Figure 7 - uploaded by wen-chang li
Content may be subject to copyright.
Early-cycle inhibition and control of multiple firing in CPG neurons. A, Voltageclamp record of a cIN, clamped at a positive potential, to illustrate a typical pattern of inward (excitatory) and outward (inhibitory) PSCs during swimming. The record shows 16 overlapped cycles of swimming (mean period, 69.5 2.1 msec), each normalized to a cycle phase of 1 and plotted twice. Synchronous, "on-cycle" EPSCs (open arrowhead) just precede the start of the ventral root burst (vr; timing adjusted for longitudinal spacing of electrodes). Early-cycle IPSCs from aINs (filled arrowheads) are delayed relative to the on-cycle excitation and are less synchronized than on-cycle EPSCs or mid-cycle IPSCs from contralateral cINs (asterisk). The shaded region shows the window for impulse firing before early-cycle inhibition. B, Examples of firing activity in two cINs during swimming shown with ventral root activity (vr). Early-cycle IPSPs are present on some cycles (e.g., arrowheads). However, the neurons fire more impulses on cycles where these are absent (open arrowheads).
Source publication
Understanding the neuronal networks in the mammal spinal cord is hampered by the diversity of neurons and their connections. The simpler networks in developing lower vertebrates may offer insights into basic organization. To investigate the function of spinal inhibitory interneurons in Xenopus tadpoles, paired whole-cell recordings were used. We sh...
Contexts in source publication
Context 1
... inhibition occurs shortly after spiking of most CPG neurons. The most obvious function for such inhibition is to limit firing. Voltage-clamp recordings from CPG neurons during fictive swimming made the timing of early-cycle IPSCs clear (Fig. 7A). The presence of early-cycle inhibition means that the win- dow during the first half of the swimming cycle in which firing is most likely to occur is very short (Fig. 7A, shaded region). Exam- ination of swimming activity in six CPG neurons (four cINs, one aIN, and one mn) showed that there was significantly less multi- ple firing on ...
Context 2
... The most obvious function for such inhibition is to limit firing. Voltage-clamp recordings from CPG neurons during fictive swimming made the timing of early-cycle IPSCs clear (Fig. 7A). The presence of early-cycle inhibition means that the win- dow during the first half of the swimming cycle in which firing is most likely to occur is very short (Fig. 7A, shaded region). Exam- ination of swimming activity in six CPG neurons (four cINs, one aIN, and one mn) showed that there was significantly less multi- ple firing on cycles when early-cycle IPSPs occurred (Fig. 7B). In five of these neurons, positive current was injected to make the IPSPs clearer and to increase the likelihood that neurons would fire ...
Context 3
... inhibition means that the win- dow during the first half of the swimming cycle in which firing is most likely to occur is very short (Fig. 7A, shaded region). Exam- ination of swimming activity in six CPG neurons (four cINs, one aIN, and one mn) showed that there was significantly less multi- ple firing on cycles when early-cycle IPSPs occurred (Fig. 7B). In five of these neurons, positive current was injected to make the IPSPs clearer and to increase the likelihood that neurons would fire more than their usual single spike per cycle. In 218 cycles with early-cycle IPSPs, there were significantly less spikes (1.16 0.37 per cycle) than in 330 cycles without early-cycle IPSPs (1.46 0.55 ...
Context 4
... and now from aINs and CPG neurons (aIN, cIN, mn, dIN), confirms that aINs provide the early-cycle inhibition in both groups of neurons. The aINs therefore appear to have two distinct roles during swimming. In CPG neurons, early-cycle in- hibition provides negative feedback that can set a narrow window to help constrain firing during swimming (Fig. 7B). In this way, inhibition from aINs may also help to synchronize the firing of CPG neurons on the same side and lead indirectly to better alter- nation between the two sides. aIN inhibition may become more important later in development when Xenopus motoneuron fire multiply on each cycle of swimming ( Sillar et al., 1991). In the case ...
Citations
... Dashed lines indicate published connectivity between V1 and V2a neurons (Kimura and Higashijima, 2019; Sengupta et al., 2021) and connections from V1 neurons to cINs (Sengupta et al., 2021) that have yet to be linked to direction-sensitive and agnostic subsets. responsible for timing and intensity control during swimming Grillner, 1987, 1988;Li et al., 2004). However, it is challenging to use this model to explain adjustments in intensity without impacting timing. ...
