Depolarization Of Motoneurons Research Articles

Chapter 15 - The phrenic neuromuscular system

The phrenic neuromuscular system consists of the phrenic motor nucleus in the mid-cervical spinal cord, the phrenic nerve, and the diaphragm muscle. This motor system helps sustain breathing throughout life, while also contributing to posture, coughing, swallowing, and speaking. The phrenic nerve contains primarily efferent phrenic axons and afferent axons from diaphragm sensory receptors but is also a conduit for autonomic fibers. On a breath-by-breath basis, rhythmic (inspiratory) depolarization of phrenic motoneurons occurs due to excitatory bulbospinal synaptic pathways. Further, a complex propriospinal network innervates phrenic motoneurons and may serve to coordinate postural, locomotor, and respiratory movements. The phrenic neuromuscular system is impacted in a wide range of neuromuscular diseases and injuries. Contemporary research is focused on understanding how neuromuscular plasticity occurs in the phrenic neuromuscular system and using this information to optimize treatments and rehabilitation strategies to improve breathing and related behaviors.

Nicotine Neurotoxicity Involves Low Wnt1 Signaling in Spinal Locomotor Networks of the Postnatal Rodent Spinal Cord.

The postnatal rodent spinal cord in-vitro is a useful model to investigate early pathophysiological changes after injury. While low dose nicotine (1 µM) induces neuroprotection, how higher doses affect spinal networks is unknown. Using spinal preparations of postnatal wild-type Wistar rat and Wnt1Cre2:Rosa26Tom double-transgenic mouse, we studied the effect of nicotine (0.5–10 µM) on locomotor networks in-vitro. Nicotine 10 µM induced motoneuron depolarization, suppressed monosynaptic reflexes, and decreased fictive locomotion in rat spinal cord. Delayed fall in neuronal numbers (including motoneurons) of central and ventral regions emerged without loss of dorsal neurons. Conversely, nicotine (0.5–1 µM) preserved neurons throughout the spinal cord and strongly activated the Wnt1 signaling pathway. High-dose nicotine enhanced expression of S100 and GFAP in astrocytes indicating a stress response. Excitotoxicity induced by kainate was contrasted by nicotine (10 µM) in the dorsal area and persisted in central and ventral regions with no change in basal Wnt signaling. When combining nicotine with kainate, the activation of Wnt1 was reduced compared to kainate/sham. The present results suggest that high dose nicotine was neurotoxic to central and ventral spinal neurons as the neuroprotective role of Wnt signaling became attenuated. This also corroborates the risk of cigarette smoking for the foetus/newborn since tobacco contains nicotine.

Neurophysiological Changes After Paired Brain and Spinal Cord Stimulation Coupled With Locomotor Training in Human Spinal Cord Injury

Neurophysiological changes that involve activity-dependent neuroplasticity mechanisms via repeated stimulation and locomotor training are not commonly employed in research even though combination of interventions is a common clinical practice. In this randomized clinical trial, we established neurophysiological changes when transcranial magnetic stimulation (TMS) of the motor cortex was paired with transcutaneous thoracolumbar spinal (transspinal) stimulation in human spinal cord injury (SCI) delivered during locomotor training. We hypothesized that TMS delivered before transspinal (TMS-transspinal) stimulation promotes functional reorganization of spinal networks during stepping. In this protocol, TMS-induced corticospinal volleys arrive at the spinal cord at a sufficient time to interact with transspinal stimulation induced depolarization of alpha motoneurons over multiple spinal segments. We further hypothesized that TMS delivered after transspinal (transspinal-TMS) stimulation induces less pronounced effects. In this protocol, transspinal stimulation is delivered at time that allows transspinal stimulation induced action potentials to arrive at the motor cortex and affect descending motor volleys at the site of their origin. Fourteen individuals with motor incomplete and complete SCI participated in at least 25 sessions. Both stimulation protocols were delivered during the stance phase of the less impaired leg. Each training session consisted of 240 paired stimuli delivered over 10-min blocks. In transspinal-TMS, the left soleus H-reflex increased during the stance-phase and the right soleus H-reflex decreased at mid-swing. In TMS-transspinal no significant changes were found. When soleus H-reflexes were grouped based on the TMS-targeted limb, transspinal-TMS and locomotor training promoted H-reflex depression at swing phase, while TMS-transspinal and locomotor training resulted in facilitation of the soleus H-reflex at stance phase of the step cycle. Furthermore, both transspinal-TMS and TMS-transspinal paired-associative stimulation (PAS) and locomotor training promoted a more physiological modulation of motor activity and thus depolarization of motoneurons during assisted stepping. Our findings support that targeted non-invasive stimulation of corticospinal and spinal neuronal pathways coupled with locomotor training produce neurophysiological changes beneficial to stepping in humans with varying deficits of sensorimotor function after SCI.

