Differential Contribution of V0 Interneurons to Execution of Rhythmic and Nonrhythmic Motor Behaviors.

Locomotion, scratching, and stabilization of the body orientation in space are basic motor functions which are critically important for animal survival. Their execution requires coordinated activity of muscles located in the left and right halves of the body. Commissural interneurons (CINs) are critical elements of the neuronal networks underlying the left-right motor coordination. V0 interneurons (characterized by the early expression of the transcription factor Dbx1) contain a major class of CINs in the spinal cord (excitatory, V0V; inhibitory, V0D), and a small subpopulation of excitatory ipsilaterally projecting interneurons. The role of V0 CINs in left-right coordination during forward locomotion was demonstrated earlier. Here, to reveal the role of glutamatergic V0 and other V0 subpopulations in control of backward locomotion, scratching, righting behavior, and postural corrections, kinematics of these movements performed by wild-type mice and knock-out mice with glutamatergic V0 or all V0 interneurons ablated were compared. Our results suggest that the functional effect of excitatory V0 neurons during backward locomotion and scratching is inhibitory, and that the execution of scratching involves active inhibition of the contralateral scratching central pattern generator mediated by excitatory V0 neurons. By contrast, other V0 subpopulations are elements of spinal networks generating postural corrections. Finally, all V0 subpopulations contribute to the generation of righting behavior. We found that different V0 subpopulations determine left-right coordination in the anterior and posterior parts of the body during a particular behavior. Our study shows a differential contribution of V0 subpopulations to diverse motor acts that provides new insight to organization of motor circuits.SIGNIFICANCE STATEMENT Commissural interneurons with their axons crossing the midline of the nervous system are critical elements of the neuronal networks underlying the left-right motor coordination. For the majority of motor behaviors, the neuronal mechanisms underlying left-right coordination are unknown. Here, we demonstrate the functional role of excitatory V0 neurons and other subpopulations of V0 interneurons in control of a number of basic motor behaviors-backward locomotion, scratching, righting behavior, and postural corrections-which are critically important for animal survival. We have shown that different subpopulations of V0 neurons determine left-right coordination in the context of different behaviors as well as in the anterior and posterior parts of the body during a particular behavior.

High Pressure and [Ca 2+ ] Produce an Inverse Modulation of Synaptic Input Strength and Network Excitability in the Rat Dentate Gyrus.

Hyperbaric environments induce the high pressure neurological syndrome (HPNS) characterized by hyperexcitability of the central nervous system (CNS) and memory impairment. Human divers and other animals experience the HPNS at pressures beyond 1.1 MPa. High pressure depresses synaptic transmission and alters its dynamics in various animal models. Medial perforant path (MPP) synapses connecting the medial entorhinal cortex with the hippocampal formation are suppressed by 50% at 10.1MPa. Reduction of synaptic inputs is paradoxically associated with enhanced ability of dentate gyrus (DG)' granule cells (GCs) to generate spikes at high pressure. This mechanism allows MPP inputs to elicit standard GC outputs at 0.1-25 Hz frequencies under hyperbaric conditions. An increased postsynaptic gain of MPP inputs probably allows diving animals to perform in hyperbaric environments, but makes them vulnerable to high intensity/frequency stimuli producing hyperexcitability. Increasing extracellular Ca2+ ([Ca2+]o) partially reverted pressure-mediated depression of MPP inputs and increased MPP's low-pass filter properties. We postulated that raising [Ca2+]o in addition to increase synaptic inputs may reduce network excitability in the DG potentially improving its function and reducing sensitivity to high intensity and pathologic stimuli. For this matter, we activated the MPP with single and 50 Hz frequency stimuli that simulated physiologic and deleterious conditions, while assessing the GC's output under various conditions of pressure and [Ca2+]o. Our results reveal that the pressure and [Ca2+]o produce an inverse modulation on synaptic input strength and network excitability. These coincident phenomena suggest a potential general mechanism of networks that adjusts gain as an inverse function of synaptic inputs' strength. Such mechanism may serve for adaptation to variable pressure and other physiological and pathological conditions and may explain the increased sensitivity to strong sensory stimulation suffered by human deep-divers and cetaceans under hyperbaric conditions.

