Single-cell and ensemble activity of lumbar intermediate and ventral horn interneurons in the spinal air-stepping cat.

We explored the relationship between population interneuronal network activation and motor output in the adult, in vivo, air-stepping, spinal cat. By simultaneously measuring the activity of large numbers of spinal interneurons, we explored ensembles of coherently firing interneurons and their relation to motor output. In addition, the networks were analyzed in relation to their spatial distribution along the lumbar enlargement for evidence of localized groups driving particular phases of the locomotor step cycle. We simultaneously recorded hindlimb EMG activity during stepping and extracellular signals from 128 channels across two polytrodes inserted within lamina V-VII of two separate lumbar segments. Results indicated that spinal interneurons participate in one of two ensembles that are highly correlated with the flexor or the extensor muscle bursts during stepping. Interestingly, less than half of the isolated single units were significantly unimodally tuned during the step cycle whereas >97% of the single units of the ensembles were significantly correlated with muscle activity. These results show the importance of population scale analysis in neural studies of behavior as there is a much greater correlation between muscle activity and ensemble firing than between muscle activity and individual neurons. Finally, we show that there is no correlation between interneurons' rostrocaudal locations within the lumbar enlargement and their preferred phase of firing or ensemble participation. These findings indicate that spinal interneurons of lamina V-VII encoding for different phases of the locomotor cycle are spread throughout the lumbar enlargement in the adult spinal cord.NEW & NOTEWORTHY We report on the ensemble organization of interneuronal activity in the spinal cord during locomotor movements and show that lumbar intermediate zone interneurons organize in two groups related to the two major phases of walking: stance and swing. Ensemble organization is also shown to better correlate with muscular output than single-cell activity, although ensemble membership does not appear to be somatotopically organized within the spinal cord.

The role of neuronal excitation and inhibition in the pre-Bötzinger complex on the cough reflex in the cat.

Brainstem respiratory neuronal network significantly contributes to cough motor pattern generation. Neuronal populations in the pre-Bötzinger complex (PreBötC) represent a substantial component for respiratory rhythmogenesis. We studied the role of PreBötC neuronal excitation and inhibition on mechanically induced tracheobronchial cough in 15 spontaneously breathing, pentobarbital anesthetized adult cats (35 mg/kg, iv initially). Neuronal excitation by unilateral microinjection of glutamate analog d,l-homocysteic acid resulted in mild reduction of cough abdominal electromyogram (EMG) amplitudes and very limited temporal changes of cough compared with effects on breathing (very high respiratory rate, high amplitude inspiratory bursts with a short inspiratory phase, and tonic inspiratory motor component). Mean arterial blood pressure temporarily decreased. Blocking glutamate-related neuronal excitation by bilateral microinjections of nonspecific glutamate receptor antagonist kynurenic acid reduced cough inspiratory and expiratory EMG amplitude and shortened most cough temporal characteristics similarly to breathing temporal characteristics. Respiratory rate decreased and blood pressure temporarily increased. Limiting active neuronal inhibition by unilateral and bilateral microinjections of GABAA receptor antagonist gabazine resulted in lower cough number, reduced expiratory cough efforts, and prolongation of cough temporal features and breathing phases (with lower respiratory rate). The PreBötC is important for cough motor pattern generation. Excitatory glutamatergic neurotransmission in the PreBötC is involved in control of cough intensity and patterning. GABAA receptor-related inhibition in the PreBötC strongly affects breathing and coughing phase durations in the same manner, as well as cough expiratory efforts. In conclusion, differences in effects on cough and breathing are consistent with separate control of these behaviors.NEW & NOTEWORTHY This study is the first to explore the role of the inspiratory rhythm and pattern generator, the pre-Bötzinger complex (PreBötC), in cough motor pattern formation. In the PreBötC, excitatory glutamatergic neurotransmission affects cough intensity and patterning but not rhythm, and GABAA receptor-related inhibition affects coughing and breathing phase durations similarly to each other. Our data show that the PreBötC is important for cough motor pattern generation, but cough rhythmogenesis appears to be controlled elsewhere.

Locomotor-related V3 interneurons initiate and coordinate muscles spasms after spinal cord injury.