Neuronal Actions of Transspinal Stimulation on Locomotor Networks and Reflex Excitability During Walking in Humans With and Without Spinal Cord Injury.

This study investigated the neuromodulatory effects of transspinal stimulation on soleus H-reflex excitability and electromyographic (EMG) activity during stepping in humans with and without spinal cord injury (SCI). Thirteen able-bodied adults and 5 individuals with SCI participated in the study. EMG activity from both legs was determined for steps without, during, and after a single-pulse or pulse train transspinal stimulation delivered during stepping randomly at different phases of the step cycle. The soleus H-reflex was recorded in both subject groups under control conditions and following single-pulse transspinal stimulation at an individualized exactly similar positive and negative conditioning-test interval. The EMG activity was decreased in both subject groups at the steps during transspinal stimulation, while intralimb and interlimb coordination were altered only in SCI subjects. At the steps immediately after transspinal stimulation, the physiological phase-dependent EMG modulation pattern remained unaffected in able-bodied subjects. The conditioned soleus H-reflex was depressed throughout the step cycle in both subject groups. Transspinal stimulation modulated depolarization of motoneurons over multiple segments, limb coordination, and soleus H-reflex excitability during assisted stepping. The soleus H-reflex depression may be the result of complex spinal inhibitory interneuronal circuits activated by transspinal stimulation and collision between orthodromic and antidromic volleys in the peripheral mixed nerve. The soleus H-reflex depression by transspinal stimulation suggests a potential application for normalization of spinal reflex excitability after SCI.

Transspinal stimulation decreases corticospinal excitability and alters the function of spinal locomotor networks.

Locomotion requires the continuous integration of descending motor commands and sensory inputs from the legs by spinal central pattern generator circuits. Modulation of spinal neural circuits by transspinal stimulation is well documented, but how transspinal stimulation affects corticospinal excitability during walking in humans remains elusive. We measured the motor evoked potentials (MEPs) at multiple phases of the step cycle conditioned with transspinal stimulation delivered at sub- and suprathreshold intensities of the spinally mediated transspinal evoked potential (TEP). Transspinal stimulation was delivered before or after transcranial magnetic stimulation during which summation between MEP and TEP responses in the surface EMG was absent or present. Relationships between MEP amplitude and background EMG activity, silent period duration, and phase-dependent EMG amplitude modulation during and after stimulation were also determined. Ankle flexor and extensor MEPs were depressed by suprathreshold transspinal stimulation when descending volleys were timed to interact with transspinal stimulation-induced motoneuron depolarization at the spinal cord. MEP depression coincided with decreased MEP gain, unaltered MEP threshold, and unaltered silent period duration. Locomotor EMG activity of bilateral knee and ankle muscles was significantly depressed during the step at which transspinal stimulation was delivered but fully recovered at the subsequent step. The results support a model in which MEP depression by transspinal stimulation occurs via subcortical or spinal mechanisms. Transspinal stimulation disrupts the locomotor output of flexor and extensor motoneurons initially, but the intact nervous system has the ability to rapidly overcome this pronounced locomotor adaptation. In conclusion, transspinal stimulation directly affects spinal locomotor centers in healthy humans.NEW & NOTEWORTHY Lumbar transspinal stimulation decreases ankle flexor and extensor motor evoked potentials (MEPs) during walking. The MEP depression coincides with decreased MEP gain, unaltered MEP threshold changes, and unaltered silent period duration. These findings indicate that MEP depression is subcortical or spinal in origin. Healthy subjects could rapidly overcome the pronounced depression of muscle activity during the step at which transspinal stimulation was delivered. Thus, transspinal stimulation directly affects the function of spinal locomotor networks in healthy humans.

