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.     

Endogenous pacemaker potentials develop into paroxysmal depolarization shifts (PDSs) with application of an epileptogenic drug

Well-known invertebrate ganglia (buccal ganglia of Helix pomatia, abdominal ganglia of Aplysia californica) were used to study the contribution of synaptic potentials, central pattern generators, and endogenously generated neuronal potentials to the development of epileptiform activity. Epileptiform activity which was induced with application of pentylenetetrazol (1 to 100 mM) or etomidate (0.12 to 1.0 mM) consisted of paroxysmal depolarization shifts (PDSs) recorded simultaneously from several identified neurons with sharp microelectrodes. With application of an epileptogenic drug, endogenous pacemaker potentials develop into PDSs. With increasing concentration of the drug, (i) amplitude of pacemaker-depolarizations and (ii) delay of pacemaker-repolarization increased progressively finally resulting in PDSs. Additionally, the activation characterists of currents shifted from between -50 and -40 mV (pacemaker potentials, control conditions) to between -100 and -40 mV (PDS, epileptic conditions). Only neurons which generated pacemaker potentials under control conditions could generate PDSs under epileptic conditions. Chemical synaptic inputs triggered or blocked pacemaker potentials as well as PDSs. Activities induced from central pattern generators were identified with simultaneous recordings from several identified neurons. The central pattern generators could trigger or block pacemaker potentials as well as PDSs. Results demonstrate that, in the used model nervous systems, pacemaker potentials which are generated by the single neurons are the physiologic basis of epileptic activity.     

A mathematical model of the activity of spinal generators of rhythmic movements

Different organization schemes concerning locomotor and scratching rhythmicity generators are analyzed by means of the mathematical simulation model. A functional group of neurons (half-centre constructed on the stochastically organized neuronal network) is the basis for generator. Some organization schemes concerning locomotor and scratching rhythmicity generators are considered, such as: two half-centres with reciprocal inhibitory connections and tonic excitatory influences on these half-centres: two half-centres with inhibitory-excitatory connections and tonic excitatory influences on one half-centre; ring structures consisting of more than two functional groups of neurons with excitatory and inhibitory connections between them. In all the schemes considered generation of rhythmic activity is possible with time characteristics similar to those of the locomotor and scratching rhythmicity. It is shown that transition from locomotor to scratching rhythmicity can be realized through simply organized actions on generator neurons. Possible principles of construction of spinal generators for locomotor and scratching movements are discussed.     

Interacting biological and electronic neurons generate realistic oscillatory rhythms

Small assemblies of neurons such as central pattern generators (CPG) are known to express regular oscillatory firing patterns comprising bursts of action potentials. In contrast, individual CPG neurons isolated from the remainder of the network can generate irregular firing patterns. In our study of cooperative behavior in CPGs we developed an analog electronic neuron (EN) that reproduces firing patterns observed in lobster pyloric CPG neurons. Using a tuneable artificial synapse we connected the EN bidirectionally to neurons of this CPG. We found that the periodic bursting oscillation of this mixed assembly depends on the strength and sign of the electrical coupling. Working with identified, isolated pyloric CPG neurons whose network rhythms were impaired, the EN/biological network restored the characteristic CPG rhythm both when the EN oscillations are regular and when they are irregular.     

The pharmacology of vertebrate spinal central pattern generators.

Central pattern generators are networks of neurons capable of generating an output pattern of spike activity in a relatively stereotyped, rhythmic pattern that has been found to underlie vital functions like respiration and locomotion. The central pattern generator for locomotion in vertebrates seems to share some basic building blocks. Activation and excitation of activity is driven by descending, sensory, and intraspinal glutamatergic neurons. NMDA receptor activation may also lead to the activation of oscillatory properties in individual neurons that depend on an array of ion channels situated in those neurons. Coordination across joints or the midline of the animal is driven primarily by glycinergic inhibition. In addition to these processes, numerous modulatory mechanisms alter the function of the central pattern generator. These include metabotropic amino acid receptors activated by rhythmic release of glutamate and GABA as well as monoamines, ACh, and peptides. Function and stability of the central pattern generator is also critically dependent on the array of ion channels found in neurons that compose these oscillators, including Ca2+ and voltage-gated K+ channels and Ca2+ channels.     

Endocannabinoid signaling in the spinal locomotor circuitry

To understand how the spinal central pattern generators produce locomotor movements, it is necessary to characterize the network's connectivity, the intrinsic properties of the constituent neurons and the modulatory mechanisms. Modulation operating within spinal locomotor networks is required for the generation of the final motor output. In this review, we have summarized how endocannabinoids released by locomotor network neurons contribute to setting the baseline locomotor frequency. They are synthesized on demand as a result of activation of mGluR1 and act as retrograde messengers to depress inhibitory synaptic transmission. We also discuss how endogenous activation of mGluR1 contributes to the normal operation of the spinal locomotor network and the underlying cellular and synaptic mechanisms.     

