@article {140660, title = {A machine-learning tool to identify bistable states from calcium imaging data.}, journal = {J Physiol}, year = {2024}, month = {2024 Mar 14}, abstract = {

Mapping neuronal activation using calcium imaging in vivo during behavioural tasks has advanced our understanding of nervous system function. In almost all of these studies, calcium imaging is used to infer spike probabilities because action potentials activate voltage-gated calcium channels and increase intracellular calcium levels. However, neurons not only fire action potentials, but also convey information via intrinsic dynamics such as by generating bistable membrane potential states. Although a number of tools for spike inference have been developed and are currently being used, no tool exists for converting calcium imaging signals to maps of cellular state in bistable neurons. Purkinje neurons in the larval zebrafish cerebellum exhibit membrane potential bistability, firing either tonically or in bursts. Several studies have implicated the role of a population code in cerebellar function, with bistability adding an extra layer of complexity to this code. In the present study, we develop a tool, CaMLSort, which uses convolutional recurrent neural networks to classify calcium imaging traces as arising from either tonic or bursting cells. We validate this classifier using a number of different methods and find that it performs well on simulated event rasters as well as real biological data that it had not previously seen. Moreover, we find that CaMLsort generalizes to other bistable neurons, such as dopaminergic neurons in the ventral tegmental area of mice. Thus, this tool offers a new way of analysing calcium imaging data from bistable neurons to understand how they participate in network computation and natural behaviours. KEY POINTS: Calcium imaging, compriising the gold standard of inferring neuronal activity, does not report cellular state in neurons that are bistable, such as Purkinje neurons in the cerebellum of larval zebrafish. We model the relationship between Purkinje neuron electrical activity and its corresponding calcium signal to compile a dataset of state-labelled simulated calcium signals. We apply machine-learning methods to this dataset to develop a tool that can classify the state of a Purkinje neuron using only its calcium signal, which works well on real data even though it was trained only on simulated data. CaMLsort (Calcium imaging and Machine Learning based tool to sort intracellular state) also generalizes well to bistable neurons in a different brain region (ventral tegmental area) in a different model organism (mouse). This tool can facilitate our understanding of how these neurons carry out their functions in a circuit.

}, issn = {1469-7793}, doi = {10.1113/JP284373}, author = {Varma, Aalok and Udupa, Sathvik and Sengupta, Mohini and Ghosh, Prasanta Kumar and Thirumalai, Vatsala} } @article {139055, title = {Predictive neural computations in the cerebellum contribute to motor planning and faster behavioral responses in larval zebrafish.}, journal = {Sci Adv}, volume = {10}, year = {2024}, month = {2024 Jan 05}, pages = {eadi6470}, abstract = {

The ability to predict the future based on past experience lies at the core of the brain{\textquoteright}s ability to adapt behavior. However, the neural mechanisms that participate in generating and updating predictions are not clearly understood. Further, the evolutionary antecedents and the prevalence of predictive processing among vertebrates are even less explored. Here, we show evidence of predictive processing via the involvement of cerebellar circuits in larval zebrafish. We presented stereotyped optic flow stimuli to larval zebrafish to evoke swims and discovered that lesioning the cerebellum abolished prediction-dependent modulation of swim latency. When expectations of optic flow direction did not match with reality, error signals arrive at Purkinje cells via the olivary climbing fibers, whereas granule cells and Purkinje cells encode signals of expectation. Strong neural representations of expectation correlate with faster swim responses and vice versa. In sum, our results show evidence for predictive processing in nonmammalian vertebrates with the involvement of cerebellum, an evolutionarily conserved brain structure.

}, keywords = {Animals, Cerebellum, Larva, Neurons, Purkinje Cells, Zebrafish}, issn = {2375-2548}, doi = {10.1126/sciadv.adi6470}, author = {Narayanan, Sriram and Varma, Aalok and Thirumalai, Vatsala} } @inbook {116718, title = {Recruitment of Motoneurons.}, booktitle = {Vertebrate Motoneurons (Series: Advances in Neurobiology)}, volume = {28}, number = {28}, year = {2022}, pages = {169-190}, publisher = {Springer}, organization = {Springer}, abstract = {

Beginning about half a century ago, the rules that determine how motor units are recruited during movement have been deduced. These classical experiments led to the formulation of the {\textquoteright}size principle{\textquoteright}. It is now clear that motoneuronal size is not the only indicator of recruitment order. In fact, motoneuronal passive, active and synaptic conductances are carefully tuned to achieve sequential recruitment. More recent studies, over the last decade or so, show that the premotor circuitry is also functionally specialized and differentially recruited. Modular sub networks of interneurons and their post-synaptic motoneurons have been shown to drive movements with varying intensities. In addition, these modular networks are under the influence of neuromodulators, which are capable of acting upon multiple motor and premotor targets, thereby altering behavioral outcomes. We discuss the recruitment patterns of motoneurons in light of these new and exciting studies.

