Neuron-glia interaction through Serotonin-BDNF-NGFR axis enables regenerative neurogenesis in Alzheimer's model of adult zebrafish brain.

It was recently suggested that supplying the brain with new neurons could counteract Alzheimer's disease (AD). This provocative idea requires further testing in experimental models in which the molecular basis of disease-induced neuronal regeneration could be investigated. We previously found that zebrafish stimulates neural stem cell (NSC) plasticity and neurogenesis in AD and could help to understand the mechanisms to be harnessed for developing new neurons in diseased mammalian brains. Here, by performing single-cell transcriptomics, we found that amyloid toxicity-induced interleukin-4 (IL4) promotes NSC proliferation and neurogenesis by suppressing the tryptophan metabolism and reducing the production of serotonin. NSC proliferation was suppressed by serotonin via down-regulation of brain-derived neurotrophic factor (BDNF)-expression in serotonin-responsive periventricular neurons. BDNF enhances NSC plasticity and neurogenesis via nerve growth factor receptor A (NGFRA)/ nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFkB) signaling in zebrafish but not in rodents. Collectively, our results suggest a complex neuron-glia interaction that regulates regenerative neurogenesis after AD conditions in zebrafish.

Functionally distinct Purkinje cell types show temporal precision in encoding locomotion.

Purkinje cells, the principal neurons of cerebellar computations, are believed to comprise a uniform neuronal population of cells, each with similar functional properties. Here, we show an undiscovered heterogeneity of adult zebrafish Purkinje cells, revealing the existence of anatomically and functionally distinct cell types. Dual patch-clamp recordings showed that the cerebellar circuit contains all Purkinje cell types that cross-communicate extensively using chemical and electrical synapses. Further activation of spinal central pattern generators (CPGs) revealed unique phase-locked activity from each Purkinje cell type during the locomotor cycle. Thus, we show intricately organized Purkinje cell networks in the adult zebrafish cerebellum that encode the locomotion rhythm differentially, and we suggest that these organizational properties may also apply to other cerebellar functions.

Motor neurons control locomotor circuit function retrogradely via gap junctions.

Motor neurons are the final stage of neural processing for the execution of motor behaviours. Traditionally, motor neurons have been viewed as the 'final common pathway', serving as passive recipients merely conveying to the muscles the final motor program generated by upstream interneuron circuits. Here we reveal an unforeseen role of motor neurons in controlling the locomotor circuit function via gap junctions in zebrafish. These gap junctions mediate a retrograde analogue propagation of voltage fluctuations from motor neurons to control the synaptic release and recruitment of the upstream V2a interneurons that drive locomotion. Selective inhibition of motor neurons during ongoing locomotion de-recruits V2a interneurons and strongly influences locomotor circuit function. Rather than acting as separate units, gap junctions unite motor neurons and V2a interneurons into functional ensembles endowed with a retrograde analogue computation essential for locomotor rhythm generation. These results show that motor neurons are not a passive recipient of motor commands but an integral component of the neural circuits responsible for motor behaviour.

Adult spinal motoneurons change their neurotransmitter phenotype to control locomotion.

A particularly essential determinant of a neuron's functionality is its neurotransmitter phenotype. While the prevailing view is that neurotransmitter phenotypes are fixed and determined early during development, a growing body of evidence suggests that neurons retain the ability to switch between different neurotransmitters. However, such changes are considered unlikely in motoneurons due to their crucial functional role in animals' behavior. Here we describe the expression and dynamics of glutamatergic neurotransmission in the adult zebrafish spinal motoneuron circuit assembly. We demonstrate that part of the fast motoneurons retain the ability to switch their neurotransmitter phenotype under physiological (exercise/training) and pathophysiological (spinal cord injury) conditions to corelease glutamate in the neuromuscular junctions to enhance animals' motor output. Our findings suggest that motoneuron neurotransmitter switching is an important plasticity-bestowing mechanism in the reconfiguration of spinal circuits that control movements.

Optogenetic activation of excitatory premotor interneurons is sufficient to generate coordinated locomotor activity in larval zebrafish.

