V2a Interneurons Research Articles

Generation of V2a Interneurons from Mouse Embryonic Stem Cells

V2a interneurons of the ventral spinal cord and hindbrain play an important role in the central pattern generators (CPGs) involved in locomotion, skilled reaching, and respiration. However, sources of V2a interneurons for in vitro studies are limited. In this study, we developed a differentiation protocol for V2a interneurons from mouse embryonic stem cells (mESCs). Cells were induced in a 2(-)/4(+) induction protocol with varying concentrations of retinoic acid (RA) and the mild sonic hedgehog (Shh) agonist purmorphamine (Pur) in order to increase the expression of V2a interneuron transcription factors (eg, Chx10). Notch signaling, which influences the commitment of p2 progenitor cells to V2a or V2b interneurons, was inhibited in cell cultures to increase the percentage of V2a interneurons. At the end of the induction period, cell commitment was assessed using quantitative real-time polymerase chain reaction, immunocytochemistry, and flow cytometry to quantify expression of transcription factors specific to V2a interneurons and the adjacent ventral spinal cord regions. Low concentrations of RA and high concentrations of Pur led to greater expression of transcription factors specific for V2a interneurons. Notch inhibition favored V2a interneuron over V2b interneuron differentiation. The protocol established in this study can be used to further elucidate the pathways involved in V2a interneuron differentiation and help produce sources of V2a interneurons for developmental neurobiology, electrophysiology, and transplantation studies.

High-efficiency motor neuron differentiation from human pluripotent stem cells and the function of Islet-1

Efficient derivation of large-scale motor neurons (MNs) from human pluripotent stem cells is central to the understanding of MN development, modelling of MN disorders in vitro and development of cell-replacement therapies. Here we develop a method for rapid (20 days) and highly efficient (~70%) differentiation of mature and functional MNs from human pluripotent stem cells by tightly modulating neural patterning temporally at a previously undefined primitive neural progenitor stage. This method also allows high-yield (>250%) MN production in chemically defined adherent cultures. Furthermore, we show that Islet-1 is essential for formation of mature and functional human MNs, but, unlike its mouse counterpart, does not regulate cell survival or suppress the V2a interneuron fate. Together, our discoveries improve the strategy for MN derivation, advance our understanding of human neural specification and MN development, and provide invaluable tools for human developmental studies, drug discovery and regenerative medicine.

Skilled reaching relies on a V2a propriospinal internal copy circuit

The precision of skilled forelimb movement has long been presumed to rely on rapid feedback corrections triggered by internally-directed copies of outgoing motor commands – but the functional relevance of inferred internal copy circuits has remained unclear. One class of spinal interneurons implicated in the control of mammalian forelimb movement, cervical propriospinal neurons (PNs), has the potential to convey an internal copy of pre-motor signals through dual innervation of forelimb-innervating motor neurons and pre-cerebellar neurons of the lateral reticular nucleus. We have examined whether the PN internal copy pathway functions in the control of goal-directed reaching. In mice, PNs include a genetically-accessible subpopulation of cervical V2a interneurons, and their targeted ablation perturbs reaching while leaving intact other elements of forelimb movement. Moreover, optogenetic activation of the PN internal copy branch recruits a rapid cerebellar feedback loop that modulates forelimb motor neuron activity and severely disrupts reaching kinematics. Our findings implicate V2a PNs as the focus of an internal copy pathway assigned to the rapid updating of motor output during reaching behavior.

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.

Locomotor Rhythm Generation Linked to the Output of Spinal Shox2 Excitatory Interneurons

Locomotion is controlled by spinal networks that generate rhythm and coordinate left-right and flexor-extensor patterning. Defined populations of spinal interneurons have been linked to patterning circuits; however, neurons comprising the rhythm-generating kernel have remained elusive. Here, we identify an ipsilaterally projecting excitatory interneuron population, marked by the expression of Shox2 that overlaps partially with V2a interneurons. Optogenetic silencing or blocking synaptic output of Shox2 interneurons (INs) in transgenic mice perturbed rhythm without an effect on pattern generation, whereas ablation of the Shox2 IN subset coinciding with the V2a population was without effect. Most Shox2 INs are rhythmically active during locomotion and analysis of synaptic connectivity showed that Shox2 INs contact other Shox2 INs, commissural neurons, and motor neurons,with preference for flexor motor neurons. Our findings focus attention on a subset of Shox2 INs that appearto participate in the rhythm-generating kernel for spinal locomotion.

