, such as the right orbital part of the superior frontal gyrus, the left supplementary motor area, the left hippocampus, and the right caudate nucleus, involved in the study is significantly decreased in the ASD group compared with the TD control group. The results of this study indicate that there are significant differences in the FC neural circuits of brain networks in the ASD group compared with the TD control group.

Reinforcing neuron extraction and spike inference in calcium imaging using deep self-supervised denoising.

Calcium imaging has transformed neuroscience research by providing a methodology for monitoring the activity of neural circuits with single-cell resolution. However, calcium imaging is inherently susceptible to detection noise, especially when imaging with high frame rate or under low excitation dosage. Here we developed DeepCAD, a self-supervised deep-learning method for spatiotemporal enhancement of calcium imaging data that does not require any high signal-to-noise ratio (SNR) observations. DeepCAD suppresses detection noise and improves the SNR more than tenfold, which reinforces the accuracy of neuron extraction and spike inference and facilitates the functional analysis of neural circuits.

The promise of the BRAIN initiative: NIH strategies for understanding neural circuit function.

New neurotechnologies fueled by the BRAIN Initiative now allow investigators to map, monitor and modulate complex neural circuits, enabling the pursuit of research questions previously considered unapproachable. Yet it is the convergence of molecular neuroscience with the new systems neuroscience that promises the greatest future advances. This is particularly true for our understanding of nervous system disorders, some of which have known molecular drivers or pathology but result in unknown perturbations in circuit function. NIH-supported research on "BRAIN Circuits" programs integrate experimental, analytic, and theoretical capabilities for analysis of specific neural circuits and their contributions to perceptions, motivations, and actions. In this review, we describe the BRAIN priority areas, review our strategy for balancing early feasibility with mature projects, and the balance of individual with team science for this 'BRAIN Circuits' program. We also highlight the diverse portfolio of techniques, species, and neural systems represented in these projects.

A Chemogenetic Tool that Enables Functional Neural Circuit Analysis

Chemogenetics enables manipulation of neuronal activity in experimental animals. While providing information about the transduced neuron expressing a ligand-activated molecule, chemogenetics does not provide understanding about the antecedent circuit that drives that neuron's activity. For current approaches, this is not feasible, because the activating molecules are not genetically encoded. The insect allatostatin/allatostatin receptor system, a highly specific, powerful inhibitory chemogenetic approach, has this advantage, because the ligand, being a peptide, is genetically encoded. We developed viral vector-based systems to express biologically active allatostatin in neurons invivo and allatostatin receptors in subpopulations of postsynaptic neurons. We demonstrate that activity-dependent release of allatostatin induces inhibition of allatostatin receptor-expressing neurons. We validate the approach in the vagal viscerosensory system where inhibitory, rather than the usual excitatory, viscerosensory input leads to sustained decreases in baroreceptor reflex sensitivity and bodyweight.

Role of the Medial Orbitofrontal Cortex and Ventral Tegmental Area in Effort-Related Responding.

The posterior subdivision of the medial orbitofrontal cortex (mOFC-p) mediates the willingness to expend effort to reach a selected goal. However, the neural circuitry through which the mOFC-p modulates effort-related function is as yet unknown. The mOFC-p projects prominently to the posterior ventral tegmental area (pVTA). Therefore, we analyzed the role of the mOFC-p and interactions with the pVTA in effort-related responding using a combination of behavioral, pharmacological, and neural circuit analysis methods in rats. Pharmacological inhibition of the mOFC-p was found to increase lever pressing for food under a progressive ratio (PR) schedule of reinforcement. These findings provide further support for a modulation of effort-related function by the mOFC-p. Then, we investigated effects of disconnecting the mOFC-p and pVTA on PR responding using unilateral pharmacological inhibition of both areas. This asymmetric intervention was also found to increase PR responding suggesting that the mOFC-p controls effort-related function through interactions with the pVTA. Possibly, a reduced excitatory mOFC-p drive on pVTA gamma-aminobutyric acid (GABA)ergic relays disinhibits VTA dopamine neurons which are known to support PR responding. Collectively, our findings suggest that the mOFC-p and pVTA are key components of a neural circuit mediating the willingness to expend effort to reach a goal.

Neural circuit analysis using a novel intersectional split intein-mediated split-Cre recombinase system

The defining features of a neuron are its functional and anatomical connections with thousands of other neurons in the brain. Together, these neurons form functional networks that direct animal behavior. Current approaches that allow the interrogation of specific populations of neurons and neural circuits rely heavily on targeting their gene expression profiles or connectivity. However, these approaches are often unable to delineate specific neuronal populations. Here, we developed a novel intersectional split intein-mediated split-Cre recombinase system that can selectively label specific types of neurons based on their gene expression profiles and structural connectivity. We developed this system by splitting Cre recombinase into two fragments with evolved split inteins and subsequently expressed one fragment under the influence of a cell type-specific promoter in a transgenic animal, and delivered the other fragment via retrograde viral gene transfer. This approach results in the reconstitution of Cre recombinase in only specific population of neurons projecting from a specific brain region or in those of a specific neuronal type. Taken together, our split intein-based split-Cre system will be useful for sophisticated characterization of mammalian brain circuits.