Navigation requires steering and propulsion, but how spinal circuits contribute to direction control during ongoing locomotion is not well understood. Here, we use drifting vertical gratings to evoke directed ‘fictive’ swimming in intact but immobilized larval zebrafish while performing electrophysiological recordings from spinal neurons. We find directed swimming involves unilateral changes in the duration of motor output and increased recruitment of motor neurons, without impacting the timing of spiking across or along the body. Voltage-clamp recordings from motor neurons reveal increases in phasic excitation and inhibition on the side of the turn. Current-clamp recordings from premotor interneurons that provide phasic excitation or inhibition reveal two types of recruitment patterns. A direction-agnostic pattern with balanced recruitment on the turning and non-turning sides is primarily observed in excitatory V2a neurons with ipsilateral descending axons, while a direction-sensitive pattern with preferential recruitment on the turning side is dominated by V2a neurons with ipsilateral bifurcating axons. Inhibitory V1 neurons are also divided into direction-sensitive and -agnostic subsets, although there is no detectable morphological distinction. Our findings support the modular control of steering and propulsion by spinal premotor circuits, where recruitment of distinct subsets of excitatory and inhibitory interneurons provide adjustments in direction while on the move.
SIGNIFICANCE STATEMENT:
Spinal circuits play an essential role in coordinating movements during locomotion. However, it is unclear how they participate in adjustments in direction that do not interfere with coordination. Here we have developed a system using larval zebrafish that allows us to directly record electrical signals from spinal neurons during ‘fictive’ swimming guided by visual cues. We find there are subsets of spinal interneurons for coordination and others that drive unilateral asymmetries in motor neuron recruitment for direction control. Our findings suggest a modular organization of spinal premotor circuits which enables uninterrupted adjustments in direction during ongoing locomotion.
... It is unknown how the tadpole midbrain interacts with the brainstem and spinal cord to avoid synchronous initiations. We suggest that the midbrain might directly or indirectly promote downstream unilateral inhibition, which limits the firing of motoneurons and other CPG interneurons (Li et al., 2004a;Koyama et al., 2016;Liu and Hale, 2017;Koyama and Pujala, 2018). This explanation is also in line with studies showing that blockade of glycinergic inhibition leads to synchrony in neonatal rats (Cowley and Schmidt, 1995) and lamprey (Cohen and Harris-Warrick, 1984). ...
Vertebrate locomotion is heavily dependent on descending control originating in the midbrain and subsequently influencing central pattern generators in the spinal cord. However, the midbrain neuronal circuitry and its connections with other brainstem and spinal motor circuits has not been fully elucidated. Basal vertebrates with very simple nervous system, like the hatchling Xenopus laevis tadpole, have been instrumental in unravelling fundamental principles of locomotion and its suspraspinal control. Here, we use behavioral and electrophysiological approaches in combination with lesions of the midbrain to investigate its contribution to the initiation and control of the tadpole swimming in response to trunk skin stimulation. None of the midbrain lesions studied here blocked the tadpole′s sustained swim behavior following trunk skin stimulation. However, we identified that distinct midbrain lesions led to significant changes in the latency and trajectory of swimming. These changes could partly be explained by the increase in synchronous muscle contractions on the opposite sides of the tadpole′s body and permanent deflection of the tail from its normal position, respectively. Furthermore, the midbrain lesions led to significant changes in the tadpole′s ability to stop swimming when it bumps head on to solid objects. We conclude that the tadpole′s embryonic trunk skin sensorimotor pathway involves the midbrain, which harbors essential neuronal circuitry to significantly contribute to the appropriate, timely and coordinated selection and execution of locomotion, imperative to the animal′s survival.
... Importantly, targeting LAR and PTPs further enhanced formation of inhibitory and excitatory synapses among human drNPC-O2derived neurons which was correlated with improved recovery of locomotion and sensorimotor integration in SCI rats. These interneurons are important for functional recovery after SCI (Trawczynski et al., 2019;Lee et al., 2020) as V1 interneurons regulate locomotion speed as well as flexor and extensor alternation (Li et al., 2004;Gosgnach et al., 2006;Falgairolle and O'Donovan, 2019), and V3 interneurons play an important role in locomotion flexibility, left-right hindlimb coordination, balanced locomotor rhythm and initiating motor movement (Zhang et al., 2008;Chopek et al., 2018;Danner et al., 2019;Lin et al., 2019). Our data also suggest that drNPC-O2derived neurons appears to form connections with the host projecting CST axons. ...