Cell-Specific Actions of a Human LHX3 Gene Enhancer During Pituitary and Spinal Cord Development

The LIM class of homeodomain protein 3 (LHX3) transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie complex pediatric syndromes featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. Here we characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in α-glycoprotein subunit -expressing cells secreting the TSHβ, LHβ, or FSHβ hormones and expressing the GATA2 and steroidogenic factor 1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module active in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression. Furthermore, these studies revealed significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice also provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system.

An asymmetric model of the spinal locomotor central pattern generator: insights from afferent stimulations

We investigated the effects of dorsal root stimulation (flexor related dL2 and extensor related dL5) on fictive locomotion evoked pharmacologically (by 5-HT+NMDA+DA) in the isolated neonatal mouse spinal cord. In our experiments, electrical stimulation produced a wide variety of effects depending on stimulation frequency, intensity, and the drug concentrations used. At stimulation intensities near threshold we were able to produce phase advances and delays that resolved within a single step cycle. During these single-cycle alterations we observed different effects after stimulating dL2 and dL5. Stimulation of dL2 during ipsilateral extension typically produced an early onset of the next flexion and termination of current extension in the ipsilateral activity with or without rhythm disturbances on the contralateral side of the cord (subject to drug concentrations and stimulus intensity). These disturbances represented a true phase resetting characterized by a full flexor phase expression independent of ending the stimulation producing the disturbance. In contrast, stimulation of dL5 often produced a complex bilateral effect starting from a brief activation of ipsilateral extension (for the stimulus duration) with a corresponding reduction in the flexor activity followed by a reactivation of ipsilateral flexor activity (a full second burst that could represent a rebound evoked by end of stimulation) and then by an enhanced next extensor burst. Our results were consistent with the previously proposed two-level architecture of the spinal central pattern generator (CPG) consisting of a top-level rhythm generator (RG) and pattern formation (PF) circuits [1,3]. The above differences in the effects of flexor and extensor afferent stimulation are consistent with the previous suggestion that the spinal CPG has an asymmetric flexor-extensor organization, so that only the flexor half-center of the rhythm generator (RG) has intrinsic rhythmogenic capabilities [2]. To further evaluate the organization of the spinal CPG and its control by flexor and extensor afferents, we extended our previous model of the CPG in the neonatal rodent spinal cord [4] by incorporating bilateral flexor and extensor afferent pathways and used this model for simulating the above effects of afferent stimulations. The extended model contains left and right half-center RGs interacting via excitatory and inhibitory commissural interneurons (CIN). The flexor half-center of each RG can intrinsically generate rhythmic bursting, while the extensor half-centers do not have intrinsic rhythmic capabilities. Each RG half-center projects to the corresponding PF population controlling the activity of the corresponding motoneuron pools. Ipsilateral neural circuits include several populations of spinal interneurons, including experimentally identified CINs, two types of V2a interneurons, and motoneurons [4]. Network interactions have been organized to be consistent with the activity of these neurons observed during spontaneous experimental deletions [4]. The model reproduces the locomotor-like rhythm with bilaterally coordinated flexor and extensor activities and the neuronal firing patterns observed in the normal conditions and during resetting and non-resetting deletions [4], as well as the experimentally evoked effects of dorsal root stimulation described above. The model proposes mechanistic explanations for the asymmetric effect of afferent stimulation and provides novel insights into the organization of the locomotor central pattern generator.

Conditionally immortalized stem cell lines from human spinal cord retain regional identity and generate functional V2a interneurons and motorneurons

IntroductionThe use of immortalized neural stem cells either as models of neural development in vitro or as cellular therapies in central nervous system (CNS) disorders has been controversial. This controversy has centered on the capacity of immortalized cells to retain characteristic features of the progenitor cells resident in the tissue of origin from which they were derived, and the potential for tumorogenicity as a result of immortalization. Here, we report the generation of conditionally immortalized neural stem cell lines from human fetal spinal cord tissue, which addresses these issues.MethodsClonal neural stem cell lines were derived from 10-week-old human fetal spinal cord and conditionally immortalized with an inducible form of cMyc. The derived lines were karyotyped, transcriptionally profiled by microarray, and assessed against a panel of spinal cord progenitor markers with immunocytochemistry. In addition, the lines were differentiated and assessed for the presence of neuronal fate markers and functional calcium channels. Finally, a clonal line expressing eGFP was grafted into lesioned rat spinal cord and assessed for survival, differentiation characteristics, and tumorogenicity.ResultsWe demonstrate that these clonal lines (a) retain a clear transcriptional signature of ventral spinal cord progenitors and a normal karyotype after extensive propagation in vitro, (b) differentiate into relevant ventral neuronal subtypes with functional T-, L-, N-, and P/Q-type Ca2+ channels and spontaneous calcium oscillations, and (c) stably engraft into lesioned rat spinal cord without tumorogenicity.ConclusionsWe propose that these cells represent a useful tool both for the in vitro study of differentiation into ventral spinal cord neuronal subtypes, and for examining the potential of conditionally immortalized neural stem cells to facilitate functional recovery after spinal cord injury or disease.

