Neural Basis Of Behavior Research Articles

Neuroethology in The Age of Gene Editing: New Tools And Novel Insights into The Molecular And Neural Basis of Behavior

Synopsis Fifty years ago, animal models studied in the lab were highly diverse, and biological insights were derived from experiments in many species. However, the pursuit of mechanistic explanations in organismal biology led to a shift in the species most commonly studied. The advent of genetic manipulations and economies of scale promoted the consolidation of research into fewer species (eg, Drosophila melanogaster, Mus musculus, Danio rerio, Caenorhabditis elegans). As a result, the tremendous variety of evolutionary adaptations across species provided insights into ultimate causes of evolution, but their proximate mechanisms have been understudied in recent decades. Within the last decade, developments in genome modifications have enabled functional genetic studies in a wide variety of species. This special issue combines papers derived from a symposium organized at the Society for Integrative and Comparative Biology conference in January 2023 in Austin, Texas. The symposium entitled “Neuroethology in the age of gene editing: New tools and novel insights into the molecular and neural basis of behavior” was convened to catalyze the transfer of knowledge and skills from researchers who have applied genome modification technologies in new model organisms to scientists who would like to bring these approaches to their own research programs. We highlight this work here, and suggest how the future of biological knowledge will be informed by these powerful experiments.

A complete biomechanical model of Hydra contractile behaviors, from neural drive to muscle to movement

How does neural activity drive muscles to produce behavior? The recent development of genetic lines in Hydra that allow complete calcium imaging of both neuronal and muscle activity, as well as systematic machine learning quantification of behaviors, makes this small cnidarian an ideal model system to understand and model the complete transformation from neural firing to body movements. To achieve this, we have built a neuromechanical model of Hydra's fluid-filled hydrostatic skeleton, showing how drive by neuronal activity activates distinct patterns of muscle activity and body column biomechanics. Our model is based on experimental measurements of neuronal and muscle activity and assumes gap junctional coupling among muscle cells and calcium-dependent force generation by muscles. With these assumptions, we can robustly reproduce a basic set of Hydra's behaviors. We can further explain puzzling experimental observations, including the dual timescale kinetics observed in muscle activation and the engagement of ectodermal and endodermal muscles in different behaviors. This work delineates the spatiotemporal control space of Hydra movement and can serve as a template for future efforts to systematically decipher the transformations in the neural basis of behavior.

Connectomics and the neural basis of behaviour.

Methods to acquire and process synaptic-resolution electron-microscopy datasets have progressed very rapidly, allowing production and annotation of larger, more complete connectomes. More accurate neuronal matching techniques are enriching cell type data with gene expression, neuron activity, behaviour and developmental information, providing ways to test hypotheses of circuit function. In a variety of behaviours such as learned and innate olfaction, navigation and sexual behaviour, connectomics has already revealed interconnected modules with a hierarchical structure, recurrence and integration of sensory streams. Comparing individual connectomes to determine which circuit features are robust and which are variable is one key research area; new work in comparative connectomics across development, experience, sex and species will establish strong links between neuronal connectivity and brain function.

Enhanced olfactory memory detection in trap-design Y-mazes allows the study of imperceptible memory traces in Drosophila.

The neural basis of behavior is identified by systematically manipulating the activity of specific neurons and screening for loss or gain of phenotype. Therefore, robust, high-scoring behavioral assays are necessary for determining the neural circuits of novel behaviors. We report a simple Y-maze design for Drosophila olfactory learning and memory assay. Memory scores in our Y-mazes are considerably better and longer-lasting than scores obtained with commonly used T-mazes. Our results suggest that trapping flies to an odor choice in a Y-maze could improve scores. We postulated that the improved scores could reveal previously undetectable memory traces, enabling the study of underlying neural mechanisms. Indeed, we identified unreported protein synthesis-dependent long-term memories (LTMs), reinforced by ingestion of (1) an aversive compound and (2) a sweet but nonnutritious sugar, both 24 h after training. We also used Y-mazes to probe how using a greater reward may change memory dynamics. Our findings predict that a greater sugar reward may extend existing memory traces or reinforce additional novel ones.

Amphibian behavioral diversity offers insights into evolutionary neurobiology.

