Brent Fedirchuk

State‐dependent hyperpolarization of voltage threshold enhances motoneurone excitability during fictive locomotion in the cat

1 Experiments were conducted on decerebrate adult cats to examine the effect of brainstem‐evoked fictive locomotion on the threshold voltage (Vth) at which action potentials were initiated in hindlimb motoneurones. Measurements of the voltage threshold of the first spike evoked by intracellular injection of depolarizing ramp currents or square pulses were compared during control and fictive locomotor conditions. The sample of motoneurones included flexor and extensor motoneurones, and motoneurones with low and high rheobase currents. 2 In all 38 motoneurones examined, action potentials were initiated at more hyperpolarized membrane potentials during fictive locomotion than in control conditions (mean hyperpolarization ‐8.0 ± 5.5 mV; range ‐1.8 to ‐26.6 mV). Hyperpolarization of Vth occurred immediately at the onset of fictive locomotion and recovered in seconds (typically < 60 s) following the termination of locomotor activity. 3 The Vth of spikes occurring spontaneously without intracellular current injection was also reduced during locomotion. 4 Superimposition of rhythmic depolarizing current pulses on current ramps in the absence of locomotion did not lower Vth to the extent seen during fictive locomotion. We suggest that Vth hyperpolarization results from an as yet undetermined neuromodulatory process operating during locomotion and is not simply the result of the oscillations in membrane potential occurring during locomotion.The hyperpolarization of Vth for action potential initiation during locomotion is a state‐dependent increase in motoneurone excitability. This Vth hyperpolarization may be a fundamental process in the generation of motoneurone activity during locomotion and perhaps other motor tasks.

Pharmacologically evoked fictive motor patterns in the acutely spinalized marmoset monkey (Callithrix jacchus)

Abstract The existence of a spinal network capable of generating rhythmic alternating activity resembling locomotion still has not been firmly established in primates, including man, although evidence for one is accumulating. The present study investigated whether it is possible to activate such a network by administration of a variety of pharmacological agents to acutely spinalized marmoset monkeys (Callithrix jacchus) in the absence of phasic afferent input to the spinal cord. Fourteen marmoset monkeys were decerebrated, spinalized, and paralyzed. The nerves supplying both hindlimbs were cut and recorded from. In 5 monkeys the effect of electrical stimulation of the brainstem was investigated before spinalization. In 3 of these monkeys, rhythmic activity alternating between extensors and flexor nerves was seen. In the 2 other monkeys only synchronized activity was elicited. In acutely spinalized monkeys, administration of l-3,4-dihydroxyphenylalanine (l-dopa; 3–4 h after treatment with nialamide) failed to evoke any rhythmic alternating activity. In contrast, administration of clonidine elicited alternating activity in all of 8 monkeys tested. In 4 of these monkeys, the activity was restricted to alternation between ipsilateral and contralateral flexor nerves, whereas alternating activity between ipsilateral flexors and extensors was also seen in the other 4 monkeys. Administration of excitatory amino acids (NMDA or NMA) also elicited rhythmic alternating activity in 7 of 10 spinalized monkeys. In 4, rhythmic alternating activity was seen between extensors and flexors on one limb as well as between ipsilateral and contralateral flexors. In 3 monkeys NMDA/NMA produced alternation between extensors and flexors of one limb without alternation between the ipsilateral and contralateral sides. Administration of noradrenaline failed to elicit any rhythmic activity, but rather completely depressed already existing activity. Administration of serotonin (5-HT) was ineffective in facilitating alternating activity in 6 of 8 monkeys and was facilitatory to rhythmic activity in the other 2. We suggest that these data provide further evidence of a network capable of eliciting rhythmic alternating activity resembling locomotion in the primate spinal cord. The network, however, seems to be more difficult to activate pharmacologically in those conditions than in other mammals. This may especially be the case in higher primates, including man.