Speeding up locomotion

The recruitment of V2a interneurons to increase swimming speed in zebrafish is incremental and is determined by the combined effect of their excitatory synaptic currents and input resistance.

The E3 Ligase Mind Bomb-1 (Mib1) Modulates Delta-Notch Signaling to Control Neurogenesis and Gliogenesis in the Developing Spinal Cord

The Notch signaling pathway is essential for neuronal and glial specification during CNS development. Mind bomb-1 (Mib1) is an E3 ubiquitin ligase that ubiquitinates and promotes the endocytosis of Notch ligands. Although Mib1 is essential for transmitting the Notch signal, it is still unclear whether it is a primary regulator of Notch ligand activity in the developing spinal cord. In Mib1 conditional knock-out mice, we observed depletion of spinal progenitors, premature differentiation of neurons, and unbalanced specification of V2 interneurons, all of which mimic the conventional Notch phenotype. In agreement with this, the reduction of progenitors in the absence of Mib1 led to a loss of both astrocytes and oligodendrocytes. Late removal of Mib1 using a drug-inducible system suppressed glial differentiation, suggesting that Mib1 continues to play a role in the formation of late progenitors mainly designated for gliogenesis. Finally, misexpression of Mib1 or Mib1 deletion mutants revealed that the ring domain of Mib1 is required for the specification of V2 interneurons in the chick neural tube. Together, these findings suggest that Mib1 is a major component of the signal-sending cells required to provide Notch ligand activity for specifying neurons and glia in the spinal cord.

Decoding the rules of recruitment of excitatory interneurons in the adult zebrafish locomotor network

Neural networks in the spinal cord transform signals from the brain into coordinated locomotor movements. An optimal adjustment of the speed of locomotion entails a precise order of recruitment of interneurons underlying excitation within these networks. However, the mechanisms encoding the recruitment threshold of excitatory interneurons have remained unclear. Here we show, using a juvenile/adult zebrafish preparation, that excitatory V2a interneurons are incrementally recruited with increased swimming frequency. The order of recruitment is not imprinted by the topography or the input resistance of the V2a interneurons. Rather, it is determined by scaling the effect of excitatory synaptic currents by the input resistance. We also show that the locomotor networks are composed of multiple microcircuits encompassing subsets of V2a interneurons and motoneurons that are recruited in a continuum with increased swimming speeds. Thus, our results provide insights into the organization and mechanisms determining the recruitment of spinal microcircuits to ensure optimal execution of locomotor movements.

Zebrafish Mnx proteins specify one motoneuron subtype and suppress acquisition of interneuron characteristics

BackgroundPrecise matching between motoneuron subtypes and the muscles they innervate is a prerequisite for normal behavior. Motoneuron subtype identity is specified by the combination of transcription factors expressed by the cell during its differentiation. Here we investigate the roles of Mnx family transcription factors in specifying the subtypes of individually identified zebrafish primary motoneurons.ResultsZebrafish has three Mnx family members. We show that each of them has a distinct and temporally dynamic expression pattern in each primary motoneuron subtype. We also show that two Mnx family members are expressed in identified VeLD interneurons derived from the same progenitor domain that generates primary motoneurons. Surprisingly, we found that Mnx proteins appear unnecessary for differentiation of VeLD interneurons or the CaP motoneuron subtype. Mnx proteins are, however, required for differentiation of the MiP motoneuron subtype. We previously showed that MiPs require two temporally-distinct phases of Islet1 expression for normal development. Here we show that in the absence of Mnx proteins, the later phase of Islet1 expression is initiated but not sustained, and MiPs become hybrids that co-express morphological and molecular features of motoneurons and V2a interneurons. Unexpectedly, these hybrid MiPs often extend CaP-like axons, and some MiPs appear to be entirely transformed to a CaP morphology.ConclusionsOur results suggest that Mnx proteins promote MiP subtype identity by suppressing both interneuron development and CaP axon pathfinding. This is, to our knowledge, the first report of transcription factors that act to distinguish CaP and MiP subtype identities. Our results also suggest that MiP motoneurons are more similar to V2 interneurons than are CaP motoneurons.