Quantitative input-output relationships between human soleus muscle spindle afferents and motoneurons

A method is described that, for the first time, allows instantaneous estimation of the Ia fiber input to human soleus motoneurons following electrical stimulation of the tibial nerve. The basis of the method is to determine the thresholds of the most and least excitable 1a fibers to electrical stimulation, and to treat the intervening thresholds as having a normal distribution about the mean; the validity of this approach is discussed. It was found that, for the same Ia fiber input, the percentage of soleus motoneurons contributing to the H (Hoffmann)-reflex differed considerably among subjects; when the results were pooled, however, there was an approximately linear relationship between Ia input and motoneuron output. Weak extension of the great toe diminished the soleus motoneuron reflex discharge in all but 2 of 16 subjects; the results for weak ankle plantarflexion were less consistent, but overall, there was a reduction in soleus motoneuron output also. The methodology should provide new insights into disorders of movement and tone, especially as it permits estimates of motoneuron depolarization to be made. NEW & NOTEWORTHY Assuming a normal distribution of Ia fiber thresholds to electrical stimulation and using the H-reflex, we determined for the first time an Ia input-α-motoneuron output relationship for the human soleus muscle. The relationship varies greatly among subjects but, overall, is approximately linear. Minimal contraction of a toe muscle alters the relationship dramatically, probably due to presynaptic inhibition of Ia fibers. Drawing on the literature, we can calculate changes in α-motoneuron membrane potential.

Histamine modulates spinal motoneurons and locomotor circuits.

Spinal motoneurons and locomotor networks are regulated by monoamines, among which, the contribution of histamine has yet to be fully addressed. The present study investigates histaminergic regulation of spinal activity, combining intra- and extracellular electrophysiological recordings from neonatal rat spinal cord in vitro preparations. Histamine dose-dependently and reversibly generated motoneuron depolarization and action potential firing. Histamine (20 µM) halved the area of dorsal root reflexes and always depolarized motoneurons. The majority of cells showed a transitory repolarization, while 37% showed a sustained depolarization maintained with intense firing. Extracellularly, histamine depolarized ventral roots (VRs), regardless of blockage of ionotropic glutamate receptors. Initial, transient glutamate-mediated bursting was synchronous among VRs, with some bouts of locomotor activity in a subgroup of preparations. After washout, the amplitude of spontaneous tonic discharges increased. No desensitization or tachyphylaxis appeared after long perfusion or serial applications of histamine. On the other hand, histamine induced single motoneuron and VR depolarization, even in the presence of tetrodotoxin (TTX). During chemically induced fictive locomotion (FL), histamine depolarized VRs. Histamine dose-dependently increased rhythm periodicity and reduced cycle amplitude until near suppression. This study demonstrates that histamine induces direct motoneuron membrane depolarization and modulation of locomotor output, indicating new potential targets for locomotor neurorehabilitation.

Modulatory and plastic effects of kinins on spinal cord networks.

Inflammatory kinins are released following spinal cord injury or neurotrauma. The effects of these kinins on ongoing locomotor activity of central pattern generator networks are unknown. In the present study, kinins were shown to have short- and long-term effects on motor networks. The short-term effects included direct depolarization of interneurons and motoneurons in the ventral horn accompanied by modulation of transient receptor potential vanilloid 1-sensitive nociceptors in the dorsal horn. Over the long-term, we observed a bradykinin-mediated effect on promoting plasticity in the spinal cord. In a model of spinal cord injury, we observed an increase in microglia numbers in both the dorsal and ventral horn and, in a microglia cell culture model, we observed bradykinin-induced expression of glial-derived neurotrophic factor. The expression and function of inflammatory mediators in the developing spinal cord remain poorly characterized. We discovered novel, short and long-term roles for the inflammatory nonapeptide bradykinin (BK) and its receptor bradykinin receptor B2 (B2R) in the neuromodulation of developing sensorimotor networks following a spinal cord injury (SCI), suggesting that BK participates in an excitotoxic cascade. Functional expression of B2R was confirmed by a transient disruptive action of BK on fictive locomotion generated by a combination of NMDA, 5-HT and dopamine. The role of BK in the dorsal horn nociceptive afferents was tested using spinal cord attached to one-hind-limb (HL) preparations. In the HL preparations, BK at a subthreshold concentration induced transient disruption of fictive locomotion only in the presence of: (1) noxious heat applied to the hind paw and (2) the heat sensing ion channel transient receptor potential vanilloid 1 (TRPV1), known to be restricted to nociceptors in the superficial dorsal horn. BK directly depolarized motoneurons and ascending interneurons in the ventrolateral funiculus. We found a key mechanism for BK in promoting long-term plasticity within the spinal cord. Using a model of neonatal SCI and a microglial cell culture model, we examined the role of BK in inducing activation of microglia and expression of glial-derived neurotrophic factor (GDNF). In the neonatal SCI model, we observed an increase in microglia numbers and increased GDNF expression restricted to microglia. In the microglia cell culture model, we observed a BK-induced increased expression of GDNF via B2R, suggesting a novel mechanism for BK spinal-mediated plasticity.