Traumatic spinal cord injury (SCI) is a leading cause of permanent neurological disabilities in young adults. Functional impairments after SCI are substantially attributed to the progressive neurodegeneration. However, regeneration of spinal specific neurons and circuit re-assembly remain challenging in the dysregulated milieu of SCI due to impaired neurogenesis and neuronal maturation by neural precursor cells (NPCs) spontaneously or in cell-based strategies. The extrinsic mechanisms that regulate neuronal differentiation and synaptogenesis in SCI are poorly understood. Here, we perform extensive in vitro and in vivo studies to unravel that SCI-induced upregulation of matrix chondroitin sulfate proteoglycans (CSPGs) impedes neurogenesis of NPCs through co-activation of two receptor protein tyrosine phosphatases, LAR and PTPσ. In adult female rats with SCI, systemic co-inhibition of LAR and PTPσ promotes regeneration of motoneurons and spinal interneurons by engrafted human directly reprogrammed caudalized NPCs and fosters their morphological maturity and synaptic connectivity within the host neural network that culminate in improved recovery of locomotion and sensorimotor integration. Our transcriptomic analysis of engrafted human NPCs in the injured spinal cord confirmed that inhibition of CSPG receptors activates a comprehensive program of gene expression in NPCs that can support neuronal differentiation, maturation, morphological complexity, signal transmission, synaptic plasticity and behavioral improvement after SCI. We uncovered that CSPG/LAR/PTPσ axis suppresses neuronal differentiation in part by blocking Wnt/β-Catenin pathway. Taken together, we provide the first evidence that CSPGs/LAR/PTPσ axis restricts neurogenesis and synaptic integration of new neurons in NPC cellular therapies for SCI. We propose targeting LAR and PTPσ receptors offers a promising clinically-feasible adjunct treatment to optimize the efficacy and neurological benefits of ongoing NPC-based clinical trials for SCI.Significance StatementTransplantation of NPCs is a promising approach for replacing damaged neurons after SCI. However, survival, neuronal differentiation, and synaptic connectivity of transplanted NPCs within remain challenging in SCI. Here, we unravel that activation of CSPG/LAR/PTPσ axis after SCI impedes the capacity of transplanted human NPCs for replacing functionally integrated neurons. Co-blockade of LAR and PTPσ is sufficient to promote re-generation of motoneurons and spinal V1 and V3 interneurons by engrafted human caudalized directly reprogrammed NPCs and facilitate their synaptic integration within the injured spinal cord. CSPG/LAR/PTPσ axis appears to suppress neuronal differentiation of NPCs by inhibiting Wnt/β-Catenin pathway. These findings identify targeting CSPG/LAR/PTPσ axis as a promising strategy for optimizing neuronal replacement, synaptic re-connectivity and neurological recovery in NPC-based strategies.
... V1 interneurons are marked by the expression of Engrailed1 (Eng1) transcription factor across vertebrates. 19,22,23 Genetic ablation of these neurons reduces locomotor speeds in both zebrafish 4 and mice, 6,24 indicating that speed regulation is a primitive function of these neurons. V1 neurons have also been implicated in flexor-extensor alternation, 25,26 and connectivity studies have suggested that these neurons additionally gate sensory signals during locomotion. ...
... V1 neurons have also been implicated in flexor-extensor alternation, 25,26 and connectivity studies have suggested that these neurons additionally gate sensory signals during locomotion. 19,23 It is unknown whether and how the motor and sensory functions of V1 neurons are organized along the longitudinal axis of the spinal cord. ...
... Next, to optimize design of our subsequent mapping experiments, we investigated the extent of V1 axonal projections in the R-C axis. V1 neurons project axons ipsilaterally and rostrally, 19,22,23 with a subset also exhibiting descending axonal branches. 19,28,29 To study morphology, we performed single-cell labeling in the Tg(eng1b:Gal4,UAS:RFP) fish line using two approaches: single-cell electroporation of fluorescently tagged dextran or micro-injection of a UAS:Dendra plasmid construct, followed by confocal imaging of single cells. ...