Recent studies have served to emphasize the unique placement of amphibians, composed of more than 8000 species, in the evolution of the brain. We provide an overview of the three amphibian orders and their respective ecologies, behaviors, and brain anatomy. Studies have probed the origins of independently evolved parental care strategies in frogs and the biophysical principles driving species-specific differences in courtship vocalization patterns. Amphibians are also important models for studying the central control of movement, especially in the context of the vertebrate origin of limb-based locomotion. By highlighting the versatility of amphibians, we hope to see a further adoption of anurans, urodeles, and gymnophionans as model systems for the evolution and neural basis of behavior across vertebrates.

A 3D adult zebrafish brain atlas (AZBA) for the digital age.

Zebrafish have made significant contributions to our understanding of the vertebrate brain and the neural basis of behavior, earning a place as one of the most widely used model organisms in neuroscience. Their appeal arises from the marriage of low cost, early life transparency, and ease of genetic manipulation with a behavioral repertoire that becomes more sophisticated as animals transition from larvae to adults. To further enhance the use of adult zebrafish, we created the first fully segmented three-dimensional digital adult zebrafish brain atlas (AZBA). AZBA was built by combining tissue clearing, light-sheet fluorescence microscopy, and three-dimensional image registration of nuclear and antibody stains. These images were used to guide segmentation of the atlas into over 200 neuroanatomical regions comprising the entirety of the adult zebrafish brain. As an open source, online (azba.wayne.edu), updatable digital resource, AZBA will significantly enhance the use of adult zebrafish in furthering our understanding of vertebrate brain function in both health and disease.

Development of a pan-neuronal genetic driver in Aedes aegypti mosquitoes

SummaryThe recent development of neurogenetic tools in Aedes aegypti mosquitoes is beginning to shed light on the neural basis of behaviors that make this species a major vector of human disease. However, we still lack a pan-neuronal expression driver—a key tool that provides genetic access to all neurons. Here, we describe our efforts to fill this gap via CRISPR/Cas9-mediated knock-in of reporters to broadly expressed neural genes and report on the generation of two strains, a Syt1:GCaMP6s strain that expresses synaptically localized GCaMP and a brp-T2A-QF2w driver strain that can be used to drive and amplify expression of any effector via the Q binary system. Both manipulations broadly and uniformly label the nervous system with only modest effects on behavior. We expect these strains to facilitate neurobiological research in Ae. aegypti mosquitoes and document both successful and failed manipulations as a roadmap for similar tool development in other non-model species.

Ex Vivo Brain Preparation to Analyze Vocal Pathways of Xenopus Frogs.

Understanding the neural basis of behavior is a challenging task for technical reasons. Most methods of recording neural activity require animals to be immobilized, but neural activity associated with most behavior cannot be recorded from an anesthetized, immobilized animal. Using amphibians, however, there has been some success in developing in vitro brain preparations that can be used for electrophysiological and anatomical studies. Here, we describe an ex vivo frog brain preparation from which fictive vocalizations (the neural activity that would have produced vocalizations had the brain been attached to the muscle) can be elicited repeatedly. When serotonin is applied to the isolated brains of male and female African clawed frogs, Xenopus laevis, laryngeal nerve activity that is a facsimile of those that underlie sex-specific vocalizations in vivo can be readily recorded. Recently, this preparation was successfully used in other species within the genus including Xenopus tropicalis and Xenopus victorianus This preparation allows a variety of techniques to be applied including extracellular and intracellular electrophysiological recordings and calcium imaging during vocal production, surgical and pharmacological manipulation of neurons to evaluate their impact on motor output, and tract tracing of the neural circuitry. Thus, the preparation is a powerful tool with which to understand the basic principles that govern the production of coherent and robust motor programs in vertebrates.

Molecular characterization of frog vocal neurons using constellation pharmacology.

