Morphologically mixed chemical/electrical synapses at axon terminals, with the electrical component formed by gap junctions, is common in the CNS of lower vertebrates. In mammalian CNS, evidence for morphologically mixed synapses has been obtained in only a few locations. Here, we used immunofluorescence approaches to examine the localization of the neuronally expressed gap junction forming protein connexin36 (Cx36) in relation to the axon terminal marker vesicular glutamate transporter-1 (vglut1) in the spinal cord and the trigeminal motor nucleus (Mo5) of rat and mouse. In adult rodents, immunolabeling for Cx36 appeared exclusively as Cx36-puncta, and was widely distributed at all rostro-caudal levels in most spinal cord laminae and in the Mo5. A high proportion of Cx36-puncta was co-localized with vglut1, forming morphologically mixed synapses on motoneurons, in intermediate spinal cord lamina, and in regions of medial lamina VII, where vglut1-containing terminals associated with Cx36 converged on neurons adjacent to the central canal. Unilateral transection of lumbar dorsal roots reduced immunolabeling of both vglut1 and Cx36 in intermediate laminae and lamina IX. Further, vglut1-terminals displaying Cx36-puncta were contacted by terminals labeled for glutamic acid decarboxylase65, which is known to be contained in presynaptic terminals on large-diameter primary afferents. Developmentally, mixed synapses begin to emerge in the spinal cord only after the second to third postnatal week and thereafter increase to adult levels. Our findings demonstrate that axon terminals of primary afferent origin form morphologically mixed synapses containing Cx36 in broadly distributed areas of adult rodent spinal cord and Mo5.

Electrical synapses formed by gap junctions containing connexin36 (Cx36) promote synchronous activity of interneurones in many regions of mammalian brain; however, there is limited information on the role of electrical synapses in spinal neuronal networks. Here we show that Cx36 is widely distributed in the spinal cord and is involved in mechanisms that govern presynaptic inhibition of primary afferent terminals. Electrophysiological recordings were made in spinal cord preparations from 8- to 11-day-old wild-type and Cx36 knockout mice. Several features associated with presynaptic inhibition evoked by conditioning stimulation of low threshold hindlimb afferents were substantially compromised in Cx36 knockout mice. Dorsal root potentials (DRPs) evoked by low intensity stimulation of sensory afferents were reduced in amplitude by 79% and in duration by 67% in Cx36 knockouts. DRPs were similarly affected in wild-types by bath application of gap junction blockers. Consistent with presynaptic inhibition of group Ia muscle spindle afferent terminals on motoneurones described in adult cats, conditioning stimulation of an adjacent dorsal root evoked a long duration inhibition of monosynaptic reflexes recorded from the ventral root in wild-type mice, and this inhibition was antagonized by bicuculline. The same conditioning stimulation failed to inhibit monosynaptic reflexes in Cx36 knockout mice. Immunofluorescence labelling for Cx36 was found throughout the dorsal and ventral horns of the spinal cord of juvenile mice and persisted in mature animals. In deep dorsal horn laminae, where interneurones involved in presynaptic inhibition of large diameter muscle afferents are located, cells were extensively dye-coupled following intracellular neurobiotin injection. Coupled cells displayed Cx36-positive puncta along their processes. Our results indicate that gap junctions formed by Cx36 in spinal cord are required for maintenance of presynaptic inhibition, including the regulation of transmission from Iamuscle spindle afferents. In addition to a role in presynaptic inhibition in juvenile animals, the persistence of Cx36 expression among spinal neuronal populations in the adult mouse suggests that the contribution of electrical synapses to integrative processes in fully mature spinal cord may be as diverse as that found in other areas of the CNS.