Spinal cord injury leads to a devastating loss of motor function and yet is accompanied by a paradoxical emergence of muscle spasms, which often involve complex muscle activation patterns across multiple joints, reciprocal muscle timing, and rhythmic clonus. We investigated the hypothesis that spasms are a manifestation of partially recovered function in spinal central pattern-generating (CPG) circuits that normally coordinate complex postural and locomotor functions. We focused on the commissural propriospinal V3 neurons that coordinate interlimb movements during locomotion and examined mice with a chronic spinal transection. When the V3 neurons were optogenetically activated with a light pulse, a complex coordinated pattern of motoneuron activity was evoked with reciprocal, crossed, and intersegmental activity. In these same mice, brief sensory stimulation evoked spasms with a complex pattern of activity very similar to that evoked by light, and the timing of these spasms was readily reset by activation of V3 neurons. Given that V3 neurons receive abundant sensory input, these results suggest that sensory activation of V3 neurons is alone sufficient to generate spasms. Indeed, when we silenced V3 neurons optogenetically, sensory evoked spasms were inhibited. Also, inhibiting general CPG activity by blocking N-methyl-d-aspartate (NMDA) receptors inhibited V3 evoked activity and associated spasms, whereas NMDA application did the opposite. Furthermore, overwhelming the V3 neurons with repeated optogenetic stimulation inhibited subsequent sensory evoked spasms, both in vivo and in vitro. Taken together, these results demonstrate that spasms are generated in part by sensory activation of V3 neurons and associated CPG circuits. NEW & NOTEWORTHY We investigated whether locomotor-related excitatory interneurons (V3) play a role in coordinating muscle spasm activity after spinal cord injury (SCI). Unexpectedly, we found that these neurons not only coordinate reciprocal motor activity but are critical for initiating spasms, as well. More generally, these results suggest that V3 neurons are important in initiating and coordinating motor output after SCI and thus provide a promising target for restoring residual motor function.

Ion Homeostasis in Rhythmogenesis: The Interplay Between Neurons and Astroglia.

Proper function of all excitable cells depends on ion homeostasis. Nowhere is this more critical than in the brain where the extracellular concentration of some ions determines neurons' firing pattern and ability to encode information. Several neuronal functions depend on the ability of neurons to change their firing pattern to a rhythmic bursting pattern, whereas, in some circuits, rhythmic firing is, on the contrary, associated to pathologies like epilepsy or Parkinson's disease. In this review, we focus on the four main ions known to fluctuate during rhythmic firing: calcium, potassium, sodium, and chloride. We discuss the synergistic interactions between these elements to promote an oscillatory activity. We also review evidence supporting an important role for astrocytes in the homeostasis of each of these ions and describe mechanisms by which astrocytes may regulate neuronal firing by altering their extracellular concentrations. A particular emphasis is put on the mechanisms underlying rhythmogenesis in the circuit forming the central pattern generator (CPG) for mastication and other CPG systems. Finally, we discuss how an impairment in the ability of glial cells to maintain such homeostasis may result in pathologies like epilepsy and Parkinson's disease.

Effect of stimulating the lumbar skin caudal to a complete spinal cord injury on hindlimb locomotion.

Sensory feedback is a potent modulator of the locomotor pattern generated by spinal networks. The purpose of this study was to assess the effect of cutaneous inputs from the back on the spinal-generated locomotor pattern. The spinal cord of six adult cats was transected at low thoracic levels. Cats were then trained to recover hindlimb locomotion. During experiments, the skin overlying lumbar vertebrae L2 to L7 was mechanically stimulated by a small calibrated clip or by manual pinching. Trials without and with cutaneous stimulation were performed at a treadmill speed of 0.4 m/s. Although manually pinching the skin completely stopped hindlimb locomotion and abolished weight support, cutaneous stimulation with the calibrated clip produced smaller effects. Specifically, more focalized cutaneous stimulation with the clip reduced flexor and extensor muscle activity and led to a more caudal positioning of the paw at contact and liftoff. Moreover, cutaneous stimulation with the clip led to a greater number of steps with improper nonplantigrade paw placements at contact and paw drag at the stance-to-swing transition. The most consistent effects on the hindlimb locomotor pattern were observed with cutaneous stimulation at midlumbar levels, from L3 to L5. The results indicate that cutaneous stimulation of the skin modulates the excitability of spinal circuits involved in generating locomotion and weight support, particularly at spinal segments thought to be critical for rhythm generation.

Locomotor recovery after spinal cord hemisection/contusion injures in bonnet monkeys: footprint testing--a minireview.