Neural networks in the spinal cord can generate locomotion in the absence of rhythmic input from higher brain structures or sensory feedback because they contain an intrinsic source of excitation. However, the molecular identity of the spinal interneurons underlying the excitatory drive within the locomotor circuit has remained unclear. Using optogenetics, we show that activation of a molecularly defined class of ipsilateral premotor interneurons elicits locomotion. These interneurons represent the excitatory module of the locomotor networks and are sufficient to produce a coordinated swimming pattern in zebrafish. They correspond to the V2a interneuron class and express the transcription factor Chx10. They produce sufficient excitatory drive within the spinal networks to generate coordinated locomotor activity. Therefore, our results define the V2a interneurons as the excitatory module within the spinal locomotor networks that is sufficient to initiate and maintain locomotor activity.

Pattern of innervation and recruitment of different classes of motoneurons in adult zebrafish.

In vertebrates, spinal circuits drive rhythmic firing in motoneurons in the appropriate sequence to produce locomotor movements. These circuits become active early during development and mature gradually to acquire the flexibility necessary to accommodate the increased behavioral repertoire of adult animals. The focus here is to elucidate how different pools of motoneurons are organized and recruited and how membrane properties contribute to their mode of operation. For this purpose, we have used the in vitro preparation of adult zebrafish. We show that different motoneuron pools are organized in a somatotopic fashion in the motor column related to the type of muscle fibers (slow, intermediate, fast) they innervate. During swimming, the different motoneuron pools are recruited in a stepwise manner from slow, to intermediate, to fast to cover the full range of locomotor frequencies seen in intact animals. The spike threshold, filtering properties, and firing patterns of the different motoneuron pools are graded in a manner that relates to their order of recruitment. Our results thus show that motoneurons in adult zebrafish are organized into distinct modules, each with defined locations, properties, and recruitment patterns tuned to precisely match the muscle properties and hence produce swimming of different speeds and modalities.

Neuroprotective gap-junction-mediated bystander transformations in the adult zebrafish spinal cord after injury.

The adult zebrafish spinal cord displays an impressive innate ability to regenerate after traumatic insults, yet the underlying adaptive cellular mechanisms remain elusive. Here, we show that while the cellular and tissue responses after injury are largely conserved among vertebrates, the large-size fast spinal zebrafish motoneurons are remarkably resilient by remaining viable and functional. We also reveal the dynamic changes in motoneuron glutamatergic input, excitability, and calcium signaling, and we underscore the critical role of calretinin (CR) in binding and buffering the intracellular calcium after injury. Importantly, we demonstrate the presence and the dynamics of a neuron-to-neuron bystander neuroprotective biochemical cooperation mediated through gap junction channels. Our findings support a model in which the intimate and dynamic interplay between glutamate signaling, calcium buffering, gap junction channels, and intercellular cooperation upholds cell survival and promotes the initiation of regeneration.

Lifelong regeneration of cerebellar Purkinje cells after induced cell ablation in zebrafish.

Zebrafish have an impressive capacity to regenerate neurons in the central nervous system. However, regeneration of the principal neuron of the evolutionary conserved cerebellum, the Purkinje cell (PC), is believed to be limited to developmental stages based on invasive lesions. In contrast, non-invasive cell type-specific ablation by induced apoptosis closely represents a process of neurodegeneration. We demonstrate that the ablated larval PC population entirely recovers in number, quickly reestablishes electrophysiological properties, and properly integrates into circuits to regulate cerebellum-controlled behavior. PC progenitors are present in larvae and adults, and PC ablation in adult cerebelli results in an impressive PC regeneration of different PC subtypes able to restore behavioral impairments. Interestingly, caudal PCs are more resistant to ablation and regenerate more efficiently, suggesting a rostro-caudal pattern of de- and regeneration properties. These findings demonstrate that the zebrafish cerebellum is able to regenerate functional PCs during all stages of the animal's life.

High cardiomyocyte diversity in human early prenatal heart development.