A computational neuro-biomechanical model of cat locomotion with two hindlimbs controlled by a loconotor spinal central pattern generator (CPG) has been developed. The 2D biomechanical model includes two three-joint hindlimbs and the trunk. Each hindlimb consists of three rigid segments connected by hinge joints and is actuated by nine muscles including two bifunctional muscles: biceps femoris posterior (BFP) and rectus femoris (RF). The architecture of spinal circuits and the locomotor CPG is based on the model of the two-level locomotor CPG (Rybak et al. 2006) consisting of the flexor and extensor half-centers and pattern formation (PF) circuits. The model of PF circuits was extended to incorporate additional interneuron populations controlling the activity of BFP and RF. The extended model of spinal circuits and the CPG allow generation of the full repertoire of muscular activity patterns including firing patterns of BFP and RF observed during fictive and normal locomotion, which are subject to afferent control of the CPG and depend on gait. The model demonstrates stable locomotion and exhibits realistic patterns of muscle activation, limb kinematics, and ground force dynamics. The model was used for investigation of the role of afferent control of the CPG for stable locomotion. Supported by NIH: R01NS048844 and R01 EB012855.

This is the first study to report on the increase in motoneurone excitability during fictive scratch in adult decerebrate cats. Intracellular recordings from antidromically identified motoneurones revealed a decrease in the voltage threshold for spike initiation ( V th ), a suppression of motoneurone afterhyperpolarization and activation of voltage‐dependent excitation at the onset of scratch. These state‐dependent changes recovered within 10–20 s after scratch and could be evoked after acute transection of the spinal cord at C1. Thus, there is a powerful intraspinal system that can quickly and reversibly re‐configure neuronal excitability during spinal network activation. Fictive scratch was evoked in spinal intact and transected decerebrate preparations by stroking the pinnae following topical curare application to the dorsal cervical spinal cord and neuromuscular block. Hyperpolarization of V th occurred (mean −5.8 mV) in about 80% of ipsilateral flexor, extensor or bifunctional motoneurones during fictive scratch. The decrease in V th began before any scratch‐evoked motoneurone activity as well as during the initial phase in which extensors are tonically hyperpolarized. The V th of contralateral extensors depolarized by a mean of +3.7 mV during the tonic contralateral extensor activity accompanying ipsilateral scratch. There was a consistent and substantial reduction of afterhyperpolarization amplitude without large increases in motoneurone conductance in both spinal intact and transected preparations. Depolarizing current injection increased, and hyperpolarization decreased the amplitude of rhythmic scratch drive potentials in acute spinal preparations indicating that the spinal scratch‐generating network can activate voltage‐dependent conductances in motoneurones. The enhanced excitability in spinal preparations associated with fictive scratch indicates the existence of previously unrecognized, intraspinal mechanisms increasing motoneurone excitability.

Central pattern generators (CPGs) located in the spinal cord produce the coordinated activation of flexor and extensor motoneurons during locomotion. Previously proposed architectures for the spinal locomotor CPG have included the classical half-center oscillator and the unit burst generator (UBG) comprised of multiple coupled oscillators. We have recently proposed another organization in which a two-level CPG has a common rhythm generator (RG) that controls the operation of the pattern formation (PF) circuitry responsible for motoneuron activation. These architectures are discussed in relation to recent data obtained during fictive locomotion in the decerebrate cat. The data show that the CPG can maintain the period and phase of locomotor oscillations both during spontaneous deletions of motoneuron activity and during sensory stimulation affecting motoneuron activity throughout the limb. The proposed two-level CPG organization has been investigated with a computational model which incorporates interactions between the CPG, spinal circuits and afferent inputs. The model includes interacting populations of spinal interneurons and motoneurons modeled in the Hodgkin-Huxley style. Our simulations demonstrate that a relatively simple CPG with separate RG and PF networks can realistically reproduce many experimental phenomena including spontaneous deletions of motoneuron activity and a variety of effects of afferent stimulation. The model suggests plausible explanations for a number of features of real CPG operation that would be difficult to explain in the framework of the classical single-level CPG organization. Some modeling predictions and directions for further studies of locomotor CPG organization are discussed.