Rostro-caudal coordination of spinal motor output is essential for locomotion. Most spinal interneurons project axons longitudinally to govern locomotor output, yet their connectivity along this axis remains unclear. In this study, we use larval zebrafish to map synaptic outputs of a major inhibitory population, V1 (Eng1+) neurons, which are implicated in dual sensory and motor functions. We find that V1 neurons exhibit long axons extending rostrally and exclusively ipsilaterally for an average of 6 spinal segments; however, they do not connect uniformly with their post-synaptic targets along the entire length of their axon. Locally, V1 neurons inhibit motor neurons (both fast and slow) and other premotor targets, including V2a, V2b, and commissural premotor neurons. In contrast, V1 neurons make robust long-range inhibitory contacts onto a dorsal horn sensory population, the commissural primary ascending neurons (CoPAs). In a computational model of the ipsilateral spinal network, we show that this pattern of short-range V1 inhibition to motor and premotor neurons underlies burst termination, which is critical for coordinated rostro-caudal propagation of the locomotor wave. We conclude that spinal network architecture in the longitudinal axis can vary dramatically, with differentially targeted local and distal connections, yielding important consequences for function.
... Secondly, aINs also form glycinergic synapses onto ipsilateral CPG neurons. The phasic inhibition appears slightly later in the swimming cycle than the on-cycle EPSPs and spiking, providing a mechanism to limit multiple firing in CPG neurons, similar to the function of Renshaw cells in the mammalian spinal cord in limiting motoneuron firing [33]. Another interesting aspect of aINs is that they express the transcription factor engrailed 1 (En1), which marks neurons with a similar function in zebrafish larvae [34] and perhaps mouse spinal cord as well [35]. ...
The spinal circuit controlling swimming locomotion in hatchling Xenopus frog tadpoles was one of the first vertebrate CPGs to be investigated in detail. Today it is one of the most completely understood CPGs in the animal kingdom. Research over the last four decades has led to a detailed understanding of the motor and interneurons that comprise the CPG, their detailed electrical properties, and synaptic connectivity. This review summarizes all aspects of the swimming system from its initiation and termination by specific sensory pathways to the switching of motor programs from swimming to struggling. We also review the intrinsic mechanisms that confer short-term motor memory via dynamic sodium pumps. After hatching there is an initial and rapid development of the swimming output in the prelude to continuous free-swimming and metamorphosis (see Chapter 8) that relies partly on the incorporation of various modulatory systems that descend from the brainstem, including aminergic and nitrergic influences.
... The analysis of the spinal circuitry responsible for orchestrating locomotion has been greatly facilitated by genetic strategies for identifying and manipulating different neuronal classes (for review, see [1]). One of the cell classes that has received considerable study is the V1 class of interneuron, which is composed of ipsilaterally projecting inhibitory interneurons that express the transcription factor engrailed-1 (En1) [2][3][4][5][6][7][8][9][10][11]. The V1 group is heterogeneous, expressing a combination of 19 transcription factors [3], and contains an estimated 50 subclasses [5] including Renshaw cells and a subset of Ia inhibitory interneurons [2]. ...
... Supporting this hypothesis, it was recently shown that the V1 inputs vary according to the motor pool they target (hip, ankle, or foot), and this also applied to limb versus epaxial or hypaxial muscles [3,10]. Furthermore, in the tadpole and the zebrafish, it has been shown that interneurons expressing En1 provide inhibition to limit the firing of motoneurons and interneurons during swimming and gate inhibition to sensory pathways [7,9]. In addition, it was shown recently that there are at least two types of V1 interneuron (slow and fast) in the zebrafish, which are selectively activated to regulate slow and fast swimming [8]. ...
... En1-positive neurons in the tadpole and the zebrafish have been shown to be involved in fast swimming [7,9]. Recently, in the zebrafish, it was shown that two classes of V1 interneurons (slow and fast) are selectively activated during slow and fast swimming, respectively [8]. ...