Mechanisms of left-right coordination in mammalian locomotor pattern generation circuits: a mathematical modeling view.

The locomotor gait in limbed animals is defined by the left-right leg coordination and locomotor speed. Coordination between left and right neural activities in the spinal cord controlling left and right legs is provided by commissural interneurons (CINs). Several CIN types have been genetically identified, including the excitatory V3 and excitatory and inhibitory V0 types. Recent studies demonstrated that genetic elimination of all V0 CINs caused switching from a normal left-right alternating activity to a left-right synchronized "hopping" pattern. Furthermore, ablation of only the inhibitory V0 CINs (V0D subtype) resulted in a lack of left-right alternation at low locomotor frequencies and retaining this alternation at high frequencies, whereas selective ablation of the excitatory V0 neurons (V0V subtype) maintained the left-right alternation at low frequencies and switched to a hopping pattern at high frequencies. To analyze these findings, we developed a simplified mathematical model of neural circuits consisting of four pacemaker neurons representing left and right, flexor and extensor rhythm-generating centers interacting via commissural pathways representing V3, V0D, and V0V CINs. The locomotor frequency was controlled by a parameter defining the excitation of neurons and commissural pathways mimicking the effects of N-methyl-D-aspartate on locomotor frequency in isolated rodent spinal cord preparations. The model demonstrated a typical left-right alternating pattern under control conditions, switching to a hopping activity at any frequency after removing both V0 connections, a synchronized pattern at low frequencies with alternation at high frequencies after removing only V0D connections, and an alternating pattern at low frequencies with hopping at high frequencies after removing only V0V connections. We used bifurcation theory and fast-slow decomposition methods to analyze network behavior in the above regimes and transitions between them. The model reproduced, and suggested explanation for, a series of experimental phenomena and generated predictions available for experimental testing.

Organization of left-right coordination of neuronal activity in the mammalian spinal cord: Insights from computational modelling.

KEY POINTS: Coordination of neuronal activity between left and right sides of the mammalian spinal cord is provided by several sets of commissural interneurons (CINs) whose axons cross the midline. Genetically identified inhibitory V0D and excitatory V0V CINs and ipsilaterally projecting excitatory V2a interneurons were shown to secure left-right alternation at different locomotor speeds. We have developed computational models of neuronal circuits in the spinal cord that include left and right rhythm-generating centres interacting bilaterally via three parallel pathways mediated by V0D , V2a-V0V and V3 neuron populations. The models reproduce the experimentally observed speed-dependent left-right coordination in normal mice and the changes in coordination seen in mutants lacking specific neuron classes. The models propose an explanation for several experimental results and provide insights into the organization of the spinal locomotor network and parallel CIN pathways involved in gait control at different locomotor speeds. ABSTRACT: Different locomotor gaits in mammals, such as walking or galloping, are produced by coordinated activity in neuronal circuits in the spinal cord. Coordination of neuronal activity between left and right sides of the cord is provided by commissural interneurons (CINs), whose axons cross the midline. In this study, we construct and analyse two computational models of spinal locomotor circuits consisting of left and right rhythm generators interacting bilaterally via several neuronal pathways mediated by different CINs. The CIN populations incorporated in the models include the genetically identified inhibitory (V0D ) and excitatory (V0V ) subtypes of V0 CINs and excitatory V3 CINs. The model also includes the ipsilaterally projecting excitatory V2a interneurons mediating excitatory drive to the V0V CINs. The proposed network architectures and CIN connectivity allow the models to closely reproduce and suggest mechanistic explanations for several experimental observations. These phenomena include: different speed-dependent contributions of V0D and V0V CINs and V2a interneurons to left-right alternation of neural activity, switching gaits between the left-right alternating walking-like activity and the left-right synchronous hopping-like pattern in mutants lacking specific neuron classes, and speed-dependent asymmetric changes of flexor and extensor phase durations. The models provide insights into the architecture of spinal network and the organization of parallel inhibitory and excitatory CIN pathways and suggest explanations for how these pathways maintain alternating and synchronous gaits at different locomotor speeds. The models propose testable predictions about the neural organization and operation of mammalian locomotor circuits.