Monoamines increase the excitability of spinal neurones in the neonatal rat by hyperpolarizing the threshold for action potential production

During fictive locomotion in the adult decerebrate cat, motoneurone excitability is increased by a hyperpolarization of the threshold potential at which an action potential is elicited (Vth). This lowering of Vth occurs at the onset of fictive locomotion, is evident for the first action potential elicited and is presumably caused by a neuromodulatory process. The present study tests the hypothesis that the monoamines serotonin (5‐HT) and noradrenaline (NA) can hyperpolarize neuronal Vth. The neonatal rat isolated spinal cord preparation and whole‐cell recording techniques were used to examine the effects of bath‐applied 5‐HT and NA on the Vth of spinal ventral horn neurones. In the majority of lumbar ventral horn neurones, 5‐HT (13/26) and NA (10/16) induced a hyperpolarization of Vth ranging from −2 to −8 mV. 5‐HT and NA had similar effects on Vth for individual neurones. This hyperpolarization of Vth was not due to a reduction of an accommodative process, and could be seen without changes in membrane potential or membrane resistence. These data reveal a previously unknown action of 5‐HT and NA, hyperpolarization of Vth of spinal neurones, a process that would facilitate both neuronal recruitment and firing.

Depression of group Ia monosynaptic EPSPs in cat hindlimb motoneurones during fictive locomotion

1 The effects of fictive locomotion on monosynaptic EPSPs recorded in motoneurones and extracellular field potentials recorded in the ventral horn were examined during brainstem‐evoked fictive locomotion in decerebrate cats. Composite homonymous and heteronymous EPSPs and field potentials were evoked by group I intensity (< = 2T) stimulation of ipsilateral hindlimb muscle nerves. Ninety‐one of the 98 monosynaptic EPSPs were reduced in amplitude during locomotion (mean depression of the 91 was to 66 % of control values); seven increased in amplitude (to a mean of 121 % of control). Twenty‐one of the 22 field potentials were depressed during locomotion (mean depression to 72 % of control). 2 All but 14 Ia EPSPs were smaller during both the flexion and extension phases of locomotion than during control. In 35 % of the cases there was < 5 % difference between the amplitudes of the EPSPs evoked during the flexion and extension phases. In 27 % of the cases EPSPs evoked during flexion were larger than those evoked during extension. The remaining 38 % of EPSPs were larger during extension. There was no relation between either the magnitude of EPSP depression or the locomotor phase in which maximum EPSP depression occurred and whether an EPSP was recorded in a flexor or extensor motoneurone. 3 The mean recovery time of both EPSP and field potential amplitudes following the end of a bout of locomotion was approximately 2 min (range, < 10 to > 300 s). 4 Motoneurone membrane resistance decreased during fictive locomotion (to a mean of 61 % of control, n= 22). Because these decreases were only weakly correlated to EPSP depression (r2= 0.31) they are unlikely to fully account for this depression. 5 The depression of monosynaptic EPSPs and group I field potentials during locomotion is consistent with the hypothesis that during fictive locomotion there is a tonic presynaptic regulation of synaptic transmission from group Ia afferents to motoneurones and interneurones. Such a reduction in neurotransmitter release would decrease group Ia monosynaptic reflex excitation during locomotion. This reduction may contribute to the tonic depression of stretch reflexes occurring in the decerebrate cat during locomotion.

Group I disynaptic excitation of cat hindlimb flexor and bifunctional motoneurones during fictive locomotion

