Neuronal Circuits Controlling Behavior: Genetic Analysis in Zebrafish
- Harold Burgess, PhD, Head, Section on Behavioral Neurogenetics
- Tripti Gupta, PhD, Staff Scientist
- Jennifer Panlilio, PhD, Postdoctoral Fellow
- Deepthi Nammi, PhD, Visiting Fellow
- Svetlana Semenova, PhD, Visiting Fellow
- Hariom Sharma, PhD, Visiting Fellow
- Daniel Bazan, BSc, Postbaccalaureate Fellow
- Grace Biddle, BSc, Postbaccalaureate Fellow
- Reid Doctor, BSc, Graduate Student
- Jennifer L. Sinclair, MSc, Zebrafish Technician
The Section on Behavioral Neurogenetics studies how, under diverse environmental contexts, the nervous system selects appropriate behavioral responses to sensory information in a way that best satisfies internal motivational objectives. We use the larval zebrafish as a model because its brain exhibits the basic architecture of the vertebrate brain but is much less complex than the mammalian brain. Despite the relative simplicity of their nervous system, zebrafish have a sophisticated repertoire of sensory-guided and internally driven behaviors. Furthermore, the optical clarity of the embryo facilitates visualization of individual neurons and their manipulation with genetic techniques. Behavior in larvae is innate and thus exhibits minimal variability between fish. Subtle alterations in behavior can therefore be robustly measured, making it possible to quickly assess the contribution of identified neurons to a variety of motor behaviors.
We focus on two aspects of behavioral regulation: the neuronal mechanisms by which sensory context regulates behavioral decisions; and the pathways that sustain changes in behavioral state. Neuronal connections that allow the brain to integrate sensory and internal-state information are established through genetic interactions during development, and are frequently disrupted by gene mutations associated with neurodevelopmental disorders. We can therefore use discoveries about sensorimotor integration pathways to understand how human disease genes disrupt brain development. To support these objectives, we develop new genetic tools and behavioral assays to probe the nexus between neuronal function and behavior at single-cell resolution.
Members of the Section on Behavioral Neurogenetics
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Left to right: Grace Biddle, Tripti Gupta, Daniel Bazan, Harold Burgess, Reid Doctor, Hariom Sharma, Jennifer Sinclair, Svetlana Semenova, Jennifer Panlilio
Neuronal pathways for auditory sensory processing
Startle responses are rapid reflexes that are triggered by sudden sensory stimuli, and which help animals defend against, or escape from, potentially threatening stimuli. In both fish and mammals, startle responses are initiated by giant reticulospinal neurons in the medulla, which receive short-latency sensory input from diverse sensory modalities. Although highly stereotyped, startle responses are nevertheless modulated by sensory context and behavioral state and are therefore an excellent system in which to study how such information is integrated for behavioral choice. In mammals, including humans, the startle response to a strong auditory stimulus can be inhibited by pre-exposure to a weak acoustic ‘prepulse,’ a form of startle modulation termed prepulse inhibition, that is diminished in several neurological conditions. Previously, we showed that, in zebrafish, as in mammals, several distinct cellular mechanisms mediate prepulse inhibition, depending on the time interval between the prepulse and the startle stimulus, with NMDA–receptor signaling playing a key role for intervals greater than 100 ms. Our work on resolving the core neuronal pathway that mediates prepulse inhibition provides a basis for probing how gene mutations linked to neurodevelopmental disorders disrupt sensory processing. NMDA–receptor mutations have been linked to both autism-spectrum disorders and schizophrenia, and, accordingly, we demonstrated that zebrafish NMDA–receptor subunit mutants have both structural brain deficits and disrupted prepulse inhibition. Recently, we also identified neurons that mediate prepulse inhibition at intervals of less than 100 ms and confirmed previous reports that a GABAergic mechanism is involved. In ongoing experiments, we are now defining how such neurons suppress startle reflexes by examining their synaptic connections with, and molecular signaling to, central neurons that initiate startle responses. We are also defining connectivity from the auditory sensory ganglion in zebrafish (the statoacoustic ganglion), in order to elucidate pathways that transmit behaviorally relevant vibration and acceleration signals to central regions that process sensory information for startle behavior, balance, and eye movements (Figure 1).
Figure 1. Morphological reconstruction of auditory sensory neurons in larval zebrafish
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After sparse labeling, we traced more than 150 statoacoustic ganglion neurons to correlate patterns of connectivity with hair-cell patches to central projection targets. In this image, neurons are colored according to peripheral target. Central projections of neurons that share a peripheral target remain closely associated.
Neural mechanisms for behavioral-state control
Over the course of the day, motivational goals change in response to both internal and external cues. At any given moment, an individual’s behavioral state strongly influences decisions on how to interact with the environment. A major goal in neuroscience is to identify the neural systems that maintain short-term behavioral states and to determine how they interact with central mechanisms for behavioral choice. We have developed several paradigms in which the behavioral state of zebrafish is temporarily altered. Our strategy is to allow larvae to remain in the arena until their motor behavior achieves a stable state, then introduce a temporary perturbation to the environment and examine changes in behavior that persist on a time-scale of several minutes after the perturbation is removed (Figure 2).
Figure 2. Paradigms for studying behavioral state control in zebrafish larvae
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To study behavioral-state control, we developed behavioral paradigms in which motor activity shows a persistent change over a timescale of minutes following a perturbation to the environment. In tonic immobility (left), an intense vibration stimulus induced a reduction in movement that lasts for around a minute after the stimulus ceases. In flow-induced arousal (right), exposure to water flow induces elevated locomotor activity that continues for several minutes, even after water movement ceases.