Dual-mode operation of neuronal networks involved in left-right alternation.

All forms of locomotion are repetitive motor activities that require coordinated bilateral activation of muscles. The executive elements of locomotor control are networks of spinal neurons that determine gait pattern through the sequential activation of motor-neuron pools on either side of the body axis. However, little is known about the constraints that link left-right coordination to locomotor speed. Recent advances have indicated that both excitatory and inhibitory commissural neurons may be involved in left-right coordination. But the neural underpinnings of this, and a possible causal link between these different groups of commissural neurons and left-right alternation, are lacking. Here we show, using intersectional mouse genetics, that ablation of a group of transcriptionally defined commissural neurons--the V0 population--leads to a quadrupedal hopping at all frequencies of locomotion. The selective ablation of inhibitory V0 neurons leads to a lack of left-right pattern at low frequencies, mixed coordination at medium frequencies, and alternation at high locomotor frequencies. When ablation is targeted to excitatory V0 neurons, left-right alternation is present at low frequencies, and hopping is restricted to medium and high locomotor frequencies. Therefore, the intrinsic logic of the central control of locomotion incorporates a modular organization, with two subgroups of V0 neurons required for the existence of left-right alternating modes at different speeds of locomotion. The two molecularly distinct sets of commissural neurons may constrain species-related naturally occurring frequency-dependent coordination and be involved in the evolution of different gaits.

Identification of minimal neuronal networks involved in flexor-extensor alternation in the mammalian spinal cord.

Neural networks in the spinal cord control two basic features of locomotor movements: rhythm generation and pattern generation. Rhythm generation is generally considered to be dependent on glutamatergic excitatory neurons. Pattern generation involves neural circuits controlling left-right alternation, which has been described in great detail, and flexor-extensor alternation, which remains poorly understood. Here, we use a mouse model in which glutamatergic neurotransmission has been ablated in the locomotor region of the spinal cord. The isolated in vitro spinal cord from these mice produces locomotor-like activity-when stimulated with neuroactive substances-with prominent flexor-extensor alternation. Under these conditions, unlike in control mice, networks of inhibitory interneurons generate the rhythmic activity. In the absence of glutamatergic synaptic transmission, the flexor-extensor alternation appears to be generated by Ia inhibitory interneurons, which mediate reciprocal inhibition from muscle proprioceptors to antagonist motor neurons. Our study defines a minimal inhibitory network that is needed to produce flexor-extensor alternation during locomotion.

Enduring medial perforant path short-term synaptic depression at high pressure.

The high pressure neurological syndrome develops during deep-diving (>1.1 MPa) involving impairment of cognitive functions, alteration of synaptic transmission and increased excitability in cortico-hippocampal areas. The medial perforant path (MPP), connecting entorhinal cortex with the hippocampal formation, displays synaptic frequency-dependent-depression (FDD) under normal conditions. Synaptic FDD is essential for specific functions of various neuronal networks. We used rat cortico-hippocampal slices and computer simulations for studying the effects of pressure and its interaction with extracellular Ca(2+) ([Ca(2+)](o)) on FDD at the MPP synapses. At atmospheric pressure, high [Ca(2+)](o) (4-6 mM) saturated single MPP field EPSP (fEPSP) and increased FDD in response to short trains at 50 Hz. High pressure (HP; 10.1 MPa) depressed single fEPSPs by 50%. Increasing [Ca(2+)](o) to 4 mM at HP saturated synaptic response at a subnormal level (only 20% recovery of single fEPSPs), but generated a FDD similar to atmospheric pressure. Mathematical model analysis of the fractions of synaptic resources used by each fEPSP during trains (normalized to their maximum) and the total fraction utilized within a train indicate that HP depresses synaptic activity also by reducing synaptic resources. This data suggest that MPP synapses may be modulated, in addition to depression of single events, by reduction of synaptic resources and then may have the ability to conserve their dynamic properties under different conditions.

Glutamatergic mechanisms for speed control and network operation in the rodent locomotor CpG.