Cardiomyocytes play key roles during cardiogenesis, but have poorly understood features, especially in prenatal stages. Here, we characterized human prenatal cardiomyocytes, 6.5-7 weeks post-conception, by integrating single-cell RNA sequencing, spatial transcriptomics, and ligand-receptor interaction information. Using a computational workflow developed to dissect cell type heterogeneity, localize cell types, and explore their molecular interactions, we identified eight types of developing cardiomyocyte, more than double compared to the ones identified in the Human Developmental Cell Atlas. These have high variability in cell cycle activity, mitochondrial content, and connexin gene expression, and are differentially distributed in the ventricles, including outflow tract, and atria, including sinoatrial node. Moreover, cardiomyocyte ligand-receptor crosstalk is mainly with non-cardiomyocyte cell types, encompassing cardiogenesis-related pathways. Thus, early prenatal human cardiomyocytes are highly heterogeneous and develop unique location-dependent properties, with complex ligand-receptor crosstalk. Further elucidation of their developmental dynamics may give rise to new therapies.

A new transgenic reporter line reveals expression of protocadherin 9 at a cellular level within the zebrafish central nervous system.

The wiring of neuronal networks is far from understood. One outstanding question is how neurons of different types link up to form subnetworks within the greater context. Cadherins have been suggested to create an inclusion code where interconnected neurons express the same subtypes. Here, we have used a CRISPR/Cas9 knock-in approach to generate a transgenic zebrafish reporter line for protocadherin 9 (pcdh9), which is predominantly expressed within the central nervous system. Expression of eGFP was detected in subsets of neurons in the cerebellum, retina and spinal cord, in both larvae and juveniles. A closer characterization of the spinal locomotor network revealed that a portion of distinct classes of both excitatory and inhibitory interneurons, as well as motor neurons, expressed pcdh9. This transgenic line could thus be used to test the cadherin network hypothesis, through electrophysiological characterization of eGFP positive cells, to show if these are synaptically connected and form a discrete network within the spinal cord.

Purkinje cells located in the adult zebrafish valvula cerebelli exhibit variable functional responses.

Purkinje cells are critically involved in processing the cerebellar functions by shaping and coordinating commands that they receive. Here, we demonstrate experimentally that in the adult zebrafish valvular part of the cerebellum, the Purkinje cells exhibited variable firing and functional responses and allowed the categorization into three firing classes. Compared with the Purkinje cells in the corpus cerebelli, the valvular Purkinje cells receive weak and occasional input from the inferior olive and are not active during locomotion. Together, our findings expand further the regional functional differences of the Purkinje cell population and expose their non-locomotor functionality.

Locomotion dependent neuron-glia interactions control neurogenesis and regeneration in the adult zebrafish spinal cord.

Physical exercise stimulates adult neurogenesis, yet the underlying mechanisms remain poorly understood. A fundamental component of the innate neuroregenerative capacity of zebrafish is the proliferative and neurogenic ability of the neural stem/progenitor cells. Here, we show that in the intact spinal cord, this plasticity response can be activated by physical exercise by demonstrating that the cholinergic neurotransmission from spinal locomotor neurons activates spinal neural stem/progenitor cells, leading to neurogenesis in the adult zebrafish. We also show that GABA acts in a non-synaptic fashion to maintain neural stem/progenitor cell quiescence in the spinal cord and that training-induced activation of neurogenesis requires a reduction of GABAA receptors. Furthermore, both pharmacological stimulation of cholinergic receptors, as well as interference with GABAergic signaling, promote functional recovery after spinal cord injury. Our findings provide a model for locomotor networks' activity-dependent neurogenesis during homeostasis and regeneration in the adult zebrafish spinal cord.

Multiple Rhythm-Generating Circuits Act in Tandem with Pacemaker Properties to Control the Start and Speed of Locomotion.

In vertebrates, specific command centers in the brain can selectively drive slow-explorative or fast-speed locomotion. However, it remains unclear how the locomotor central pattern generator (CPG) processes descending drive into coordinated locomotion. Here, we reveal, in adult zebrafish, a logic of the V2a interneuron rhythm-generating circuits involving recurrent and hierarchical connectivity that acts in tandem with pacemaker properties to provide an ignition and gear-shift mechanism to start locomotion and change speed. A comprehensive mapping of synaptic connections reveals three recurrent circuit modules engaged sequentially to increase locomotor speed. The connectivity between V2a interneurons of different modules displayed a clear asymmetry in favor of connections from faster to slower modules. The interplay between V2a interneuron pacemaker properties and their organized connectivity provides a mechanism for locomotor initiation and speed control. Thus, our results provide mechanistic insights into how the spinal CPG transforms descending drive into locomotion and align its speed with the initial intention.