Comparison between ventral spinocerebellar and rubrospinal activities during locomotion in the cat

During fictive locomotion of the thalamic cat, rhythmic activity related to the efferent discharges in hindlimb nerves was found in rubrospinal neurons (Arshavsky et al., this issue). Since the movements were abolished by curarization, this modulation could not result from rhythmic peripheral inputs and had therefore a central origin. Taking into account the existence of spinal generators, it was suggested that ascending pathways transmit rhythmic activity from these spinal centers to the supraspinal ones. Preliminary results have been obtained for neurons of the ventral spinocerebellar tract (VSCT) recorded during fictive locomotion: (1) their discharge is rhythmically modulated at the periodicity of the locomotor rhythm; (2) their discharge pattern can be complex and variable in relation with the complexity and variability of the pattern of efferent activity in various muscle nerves of the ipsilateral hindlimb; (3) their responses to phasic afferent stimulation of the ipsilateral hindlimb are modulated in parallel with their locomotor-related activity. These results show that VSCT neurons convey information on the central spinal activity during locomotion, and suggest that these neurons contribute to the activity of lumbar-projecting rubrospinal neurons which have similar characteristics.     

Irregular Breathing in Mice following Genetic Ablation of V2a Neurons

Neural networks called central pattern generators (CPGs) generate repetitive motor behaviors such as locomotion and breathing. Glutamatergic neurons are required for the generation and inhibitory neurons for the patterning of the motor activity associated with repetitive motor behaviors. In the mouse, glutamatergic V2a neurons coordinate the activity of left and right leg CPGs in the spinal cord enabling mice to generate an alternating gait. Here, we investigate the role of V2a neurons in the neural control of breathing, an essential repetitive motor behavior. We find that, following the ablation of V2a neurons, newborn mice breathe at a lower frequency. Recordings of respiratory activity in brainstem-spinal cord and respiratory slice preparations demonstrate that mice lacking V2a neurons are deficient in central respiratory rhythm generation. The absence of V2a neurons in the respiratory slice preparation can be compensated for by bath application of neurochemicals known to accelerate the breathing rhythm. In this slice preparation, V2a neurons exhibit a tonic firing pattern. The existence of direct connections between V2a neurons in the medial reticular formation and neurons of the pre-B?tzinger complex indicates that V2a neurons play a direct role in the function of the respiratory CPG in newborn mice. Thus, neurons of the embryonic V2a lineage appear to have been recruited to neural networks that control breathing and locomotion, two prominent CPG-driven, repetitive motor behaviors.     

Proctolin activates an inward current whose voltage dependence is modified by extracellular Ca2+.

The pentapeptide proctolin modulates the activity of the rhythmic pattern generators in the crustacean stomatogastric nervous system. Proctolin strongly excites the lateral pyloric and the inferior cardiac neurons of the stomatogastric ganglion (STG), causing them to fire extended high-frequency bursts of action potentials (Hooper and Marder, 1987; Nusbaum and Marder, 1989a,b). We now report that proctolin depolarizes these cells maximally at membrane potentials close to the threshold for action potential generation. In voltage clamp, proctolin evokes an inward current, carried at least partially by Na+, that shows strong outward rectification. Removal of extracellular Ca2+ markedly increases the amplitude of the proctolin-evoked current and linearizes its current-voltage curve. The properties of the proctolin current make it ideally suited to contribute to the activity-dependent modulation of the pyloric network of the STG.     

Dynamic control of a central pattern generator circuit: a computational model of the snail feeding network

Central pattern generators (CPGs) are networks underlying rhythmic motor behaviours and they are dynamically regulated by neuronal elements that are extrinsic or intrinsic to the rhythmogenic circuit. In the feeding system of the pond snail, Lymnaea stagnalis, the extrinsic slow oscillator (SO) interneuron controls the frequency of the feeding rhythm and the N3t (tonic) has a dual role; it is an intrinsic CPG interneuron, but it also suppresses CPG activity in the absence of food, acting as a decision-making element in the feeding circuit. The firing patterns of the SO and N3t neurons and their synaptic connections with the rest of the CPG are known, but how these regulate network function is not well understood. This was investigated by building a computer model of the feeding network based on a minimum number of cells (N1M, N2v and N3t) required to generate the three-phase motor rhythm together with the SO that was used to activate the system. The intrinsic properties of individual neurons were represented using two-compartment models containing currents of the Hodgkin-Huxley type. Manipulations of neuronal activity in the N3t and SO neurons in the model produced similar quantitative effects to food and electrical stimulation in the biological network indicating that the model is a useful tool for studying the dynamic properties of the feeding circuit. The model also predicted novel effects of electrical stimulation of two CPG interneurons (N1M and N2v). When tested experimentally, similar effects were found in the biological system providing further validation of our model.     