Locomotion is a fundamental motor act that, to a large degree, is controlled by central pattern-generating (CPG) networks in the spinal cord. Glutamate is thought to be responsible for most of the excitatory input to and the excitatory activity within the locomotor CPG. However, previous studies in mammals have produced conflicting results regarding the necessity and role of the different ionotropic glutamate receptors (GluRs) in the CPG function. Here, we use electrophysiological and pharmacological techniques in the in vitro neonatal mouse lumbar spinal cord to investigate the role of a broad range of ionotropic GluRs in the control of locomotor speed and intrinsic locomotor network function. We show that non-NMDA (non-NMDARs) and NMDA receptor (NMDAR) systems may independently mediate locomotor-like activity and that these receptors set different speeds of locomotor-like activity through mechanisms acting at various network levels. AMPA and kainate receptors are necessary for generating the highest locomotor frequencies. For coordination, NMDARs are more important than non-NMDARs for conveying the rhythmic signal from the network to the motor neurons during long-lasting and steady locomotor activity. This study reveals that a diversity of ionotropic GluRs tunes the network to perform at different locomotor speeds and provides multiple levels for potential regulation and plasticity.

Excitatory components of the mammalian locomotor CPG.

Locomotion in mammals is to a large degree controlled directly by intrinsic spinal networks, called central pattern generators (CPGs). The overall function of these networks is governed by interaction between inhibitory and excitatory neurons. In the present review, we will discuss recent findings addressing the role of excitatory synaptic transmission for network function including the role of specific excitatory neuronal populations in coordinating muscle activity and in generating rhythmic activity.

Superior survival and durability of neurons and astrocytes on 3-dimensional aragonite biomatrices.

Current needs of central nervous system therapy urge for the identification of scaffolds supporting the generation and long-term maintenance of healthy and functional neuronal tissue. We compared for the first time the viability of hippocampal neurons and astrocytes grown on conventional 2-dimensional (2D) conditions with that of cells grown on an aragonite bioactive 3-dimensional (3D) scaffold prepared from coralline exoskeleton. Cultures in 3D showed significantly lower mortality rate and higher neurons/astrocytes ratio than 2D cultures. Moreover, whereas cell survival in 2D was arrested in the absence of the supporting substrates poly-D-lysine and laminin, these substrates had negligible effect on the 3D cultures. Furthermore, aragonite matrices supported cell survival and growth under conditions of calcium and nutrients deprivation, whereas in 2D such treatments led to death of all neurons and of almost all astrocytes. To show that the aragonite matrices are permissive for neural cells also in vivo, aragonite matrices having no substrate coating grafted into postnatal rat cortex were invaded by neurons growing on the surface and in multilayer structures resembling those seen in the 3D culture in vitro. Hence, culture of neurons and astrocytes on 3D aragonite coralline matrices is a novel mean for production of stable neuronal tissue, with significant implication to the field of neuronal tissue restoration.

Microglia activated by IL-4 or IFN-gamma differentially induce neurogenesis and oligodendrogenesis from adult stem/progenitor cells.

Cell renewal in the adult central nervous system (CNS) is limited, and is blocked in inflammatory brain conditions. We show that both neurogenesis and oligodendrogenesis of adult neural progenitor cells in mice are blocked by inflammation-associated (endotoxin-activated) microglia, but induced by microglia activated by cytokines (IL-4 or low level of IFN-gamma) associated with T-helper cells. Blockage was correlated with up-regulation of microglial production of tumor necrosis factor-alpha. The effect induced by IL-4-activated microglia was mediated, at least in part, by insulin-like growth factor-I. The IL-4-activated microglia showed a bias towards oligodendrogenesis whereas the IFN-gamma-activated microglia showed a bias towards neurogenesis. It thus appears that microglial phenotype critically affects their ability to support or impair cell renewal from adult stem cell.

Activation of microglia by aggregated beta-amyloid or lipopolysaccharide impairs MHC-II expression and renders them cytotoxic whereas IFN-gamma and IL-4 render them protective.