Identification and characterization of neuronal cell classes in motor circuits are essential for understanding the neural basis of behavior. It is a challenging task, especially in a non-genetic-model organism, to identify cell-specific expression of functional macromolecules. Here, we performed constellation pharmacology, calcium imaging of dissociated neurons to pharmacologically identify functional receptors expressed by vocal neurons in adult male and female African clawed frogs, Xenopus laevis. Previously we identified a population of vocal neurons called fast trill neurons (FTNs) in the amphibian parabrachial nucleus (PB) that express N-methyl-d-aspartate (NMDA) receptors and GABA and/or glycine receptors. Using constellation pharmacology, we identified four cell classes of putative fast trill neurons (pFTNs, responsive to both NMDA and GABA/glycine applications). We discovered that some pFTNs responded to the application of substance P (SP), acetylcholine (ACh), or both. Electrophysiological recordings obtained from FTNs using an ex vivo preparation verified that SP and/or ACh depolarize FTNs. Bilateral injection of ACh, SP, or their antagonists into PBs showed that ACh receptors are not sufficient but necessary for vocal production, and SP receptors play a role in shaping the morphology of vocalizations. Additionally, we discovered that the PB of adult female X. laevis also contains all the subclasses of neurons at a similar frequency as in males, despite their sexually distinct vocalizations. These results reveal novel neuromodulators that regulate X. laevis vocal production and demonstrate the power of constellation pharmacology in identifying the neuronal subtypes marked by functional expression of cell-specific receptors in non-genetic-model organisms.NEW & NOTEWORTHY Molecular profiles of neurons are critical for understanding the neuronal functions, but their identification is challenging especially in non-genetic-model organisms. Here, we characterized the functional expression of membrane macromolecules in vocal neurons of African clawed frogs, Xenopus laevis, using a technique called constellation pharmacology. We discovered that receptors for acetylcholine and/or substance P are expressed by some classes of vocal neurons, and their activation plays a role in the production of normal vocalizations.

Robotic Stimulation of Freely Moving <italic>Drosophila</italic> Larvae Using a 3D-Printed Micro Force Sensor

The Drosophila larva is an excellent model organism in biology for studying the mechanisms of sensory mechanotransduction and the neural basis of behavior. This paper reports an automated robotic micromanipulation system capable of force-controlled mechanical stimulation and locomotion behavior analysis of freely moving Drosophila larvae, which improves the force regulation accuracy and larva operation consistency over conventional manual manipulation. A three-dimensionally-printed, three-axis micro force sensor was developed and integrated into the robotic system to measure the contact force between the tip of an end-effector and the larva head, and an adaptive fuzzy proportional-integral-derivative (PID) controller was proposed for closed-loop control of the contact force. The three-axis force sensor was also employed to monitor lateral disturbances to the contact force caused by small larva head movements, and thus validate the effectiveness of larva stimulation. The robotic system performed automated larva stimulation and locomotion analysis at a speed of four larvae per minute, and was applied to quantify the correlation between the applied contact force direction/magnitude and the larva reorientation behavior. With its high accuracy and efficiency, this system will greatly facilitate large-scale studies of mechanosensory behaviors in Drosophila larva.

Full reconstruction of large lobula plate tangential cells in Drosophila from a 3D EM dataset.

With the advent of neurogenetic methods, the neural basis of behavior is presently being analyzed in more and more detail. This is particularly true for visually driven behavior of Drosophila melanogaster where cell-specific driver lines exist that, depending on the combination with appropriate effector genes, allow for targeted recording, silencing and optogenetic stimulation of individual cell-types. Together with detailed connectomic data of large parts of the fly optic lobe, this has recently led to much progress in our understanding of the neural circuits underlying local motion detection. However, how such local information is combined by optic flow sensitive large-field neurons is still incompletely understood. Here, we aim to fill this gap by a dense reconstruction of lobula plate tangential cells of the fly lobula plate. These neurons collect input from many hundreds of local motion-sensing T4/T5 neurons and connect them to descending neurons or central brain areas. We confirm all basic features of HS and VS cells as published previously from light microscopy. In addition, we identified the dorsal and the ventral centrifugal horizontal, dCH and vCH cell, as well as three VSlike cells, including their distinct dendritic and axonal projection area.

A Wireless Head-mountable Device with Tapered Optical Fiber-coupled Laser Diode for Light Delivery in Deep Brain Regions.