Xenopus embryonic spinal neurons recorded in situ with patch‐clamp electrodes – conditional oscillators after all?

The central pattern generator for swimming Xenopus embryo is organized as two half-centres linked by reciprocal inhibition. Microelectrode recordings suggest that Xenopus neurons are poorly excitable, necessitating a key role for postinhibitory rebound in the operation of the central pattern generator. However the Xenopus central pattern generator seems unusual in that the component neurons apparently have no intrinsic or conditional rhythmogenic properties. We have re-examined the firing properties of Xenopus embryo spinal neurons by making patch-clamp recordings in situ from intact spinal cord. Recordings made from 99 neurons were divided into three groups. Central pattern generator neurons overwhelmingly (44/51) fired trains of action potentials in response to current injection. Just over half of the sensory interneurons (13/22) also fired trains of action potentials. Neurons that received no synaptic inputs during swimming mostly fired just one or two action potentials (22/26). Thirty-four neurons were identified morphologically. Commissural (8/12) and descending (6/6) interneurons, key components of the spinal central pattern generator, fired repetitive trains of action potentials during current injection. Neurons that were not part of the central pattern generator did not demonstrate this preponderance for repetitive firing. Analysis of the interspike intervals during current injection revealed that the majority of central pattern generators, descending and commissural interneurons, could readily fire at frequencies up to twice that of swimming. We suggest that Xenopus neurons can be considered as conditional oscillators: in the presence of unpatterned excitation they exhibit an ability to fire rhythmically. This property makes the Xenopus embryonic central pattern generator more similar to other model central pattern generators than has hitherto been appreciated.     

Self‐organization of repetitive spike patterns in developing neuronal networks in vitro

The appearance of spontaneous correlated activity is a fundamental feature of developing neuronal networks in vivo and in vitro. To elucidate whether the ontogeny of correlated activity is paralleled by the appearance of specific spike patterns we used a template‐matching algorithm to detect repetitive spike patterns in multi‐electrode array recordings from cultures of dissociated mouse neocortical neurons between 6 and 15 days in vitro (div). These experiments demonstrated that the number of spiking neurons increased significantly between 6 and 15 div, while a significantly synchronized network activity appeared at 9 div and became the main discharge pattern in the subsequent div. Repetitive spike patterns with a low complexity were first observed at 8 div. The number of repetitive spike patterns in each dataset as well as their complexity and recurrence increased during development in vitro. The number of links between neurons implicated in repetitive spike patterns, as well as their strength, showed a gradual increase during development. About 8% of the spike sequences contributed to more than one repetitive spike patterns and were classified as core patterns. These results demonstrate for the first time that defined neuronal assemblies, as represented by repetitive spike patterns, appear quite early during development in vitro, around the time synchronized network burst become the dominant network pattern. In summary, these findings suggest that dissociated neurons can self‐organize into complex neuronal networks that allow reliable flow and processing of neuronal information already during early phases of development.     

Impaired dendritic inhibition leads to epileptic activity in a computer model of CA3

Temporal lobe epilepsy (TLE) is a common type of epilepsy with hippocampus as the usual site of origin. The CA3 subfield of hippocampus is reported to have a low epileptic threshold and hence initiates the disorder in patients with TLE. This study computationally investigates how impaired dendritic inhibition of pyramidal cells in the vulnerable CA3 subfield leads to generation of epileptic activity. A model of CA3 subfield consisting of 800 pyramidal cells, 200 basket cells (BC) and 200 Oriens—Lacunosum Moleculare (O‐LM) interneurons was used. The dendritic inhibition provided by O‐LM interneurons is reported to be selectively impaired in some TLEs. A step‐wise approach is taken to investigate how alterations in network connectivity lead to generation of epileptic patterns. Initially, dendritic inhibition alone was reduced, followed by an increase in the external inputs received at the distal dendrites of pyramidal cells, and finally additional changes were made at the synapses between all neurons in the network. In the first case, when the dendritic inhibition of pyramidal cells alone was reduced, the local field potential activity changed from a theta‐modulated gamma pattern to a prominently gamma frequency pattern. In the second case, in addition to this reduction of dendritic inhibition, with a simultaneous large increase in the external excitatory inputs received by pyramidal cells, the basket cells entered a state of depolarization block, causing the network to generate a typical ictal activity pattern. In the third case, when the dendritic inhibition onto the pyramidal cells was reduced and changes were simultaneously made in synaptic connectivity between all neurons in the network, the basket cells were again observed to enter depolarization block. In the third case, impairment of dendritic inhibition required to generate an ictal activity pattern was lesser than the two previous cases. Moreover, the ictal like activity began earlier in the third case. Hence, our study suggests that greater synaptic plasticity occurring in the whole network due to increase in reception of external excitatory inputs (due to impaired dendritic inhibition) makes the network more susceptible to generation of epileptic activity. © 2015 Wiley Periodicals, Inc.     