}, keywords = {Humans, Interneurons, Motor Neurons, Movement}, isbn = {9783031071669}, doi = {10.1007/978-3-031-07167-6_8}, author = {Thirumalai, Vatsala and Jha, Urvashi} } @article {97224, title = {Gjd2b-mediated gap junctions promote glutamatergic synapse formation and dendritic elaboration in Purkinje neurons.}, journal = {Elife}, volume = {10}, year = {2021}, month = {2021 Aug 04}, abstract = {

Gap junctions between neurons serve as electrical synapses, in addition to conducting metabolites and signaling molecules. During development, early-appearing gap junctions are thought to prefigure chemical synapses, which appear much later. We present evidence for this idea at a central, glutamatergic synapse and provide some mechanistic insights. Loss or reduction in the levels of the gap junction protein Gjd2b decreased the frequency of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in cerebellar Purkinje neurons (PNs) in larval zebrafish. Ultrastructural analysis in the molecular layer showed decreased synapse density. Further, mEPSCs had faster kinetics and larger amplitudes in mutant PNs, consistent with their stunted dendritic arbors. Time-lapse microscopy in wild type and mutant PNs reveals that Gjd2b puncta promote the elongation of branches and that CaMKII may be a critical mediator of this process. These results demonstrate that Gjd2b-mediated gap junctions regulate glutamatergic synapse formation and dendritic elaboration in PNs.

}, issn = {2050-084X}, doi = {10.7554/eLife.68124}, author = {Sitaraman, Sahana and Yadav, Gnaneshwar and Agarwal, Vandana and Jabeen, Shaista and Verma, Shivangi and Jadhav, Meha and Thirumalai, Vatsala} } @article {99936, title = {High behavioral variability mediated by altered neuronal excitability in mutant zebrafish.}, journal = {eNeuro}, year = {2021}, month = {2021 Sep 17}, abstract = {

Autism spectrum disorders (ASDs) are characterized by abnormal behavioral traits arising from neural circuit dysfunction. While a number of genes have been implicated in ASDs, in most cases, a clear understanding of how mutations in these genes lead to circuit dysfunction and behavioral abnormality is absent. The () gene is one such gene, associated with ASDs, intellectual disability and a range of other neurodevelopmental conditions. Yet, the role of AUTS2 in neural development and circuit function is not at all known. Here, we undertook functional analysis of Auts2a, the main homolog of AUTS2 in zebrafish, in the context of the escape behavior. Escape behavior in wild type zebrafish is critical for survival and is therefore, reliable, rapid, and has well-defined kinematic properties. mutant zebrafish are viable, have normal gross morphology and can generate escape behavior with normal kinematics. However, the behavior is unreliable and delayed, with high trial-to-trial variability in the latency. Using calcium imaging we probed the activity of Mauthner neurons during otic vesicle stimulation and observed lower probability of activation and reduced calcium transients in the mutants. With direct activation of Mauthner by antidromic stimulation, the threshold for activation in mutants was higher than that in wild type, even when inhibition was blocked. Taken together, these results point to reduced excitability of Mauthner neurons in auts2a mutant larvae leading to unreliable escape responses. Our results show a novel role for Auts2a in regulating neural excitability and reliability of behavior.AUTS2 is one among recently identified autism susceptibility candidate genes, whose function in neuronal circuits is unclear. Using zebrafish as a model organism, we probe the function of Auts2a (homolog of mammalian AUTS2) at the cellular, network and behavioral levels. The escape behavior of Auts2a mutant zebrafish is highly variable with normal short latency escapes, long latency escapes and total failures across trials in the same fish. This occurs because neuronal excitability is inappropriately set in the Mauthner neurons of mutants leading to large trial-to-trial variability in responses. The behavioral variability is fully explained by variability in firing action potentials in the Mauthner neuron, providing an integrative understanding of how behavioral variability arises from mutations at the genetic level.