A computational model of the mammalian spinal cord circuitry incorporating a two-level central pattern generator (CPG) with separate half-center rhythm generator (RG) and pattern formation (PF) networks is reviewed. The model consists of interacting populations of interneurons and motoneurons described in the Hodgkin-Huxley style. Locomotor rhythm generation is based on a combination of intrinsic (persistent sodium current dependent) properties of excitatory RG neurons and reciprocal inhibition between the two half-centers comprising the RG. The two-level architecture of the CPG was suggested from an analysis of deletions (spontaneous omissions of activity) and the effects of afferent stimulation on the locomotor pattern and rhythm observed during fictive locomotion in the cat. The RG controls the activity of the PF network that in turn defines the rhythmic pattern of motoneuron activity. The model produces realistic firing patterns of two antagonist motoneuron populations and generates locomotor oscillations encompassing the range of cycle periods and phase durations observed during cat locomotion. A number of features of the real CPG operation can be reproduced with separate RG and PF networks, which would be difficult if not impossible to demonstrate with a classical single-level CPG. The two-level architecture allows the CPG to maintain the phase of locomotor oscillations and cycle timing during deletions and during sensory stimulation. The model provides a basis for functional identification of spinal interneurons involved in generation and control of the locomotor pattern.

The mammalian spinal cord contains a locomotor central pattern generator (CPG) that can produce alternating rhythmic activity of flexor and extensor motoneurones in the absence of rhythmic input and proprioceptive feedback. During such fictive locomotor activity in decerebrate cats, spontaneous omissions of activity occur simultaneously in multiple agonist motoneurone pools for a number of cycles. During these 'deletions', antagonist motoneurone pools usually become tonically active but may also continue to be rhythmic. The rhythmic activity that re-emerges following a deletion is often not phase shifted. This suggests that some neuronal mechanism can maintain the locomotor period when motoneurone activity fails. To account for these observations, a simplified computational model of the spinal circuitry has been developed in which the locomotor CPG consists of two levels: a half-centre rhythm generator (RG) and a pattern formation (PF) network, with reciprocal inhibitory interactions between antagonist neural populations at each level. The model represents a network of interacting neural populations with single interneurones and motoneurones described in the Hodgkin-Huxley style. The model reproduces the range of locomotor periods and phase durations observed during real locomotion in adult cats and permits independent control of the level of motoneurone activity and of step cycle timing. By altering the excitability of neural populations within the PF network, the model can reproduce deletions in which motoneurone activity fails but the phase of locomotor oscillations is maintained. The model also suggests criteria for the functional identification of spinal interneurones involved in the mammalian locomotor pattern generation.

A computational model of the mammalian spinal cord circuitry incorporating a two‐level central pattern generator (CPG) with separate half‐centre rhythm generator (RG) and pattern formation (PF) networks has been developed from observations obtained during fictive locomotion in decerebrate cats. Sensory afferents have been incorporated in the model to study the effects of afferent stimulation on locomotor phase switching and step cycle period and on the firing patterns of flexor and extensor motoneurones. Here we show that this CPG structure can be integrated with reflex circuits to reproduce the reorganization of group I reflex pathways occurring during locomotion. During the extensor phase of fictive locomotion, activation of extensor muscle group I afferents increases extensor motoneurone activity and prolongs the extensor phase. This extensor phase prolongation may occur with or without a resetting of the locomotor cycle, which (according to the model) depends on the degree to which sensory input affects the RG and PF circuits, respectively. The same stimulation delivered during flexion produces a temporary resetting to extension without changing the timing of following locomotor cycles. The model reproduces this behaviour by suggesting that this sensory input influences the PF network without affecting the RG. The model also suggests that the different effects of flexor muscle nerve afferent stimulation observed experimentally (phase prolongation versus resetting) result from opposing influences of flexor group I and II afferents on the PF and RG circuits controlling the activity of flexor and extensor motoneurones. The results of modelling provide insights into proprioceptive control of locomotion.