Different proctolin neurons elicit distinct motor patterns from a multifunctional neuronal network.

Distinct motor patterns are selected from a multifunctional neuronal network by activation of different modulatory projection neurons. Subsets of these projection neurons can contain the same neuromodulator(s), yet little is known about the relative influence of such neurons on network activity. We have addressed this issue in the stomatogastric nervous system of the crab Cancer borealis. Within this system, there is a neuronal network in the stomatogastric ganglion (STG) that produces many versions of the pyloric and gastric mill rhythms. These different rhythms result from activation of different projection neurons that innervate the STG from neighboring ganglia and modulate STG network activity. Three pairs of these projection neurons contain the neuropeptide proctolin. These include the previously identified modulatory proctolin neuron and modulatory commissural neuron 1 (MCN1) and the newly identified modulatory commissural neuron 7 (MCN7). We document here that each of these neurons contains a unique complement of cotransmitters and that each of these neurons elicits a distinct version of the pyloric motor pattern. Moreover, only one of them (MCN1) also elicits a gastric mill rhythm. The MCN7-elicited pyloric rhythm includes a pivotal switch by one STG network neuron from playing a minor to a major role in motor pattern generation. Therefore, modulatory neurons that share a peptide transmitter can elicit distinct motor patterns from a common target network.     

Suppressive control of the crustacean pyloric network by a pair of identified interneurons. I. Modulation of the motor pattern

A pair of identified neuromodulatory neurons, the pyloric suppressor (PS) neurons, can individually and strongly modify the activity of the pyloric network in the stomatogastric nervous system of the lobster Homarus gammarus. The PS neurons are identified by the location of their somata in the inferior ventricular nerve, their axonal projections, and their effects on pyloric network activity in vitro. Discharge of a PS neuron evokes large EPSPs in the pyloric dilator (PD) neurons and a long-lasting cessation of rhythmic activity in the neurons that control movements of the pyloric filter: PD, lateral pyloric (LP), and pyloric (PY). This cessation of rhythmic activity can outlast by several 10s of seconds a brief discharge of PS lasting only a few seconds. The different neurons of the pyloric filter do not exhibit the same sensitivity to the suppressive effects of PS, with the LP neuron being the most sensitive. Tonic discharge in PS induces graded alterations in the pyloric pattern, depending on its firing frequency. At low (less than 5 Hz) discharge frequencies, PS provokes changes in phase relationships and duration of bursting in pyloric neurons. A slight increase in PS frequency suppresses the rhythmic activity of some pyloric neurons, resulting in a switch from a triphasic to a biphasic pattern. At higher (greater than 10 Hz) PS firing frequencies, rhythmic activity in all the pyloric neurons, including the pacemakers (PD, anterior burster), is abolished, except in cells (ventricular dilator, inferior cardiac) controlling the pyloric valve. We conclude that a central pattern generator is not only subject to activating modulatory control, but may also be the target of suppressive inputs that are themselves able to provoke functional reconfigurations of the network.     

Chaotic pattern transitions in pulse neural networks.

In models of associative memory composed of pulse neurons, chaotic pattern transitions where the pattern retrieved by the network changes chaotically were found. The network is composed of multiple modules of pulse neurons, and when the inter-module connection strength decreased, the stability of pattern retrieval changed from stable to chaotic. It was found that the mixed pattern of stored patterns plays an important role in chaotic pattern transitions.     