}, issn = {2373-2822}, doi = {10.1523/ENEURO.0493-20.2021}, author = {Jha, Urvashi and Kondrychyn, Igor and Korzh, Vladimir and Thirumalai, Vatsala} } @article {73638, title = {Neuromodulatory Selection of Motor Neuron Recruitment Patterns in a Visuomotor Behavior Increases Speed.}, journal = {Curr Biol}, year = {2020}, month = {2020 Feb 20}, abstract = {

Animals generate locomotion at different speeds to suit their behavioral needs. Spinal circuits generate locomotion at these varying speeds by sequential activation of different spinal interneurons and motor neurons. Larval zebrafish can generate slow swims for prey capture and exploration by activation of secondary motor neurons and much faster and vigorous swims during escape and struggle via additional activation of primary motor neurons. Neuromodulators are known to alter the motor output of spinal circuits, but their precise role in speed regulation is not well\ understood. Here, in the context of optomotor response (OMR), an innate evoked locomotor behavior, we show that dopamine (DA) provides an additional layer to regulation of swim speed in larval zebrafish. Activation of D1-like receptors increases swim speed during OMR in free-swimming larvae. By analyzing tail bend kinematics in head-restrained larvae, we show that the increase in speed is actuated by larger tail bends. Whole-cell patch-clamp recordings from motor neurons reveal that, during OMR, typically only secondary motor neurons are active, whereas primary motor neurons are quiescent. Activation of D1-like receptors increases intrinsic excitability and excitatory synaptic drive in primary and secondary motor neurons. These actions result in greater recruitment of motor neurons during OMR. Our findings provide an example of neuromodulatory reconfiguration of spinal motor neuron speed modules where members are selectively recruited and motor drive is increased to effect changes in locomotor speed. VIDEO ABSTRACT.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2019.12.064}, author = {Jha, Urvashi and Thirumalai, Vatsala} } @article {55794, title = {Building behaviors, one layer at a time.}, journal = {Elife}, volume = {8}, year = {2019}, month = {2019 Apr 04}, abstract = {

New interneurons are added in the hindbrain to support more complex movements as young zebrafish get older.

}, issn = {2050-084X}, doi = {10.7554/eLife.46375}, author = {Wyart, Claire and Thirumalai, Vatsala} } @article {55795, title = {Contributions of the Cerebellum for Predictive and Instructional Control of Movement.}, journal = {Curr Opin Physiol}, volume = {8}, year = {2019}, month = {2019 Apr}, pages = {146-151}, abstract = {

The cerebellum with its layered structure and stereotyped and conserved connectivity has long puzzled neurobiologists. While it is well established that the cerebellum functions in regulating balance, motor coordination and motor learning, how it achieves these end results has not been very clear. Recent technical advances have made it possible to tease apart the contributions of cerebellar cell types to movement in behaving animals. We review these studies focusing on the three major cerebellar cell types, namely: granule cells, Purkinje neurons and the cells of the deep cerebellar nuclei. Further, we also review our current understanding of cortico-cerebellar and basal ganglia-cerebellar interactions that play vital roles in motor planning and motor learning.

}, issn = {2468-8673}, doi = {10.1016/j.cophys.2019.01.011}, author = {Narayanan, Sriram and Thirumalai, Vatsala} } @article {43855, title = {The interplay between electrical and chemical synaptogenesis.}, journal = {J Neurophysiol}, year = {2018}, month = {2018 Aug 01}, abstract = {

Neurons communicate with each other via electrical or chemical synaptic connections. The pattern and strength of connections between neurons is critical for generating appropriate output. What mechanisms govern the formation of electrical and/or chemical synapses between two neurons? Recent studies indicate that common molecular players could regulate the formation of both these classes of synapses. In addition, electrical and chemical synapses can mutually co-regulate each other{\textquoteright}s formation. Electrical activity, generated spontaneously by the nervous system, or initiated from sensory experience, plays an important role in this process, leading to the selection of appropriate connections and the elimination of inappropriate ones. In this review, we discuss recent studies that shed light on the formation and developmental interactions of chemical and electrical synapses.

}, issn = {1522-1598}, doi = {10.1152/jn.00398.2018}, author = {Jabeen, Shaista and Thirumalai, Vatsala} } @article {17945, title = {Loss of the Habenula Intrinsic Neuromodulator Kisspeptin1 Affects Learning in Larval Zebrafish.}, journal = {eNeuro}, volume = {4}, year = {2017}, month = {2017 May-Jun}, abstract = {