In intact animals and humans, increases in locomotor speed are usually associated with decreases in step cycle duration. Most data indicate that the locomotor central pattern generator (CPG) shortens cycle duration mainly by shortening the durations of extensor rather than flexor phases of the step cycle. Here we report that in fictive locomotion elicited by electrical stimulation of the midbrain locomotor region (MLR) in the cat, spontaneous variations in cycle duration were due more to changes in flexor rather than extensor phase durations in 22 of 31 experiments. The locomotor CPG is therefore not inherently extensor- or flexor-biased. We coined the term "dominant" to designate the phase (flexion or extension) showing the larger variation. In a simple half-center oscillator model, experimental phase duration plots were fitted well by adjusting two parameters that corresponded to background drive ("bias") and sensitivity ("gain") of the oscillator's timing elements. By analogy we argue that variations in background drive to the neural timing elements of the CPG could produce larger variations in phase duration in the half-center receiving the lower background drive, i.e., background drive may determine which half-center is dominant. The fact that data from normal cats were also fitted well by the model indicates that sensory input and central drive combine to determine locomotor phase durations. We conclude that there is a considerable flexibility in the control of phase durations in MLR-induced fictive locomotion. We posit that this may be explained by changes in background excitation of neural timing elements in the locomotor CPG.

Reflex actions of muscle afferents in hindlimb flexor nerves were examined on ipsilateral motoneurone activity recorded in peripheral nerves during midbrain stimulation-evoked fictive locomotion and during fictive scratch in decerebrate cats. Trains of stimuli (15-30 shocks at 200 Hz) were delivered during the flexion phase at intensities sufficient to activate both group I and II afferents (5 times threshold, T). In many preparations tibialis anterior (TA) nerve stimulation terminated ongoing flexion and reset the locomotor cycle to extension (19/31 experiments) while extensor digitorum longus (EDL) stimulation increased and prolonged the ongoing flexor phase activity (20/33 preparations). The effects of sartorius, iliopsoas and peroneus longus muscle afferent stimulation were qualitatively similar to those of EDL nerve. Resetting to extension was seen only with higher intensity stimulation (5 T) while ongoing flexor activity was often enhanced at group I intensity (2T) stimulation. The effects of flexor nerve stimulation were qualitatively similar during fictive scratch. Reflex reversals were consistently observed in some fictive locomotor preparations. In those cases, EDL stimulation produced a resetting to extension and TA stimulation prolonged the ongoing flexion phase. Occasionally reflex reversals occurred spontaneously during only one of several stimulus presentations. The variable and opposite actions of flexor afferents on the locomotor step cycle indicate the existence of parallel spinal reflex pathways. A hypothetical organization of reflex pathways from flexor muscle afferents to the spinal pattern generator networks with competing actions of group I and group II afferents on the flexor and extensor portions of this central circuitry is proposed.

We examined the features of spontaneous deletions of bursts of motoneuron activity that can occur within otherwise rhythmic alternating flexor and extensor activity during fictive locomotion and scratch in adult decerebrate cats. Deletions of activity were observed both in hindlimb flexor and extensor motoneuron pools during brain stem-stimulation-evoked fictive locomotion but only in extensors during fictive scratch. Paired intracellular motoneuron recordings showed that deletions reduced the depolarization of homonymous motoneurons in qualitatively similar ways. Differences occurred in the extent to which activity in synergist motoneuron pools operating at other joints within the limb was reduced during deletions. The timing of the rhythmic activity that followed a deletion was often at an integer multiple of the preexisting locomotor or scratch cycle period. This maintenance of cycle period was also seen during deletions in which there was a complete failure of motoneuron depolarization. The activity of antagonist motoneurons was usually sustained during deletions with some rhythmic modulation at intervals of the preexisting cycle period. We discuss an organization of the central pattern generator for locomotion and scratch that functions as a single rhythm generator with separate and multiple pattern formation modules for controlling the hyper- and depolarization of subsets of motoneurons within the limb.