In the mouse spinal cord, V1 interneurons are a heterogeneous population of inhibitory spinal interneurons that have been implicated in regulating the frequency of the locomotor rhythm and in organizing flexor and extensor alternation. By introducing archaerhodopsin into engrailed-1-positive neurons, we demonstrate that the function of V1 neurons in locomotor-like activity is more complex than previously thought. In the whole cord, V1 hyperpolarization increased the rhythmic synaptic drive to flexor and extensor motoneurons, increased the spiking in each cycle, and slowed the locomotor-like rhythm. In the hemicord, V1 hyperpolarization accelerated the rhythm after an initial period of tonic activity, implying that a subset of V1 neurons are active in the hemicord, which was confirmed by calcium imaging. Hyperpolarizing V1 neurons resulted in an equalization of the duty cycle in flexor and extensors from an asymmetrical pattern in control recordings in which the extensor bursts were longer than the flexor bursts. Our results suggest that V1 interneurons are composed of several subsets with different functional roles. Furthermore, during V1 hyperpolarization, the default state of the locomotor central pattern generator (CPG) is symmetrical, with antagonist motoneurons each firing with an approximately 50% duty cycle. We hypothesize that one function of the V1 population is to set the burst durations of muscles to be appropriate to their biomechanical function and to adapt to the environmental demands, such as changes in locomotor speed.
... V1 neurons are one class of neurons that are defined by the expression of En1. V1 neurons are ipsilaterally projecting inhibitory neurons in vertebrates thus far examined [26][27][28][29] . In larval zebrafish and frog tadpoles, these neurons generally fire in phase with MNs located nearby during swimming and are proposed to provide in-phase inhibition to CPG and motor neurons to help terminate the firing of the target neurons in each cycle during swimming 27,28 . ...
... V1 neurons are ipsilaterally projecting inhibitory neurons in vertebrates thus far examined [26][27][28][29] . In larval zebrafish and frog tadpoles, these neurons generally fire in phase with MNs located nearby during swimming and are proposed to provide in-phase inhibition to CPG and motor neurons to help terminate the firing of the target neurons in each cycle during swimming 27,28 . In this scheme, inactivation of V1 neurons would be expected to prolong firings of the CPG and motor neurons in each cycle and, consequently, prolong the cycle period. ...
... e Histogram of spike timings of fast-type V1 neurons during fast (50-65 Hz) swim (1387 swimming cycles from 29 cells). f Histogram of spike timings of slow-type V1 neurons during slow (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) Hz) swim (5782 swimming cycles from 10 cells). g Schematic diagram of the Kaede photo-conversion experiment (top). ...
During fast movements in vertebrates, slow motor units are thought to be deactivated due to the mechanical demands of muscle contraction, but the associated neuronal mechanisms for this are unknown. Here, we perform functional analyses of spinal V1 neurons by selectively killing them in larval zebrafish, revealing two functions of V1 neurons. The first is the long-proposed role of V1 neurons: they play an important role in shortening the cycle period during swimming by providing in-phase inhibition. The second is that V1 neurons play an important role in the selection of active sets of neurons. We show that strong inhibitory inputs coming from V1 neurons play a crucial role in suppressing the activities of slow-type V2a and motor neurons, and, consequently, of slow muscles during fast swimming. Our results thus highlight the critical role of spinal inhibitory neurons for silencing slow-component neurons during fast movements.
... ( Higashijima et al., 2004;Li et al., 2004;Benito-Gonzalez and Alvarez, 2012;Stam et al., 2012). 509 ...
... ( Higashijima et al., 2004;Li et al., 2004;Benito-Gonzalez and Alvarez, 2012;Stam et al., 2012). 509 ...
