PhD candidate, Hong Kong University of Science and Technology
In vivo formation of the functional connectivity within spinal cord requires Ca2+ signalling
In vertebrates, two key areas underlying locomotion are the hindbrain and spinal cord. In the vertebrate model zebrafish, both of these structures display a relatively simple architecture, which greatly facilitate its use for neuroscience research.
During the early development of zebrafish, the motor neurons in the spinal cord begin to display spontaneous and stochastic Ca2+ activity, which coincides with the generation of Ca2+ transients in the muscle cells and spontaneous coiling of the trunk. Studies have shown that such spontaneous activity is not affected by hindbrain lesion, indicating that the patterned activity may be initiated from an endogenous network in the spinal cord. As development proceeds, the immature coilings develops into an organized form of swimming, and this transition requires the establishment of a synchronized, correlated connectivity within the spinal network and with the developing cells of the myotome.
Ca2+ is a versatile and universal second messenger, which regulates neuronal differentiation and proliferation, as well as axon growth and guidance. Although most of the previous work on Ca2+ signaling in neural development has been limited to in vitro experiments, relatively few studies have explored its role in the formation of the neural circuitry in an intact, developing vertebrate.
In this study, I demonstrated that nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca2+ release via two-pore channel type 2 (TPC2) from the acidic stores is required for the regulation of the transition from the sporadic and uncorrelated, to the rhythmic, ipsilaterally correlated and contralaterally anti-correlated activity of the zebrafish embryonic spinal circuitry in vivo. Using a double-transgenic line that expresses a genetically encoded Ca2+ indicator specifically in a subset of motor neurons, the frequency, amplitude, and duration of the Ca2+ transients in the motor neurons were shown to be disrupted after TPC2 inhibition via molecular, genetic or pharmacological means.
Combining with my previous observations that the muscle development as well as the motility of the zebrafish embryos were severely compensated after TPC2 inhibition, my new data therefore suggest a novel function for NAADP/TPC2/Ca2+ signaling in the development, coordination, and maturation of the spinal network required for the establishment of early coordinated muscle-mediated locomotory behavioral patterns.
Abstract: Developing neural networks display spontaneous and correlated rhythmic bursts of action potentials that are essential for circuit refinement. In the spinal cord, it is poorly understood how correlated activity is acquired and how its emergence relates to the formation of the spinal central pattern generator (CPG), the circuit that mediates rhythmic behaviors like walking and swimming. It is also unknown whether early, uncorrelated activity is necessary for the formation of the coordinated CPG.Time-lapse imaging in the intact zebrafish embryo with the genetically encoded calcium indicator GCaMP3 revealed a rapid transition from slow, sporadic activity to fast, ipsilaterally correlated, and contralaterally anticorrelated activity, characteristic of the spinal CPG. Ipsilateral correlations were acquired through the coalescence of local microcircuits. Brief optical manipulation of activity with the light-driven pump halorhodopsin revealed that the transition to correlated activity was associated with a strengthening of ipsilateral connections, likely mediated by gap junctions. Contralateral antagonism increased in strength at the same time. The transition to coordinated activity was disrupted by long-term optical inhibition of sporadic activity in motoneurons and ventral longitudinal descending interneurons and resulted in more neurons exhibiting uncoordinated activity patterns at later time points.These findings show that the CPG in the zebrafish spinal cord emerges directly from a sporadically active network as functional connectivity strengthens between local and then more distal neurons. These results also reveal that early, sporadic activity in a subset of ventral spinal neurons is required for the integration of maturing neurons into the coordinated CPG network.
Pub.: 27 Dec '11, Pinned: 01 Aug '17
Abstract: Animal behaviors are generated by well-coordinated activation of neural circuits. In zebrafish, embryos start to show spontaneous muscle contractions at 17 to 19 h postfertilization. To visualize how motor circuits in the spinal cord are activated during this behavior, we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the genetically encoded calcium indicator GCaMP, and created transgenic zebrafish carrying the GCaMP-HS gene downstream of the Gal4-recognition sequence, UAS (upstream activation sequence). Then we performed a gene-trap screen and identified the SAIGFF213A transgenic fish that expressed Gal4FF, a modified version of Gal4, in a subset of spinal neurons including the caudal primary (CaP) motor neurons. We conducted calcium imaging using the SAIGFF213A; UAS:GCaMP-HS double transgenic embryos during the spontaneous contractions. We demonstrated periodic and synchronized activation of a set of ipsilateral motor neurons located on the right and left trunk in accordance with actual muscle movements. The synchronized activation of contralateral motor neurons occurred alternately with a regular interval. Furthermore, a detailed analysis revealed rostral-to-caudal propagation of activation of the ipsilateral motor neuron, which is similar to but much slower than the rostrocaudal delay observed during swimming in later stages. Our study thus demonstrated coordinated activities of the motor neurons during the first behavior in a vertebrate. We propose the GCaMP technology combined with the Gal4FF-UAS system is a powerful tool to study functional neural circuits in zebrafish.
Pub.: 09 Mar '11, Pinned: 01 Aug '17
Abstract: Neuronal motility is a fundamental feature that underlies the development, regeneration, and plasticity of the nervous system. Two major developmental events--directed migration of neuronal precursor cells to the proper positions and guided elongation of axons to their target cells--depend on large-scale neuronal motility. At a finer scale, motility is also manifested in many aspects of neuronal structures and functions, ranging from differentiation and refinement of axonal and dendritic morphology during development to synapse remodeling associated with learning and memory in the adult brain. As a primary second messenger that conveys the cytoplasmic actions of electrical activity and many neuroactive ligands, Ca(2+) plays a central role in the regulation of neuronal motility. Recent studies have revealed common Ca(2+)-dependent signaling pathways that are deployed for regulating cytoskeletal dynamics associated with neuronal migration, axon and dendrite development and regeneration, and synaptic plasticity.