In cat and humans, contact between an obstacle and the dorsum of the foot evokes the stumbling corrective reaction (reflex) that lifts the foot to avoid falling. This reflex can also be evoked by short trains of stimuli to the cutaneous superficial peroneal (SP) nerve in decerebrate cats during the flexion phase of fictive locomotion. Here we examine intracellular events in hindlimb motoneurons accompanying stumbling correction. SP stimulation delivered during the flexion phase excites knee flexor motoneurons at short latency [minimum excitatory postsynaptic potential (EPSP) latency 1.8 ms; mean 2.7 ms]. Although a similar short latency excitation occurs in ankle extensors (mean latency, 2.8 ms), recruitment is delayed until successive shocks in the stimulus train overcome the locomotor-related hyperpolarization of ankle extensors. In ankle flexor motoneurons, SP stimulation evokes an inhibition (mean latency, 2.7 ms) that briefly reduces or stops their firing during the flexion phase. There is a phase-dependent modulation of SP-evoked EPSP amplitude as well as latency during locomotion. However, the more obvious change in SP reflex pathways with the onset of fictive locomotion is the reduced inhibition of ankle extensor motoneurons and the increased inhibition of ankle flexors. These results show that the characteristic pattern of hindlimb motoneuron activation during SP nerve-evoked stumbling correction results from 1) di- and trisynaptic excitation of knee flexor and ankle extensor motoneurons; 2) increased inhibitory postsynaptic potentials in ankle flexors and a suppression of inhibition in extensors, 3) sculpting of the short-latency SP postsynaptic effects by motoneuron membrane potential, and 4) longer latency excitatory effects that are likely evoked by lumbar interneurons involved in the generation of fictive locomotion.

An obstacle contacting the dorsal surface of a cat's hind foot during the swing phase of locomotion evokes a reflex ( the stumbling corrective reaction) that lifts the foot and extends the ankle to avoid falling. We show that the same sequence of ipsilateral hindlimb motoneuron activity can be evoked in decerebrate cats during fictive locomotion. As recorded in the peripheral nerves, twice threshold intensity stimulation of the cutaneous superficial peroneal (SP) nerve during the flexion phase produced a very brief excitation of ankle flexors ( e. g., tibialis anterior and peroneus longus) that was followed by an inhibition for the duration of the stimulus train ( 10 - 25 shocks, 200 Hz). Extensor digitorum longus was always, and hip flexor ( sartorius) activity was sometimes, inhibited during SP stimulation. At the same time, knee flexor and the normally quiescent ankle extensor motoneurons were recruited ( mean latencies 4 and 16 ms) with SP stimulation during fictive stumbling correction. After the stimulus train, ankle extensor activity fell silent, and there was an excitation of hip, knee, and ankle flexors. The ongoing flexion phase was often prolonged. Hip extensors were also recruited in some fictive stumbling trials. Only the SP nerve was effective in evoking stumbling correction. Delivered during extension, SP stimulus trains increased ongoing extensor motoneuron activity as well as increasing ipsilateral hip, knee, and ankle hindlimb flexor activity in the subsequent step cycle. The fictive stumbling corrective reflex seems functionally similar to that evoked in intact, awake animals and involves a fixed pattern of short-latency reflexes as well as actions evoked through the lumbar circuitry responsible for the generation of rhythmic alternating locomotion.

Extracellular recordings from interneurons located in the L4 spinal segment were made during fictive locomotion produced by electrical stimulation of the mesencephalic locomotor region (MLR) in the paralysed decerebrate cat. Only interneurons within the L4 segment which received group II input from quadriceps, sartorius or the pretibial flexor muscle afferents and which had axonal projections to motor nuclei in L7 were selected for analysis. During the fictive step cycle two thirds of these interneurons fired action potentials during the time of activity in the ipsilateral hindlimb flexor neurograms. These cells were also less responsive to peripheral input during the extension phase of the fictive locomotion cycle. The remaining one third of the interneurons examined were not rhythmically active during locomotion. The possible contributions of the midlumbar interneurons to motoneuron activity during locomotion are discussed.