Spinal Cord Injury Induces Serotonin Supersensitivity without Increasing Intrinsic Excitability of Mouse V2a Interneurons

Denervation-induced plastic changes impair locomotor recovery after spinal cord injury (SCI). Spinal motoneurons become hyperexcitable after SCI, but the plastic responses of locomotor network interneurons (INs) after SCI have not been studied. Using an adult mouse SCI model, we analyzed the effects of complete spinal cord lesions on the intrinsic electrophysiological properties, excitability, and neuromodulatory responses to serotonin (5-HT) in mouse lumbar V2a spinal INs, which help regulate left-right alternation during locomotion. Four weeks after SCI, V2a INs showed almost no changes in baseline excitability or action potential properties; the only parameter that changed was a reduced input resistance. However, V2a INs became 100- to 1000-fold more sensitive to 5-HT. Immunocytochemical analysis showed that SCI caused a coordinated loss of serotonergic fibers and the 5-HT transporter (SERT). Blocking the SERT with citalopram in intact mice did not increase 5-HT sensitivity to the level seen after SCI. SCI also evoked an increase in 5-HT(2C) receptor cluster number and intensity, suggesting that several plastic changes cooperate in increasing 5-HT sensitivity. Our results suggest that different components of the spinal neuronal network responsible for coordinating locomotion are differentially affected by SCI, and highlight the importance of understanding these changes when considering therapies targeted at functional recovery.

Neuronal activity in the isolated mouse spinal cord during spontaneous deletions in fictive locomotion: insights into locomotor central pattern generator organization

We explored the organization of the spinal central pattern generator (CPG) for locomotion by analysing the activity of spinal interneurons and motoneurons during spontaneous deletions occurring during fictive locomotion in the isolated neonatal mouse spinal cord, following earlier work on locomotor deletions in the cat. In the isolated mouse spinal cord, most spontaneous deletions were non-resetting, with rhythmic activity resuming after an integer number of cycles. Flexor and extensor deletions showed marked asymmetry: flexor deletions were accompanied by sustained ipsilateral extensor activity, whereas rhythmic flexor bursting was not perturbed during extensor deletions. Rhythmic activity on one side of the cord was not perturbed during non-resetting spontaneous deletions on the other side, and these deletions could occur with no input from the other side of the cord. These results suggest that the locomotor CPG has a two-level organization with rhythm-generating (RG) and pattern-forming (PF) networks, in which only the flexor RG network is intrinsically rhythmic. To further explore the neuronal organization of the CPG, we monitored activity of motoneurons and selected identified interneurons during spontaneous non-resetting deletions. Motoneurons lost rhythmic synaptic drive during ipsilateral deletions. Flexor-related commissural interneurons continued to fire rhythmically during non-resetting ipsilateral flexor deletions. Deletion analysis revealed two classes of rhythmic V2a interneurons. Type I V2a interneurons retained rhythmic synaptic drive and firing during ipsilateral motor deletions, while type IIV2a interneurons lost rhythmic synaptic input and fell silent during deletions. This suggests that the type I neurons are components of the RG, whereas the type II neurons are components of the PF network.We propose a computational model of the spinal locomotor CPG that reproduces our experimental results. The results may provide novel insights into the organization of spinal locomotor networks.

The LIM-Homeodomain Protein Islet Dictates Motor Neuron Electrical Properties by Regulating K+ Channel Expression

SummaryNeuron electrical properties are critical to function and generally subtype specific, as are patterns of axonal and dendritic projections. Specification of motoneuron morphology and axon pathfinding has been studied extensively, implicating the combinatorial action of Lim-homeodomain transcription factors. However, the specification of electrical properties is not understood. Here, we address the key issues of whether the same transcription factors that specify morphology also determine subtype specific electrical properties. We show that Drosophila motoneuron subtypes express different K+ currents and that these are regulated by the conserved Lim-homeodomain transcription factor Islet. Specifically, Islet is sufficient to repress a Shaker-mediated A-type K+ current, most likely due to a direct transcriptional effect. A reduction in Shaker increases the frequency of action potential firing. Our results demonstrate the deterministic role of Islet on the excitability patterns characteristic of motoneuron subtypes.