Transient BK outward current enhances motoneurone firing rates during Drosophila larval locomotion.

We combine in situ electrophysiology with genetic manipulation in Drosophila larvae aiming to investigate the role of fast calcium-activated potassium currents for motoneurone firing patterns during locomotion. We first demonstrate that slowpoke channels underlie fast calcium-activated potassium currents in these motoneurones. By conducting recordings in semi-intact animals that produce crawling-like movements, we show that slowpoke channels are required specifically in motoneurones for maximum firing rates during locomotion. Such enhancement of maximum firing rates occurs because slowpoke channels prevent depolarization block by limiting the amplitude of motoneurone depolarization in response to synaptic drive. In addition, slowpoke channels mediate a fast afterhyperpolarization that ensures the efficient recovery of sodium channels from inactivation during high frequency firing. The results of the present study provide new insights into the mechanisms by which outward conductances facilitate neuronal excitability and also provide direct confirmation of the functional relevance of precisely regulated slowpoke channel properties in motor control. A large number of voltage-gated ion channels, their interactions with accessory subunits, and their post-transcriptional modifications generate an immense functional diversity of neurones. Therefore, a key challenge is to understand the genetic basis and precise function of specific ionic conductances for neuronal firing properties in the context of behaviour. The present study identifies slowpoke (slo) as exclusively mediating fast activating, fast inactivating BK current (ICF ) in larval Drosophila crawling motoneurones. Combining in vivo patch clamp recordings during larval crawling with pharmacology and targeted genetic manipulations reveals that ICF acts specifically in motoneurones to sculpt their firing patterns in response to a given input from the central pattern generating (CPG) networks. First, ICF curtails motoneurone postsynaptic depolarizations during rhythmical CPG drive. Second, ICF is activated during the rising phase of the action potential and mediates a fast afterhyperpolarization. Consequently, ICF is required for maximal intraburst firing rates during locomotion, probably by allowing recovery from inactivation of fast sodium channels and decreased potassium channel activation. This contrasts the common view that outward conductances oppose excitability but is in accordance with reports on transient BK and Kv3 channel function in multiple types of vertebrate neurones. Therefore, our finding that ICF enhances firing rates specifically during bursting patterns relevant to behaviour is probably of relevance to all brains.

Delayed depolarization and firing behavior of human motoneurons during voluntary muscle contractions.

The firing behavior of motoneurons is governed by the interaction between the intrinsic properties of motoneurons and the synaptic inputs that they receive. In particular, long-lasting after-hyperpolarization (AHP) following each motoneuron spike and thus decreasing motoneuron excitability immediately after spike was found to be a key mechanism in controlling of interspike interval (ISI) duration (Eccles et al., 1958; Kernell, 1965). As a result, motoneurons commonly fire single discharges at rather low firing rates, especially during gentle voluntary muscle contractions and postural tasks. However, some motoneurons, under conditions of weak synaptic drive, can fire double discharges (doublets) with very short intradoublet ISIs (up to a few ms). Each doublet is commonly followed by a prolonged post-doublet (interdoublet) ISI resulting from the AHP summation (Calvin and Schwindt, 1972). Two types of doublets have been observed: single and repetitive ones. Single doublets have been reported many times in cat and human motoneurons (e.g., Eccles and Hoff, 1932; Hoff and Grant, 1944; Denslow, 1948; Calvin and Schwindt, 1972; Kudina, 1974; Kudina and Churikova, 1990; Kirkwood and Munson, 1996; Piotrkiewicz et al., 2008; Stephenson and Maluf, 2010). Repetitive doublets have also been demonstrated in some of the reports above, but as a rule by the way; and only a few studies have directly addressed this topic (Bawa and Calancie, 1983; Kudina and Alexeeva, 1992; Kudina and Andreeva, 2010). However, a number of questions are still unanswered, in particular the question of whether there are certain muscles and (or) certain motoneurons for which repetitive doublet firing is likely to appear. This question will be taken up below based on an analysis of doublet firing in different limb and trunk muscles in healthy humans. With regard to the origin of this surprising firing, currently, the short delayed depolarization (DD) appearing as a “hump” on the falling phase of an action potential, preceding the AHP (Granit et al., 1963; Kernell, 1964; Nelson and Burke, 1967; Kernell, 2006) and thus shortly increasing MN excitability is widely accepted as the mechanism responsible for single doublets in cat (Calvin and Schwindt, 1972; Kirkwood and Munson, 1996; see also Kernell, 2006), and human motoneurons (Kudina, 1974; Bawa and Calancie, 1983; Kudina and Alexeeva, 1992; Bawa and Lemon, 1993; Garland and Griffin, 1999; Piotrkiewicz et al., 2008; Stephenson and Maluf, 2010). As to the origin of repetitive doublets, the question still remains open. At first sight, one could assume that they have the same underlying mechanism like single doublets. However, as known from findings by Granit et al. (1963) and Calvin (1975), in cat motoneurons, the hump-DD changes systematically during rhythmic firing evoked by intracellular depolarization, becoming smaller with each successive spike. Therefore, as may be expected, in firing motoneurons the DD can reach the threshold only occasionally and generate single doublets only. Consequently, it might be suggested that the DD alone can hardly play a pivotal role in producing repetitive doublets. In our recent studies (Kudina and Andreeva, 2010, 2013), we hypothesized that the mechanism underlying repetitive doublet firing in human motoneurons during voluntary muscle contraction is complex, including the hump-DD as the primary determinant that possibly becomes persistent due to a plateau potential probably activated in parallel with a common synaptic input. The assumption about a plateau potential role in repetitive doublet mechanisms was inferred from the comparison between essential characteristics of human motor unit (MU) repetitive doublet firing and properties of cat motoneuron firing in the presence of a plateau potential (e.g., Conway et al., 1988). If this assumption is true, then the question arises what are the DD characteristics which become persistent during repetitive doublet firing. The question will be considered below.