Spinal motor neurons and the peripheral muscle fibers they innervate form discrete motor units that execute movements of varying force and speed. Subsets of spinal motor neurons also exhibit axon collaterals that influence motor output centrally. Here, we have used in vivo imaging to anatomically characterize the central and peripheral innervation patterns of axial motor units in larval zebrafish. Using early born primary motor neurons and their division of epaxial and hypaxial muscle into four distinct quadrants as a reference, we define three distinct types of later born secondary motor units. The largest are m-type units, which innervate deeper fast-twitch muscle fibers via medial nerves. Next in size are ms-type secondaries, which innervate superficial fast-twitch and slow fibers via medial and septal nerves, followed by s-type units, which exclusively innervate superficial slow muscle fibers via septal nerves. All types of secondaries innervate up to four axial quadrants. Central axon collaterals are found in subsets of primaries based on soma position and predominantly in secondary fast-twitch units (m, ms) with increasing likelihood based on number of quadrants innervated. Collaterals are labeled by synaptophysin-tagged fluorescent proteins, but not PSD95, consistent with their output function. Also, PSD95 dendrite labeling reveals that larger motor units receive more excitatory synaptic input. Collaterals are largely restricted to the neuropil, however perisomatic connections are observed between motor units. These observations suggest that recurrent interactions are dominated by motor neurons recruited during stronger movements and set the stage for functional investigations of recurrent motor circuitry in larval zebrafish.
... Do dINs potentially possess molecular characteristics that could be used to link them to similar excitatory interneurons in the locomotion CPGs in other vertebrates? Apart from using immunocytochemical methods to reveal neurons with different neurotransmitters, several molecular markers have been used in the past to identify the sensory Rohon-Beard (RB) cells, MNs, the ascending inhibitory interneurons in Xenopus tadpoles (Borodinsky et al., 2004;Li et al., 2004a). Unfortunately, proper double-labeling was generally lacking to confirm the specificity for these markers in segregating neuronal groups defined anatomically or functionally, although the anatomical identification of RB cells is reliable. ...
Vertebrate central pattern generators (CPGs) controlling locomotion contain neurons which provide the excitation that drives and maintains network rhythms. In a simple vertebrate, the developing Xenopus tadpole, we study the role of excitatory descending neurons with ipsilateral projecting axons (descending interneurons, dINs) in the control of swimming rhythms. In tadpoles with both intact central nervous system (CNS) and transections in the hindbrain, exciting some individual dINs in the caudal hindbrain region could start swimming repeatedly. Analyses indicated the recruitment of additional dINs immediately after such evoked dIN spiking and prior to swimming. Excitation of dINs can therefore be sufficient for the initiation of swimming. These “powerful” dINs all possessed both ascending and descending axons. However, their axon projection lengths were not different from those of other excitatory dINs at similar locations. The dorsoventral position of dINs, as a population, significantly better matched that of cells marked by immunocytochemistry for the transcription factor CHX10 than other known neuron types in the ventral hindbrain and spinal cord. The comparison suggests that the excitatory interneurons including dINs are CHX10-positive, in agreement with CHX10 as a marker for excitatory neurons with ipsilateral projections in the spinal cord and brainstem of other vertebrates. Overall, our results further demonstrate the key importance of dINs in driving tadpole swimming rhythms.
... Neural pathways conveying CD have been delineated in a diverse array of species (Dale and Cullen, 2017;Davis et al., 1973;Fee et al., 1997;Schneider et al., 2014;Sommer and Wurtz, 2002;Yang et al., 2008). Neural recordings of the CD signal itself, however, have mostly been performed in non-mammalian species, including crickets, sea slugs, crayfish, tadpoles, and electric fish (Evans et al., 2003;Kirk and Wine, 1984;Li et al., 2004;Poulet and Hedwig, 2006;Requarth and Sawtell, 2014). The relatively small and simple nervous systems of these species have allowed for the isolation of neurons that carry or receive CD signals and identify their relationship to behavior. ...
In week-old rats, somatosensory input arises predominantly from external stimuli or from sensory feedback (reafference) associated with myoclonic twitches during active sleep. A previous study suggested that the brainstem motor structures that produce twitches also send motor copies (or corollary discharge, CD) to the cerebellum. We tested this possibility by recording from two precerebellar nuclei-the inferior olive (IO) and lateral reticular nucleus (LRN). In most IO and LRN neurons, twitch-related activity peaked sharply around twitch onset, consistent with CD. Next, we identified twitch-production areas in the midbrain that project independently to the IO and LRN. Finally, we blocked calcium-activated slow potassium (SK) channels in the IO to explain how broadly tuned brainstem motor signals can be transformed into precise CD signals. We conclude that the precerebellar nuclei convey a diversity of sleep-related neural activity to the developing cerebellum to enable processing of convergent input from CD and reafferent signals.