While studying defensive responses to auditory stimuli, we noted that cues of about an order of magnitude larger than those needed to provoke startle responses drove an unexpected freezing behavior, especially when repeatedly presented over a short period of time. In many species, overwhelming stimuli elicit a behavior known as tonic immobility, an ultimate response to inescapable threat. In humans, the experience of tonic immobility correlates with development of the post-traumatic stress disorder, yet very little is known about its underlying neural basis. Leveraging our library of Gal4 (a tool to modulate gene activity)–transgenic lines, we performed a screen to discover neurons that are required for such behavior in zebrafish. We isolated a cluster of neurons in the prepontine tegmentum that are necessary for sustained immobility after an intense auditory stimulus. By manually isolating and performing RNA-seq on these neurons, we found that they express several stress-associated neuropeptides, including markers that make them likely homologs of part of the mammalian parabrachial complex, an area recently implicated in responses to noxious stimuli. Our screen also demonstrated a central role for cerebellar signaling in tonic immobility, and we found a direct projection from Purkinje neurons to the prepontine neurons, similar to recent work showing that a subset of Purkinje neurons in mammals also directly project to the parabrachial nucleus. The study identified, for the first time, a cellular pathway that mediates tonic immobility and suggests that the parabrachial complex has a deep evolutionary history in mediating defensive behavior [Reference 1].
A second paradigm that we used to study behavioral state control is flow-induced arousal. After exposure to a brief water flow stimulus, zebrafish larvae show elevated motor activity and sensory responsiveness that persists for several minutes after termination of water movement [Yokogawa T, Hannan MC, Burgess HA, J Neurosci 2012;32:15205]. We showed that the serotonergic raphe nucleus regulates sensory responsiveness during this state, but the neural basis for hyperactivity was not known. Recently, we screened Gal4 lines to identify neurons that are required for increased motor activity during flow-induced arousal, and assessed effects of pharmacological manipulations on this behavior. We are currently zeroing in on relevant neurons by selectively laser-ablating neurons in a transgenic line that labels neurons required for flow-induced arousal.
Zebrafish models of neurodevelopmental disorders
We collaborate widely with clinicians to generate and characterize zebrafish models for mutations discovered in humans (often through exome-sequencing) that are likely to have a neurodevelopmental origin. We use the CRISPR/Cas9 system to generate lesions in zebrafish genes that are homologous to those disrupted in the human disorders. We then apply behavioral analysis, transcriptomics, and voxel-based morphometry as part of a broad phenotyping strategy. To promote rigorous use of zebrafish neurological disease models, we wrote a critical review outlining advantages and limitations of the zebrafish system [References 2, 3].
Our work with brain morphometry arose from earlier studies in which we generated several hundred new Gal4 and Cre lines in order to provide genetic accessibility to neurons of interest. A unique feature of brain imaging in zebrafish is the ability to visualize the total architecture of the brain while simultaneously recording the position and morphology of every constituent labeled neuron. To make these transgenic lines accessible to the broader research community, we performed whole-brain imaging for each line, then registered the image of each line to the same reference brain. In collaboration with Nicholas Polys, we then developed an online brain atlas that enables researchers to quickly visualize the larval brain and locate transgenic lines to aid experiments. Such powerful visualization tools facilitate integrated analysis of reconstructed neuronal morphology in the context of the three-dimensional anatomy of the brain. Then, in order to build a brain atlas, we optimized a protocol that permits highly precise brain registration [Reference 4].
We showed that such high precision of alignment permits statistically robust whole-brain analysis of neuronal composition and morphology in zebrafish mutant models, pinpointing brain regions with changes that are difficult to detect visually. The technique can be applied to almost any zebrafish neurodevelopmental model, thereby enabling robust and quantitative detection of subtle changes in brain structure or composition. We used this method to test brain structure and composition in zebrafish that carry mutations in genes that are homologous to human genes known to be disrupted in a variety of neurodevelopmental disorders, including autism and intellectual disability. Through this work, we aim to provide insight into the fundamental molecular and cellular processes associated with each disorder.
Publications
- Bhandiwad AA, Chu NC, Semenova SA, Holmes GA, Burgess HA. A cerebellar-prepontine circuit for tonic immobility triggered by an inescapable threat. Sci Adv 2022 8(39):eabo0549.
- Burgess HA, Burton EA. A critical review of zebrafish neurological disease models – 1. The premise: neuroanatomical, cellular, and genetic homology, and experimental tractability. Oxf Open Neurosci 2023 kvac018.
- Burton EA, Burgess HA. A critical review of zebrafish neurological disease models – 2. Application: functional and neuroanatomical phenotyping strategies and chemical screens. Oxf Open Neurosci 2023 kvac019.
- Bhandiwad AA, Gupta T, Subedi A, Heigh V, Holmes GA, Burgess HA. Brain imaging and registration in larval zebrafish. Methods Mol Biol 2024 2707:141–153.
Collaborators
- Edward A. Burton, MD, DPhil, FRCP, Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh, Pittsburgh, PA
- Thomas E. Dever, PhD, Section on Protein Biosynthesis, NICHD, Bethesda, MD
- Todd S. Macfarlan, PhD, Section on Mammalian Epigenome Reprogramming, NICHD, Bethesda, MD
- Anne O'Donnell Luria, MD, PhD, Mendelian Genomics Research Center, Broad Institute of MIT, and Harvard, Cambridge, MA
- Nicholas Polys, PhD, Virginia Tech, Blacksburg, VA
- Howard Sirotkin, PhD, Stony Brook University, Stony Brook, NY
- Lonnie P. Wollmuth, PhD, Stony Brook University, Stony Brook, NY
Contact
For more information, email haroldburgess@mail.nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/burgess.