Spinal cord injuries usually produce loss or impairment of sensory, motor and reflex function below the level of damage. In the absence of functional regeneration or manipulations that promote regeneration, spontaneous improvements in motor functions occur due to the activation of multiple compensatory mechanisms in animals and humans following the partial spinal cord injury. Many studies were performed on quantitative evaluation of locomotor recovery after induced spinal cord injury in animals using behavioral tests and scoring techniques. Although few studies on rodents have led to clinical trials, it would appear imperative to use nonhuman primates such as macaque monkeys in order to relate the research outcomes to recovery of functions in humans. In this review, we will discuss some of our research evidences concerning the degree of spontaneous recovery in bipedal locomotor functions of bonnet monkeys that underwent spinal cord hemisection/contusion lesions. To our knowledge, this is the first report to discuss on the extent of spontaneous recovery in bipedal locomotion of macaque monkeys through the application of footprint analyzing technique. In addition, the results obtained were compared with the published data on recovery of quadrupedal locomotion of spinally injured rodents. We propose that the mechanisms underlying spontaneous recovery of functions in spinal cord lesioned monkeys may be correlated to the mature function of spinal pattern generator for locomotion under the impact of residual descending and afferent connections. Moreover, based on analysis of motor functions observed in locomotion in these subjected monkeys, we understand that spinal automatism and development of responses by afferent stimuli from outside the cord could possibly contribute to recovery of paralyzed hindlimbs. This report also emphasizes the functional contribution of progressive strengthening of undamaged nerve fibers through a collateral sprouts/synaptic plasticity formed in partially lesioned cord of monkeys.

Perinatal inflammation and gestational intermittent hypoxia disturbs respiratory rhythm generation and long-term facilitation in vitro: Partial protection by acute minocycline.

Perinatal inflammation triggers breathing disturbances early in life and affects the respiratory adaptations to challenging conditions, including the generation of amplitude long-term facilitation (LTF) by acute intermittent hypoxia (AIH). Some of these effects can be avoided by anti-inflammatory treatments like minocycline. Since little is known about the effects of perinatal inflammation on the inspiratory rhythm generator, located in the preBötzinger complex (preBötC), we tested the impact of acute lipopolysaccharide (LPS) systemic administration (sLPS), as well as gestational LPS (gLPS) and gestational chronic IH (gCIH), on respiratory rhythm generation and its long-term response to AIH in a brainstem slice preparation from neonatal mice. We also evaluated whether acute minocycline administration could influence these effects. We found that perinatal inflammation induced by sLPS or gLPS, as well as gCIH, modulate the frequency, signal-to-noise ratio and/or amplitude (and their regularity) of the respiratory rhythm recorded from the preBötC in the brainstem slice. Moreover, all these perinatal conditions inhibited frequency LTF and amplitude long-term depression (LTD); gCIH even induced frequency LTD of the respiratory rhythm after AIH. Some of these alterations were not observed in slices pre-treated in vitro with minocycline, when compared with slices obtained from naïve pups, suggesting that ongoing inflammatory conditions affect respiratory rhythm generation and its plasticity. Thus, it is likely that alterations in the inspiratory rhythm generator and its adaptive responses could contribute to the respiratory disturbances observed in neonates that suffered from perinatal inflammatory challenges.

Mouse incising central pattern generator: Characteristics and modulation by pain.

INTRODUCTION: Vertebrate incising and chewing are controlled by a set of neurons comprising the central pattern generator (CPG) for mastication. Mandibular positioning and force generation to perform these tasks is complex and requires coordination of multiple jaw opening and closing muscle compartments located in muscles on both sides of the jaw. The purpose of this study was to determine the characteristics of the CPG by recording mouse incising forces in the home cage environment to evaluate changes in force characteristics with incising frequency and force direction. A second purpose was to evaluate the effects of jaw closing muscle pain on CPG output parameters. METHODS: Digitized incising forces were recorded for approximately 24 h using a 3-dimensional force transducer attached to solid food chow. Male and female CD-1 mice were evaluated during their last (fourth) baseline assessment and seven days after a second acidic saline injection into the left masseter muscle when maximum pain was experienced. Incising force resultants were calculated from the three axes data and force parameters were assessed including inter-peak intervals (IPI), peak amplitude, load time and unload time. Multiple regression analyses were conducted to identify incising episodes that had parameters of force that were significantly correlated (p < 0.001). These incising episodes were considered to represent the output of the CPG with a steady state of incoming sensory afferent inputs. Incising parameters were evaluated for each of the discrete incising frequencies (4.6, 5.3, 6.2, 7.6 Hz) and the predominant force directions: jaw closing (-Z), jaw retrusion (+X) and jaw protrusion (-X). RESULTS: A significant correlation between incising frequency (IPI) and the load time was observed. A significant decrease in peak amplitude was observed with higher incising frequency while the load rate significantly increased. The force peak amplitude and load rates were found to be statistically different when the force direction was considered, with smaller peak amplitudes and smaller load rates found in the jaw closing direction. The effect of pain on incising was to reduce the peak amplitude and load rate of incising compared to the baseline condition at lower incising frequencies. CONCLUSIONS: Like the central pattern generator for locomotion, the CPG for incising controls rhythmicity, peak amplitude and force load duration/rate. However, unlike the CPG for locomotion, the amplitude of incising force decreases as the frequency increases. During incising, load rate increases with faster rhythm and is consistent with the recruitment of larger motor units. Muscle pain reduced the excitatory drive of the CPG on motoneurons and provides further support of the Pain Adaptation Model.