Learning how to actively avoid a predictable threat involves two steps: recognizing the cue that predicts upcoming punishment and learning a behavioral response that will lead to avoidance. In zebrafish, ventral habenula (vHb) neurons have been proposed to participate in both steps by encoding the expected aversiveness of a stimulus. vHb neurons increase their firing rate as expectation of punishment grows but reduce their activity as avoidance learning occurs. This leads to changes in the activity of raphe neurons, which are downstream of the vHb, during learning. How vHb activity is regulated is not known. Here, we ask whether the neuromodulator Kisspeptin1, which is expressed in the ventral habenula together with its receptor, could be involved. Kiss1 mutants were generated with CRISPR/Cas9 using guide RNAs targeted to the signal sequence. Mutants, which have a stop codon upstream of the active Kisspeptin1 peptide, have a deficiency in learning to avoid a shock that is predicted by light. Electrophysiology indicates that Kisspeptin1 has a concentration-dependent effect on vHb neurons: depolarizing at low concentrations and hyperpolarizing at high concentrations. Two-photon calcium imaging shows that mutants have reduced raphe response to shock. These data are consistent with the hypothesis that Kisspeptin1 modulates habenula neurons as the fish learns to cope with a threat. Learning a behavioral strategy to overcome a stressor may thus be accompanied by physiological change in the habenula, mediated by intrinsic neuromodulation.

}, issn = {2373-2822}, doi = {10.1523/ENEURO.0326-16.2017}, author = {Lupton, Charlotte and Sengupta, Mohini and Cheng, Ruey-Kuang and Chia, Joanne and Thirumalai, Vatsala and Jesuthasan, Suresh} } @article {19198, title = {Transcriptional Complexity and Distinct Expression Patterns of auts2 Paralogs in Danio rerio.}, journal = {G3 (Bethesda)}, year = {2017}, month = {2017 Jun 16}, abstract = {

Several genes that have been implicated in autism spectrum disorders are large and have multiple transcripts. Neurons are especially enriched with longer transcripts compared to non-neural cell types. The human autism susceptibility candidate 2 (AUTS2) gene is ~1.2 Mb long and is implicated in a number of neurological disorders including autism, intellectual disability, addiction and developmental delay. Recent studies show AUTS2 to be important for activation of transcription of neural specific genes, neuronal migration and neurite outgrowth. However, much remains to be understood regarding the transcriptional complexity and the functional roles of AUTS2 in neurodevelopment. Zebrafish provide an excellent model system for studying both these questions. We undertook genomic identification and characterization of auts2 and its paralogous genes in zebrafish. There are four auts2 family genes in zebrafish: auts2a, auts2b, fbrsl1 and fbrs The absence of complete annotation of their structures hampers functional studies. We present evidence for transcriptional complexity of these four genes mediated by alternative splicing and alternative promoter usage. Furthermore, the expression of the various paralogs is tightly regulated both spatially and developmentally. Our findings suggest that auts2 paralogs serve distinct functions in the development and functioning of target tissues.

}, issn = {2160-1836}, doi = {10.1534/g3.117.042622}, author = {Kondrychyn, Igor and Robra, Lena and Thirumalai, Vatsala} } @article {7038, title = {The Intracellular Signaling Molecule Darpp-32 Is a Marker for Principal Neurons in the Cerebellum and Cerebellum-Like Circuits of Zebrafish.}, journal = {Front Neuroanat}, volume = {10}, year = {2016}, month = {2016}, pages = {81}, abstract = {

The dopamine and cAMP regulated phosphoprotein of apparent molecular weight 32 kDa (Darpp-32) is an inhibitory subunit of protein phosphatase-1 (PP-1). Darpp-32 activity is regulated by multiple ligand-activated G-protein coupled receptors (GPCRs). This protein is coded for by the protein phosphatase-1 regulatory subunit 1b (ppp1r1b) gene. Here, we provide experimental evidence for the presence of multiple isoforms of ppp1r1b in zebrafish. We show that these isoforms are differentially expressed during development with the full-length isoform being maternally deposited. Next, with a custom polyclonal antibody generated against the full-length protein, we show that in the adult, Darpp-32 is strongly expressed in principal neurons of the cerebellum and cerebellum-like circuits. These include Purkinje neurons in the cerebellum, Type-I neurons in the optic tectum, and crest cells in the medial octavolateralis nucleus (MON). We confirmed the identity of these neurons through their colocalization with Parvalbumin 7 immunoreactivity. Darpp-32 is seen in the somata and dendrites of these neurons with faint staining in the axons. In all of these regions, Darpp-32-immunoreactive cells were in close proximity to tyrosine hydroxylase (TH) immunoreactive puncta indicating the presence of direct catecholaminergic input to these neurons. Darpp-32 immunoreactivity was seen in Purkinje neurons as early as 3 days post-fertilization (dpf) when Purkinje neurons are first specified. In sum, we show that Darpp-32, a signaling integrator, is a specific marker of principal neurons in the cerebellum and cerebellum-like circuits in zebrafish.

}, issn = {1662-5129}, doi = {10.3389/fnana.2016.00081}, author = {Robra, Lena and Thirumalai, Vatsala} }