Large-Scale Analysis of the Diversity and Complexity of the Adult Spinal Cord Neurotransmitter Typology.

The development of nervous system atlases is a fundamental pursuit in neuroscience, since they constitute a fundamental tool to improve our understanding of the nervous system and behavior. As such, neurotransmitter maps are valuable resources to decipher the nervous system organization and functionality. We present here the first comprehensive quantitative map of neurons found in the adult zebrafish spinal cord. Our study overlays detailed information regarding the anatomical positions, sizes, neurotransmitter phenotypes, and the projection patterns of the spinal neurons. We also show that neurotransmitter co-expression is much more extensive than previously assumed, suggesting that spinal networks are more complex than first recognized. As a first direct application, we investigated the neurotransmitter diversity in the putative glutamatergic spinal V2a-interneuron assembly. These studies shed new light on the diverse and complex functions of this important interneuron class in the neuronal interplay governing the precise operation of the central pattern generators.

Spinal cholinergic interneurons differentially control motoneuron excitability and alter the locomotor network operational range.

While cholinergic neuromodulation is important for locomotor circuit operation, the specific neuronal mechanisms that acetylcholine employs to regulate and fine-tune the speed of locomotion are largely unknown. Here, we show that cholinergic interneurons are present in the zebrafish spinal cord and differentially control the excitability of distinct classes of motoneurons (slow, intermediate and fast) in a muscarinic dependent manner. Moreover, we reveal that m2-type muscarinic acetylcholine receptors (mAChRs) are present in fast and intermediate motoneurons, but not in the slow motoneurons, and that their activation decreases neuronal firing. We also reveal a strong correlation between the muscarinic receptor configuration on motoneurons and the ability of the animals to locomote at different speeds, which might serve as a plasticity mechanism to alter the operational range of the locomotor networks. These unexpected findings provide new insights into the functional flexibility of motoneurons and how they execute locomotion at different speeds.

Complementary expression of calcium binding proteins delineates the functional organization of the locomotor network.

Neuronal networks in the spinal cord generate and execute all locomotor-related movements by transforming descending signals from supraspinal areas into appropriate rhythmic activity patterns. In these spinal networks, neurons that arise from the same progenitor domain share similar distribution patterns, neurotransmitter phenotypes, morphological and electrophysiological features. However, subgroups of them participate in different functionally distinct microcircuits to produce locomotion at different speeds and of different modalities. To better understand the nature of this network complexity, here we characterized the distribution of parvalbumin (PV), calbindin D-28 k (CB) and calretinin (CR) which are regulators of intracellular calcium levels and can serve as anatomical markers for morphologically and potential functionally distinct neuronal subpopulations. We observed wide expression of CBPs in the adult zebrafish, in several spinal and reticulospinal neuronal populations with a diverse neurotransmitter phenotype. We also found that several spinal motoneurons express CR and PV. However, only the motoneuron pools that are responsible for generation of fast locomotion were CR-positive. CR can thus be used as a marker for fast motoneurons and might potentially label the fast locomotor module. Moreover, CB was mainly observed in the neuronal progenitor cells that are distributed around the central canal. Thus, our results suggest that during development the spinal neurons utilize CB and as the neurons mature and establish a neurotransmitter phenotype they use CR or/and PV. The detailed characterization of CBPs expression, in the spinal cord and brainstem neurons, is a crucial step toward a better understanding of the development and functionality of neuronal locomotor networks.

Sexual dimorphisms in swimming behavior, cerebral metabolic activity and adrenoceptors in adult zebrafish (Danio rerio).