'Protective autoimmunity' refers to a well-controlled anti-self response that helps the body resist neurodegeneration. The response is mediated by autoimmune T cells, which produce cytokines and growth factors. Using an in vitro assay of hippocampal slices, we show that the cytokines interferon-gamma and (especially) interleukin-4, characteristic of pro-inflammatory and anti-inflammatory T cells, respectively, can make microglia neuroprotective. Aggregated beta-amyloid, like bacterial cell wall-derived lipopolysaccharide, rendered the microglia cytotoxic. Cytotoxicity was correlated with a signal transduction pathway that down-regulates expression of class-II major histocompatibility proteins (MHC-II) through the MHC-II-transactivator and the invariant chain. Protection by interleukin-4 was attributed to down-regulation of tumor necrosis factor-alpha and up-regulation of insulin-like growth factor I. These findings suggest that beneficial or harmful expression of the local immune response in the damaged CNS depends on how microglia interpret the threat, and that a well-regulated T-cell-mediated response enables microglia to alleviate rather than exacerbate stressful situations in the CNS.

Enhanced excitability compensates for high-pressure-induced depression of cortical inputs to the hippocampus.

High pressure (>1.0 MPa) induces the high-pressure neurological syndrome (HPNS) characterized by increased excitability of the CNS and cognitive impairments involving memory disorders. The perforant-path transfer of cortical information to the hippocampal formation is important for memory acquisition. High pressure may alter information transfer in this connection. We used rat corticohippocampal slices for studying the effect of pressure on the transfer function between synaptic inputs from the medial perforant path (MPP) and spike generation by granule cells (GC) of the dentate gyrus. High pressure (10.1 MPa) reduced single MPP field excitatory postsynaptic potential (fEPSP) amplitude and slope by nearly 50%. Field antidromic action potentials (AAPs) elicited by stimulation of GC axons, and population spike (PS) generation by the pressure-depressed MPP fEPSP were not significantly altered at hyperbaric conditions. Nevertheless the relationship PS/fEPSP increased at high pressure, indicating dendritic hyperexcitability in the GC. PSs elicited by paired-pulse MPP fEPSPs at 10- to 200-ms interstimulus intervals and PS generated by trains of five fEPSPs at 25 Hz were also not affected in spite of severe pressure-induced synaptic depression. Similarly, trains of AAPs at 25-50 Hz were not significantly changed. Trains of fEPSPs at higher frequency (50 Hz), however, induced additional spikes at high pressure, indicating pressure disruption of the regular low-pass filter properties of the DG. Such effect was closely mimicked by partial blockade of GABAA inhibition. High pressure depresses synaptic activity while increases excitability in the neuronal dendrites but not in the axons. This mechanism, allowing neuronal communication at low input signals, may partially cope with pressure effects at the low frequency range (<25 Hz) but losses reliability at higher frequencies (>50 Hz).

Modulation of rat corticohippocampal synaptic activity by high pressure and extracellular calcium: single and frequency responses.

High pressure (>1.5 MPa) induces a series of disturbances of the nervous system that are generically termed high-pressure nervous syndrome (HPNS). HPNS is characterized by motor and cognitive impairments. The neocortex and the hippocampus are presumably involved in this last disorder. The medial perforant path (MPP) synapse onto the granule cells of the dentate gyrus is the main connection between these structures. We have studied high-pressure (HP) effects on single and frequency response of this synapse. Since effects of HP on various synapses were mimicked by reducing [Ca2+]o, results under these conditions were compared. Medial perforant path-evoked field excitatory postsynaptic potentials (fEPSPs) were recorded from granule cells in rat brain slices. Slices were exposed to high pressure of helium (0.1-10.1 MPa) at 30 degrees C. HP depressed single fEPSPs by 35 and 55% at 5.1 and 10.1 MPa, respectively, and increased paired-pulse facilitation (PPF) at 10- to 40-ms inter-stimulus intervals. Frequency-dependent depression (FDD) was enhanced by HP during trains of stimuli at 50 but not at 25 Hz. Depression of single fEPSPs by reduction of [Ca2+]o from 2 mM control to 1 mM at normal pressure was equivalent to the effect of 10.1 MPa at control [Ca2+]o. However, this low [Ca2+]o induced greater enhancement of PPF, and in contrast, turned FDD at 25-50 Hz into frequency-dependent potentiation. These results suggest that HP depresses single synaptic release by reducing Ca2+ entry, whereas slowing of synaptic frequency response is independent of Ca2+. These findings increase our understanding of HPNS experienced by deep divers.