In the present study we sought to find interneurones responsible for the group I-evoked disynaptic excitation of hindlimb extensor motoneurones that occurs during fictive locomotion. Locomotion was produced by stimulation of the mesencephalic locomotor region (MLR) in decerebrate paralysed cats in which activation of ankle extensor group I afferents evoked a disynaptic excitation of motoneurones during the extension phase of fictive locomotion. Extracellular recordings were used to locate interneurones fulfilling all, or five of the six following criteria: (i) weak or no response to stimulation of extensor group I afferents in the absence of locomotion; (ii) strong group I activation during locomotion; (iii) group I activation at monosynaptic latencies; (iv) strong group I activation during only the extensor phase of locomotion; and (v) antidromic activation from the extensor motor nuclei; but (vi) no antidromic activation from rostral spinal segments. Candidate excitatory interneurones were located in mid to caudal parts of the L7 segments in areas where monosynaptic field potentials were evoked by group I afferents, within 2 mm of the stimulation site in the ventral horn from which they were antidromically activated. All were activated during extension by stimulation of group I afferents in extensor nerves. In the absence of peripheral nerve stimulation, six of the seven candidate excitatory interneurones were rhythmically active with maximal activation during the extension phase of fictive locomotion. Rhythmic activity during extension was also seen in five additional interneurones located near candidate interneurones but not activated by group I strength stimulation of the tested nerves. We suggest that the lumbosacral interneurones located in the intermediate laminae that can be activated by extensor group I afferents during the extension phase are a previously unknown population of interneurones, and may mediate group I-evoked disynaptic excitation of extensor motoneurones. Their rhythmic activity suggests that they also provide central excitatory drive to extensor motoneurones during locomotion.

Inspiratory drive to hypoglossal (XII) motoneurones is mediated predominantly via AMPA receptors (Funk et al. 1993). The effect of inhibition upon inspiratory drive currents, and its functional significance for respiratory-related motor output is unclear. Here, we describe phasic inhibition in XII motoneurons, its effect on inspiratory drive currents, and modulation of this inhibition by protein kinase A (PKA).

During fictive locomotion the excitability of adult cat lumbar motoneurones is increased by a reduction (a mean hyperpolarization of approximately 6.0 mV) of voltage threshold (Vth) for action potential (AP) initiation that is accompanied by only small changes in AP height and width. Further examination of the experimental data in the present study confirms that Vth lowering is present to a similar degree in both the hyperpolarized and depolarized portions of the locomotor step cycle. This indicates that Vth reduction is a modulation of motoneurone membrane currents throughout the locomotor state rather than being related to the phasic synaptic input within the locomotor cycle. Potential ionic mechanisms of this locomotor-state-dependent increase in excitability were examined using three five-compartment models of the motoneurone innervating slow, fast fatigue resistant and fast fatigable muscle fibres. Passive and active membrane conductances were set to produce input resistance, rheobase, afterhyperpolarization (AHP) and membrane time constant values similar to those measured in adult cat motoneurones in non-locomoting conditions. The parameters of 10 membrane conductances were then individually altered in an attempt to replicate the hyperpolarization of Vth that occurs in decerebrate cats during fictive locomotion. The goal was to find conductance changes that could produce a greater than 3 mV hyperpolarization of Vth with only small changes in AP height (< 3 mV) and width (< 1.2 ms). Vth reduction without large changes in AP shape could be produced either by increasing fast sodium current or by reducing delayed rectifier potassium current. The most effective Vth reductions were achieved by either increasing the conductance of fast sodium channels or by hyperpolarizing the voltage dependency of their activation. These changes were particularly effective when localized to the initial segment. Reducing the conductance of delayed rectifier channels or depolarizing their activation produced similar but smaller changes in Vth. Changes in current underlying the AHP, the persistent Na(+) current, three Ca(2+) currents, the "h" mixed cation current, the "A" potassium current and the leak current were either ineffective in reducing Vth or also produced gross changes in the AP. It is suggested that the increased excitability of motoneurones during locomotion could be readily accomplished by hyperpolarizing the voltage dependency of fast sodium channels in the axon hillock by a hitherto unknown neuromodulatory action.