Sexually dimorphic behaviors and brain sex differences, not only restricted to reproduction, are considered to be evolutionary preserved. Specifically, anxiety related behavioral repertoire is suggested to exhibit sex-specific characteristics in rodents and primates. The present study investigated whether behavioral responses to novelty, have sex-specific characteristics in the neurogenetic model organism zebrafish (Danio rerio), lacking chromosomal sex determination. For this, aspects of anxiety-like behavior (including reduced exploration, increased freezing behavior and erratic movement) of male and female adult zebrafish were tested in a novel tank paradigm and after habituation. Male and female zebrafish showed significant differences in their swimming activity in response to novelty, with females showing less anxiety spending more time in the upper tank level. When fish have habituated, regional cerebral glucose uptake, an index of neuronal activity, and brain adrenoceptors' (ARs) expression (α2-ARs and ß-ARs) were determined using in vivo 2-[(14)C]-deoxyglucose methodology and in vitro neurotransmitter receptors quantitative autoradiography, respectively. Intriguingly, females exhibited higher glucose utilization than males in hypothalamic brain areas. Adrenoceptor's expression pattern was dimorphic in zebrafish telencephalic, preoptic, hypothalamic nuclei, central gray, and cerebellum, similarly to birds and mammals. Specifically, the lateral zone of dorsal telencephalon (Dl), an area related to spatial cognition, homologous to the mammalian hippocampus, showed higher α2-AR densities in females. In contrast, male cerebellum included higher densities of ß-ARs in comparison to female. Taken together, our data demonstrate a well-defined sex discriminant cerebral metabolic activity and ARs' pattern in zebrafish, possibly contributing to male-female differences in the swimming behavior.

A Hardwired Circuit Supplemented with Endocannabinoids Encodes Behavioral Choice in Zebrafish.

Animals constantly make behavioral choices to facilitate moving efficiently through their environment. When faced with a threat, animals make decisions in the midst of other ongoing behaviors through a context-dependent integration of sensory stimuli. In vertebrates, the mechanisms underlying behavioral selection are poorly understood. Here, we show that ongoing swimming in zebrafish is suppressed by escape. The selection of escape over swimming is mediated by switching between two distinct motoneuron pools. A hardwired circuit mediates this switch by acting as a clutch-like mechanism to disengage the swimming motoneuron pool and engage the escape motoneuron pool. Threshold for escape initiation is lowered and swimming suppression is prolonged by endocannabinoid neuromodulation. Thus, our results reveal a novel cellular mechanism involving a hardwired circuit supplemented with endocannabinoids acting as a clutch-like mechanism to engage/disengage distinct motor pools to ensure behavioral selection and a smooth execution of motor action sequences in a vertebrate system.

Separate microcircuit modules of distinct v2a interneurons and motoneurons control the speed of locomotion.

Spinal circuits generate locomotion with variable speed as circumstances demand. These circuits have been assumed to convey equal and uniform excitation to all motoneurons whose input resistance dictates their activation sequence. However, the precise connectivity pattern between excitatory premotor circuits and the different motoneuron types has remained unclear. Here, we generate a connectivity map in adult zebrafish between the V2a excitatory interneurons and slow, intermediate, and fast motoneurons. We show that the locomotor network does not consist of a uniform circuit as previously assumed. Instead, it can be deconstructed into three separate microcircuit modules with distinct V2a interneuron subclasses driving slow, intermediate, or fast motoneurons. This modular design enables the increase of locomotor speed by sequentially adding microcircuit layers from slow to intermediate and fast. Thus, this principle of organization of vertebrate spinal circuits represents an intrinsic mechanism to increase the locomotor speed by incrementally engaging different motor units.

Principles governing recruitment of motoneurons during swimming in zebrafish.

Locomotor movements are coordinated by a network of neurons that produces sequential muscle activation. Different motoneurons need to be recruited in an orderly manner to generate movement with appropriate speed and force. However, the mechanisms governing recruitment order have not been fully clarified. Using an in vitro juvenile/adult zebrafish brainstem-spinal cord preparation, we found that motoneurons were organized into four pools with specific topographic locations and were incrementally recruited to produce swimming at different frequencies. The threshold of recruitment was not dictated by the input resistance of motoneurons, but was instead set by a combination of specific biophysical properties and the strength of the synaptic currents. Our results provide insights into the cellular and synaptic computations governing recruitment of motoneurons during locomotion.