Pub.: 20 Oct '07, Pinned: 17 Aug '17
Abstract: We recently demonstrated a critical role for two-pore channel type 2 (TPC2)-mediated Ca(2+) release during the differentiation of slow (skeletal) muscle cells (SMC) in intact zebrafish embryos, via the introduction of a translational-blocking morpholino antisense oligonucleotide (MO). Here, we extend our study and demonstrate that knockdown of TPC2 with a non-overlapping splice-blocking MO, knockout of TPC2 (via the generation of a tpcn2(dhkz1a) mutant line of zebrafish using CRISPR/Cas9 gene-editing), or the pharmacological inhibition of TPC2 action with bafilomycin A1 or trans-ned-19, also lead to a significant attenuation of SMC differentiation, characterized by a disruption of SMC myofibrillogenesis and gross morphological changes in the trunk musculature. When the morphants were injected with tpcn2-mRNA or were treated with IP3/BM or caffeine (agonists of the inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR), respectively), many aspects of myofibrillogenesis and myotomal patterning (and in the case of the pharmacological treatments, the Ca(2+) signals generated in the SMCs), were rescued. STED super-resolution microscopy revealed a close physical relationship between clusters of RyR in the terminal cisternae of the SR, and TPC2 in lysosomes, with a mean estimated separation of ~52-87nm. Our data therefore add to the increasing body of evidence, which indicate that localized Ca(2+) release via TPC2 might trigger the generation of more global Ca(2+) release from the sarcoplasmic reticulum via Ca(2+)-induced Ca(2+) release.
Pub.: 10 Apr '17, Pinned: 01 Aug '17
Abstract: General mechanisms of motor network development have often been examined in the spinal cord because of its relative simplicity when compared to higher parts of the brain. Indeed, most of our current understanding of motor pattern generation comes from work in the lower vertebrate spinal cord. Nevertheless, very little is known about the initial stages of motor network formation and the interplay between genes and electrical activity. Recent research has led to the establishment of the zebrafish as a key model system to study the genetics of neural development. The spinal cord of zebrafish is amenable to optical and electrophysiological analysis of neuronal activity even at the earliest embryonic stages when the network is immature. The combination of physiology and genetics in the same animal model should lead to insights into the basic mechanisms of motor circuit formation. This paper reviews recent work on the development of zebrafish motor activity and discusses them in the context of the current knowledge of embryonic and larval zebrafish spinal cord morphology and physiology.
Pub.: 06 Feb '08, Pinned: 17 Aug '17
Abstract: It is well known that slow and fast muscles are used for long-term sustained movement and short bursts of activity, respectively, in adult animal behaviors. However, the contribution of the slow and fast muscles in early animal movement has not been thoroughly explored. In wild-type zebrafish embryos, tactile stimulation induces coilings consisting of 1-3 alternating contractions of the trunk and tail at 24 hours postfertilization (hpf) and burst swimming at 48 hpf. But, embryos defective in flightless I homolog (flii), which encodes for an actin-regulating protein, exhibit normal coilings at 24 hpf that is followed by significantly slower burst swimming at 48 hpf. Interestingly, actin fibers are disorganized in mutant fast muscle but not in mutant slow muscle, suggesting that slower swimming at 48 hpf is attributable to defects of the fast muscle tissue. In fact, perturbation of the fast muscle contractions by eliminating Ca(2+) release only in fast muscle resulted in normal coilings at 24 hpf and slower burst swimming at 48 hpf, just as flii mutants exhibited. In contrast, specific inactivation of slow muscle by knockdown of the slow muscle myosin genes led to complete loss of coilings at 24 hpf, although normal burst swimming was retained by 48 hpf. These findings indicate that coilings at 24 hpf is mediated by slow muscle only, whereas burst swimming at 48 hpf is executed primarily by fast muscle. It is consistent with the fact that differentiation of fast muscle follows that of slow muscle. This is the first direct demonstration that slow and fast muscles have distinct physiologically relevant contribution in early motor development at different stages.
Pub.: 11 May '11, Pinned: 17 Aug '17
Abstract: The anatomy of the developing zebrafish spinal cord is relatively simple but, despite this simplicity, it generates a sequence of three patterns of locomotive behaviors. The first behavior exhibited is spontaneous movement, then touch-evoked coiling, and finally swimming. Previous studies in zebrafish have suggested that spontaneous movements occur independent of supraspinal input and do not require chemical neurotransmission, while touch-evoked coiling and swimming depend on glycinergic neurotransmission as well as supraspinal input. In contrast, studies in other vertebrate preparations have shown that spontaneous movement requires glycine and other neurotransmitters and that later behaviors do not require supraspinal input. Here, we use lesion analysis combined with high-speed kinematic analysis to re-examine the role of glycine and supraspinal input in each of the three behaviors. We find that, similar to other vertebrate preparations, supraspinal input is not essential for spontaneous movement, touch-evoked coiling, or swimming behavior. Moreover, we find that blockade of glycinergic neurotransmission decreases the rate of spontaneous movement and impairs touch-evoked coiling and swimming, suggesting that glycinergic neurotransmission plays critical yet distinct roles for individual patterns of locomotive behaviors.
Pub.: 14 Feb '06, Pinned: 17 Aug '17
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