Recruitment of Polysynaptic Connections Underlies Functional Recovery of a Neural Circuit after Lesion.

The recruitment of additional neurons to neural circuits often occurs in accordance with changing functional demands. Here we found that synaptic recruitment plays a key role in functional recovery after neural injury. Disconnection of a brain commissure in the nudibranch mollusc, Tritonia diomedea, impairs swimming behavior by eliminating particular synapses in the central pattern generator (CPG) underlying the rhythmic swim motor pattern. However, the CPG functionally recovers within a day after the lesion. The strength of a spared inhibitory synapse within the CPG from Cerebral Neuron 2 (C2) to Ventral Swim Interneuron B (VSI) determines the level of impairment caused by the lesion, which varies among individuals. In addition to this direct synaptic connection, there are polysynaptic connections from C2 and Dorsal Swim Interneurons to VSI that provide indirect excitatory drive but play only minor roles under normal conditions. After disconnecting the pedal commissure (Pedal Nerve 6), the recruitment of polysynaptic excitation became a major source of the excitatory drive to VSI. Moreover, the amount of polysynaptic recruitment, which changed over time, differed among individuals and correlated with the degree of recovery of the swim motor pattern. Thus, functional recovery was mediated by an increase in the magnitude of polysynaptic excitatory drive, compensating for the loss of direct excitation. Since the degree of susceptibility to injury corresponds to existing individual variation in the C2 to VSI synapse, the recovery relied upon the extent to which the network reorganized to incorporate additional synapses.

How do neuroanatomical changes in individuals with chronic pain result in the constant perception of pain?

Since the advent of anatomical brain imaging analysis techniques, numerous reports have shown altered regional brain anatomy in individuals with various chronic pain conditions. While early reports of increased regional brain volumes in taxi drivers and pianists were simply interpreted as responses to excessive use, the mechanisms responsible for anatomical changes associated with chronic pain are not so straightforward. The main aim of this paper is to explore the potential underlying cellular changes responsible for change in gross brain anatomy in individuals with chronic pain, in particular pain following nervous system damage. Determining the basis of these changes may provide a platform for development of targeted, personalized and ultimately more effective treatment regimens.

Spinal cord injury as a trigger to develop periodic leg movements during sleep: an evolutionary perspective.

The primary trigger to periodic limb movement (PLM) during sleep is still unknown. Its association with the restless legs syndrome (RLS) is established in humans and was reported in spinal cord injury (SCI) patients classified by the American Spinal Injury Association (ASIA) as A. Its pathogenesis has not been completely unraveled, though recent advances might enhance our knowledge about those malfunctions. PLM association with central pattern generator (CPG) is one of the possible pathologic mechanisms involved. This article reviewed the advances in PLM and RLS genetics, the evolution of CPG functioning, and the neurotransmitters involved in CPG, PLM and RLS. We have proposed that SCI might be a trigger to develop PLM.

Spinal cord injury as a trigger to develop periodic leg movements during sleep: an evolutionary perspective / Lesão medular como gatilho para desenvolver movimentos periódicos das pernas durante o sono: uma perspectiva evolutiva

The primary trigger to periodic limb movement (PLM) during sleep is still unknown. Its association with the restless legs syndrome (RLS) is established in humans and was reported in spinal cord injury (SCI) patients classified by the American Spinal Injury Association (ASIA) as A. Its pathogenesis has not been completely unraveled, though recent advances might enhance our knowledge about those malfunctions. PLM association with central pattern generator (CPG) is one of the possible pathologic mechanisms involved. This article reviewed the advances in PLM and RLS genetics, the evolution of CPG functioning, and the neurotransmitters involved in CPG, PLM and RLS. We have proposed that SCI might be a trigger to develop PLM.

O gatilho principal para o desenvolvimento de movimentos periódicos dos membros (MPM) durante o sono ainda é desconhecido. A associação entre o MPM e a síndrome das pernas inquietas (SPI) em seres humanos já foi previamente estabelecida e relatada em pacientes com lesão medular (LM), classificados pela American Spinal Injury Association (ASIA) como A. A patogênese do MPM não foi completamente desvendada, apesar de avanços recentes poderem ampliar o conhecimento sobre essas disfunções. Um dos possíveis mecanismos patológicos envolvidos é o gerador de padrão central (GPC). Este artigo revisou os avanços na genética do MPM e da SPI, a evolução do funcionamento do GPC e os neurotransmissores relacionados ao GPC, ao MPM e à SPI. Foi proposta a hipótese de que a LM poderia ser um gatilho para deflagrar os MPM.