5-Hydroxytryptamine 5HT2C Receptors Form a Protein Complex with N-Methyl-d-aspartate GluN2A Subunits and Activate Phosphorylation of Src Protein to Modulate Motoneuronal Depolarization

N-Methyl-D-aspartate (NMDA)-gated ion channels are known to play a critical role in motoneuron depolarization, but the molecular mechanisms modulating NMDA activation in the spinal cord are not well understood. This study demonstrates that activated 5HT2C receptors enhance NMDA depolarizations recorded electrophysiologically from motoneurons. Pharmacological studies indicate involvement of Src tyrosine kinase mediates 5HT2C facilitation of NMDA. RT-PCR analysis revealed edited forms of 5HT2C were present in mammalian spinal cord, indicating the availability of G-protein-independent isoforms. Spinal cord neurons treated with the 5HT2C agonist MK 212 showed increased Src(Tyr-416) phosphorylation in a dose-dependent manner thus verifying that Src is activated after treatment. In addition, 5HT2C antagonists and tyrosine kinase inhibitors blocked 5HT2C-mediated Src(Tyr-416) phosphorylation and also enhanced NMDA-induced motoneuron depolarization. Co-immunoprecipitation of synaptosomal fractions showed that GluN2A, 5HT2C receptors, and Src tyrosine kinase form protein associations in synaptosomes. Moreover, immunohistochemical analysis demonstrated GluN2A and 5HT2C receptors co-localize on the processes of spinal neurons. These findings reveal that a distinct multiprotein complex links 5-hydroxytryptamine-activated intracellular signaling events with NMDA-mediated functional activity.

Development of synaptic transmission to respiratory motoneurons

Respiratory motoneurons provide the exclusive drive to respiratory muscles and therefore are a key relay between brainstem neural circuits that generate respiratory rhythm and respiratory muscles that control moment of gases into and out of the airways and lungs. This review is focused on postnatal development of fast ionotropic synaptic transmission to respiratory motoneurons, with a focus on hypoglossal motoneurons (HMs). Glutamatergic synaptic transmission to HMs involves activation of both non-NMDA and NMDA receptors and during the postnatal period co-activation of these receptors located at the same synapse may occur. Further, the relative role of each receptor type in inspiratory-phase motoneuron depolarization is dependent on the type of preparation used (in vitro versus in vivo; neonatal versus adult). Respiratory motoneurons receive both glycinergic and GABAergic inhibitory synaptic inputs. During inspiration phrenic and HMs receive concurrent excitatory and inhibitory synaptic inputs. During postnatal development in HMs GABAergic and glycinergic synaptic inputs have slow kinetics and are depolarizing and with postnatal development they become faster and hyperpolarizing. Additionally shunting inhibition may play an important role in synaptic processing by respiratory motoneurons.