Optogenetics sets new experimental paradigms that can reveal cell type-specific contributions on the neural basis of behavior. Since most of the available systems for this purpose are based on approaches that tether animals to a set of cables, recent research activities have been focused on minimizing external factors that can alter animal movements. Current wireless optogenetic systems are based on waveguide-coupled LED and implanted LEDs. However, each configuration separately suffers from significant limitations, such as low coupling efficiency, penetration depth and invasiveness of waveguide-coupled LED, and local heat generated by implanted μLEDs. This work presents a novel wireless head-mountable stimulating system for a wide-volume light delivery. The device couples the output of a semiconductor laser diode (LD) to a tapered optical fiber (TF) on a wireless platform. The LD-TF coupling was engineered by setting up far-field analysis, which allows the full exploitation of the mode division demultiplexing properties of TFs. The output delivered light along the tapered segment is capable of stimulating structures of depths up to ~2mm. TFs are tapered to a gradual taper angle (2° to 10°) that ends with a sharp tip (~500 nm) for smooth insertion and less invasiveness. Thus, the proposed system extends the capabilities of wireless optogenetic by offering a novel solution for wide volume light delivery in deep brain regions.

Opinion on monoaminergic contributions to traits and temperament.

This article critically reviews evidence relating temperamental traits and personality factors to the monoamine neurotransmitters, especially dopamine and serotonin. The genetic evidence is not yet considered to be conclusive and it is argued that basic neuroscience research on the neural basis of behaviour in experimental animals should be taken more into account. While questionnaire and lexical methodology including the 'Five Factor' theory has been informative (mostly for the traits relevant to social functioning, i.e. personality), biologically oriented approaches should be employed with more objective, theoretically grounded measures of cognition and behaviour, combined with neuroimaging and psychopharmacology, where appropriate. This strategy will enable specific functions of monoamines and other neuromodulators such as acetylcholine and neuropeptides (such as orexin) to be defined with respect to their roles in modulating activity in specific neural networks-leading to a more realistic definition of their interactive roles in complex, biologically based traits (i.e. temperament).This article is part of the theme issue 'Diverse perspectives on diversity: multi-disciplinary approaches to taxonomies of individual differences'.

A Reward-Based Behavioral Platform to Measure Neural Activity during Head-Fixed Behavior.

Understanding the neural computations that contribute to behavior requires recording from neurons while an animal is behaving. This is not an easy task as most subcellular recording techniques require absolute head stability. The Go/No-Go sensory task is a powerful decision-driven task that enables an animal to report a binary decision during head-fixation. Here we discuss how to set up an Ardunio and Python based platform system to control a Go/No-Go sensory behavior paradigm. Using an Arduino micro-controller and Python-based custom written program, a reward can be delivered to the animal depending on the decision reported. We discuss the various components required to build the behavioral apparatus that can control and report such a sensory stimulus paradigm. This system enables the end user to control the behavioral testing in real-time and therefore it provides a strong custom-made platform for probing the neural basis of behavior.

Current techniques for high-resolution mapping of behavioral circuits in Drosophila.

Understanding behavior requires unraveling the mysteries of neurons, glia, and their extensive connectivity. Drosophila has emerged as an excellent organism for studying the neural basis of behavior. This can be largely attributed to the extensive effort of the fly community to develop numerous sophisticated genetic tools for visualizing, mapping, and manipulating behavioral circuits. Here, we attempt to highlight some of the new reagents, techniques and approaches available for dissecting behavioral circuits in Drosophila. We focus on detailing intersectional strategies such as the Flippase-induced intersectional Gal80/Gal4 repression (FINGR), because of the tremendous potential they possess for mapping the minimal number of cells required for a particular behavior. The logic and strategies outlined in this review should have broad applications for other genetic model organisms.

基于个体差异的大样本脑影像数据在心理学研究中的应用

Psychological research frequently uses cognitive neuroscience technologies to reveal the brain mechanisms underlying the psychological structure of personality, cognition, and emotion. However, previous research has often been constrained by small samples and other features of the experimental design and data analysis. Additionally, individual differences in behavioral responses and cognitive or emotional processing have often been considered confounding factors or random error, so they are usually controlled in data analysis. Human activity is the result of interactions between genes, environment, and the brain. Therefore, the investigation of environmental factors and individual life experiences, the collection of large samples of behavior, and the use of multimodal brain imaging data measuring cognition, emotion, and personality can help to address several questions from an individual differences perspective: (1) the neural basis of personality; (2) the brain mechanisms of emotion; (3) the brain mechanisms of cognition; (4) the construction of a model to predict interactions between the environment, personality, the brain, and behavior. This article summarizes recent domestic and international studies on the relationship between brain imaging and behavior within a large sample, and it is a good way to understand the neural basis of behavior, personality, emotions, and cognitive ability from the perspective of individual differences.

Imaging the Dynamics of Neocortical Population Activity in Behaving and Freely Moving Mammals.