Patterns of presynaptic activity and synaptic strength interact to produce motor output

Motor neuron activity is coordinated by premotor networks into a functional motor pattern by complex patterns of synaptic drive. These patterns combine both the temporal pattern of spikes of the premotor network and the profiles of synaptic strengths (i.e., conductances). Given the complexity of premotor networks in vertebrates, it has been difficult to ascertain the relative contributions of temporal patterns and synaptic strength profiles to the motor patterns observed in these animals. Here, we use the leech (Hirudo sp.) heartbeat central pattern generator (CPG), in which we can measure both the temporal pattern and the synaptic strength profiles of the entire premotor network and the motor outflow in individual animals. In this system, a series of motor neurons all receive input from the same premotor interneurons of the CPG but must be coordinated differentially to produce a functional pattern. These properties allow a theoretical and experimental dissection of the rules that govern how temporal patterns and synaptic strength profiles are combined in motor neurons so that functional motor patterns emerge, including an analysis of the impact of animal-to-animal variation in input to such variation in output. In the leech, segmental heart motor neurons are coordinated alternately in a synchronous and peristaltic pattern. We show that synchronous motor patterns result from a nearly synchronous premotor temporal pattern produced by the leech heartbeat CPG. For peristaltic motor patterns, the staggered premotor temporal pattern determines the phase range over which segmental motor neurons can fire while synaptic strength profiles define the intersegmental motor phase progression realized.     

Organization of left–right coordination in the mammalian locomotor network

Neuronal circuits involved in left–right coordination are a fundamental feature of rhythmic locomotor movements. These circuits necessarily include commissural interneurons (CINs) that have axons crossing the midline of the spinal cord. The properties of CINs have been described in some detail in the spinal cords of a number of aquatic vertebrates including the Xenopus tadpole and the lamprey. However, their function in left–right coordination of limb movements in mammals is poorly understood. In this review we describe the present understanding of commissural pathways in the functioning of spinal cord central pattern generators (CPGs). The means by which reciprocal inhibition and integration of sensory information are maintained in swimming vertebrates is described, with similarities between the three basic populations of commissural interneurons highlighted. The subsequent section concentrates on recent evidence from mammalian limbed preparations and specifically the isolated spinal cord of the neonatal rat. Studies into the role of CPG elements during drug-induced locomotor-like activity have afforded a better understanding of the location of commissural pathways, such that it is now possible, using whole cell patch clamp, to record from anatomically defined CINs located in the rhythm-generating region of the lumbar segments. Initial results would suggest that the firing pattern of these neurons shows a greater diversity than that previously described in swimming central pattern generators. Spinal CINs play an important role in the generation of locomotor output. Increased knowledge as to their function in producing locomotion is likely to provide valuable insights into the spinal networks required for postural control and walking.     

Active cortical innervation protects striatal neurons from slow degeneration in culture

Spiny striatal GABAergic neurons receive most of their excitatory input from the neocortex. In culture, striatal neurons form inhibitory connections, but the lack of intrinsic excitatory afferents prevents the development of spontaneous network activity. Addition of cortical neurons to the striatal culture provides the necessary excitatory input to the striatal neurons, and in the presence of these neurons, striatal cultures do express spontaneous network activity. We have confirmed that cortical neurons provide excitatory drive to striatal neurons in culture using paired recording from cortical and striatal neurons. In the presence of tetrodotoxin (TTX), which blocks action potential discharges, the connections between cortical and striatal neurons are still formed, and in fact synaptic currents generated between them when TTX is removed are far larger than in control, undrugged cultures. Interestingly, the continuous presence of TTX in the co-culture caused striatal cell death. These observations indicate that the mere presence of cortical neurons is not sufficient to preserve striatal neurons in culture, but their synchronous activity, triggered by cortical excitatory synapses, is critical for the maintenance of viability of striatal neurons. These results have important implications for understanding the role of activity in neurodegenerative diseases of the striatum.     

Activation of substantia nigra pars reticulata neurons: role in the initiation and behavioral expression of kindled seizures

Numerous studies have implicated the substantia nigra pars reticulata (SNR) in the initiation and behavioral expression of kindled seizures. In immobilized, amygdala-kindled animals, SNR neurons have been shown to enter an intense burst-firing pattern during afterdischarge (AD). Taken together these findings raised the possibility that the SNR facilitates the expression of kindled seizures by directly propagating seizure activity into target structures. In this study we examined the relationship between activation of SNR neurons and the electrical (EEG) and behavioral (clonic motor) expression of kindled seizures using both immobilized and unrestrained animals. The principal findings were that: (1) in both immobilized and unrestrained animals the SNR neurons of kindled, but not control, animals were recruited into a burst-firing pattern during AD; (2) the onset of burst-firing was delayed until after the onset of AD; and (3) the onset of burst-firing was not correlated with the onset of rhythmic motor seizure activity. These findings support the idea that the development of kindling is associated with recruitment of SNR neurons into a seizure propagating network. However, these data suggest that activation of SNR neurons is not necessary for the expression of clonic motor activity and does not lower seizure threshold.