The effects of polyamine agonists and antagonists on N-methyl- d-aspartate-induced depolarizations of amphibian motoneurons in situ

Polyamines have been found to reduce proton inhibition of isolated N-methyl- d-aspartate (NMDA) channels recorded in vitro. This study examines the role of polyamine modulation of motoneuronal excitation in situ, with an emphasis on possible interactions with NMDA-mediated depolarization of motoneurons and receptor mediated modulation of NMDA receptors by l-glutamate and serotonin (5-HT). Motoneuron membrane potential changes were electrotonically recorded in situ from the ventral root of isolated, hemisected amphibian spinal cords using sucrose gap techniques. The methods provided highly stable recordings (< 1.0%) of membrane potential changes upon application of NMDA. Spermine, but not spermidine, enhanced NMDA-induced depolarization of motoneurons with and without Mg 2+ present in the superfusate but had no significant effect on either (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, an mGLU receptor agonist) or 5-HT2B-G-protein receptor mediated enhancement of Mg 2+ blocked NMDA-induced activity. The polyamine antagonist arcaine or the allosteric modulator ifenprodil had no effect on NMDA-induced changes in motoneuron membrane potentials recorded in situ but blocked the effects of spermine. Synthalin did not block spermine enhancement of NMDA-induced depolarization of motoneurons but mimicked Mg 2+ block of the NMDA channel. The data provide evidence that the proton block of the NMDA receptor is maximized in frog motoneurons in situ and also for a spermine specific polyamine site on native NMDA receptors of motoneurons that can enhance NMDA-induced depolarization when activated. Polyamines do not appear to be constitutively active at the motoneurons recorded since polyamine antagonists had no effect on either membrane depolarization or modulation of NMDA receptors.

Participation of receptors of the NMDA type in regulation by glutamate of alimentary motor program of the freshwater mollusc Lymnaea stagnalis

A possible participation of receptors of the NMDA type in regulation by glutamate of the Lymnaea stagnalis feeding program was studied in electrophysiological experiments. The specific antagonist of receptors of the N-methyl-D-aspartate (NMDA) type MK-801 has been shown to turn off the endogenous generation of the standard three-phase rhythm or the two-phase rhythm. Stimulation of receptors of this type by their specific agonist, NMDA, on the contrary, increased frequency of the alimentary rhythm and transformed it into the two-phase one. All NMDA effects are eliminated by MK-801. Apart from action on generation of central feeding rhythms, ligands of receptors of the NMDA type changes the tonical level of depolarization and activity of the alimentary circuit motoneurons. MK-801 decreased the initial level of the motoneuron B4 activity and inhibited the excitatory effect both of NMDA and of glutamate itself. There are also obtained data in favor of that earlier reported effect of transformation of the inhibitory response of neurons B4 to glutamate into the excitatory one at action of nitric oxide (NO) donors can be mediated by the specific NO effect on the activity of receptors of the NMDA type. The blocker of NMDA receptors MK-801 has been shown to inhibit the effect of transformation of the response to glutamate. The NO donor nitroprusside enhanced essentially the NMDA excitatory action, while the NO acceptor oral PTIO decreased it. The results obtained with use of ODQ, the blocker of NO-sensitive guanylyl cyclase (GC), allow thinking that effect of NO on activity of the NMDA receptors of the pond snail feeding program can be realized through the metabolic pathway GC--cGMP. On the whole, the obtained results show the pond snail receptors of the NMDA type to participate in generation and rearragement of rhythmical alimentary programs in the tonical excitatory effect on the feeding program motoneurons in the NO-dependent transformation of the glutamate response.

Pharmacological Analysis of Tonic Activity in Motoneurons During Stick Insect Walking