The development of functional imaging techniques applicable to neuroscience and covering a wide range of spatial and temporal scales has greatly facilitated the exploration of the relationships between cognition, behaviour and electrical brain activity. For mammals, the neocortex plays a particularly profound role in generating sensory perception, controlling voluntary movement, higher cognitive functions and planning goal-directed behaviours. Since these remarkable functions of the neocortex cannot be explored in simple model preparations or in anesthetised animals, the neural basis of behaviour must be explored in awake behaving subjects. Because neocortical function is highly distributed across many rapidly interacting regions, it is essential to measure spatiotemporal dynamics of cortical activity in real-time. Extensive work in anesthetised mammals has shown that in vivo Voltage-Sensitive Dye Imaging (VSDI) reveals the neocortical population membrane potential dynamics at millisecond temporal resolution and subcolumnar spatial resolution. Here, we describe recent advances indicating that VSDI is also already well-developed for exploring cortical function in behaving monkeys and mice. The first animal model, the non-human primate, is well-suited for fundamental exploration of higher-level cognitive function and behavior. The second animal model, the mouse, benefits from a rich arsenal of molecular and genetic technologies. In the monkey, imaging from the same patch of cortex, repeatedly, is feasible for a long period of time, up to a year. In the rodent, VSDI is applicable to freely moving and awake head-restrained mice. Interactions between different cortical areas and different cortical columns can therefore now be dynamically mapped through VSDI and related to the corresponding behaviour. Thus by applying VSDI to mice and monkeys one can begin to explore how behaviour emerges from neuronal activity in neuronal networks residing in different cortical areas.

Wireless Optogenetics: An Exploration of Portable Microdevices for Small Animal Photostimulation

Preclinical research in optogeneticneuromodulation in small laboratory animals allows far greater control of neural circuitry. This precision provides an enhanced opportunity for understanding the neural basis of behavior. However, behavioral neuroscience research is limited by conventional benchtop optogenetic systems. By necessity, the animal is tethered to the light source external to the testing environment. Portable optogeneticmicrodevices enhance the potential for valid behavioral testing in naturalistic conditions by eliminating tethering and enabling free and unrestricted movement. This paper reviews recent advances in the development of portable optogeneticmicrodevices supported by wireless power transfer. Light sources and fiber coupling are common problems in optogenetic systems and are addressed. Device designs and parameters are summarized, along with advances in component technology for energy storage and distribution that make these devices possible.

Optogenetics: illuminating the neural bases of rodent behavior

In vivo optogenetics has provided researchers with the ability to delve deeper into the neural basis of behavior by driving cell-type specific circuit connections within and between brain regions. The diverse toolbox available for circuit- and cell-specific manipula - tions is ever growing. Using these tools in conjunction with established and novel genetic and behavioral methods, neuroscience research has experienced an explosion in the understanding of the roles of specific cell subtypes in behavior. This review aims to outline recent advances in in vivo optogenetic tools for manipulation of behavior related to movement, pain and sensation, motivation, reward, emotion, learning, sleep, and epilepsy.

Animal-oriented virtual environments: illusion, dilation, and discovery

As a research tool, virtual environments (VEs) hold immense promise for brain scientists. Yet to fully realize this potential in non-human systems, theoretical and conceptual perspectives must be developed. When selectively coupled to nervous systems, virtual environments can help us better understand the functional architecture of animals’ brains during naturalistic behaviors. While this will no doubt allow us to further our understanding of the neural basis of behavior, there is also an opportunity to uncover the diversity inherent in brain activity and behavior. This is due to two properties of virtual environments: the ability to create sensory illusions, and the ability to dilate space and/or time. These and other potential manipulations will be characterized as the effects of virtuality. In addition, the systems-level outcomes of virtual environment enhanced perception will be discussed in the context of the uncanny valley and other expected relationships between emotional valence, cognition, and training. These effects and their usefulness for brain science will be understood in the context of three types of neurobehavioral phenomena: sensorimotor integration, spatial navigation, and interactivity. For each of these behaviors, a combination of illusory and space/time dilation examples will be reviewed. Once these examples are presented, the implications for improving upon virtual models for more directly inducing the mental phenomena of illusion and space/time dilation will be considered. To conclude, future directions for integrating the use of VEs into a strategy of broader biological inquiry will be presented.