Origin of excitation underlying locomotion in the spinal circuit of zebrafish

Neural circuits in the spinal cord transform instructive signals from the brain into well-coordinated locomotor movements by virtue of rhythm-generating components. Although evidence suggests that excitatory interneurons are the essence of locomotor rhythm generation, their molecular identity and the assessment of their necessity have remained unclear. Here we show, using larval zebrafish, that V2a interneurons represent an intrinsic source of excitation necessary for the normal expression of the locomotor rhythm. Acute and selective ablation of these interneurons increases the threshold of induction of swimming activity, decreases the burst frequency, and alters the coordination of the rostro-caudal propagation of activity. Thus, our results argue that V2a interneurons represent a source of excitation that endows the spinal circuit with the capacity to generate locomotion.

A comparison of serotonin neuromodulation of mouse spinal V2a interneurons using perforated patch and whole cell recording techniques

Whole cell recordings (WCRs) are frequently used to study neuronal properties, but may be problematic when studying neuromodulatory responses, due to dialysis of the cell's cytoplasm. Perforated patch recordings (PPR) avoid cellular dialysis and might reveal additional modulatory effects that are lost during WCR. We have previously used WCR to characterize the responses of the V2a class of Chx10-expressing neurons to serotonin (5-HT) in the neonatal mouse spinal cord (Zhong et al., 2010). Here we directly compare multiple aspects of the responses to 5-HT using WCR and PPR in Chx10-eCFP neurons in spinal cord slices from 2 to 4 day old mice. Cellular properties recorded in PPR and WCR were similar, but high-quality PP recordings could be maintained for significantly longer. Both WCR and PPR cells could respond to 5-HT, and although neurons recorded by PPR showed a significantly greater response to 5-HT in some parameters, the absolute differences between PPR and WCR were small. We conclude that WCR is an acceptable recording method for short-term recordings of neuromodulatory effects, but the less invasive PPR is preferable for detailed analyses and is necessary for stable recordings lasting an hour or more.

Frequency-dependent recruitment of V2a interneurons during fictive locomotion in the mouse spinal cord

The principles governing the recruitment of interneurons during acceleration in vertebrate locomotion are not known. In the mouse, the V2a spinal interneurons are dispensable for left-right coordination at low locomotor frequencies, but their function is essential for maintaining left-right coordination at high frequencies. Here we explore the mechanisms driving this frequency-dependent role, using four methods to determine how V2a interneurons are recruited at different locomotor frequencies. We show that half the V2a interneurons receive rhythmic locomotor synaptic drive which increases with cycle frequency, recruiting more of the neurons to fire at higher frequencies. The other V2a interneurons do not receive locomotion-related synaptic drive, and are not recruited into the locomotor network at any frequency. The increased role of V2a interneurons at higher locomotor frequencies arises from increased synaptic drive to recruit subthreshold oscillating V2a neurons, and not from recruitment of a second set of silent V2a interneurons.

Sox1 Is Required for the Specification of a Novel p2-Derived Interneuron Subtype in the Mouse Ventral Spinal Cord

During mouse development, the ventral spinal cord becomes organized into five progenitor domains that express different combinations of transcription factors and generate different subsets of neurons and glia. One of these domains, known as the p2 domain, generates two subtypes of interneurons, V2a and V2b. Here we have used genetic fate mapping and loss-of-function analysis to show that the transcription factor Sox1 is expressed in, and is required for, a third type of p2-derived interneuron, which we named V2c. These are close relatives of V2b interneurons, and, in the absence of Sox1, they switch to the V2b fate. In addition, we show that late-born V2a and V2b interneurons are heterogeneous, and subsets of these cells express the transcription factor Pax6. Our data demonstrate that interneuron diversification in the p2 domain is more complex than previously thought and directly implicate Sox1 in this process.

Functional organization of V2a‐related locomotor circuits in the rodent spinal cord

Studies of mammalian locomotion have been greatly facilitated by the use of the isolated rodent spinal cord preparation that retains the locomotor circuits needed to execute the movement. Physiological and molecular genetic experiments in this preparation have started to unravel the basic circuit organization responsible for walking in mammals. Here, we review these experiments with a focus on the functional role of excitatory V2a interneurons in the mammalian locomotor network. With regard to these neurons and other network structures we also discuss similarities and differences between the mammalian walking central pattern generator (CPG) and the fish swimming CPG.