Stick insect middle leg (mesothoracic) motoneurons receive tonic excitatory input during front leg stepping on a treadmill. We studied the pharmacology of this excitatory input to the motoneurons during single-legged treadmill walking (in situ). During bath application of drugs restricted to the mesothoracic ganglion, activity in motoneurons contralateral to the stepping front leg was recorded from neuropilar processes. Application of the cholinergic antagonist atropine reduced the tonic depolarization amplitude. These results were compared with findings in acutely dissociated motoneuron cell bodies (in vitro) under whole cell voltage-clamp conditions. The presence of an acetylcholine-induced current in situ was supported by the finding of an acetylcholine evoked biphasic inward current with a sustained component that could be blocked by atropine. In situ the tonic depolarization was generally increased by application of the neuro-modulator octopamine and decreased by its antagonist mianserin. In vitro, however, octopamine reduced the inward current evoked by acetylcholine application to motoneurons. Intracellular application of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) into motoneurons in situ revealed a dependence of the tonic depolarization on Ca(2+) and application of the membrane-permeable cAMP analogue 8-bromo-cAMP increased the tonic depolarization. In contrast, 8-bromo-cAMP reduced the inward current evoked by acetylcholine application to motoneurons in vitro. We conclude that during walking, acetylcholine contributes to mediating the tonic depolarization possibly by acting on atropine-sensitive receptors on motoneurons. Octopamine that is released during walking increases the tonic depolarization. This increase, however, is not based on modulation of cholinergic action on motoneurons but rather on effects on premotor neurons. Both, Ca(2+) and cAMP are likely second messengers involved in mediating the tonic depolarization, but whereas Ca(2+) acts in motoneurons, cAMP does not appear to mediate a cholinergic depolarization in motoneurons.

Force Regulation of Ankle Extensor Muscle Activity in Freely Walking Cats

To gain insight into the relative importance of force feedback to ongoing ankle extensor activity during walking in the conscious cat, we isolated the medial gastrocnemius muscle (MG) by denervating the other ankle extensors and measured the magnitude of its activity at different muscle lengths, velocities, and forces accomplished by having the animals walk up and down a sloped pegway. Mathematical models of proprioceptor dynamics predicted afferent activity and revealed that the changes in muscle activity under our experimental conditions were strongly correlated with Ib activity and not consistently associated with changes in Ia or group II activity. This allowed us to determine the gains within the force feedback pathway using a simple model of the neuromuscular system and the measured relationship between MG activity and force. Loop gain increased with muscle length due to the intrinsic force-length property of muscle. The gain of the pathway that converts muscle force to motoneuron depolarization was independent of length. To better test for a causal relationship between modulation of force feedback and changes in muscle activity, a second set of experiments was performed in which the MG muscle was perturbed during ground contact of the hind foot by dropping or lifting the peg underfoot. Collectively, these investigations support a causal role for force feedback and indicate that about 30% of the total muscle activity is due to force feedback during level walking. Force feedback's role increases during upslope walking and decreases during downslope walking, providing a simple mechanism for compensating for changes in terrain.

Evidence for increased activation of persistent inward currents in individuals with chronic hemiparetic stroke.

Despite the prevalence of hyperactive stretch reflexes in the paretic limbs of individuals with chronic hemiparetic stroke, the fundamental pathophysiological mechanisms responsible for their expression remain poorly understood. This study tests whether the manifestation of hyperactive stretch reflexes following stroke is related to the development of persistent inward currents (PICs) leading to hyperexcitability of motoneurons innervating the paretic limbs. Because repetitive volleys of 1a afferent feedback can elicit PICs, this investigation assessed motoneuronal excitability by evoking the tonic vibration reflex (TVR) of the biceps muscle in 10 awake individuals with chronic hemiparetic stroke and measuring the joint torque and electromyographic (EMG) responses of the upper limbs. Elbow joint torque and the EMG activity of biceps, brachioradialis, and the long and lateral heads of triceps brachii were recorded during 8 s of 112-Hz biceps vibration (evoking the TVR) and for 5 s after cessation of stimulation. Repeated-measures ANOVA tests revealed significantly (P <or= 0.05) greater increases in elbow flexion torque and EMG activity in the paretic as compared with the nonparetic limbs, both during and up to 5 s following biceps vibration. The finding of these augmentations exclusively in the paretic limb suggests that contralesional motoneurons may become hyperexcitable and readily invoke PICs following stroke. An enhanced tendency to evoke PICs may be due to an increased subthreshold depolarization of motoneurons, an increased monoaminergic input from the brain stem, or both.

Activity-dependent repression of muscle genes by NFAT.

Adult skeletal muscles retain an adaptive capacity to switch between slow- and fast-twitch properties that largely depend on motoneuron activity. The NFAT (nuclear factor of activated T cells) family of calcium-dependent transcription factors has been implicated in the up-regulation of genes encoding slow contractile proteins in response to slow-patterned motoneuron depolarization. Here, we demonstrate an unexpected, novel function of NFATc1 in slow-twitch muscles. Using the troponin I fast (TnIf) intronic regulatory element (FIRE), we identified sequences that down-regulate its function selectively in response to patterns of electrical activity that mimic slow motoneuron firing. A bona fide NFAT binding site in the TnIf FIRE was identified by site-directed mutations and by electrophoretic mobility and supershift assays. The activity-dependent transcriptional repression of FIRE is mediated through this NFAT site and, importantly, its mutation did not alter the up-regulation of TnIf transcription by fast-patterned activity. siRNA-mediated knockdown of NFATc1 in adult muscles resulted in ectopic activation of the FIRE in the slow soleus, without affecting enhancer activity in the fast extensor digitorum longus muscle. These findings demonstrate that NFAT can function as a repressor of fast contractile genes in slow muscles and they exemplify how an activity pattern can increase or decrease the expression of distinct contractile genes in a use-dependent manner as to enhance phenotypic differences among fiber types or induce adaptive changes in adult muscles.

Changes in sensory-evoked synaptic activation of motoneurons after spinal cord injury in man

Following spinal cord injury (SCI), prolonged muscle spasms are readily triggered by brief sensory stimuli. Animal and indirect human studies have shown that a substantial portion of the depolarization of motoneurons during a muscle spasm comes from the activation of persistent inward currents (PICs). The brief (single pulse) sensory stimuli that trigger the PICs and muscle spasms in chronically spinalized animals evoke excitatory post-synaptic potentials (EPSPs) that are broadened to more than 500 ms, the duration of depolarization required to activate a PIC in the motoneuron. Thus, in humans, we investigated if post-synaptic potentials (PSPs) evoked from brief (<20 ms) sensory stimulation are changed after SCI and if they are broadened to > or =500 ms to more readily activate motoneuron PICs and muscle spasms. To estimate both the shape and duration of PSPs in human subjects we used peristimulus frequencygrams (PSFs), which are plots of the instantaneous firing frequency of tonically active single motor units that are time-locked to the occurrence of the sensory stimulus. PSFs in response to cutaneomuscular stimulation of the medial arch or toe of the foot, a sensory stimulus that readily triggers muscle spasms, were compared between non-injured control subjects and in spastic subjects with chronic (>1 year), incomplete SCI. In non-injured controls, a single shock or brief (<20 ms) train of cutaneomuscular stimulation produced PSFs consisting of a 300 ms increase in firing rate above baseline with an interposed period of reduced firing. Parallel intracellular experiments in motoneurons of adult rats revealed that a 300 ms EPSP with a fast intervening inhibitory PSP (IPSP) reproduced the PSF recorded in non-injured subjects. In contrast, the same brief sensory stimulation in subjects with chronic SCI produced PSFs of comparatively long duration (1200 ms) with no evidence for IPSP activation, as reflected by a lack of reduced firing rates after the onset of the PSF. Thus, unlike non-injured controls, the motoneurons of subjects with chronic SCI are activated by very long periods of pure depolarization from brief sensory activation. It is likely that these second-long EPSPs securely recruit slowly activating PICs in motoneurons that are known to mediate, in large part, the many seconds-long activation of motoneurons during involuntary muscle spasms.

Pudendal nerve stimulation, interneurons post-discharge and delayed depolarization in hind limb motoneurons of the female cat

The present experiments were done in the spinal female cat. In a prior work in the decerebrate female cat, stimulation of the sensory pudendal nerve (SPN) induced a depolarizing wave (LD) in hind limb motoneurons that outlasted the stimulus by up to 6 s. LD triggered self-sustained motoneuron firing (bistability). An intrinsic potential underlies bistable firing, which, in the cat, depends on two main factors; first, the integrity of pathways descending from the brain stem to the spinal cord and, second, the membrane potential of the motoneuron just before the stimulus; at high resting potential, excitatory short-lasting inputs induce transient but no sustained firing. Thus, no bistability occurs in the spinal cat or in hyperpolarized motoneurons of the decerebrate cat. LD might be an intrinsic potential that could also be absent in the spinal cat, or an extrinsic (synaptic) potential induced by spinal interneurons. In the latter case, the interneurons generating LD should show post-discharge as prolonged as LD. LD was produced in spinal cats and its amplitude did not change or increase slightly during hyperpolarizing pulses, which suggests that LD might be a synaptic response. Interneurons showing post-discharge to train of stimulation to SPN were located 100–200 μm above the pools of hind limb motoneurons. Some post-discharges were as prolonged as LD. We conclude that LD might be a synaptic response to local interneurons.