1 The incidence of short latency excitation of motoneurones innervating flexor and bifunctional muscles evoked by group I intensity (≤ 2 × threshold) electrical stimulation of hindlimb muscle nerves was investigated during fictive locomotion in decerebrate cats. Intracellular recordings were made from hindlimb motoneurones in which action potentials were blocked by intracellular diffusion of a lidocaine (lignocaine) derivative (QX‐314) and fictive locomotion was evoked by electrical stimulation of the midbrain. 2 Few motoneurones (16%) received group I‐evoked oligosynaptic excitation in the absence of fictive locomotion. During fictive locomotion 39/44 (89%) motoneurones innervating ankle, knee or hip flexor muscles and 18/28 (64%) motoneurones innervating bifunctional muscles received group I‐evoked oligosynaptic EPSPs. In flexor motoneurones, locomotor‐dependent excitation was present in both step cycle phases but largest during flexion. In bifunctional motoneurones, EPSPs were often largest at the transition between flexion and extension phases. 3 Activation of homonymous afferents most consistently evoked the largest locomotor‐dependent excitation (amplitude up to 4.6 mV), but in some cases stimulation of heteronymous flexor or bifunctional muscle nerves evoked large EPSPs. EPSP amplitude became maximal as stimulation intensity was increased to about twice threshold. This suggests that tendon organ afferents can evoke group I EPSPs during locomotion. The EPSPs resulting from brief, small stretches of extensor digitorum longus tendons indicate that group Ia muscle spindle afferents can also evoke the group I excitation of flexors. Stimulation of extensor group I afferents did not result in excitation of flexor motoneurones. 4 The mean latency of locomotor‐dependent group I excitation in flexor and bifunctional motoneurones was 1.64 ± 0.16 ms, indicating a path consisting of a single interneurone interposed between group I afferents and motoneurones innervating flexor and bifunctional muscles. This disynaptic excitation is analogous to that recorded in extensor motoneurones and evoked from extensor group I afferents during locomotion. Differences in the phase dependence and sources of group I excitation to flexor and extensor motoneurones during locomotion suggest the existence of separate groups of excitatory interneurones exciting flexor and extensor motoneurones. 5 The wide distribution of group I disynaptic excitation in motoneurones innervating extensor, flexor and bifunctional muscles acting on hip, knee and ankle joints suggests that these pathways can play an important role in the reinforcement of ongoing locomotor activity throughout the limb.

The excitability of lumbar motoneurones in the neonatal rat is increased by a hyperpolarization of their voltage threshold for activation by descending serotonergic fibres

Previous work has shown there is an increase in motoneurone excitability produced by hyperpolarization of the threshold potential at which an action potential is elicited (Vth) at the onset, and throughout brainstem‐induced fictive locomotion in the decerebrate cat. This represents a transient facilitation in the membrane potential for activation dependent on the presence of fictive locomotion. The present study tests the hypothesis that a similar neuromodulatory mechanism facilitating neuronal recruitment also exists in the neonatal rat, and the endogenous pathway mediating the Vth hyperpolarization can be activated by electrical stimulation of the neonatal brainstem. Isolated brainstem–spinal cord preparations from 1‐ to 5‐day‐old neonatal rats, and whole‐cell recording techniques were used to examine the patterns of ventral root (VR) activity produced, and the effect of electrical stimulation of the ventromedial medulla on lumbar spinal neurones. Hyperpolarization of Vth was seen in 10/11 (range –2 to –18 mV) neurones recorded during locomotor‐like VR activity, and appeared analogous to the locomotor‐dependent Vth hyperpolarization previously described in the cat. However, in the present study, Vth hyperpolarization was also seen during electrical brainstem stimulation that evoked alternating, rhythmic, or tonic VR activity, or failed to evoke VR activity. Thirty‐six of 71 neurones were antidromically identified as lumbar motoneurones and 33/36 showed a hyperpolarization of Vth (–2 to –14 mV) during electrical brainstem stimulation. Of the unidentified lumbar ventral horn neurones, 31/35 also showed hyperpolarization of Vth (–2 to –20 mV) during brainstem stimulation. The hyperpolarization of Vth and VR activity induced by brainstem stimulation was reversibly blocked by cooling of the cervical cord, indicating it is mediated by descending fibres, and application of the serotonergic antagonist ketanserin to the spinal cord was effectively able to block the brainstem‐evoked hyperpolarization of Vth. These results demonstrate a previously unknown action of the endogenous descending serotonergic system to facilitate spinal motoneuronal recruitment and firing by inducing a hyperpolarization of Vth. This modulatory process can be examined in the neonatal rat brainstem–spinal cord preparation without the requirement for ongoing locomotor activity.

A modelling study of locomotion‐induced hyperpolarization of voltage threshold in cat lumbar motoneurones

During fictive locomotion the excitability of adult cat lumbar motoneurones is increased by a reduction (a mean hyperpolarization of ≈6.0 mV) of voltage threshold (Vth) for action potential (AP) initiation that is accompanied by only small changes in AP height and width. Further examination of the experimental data in the present study confirms that Vth lowering is present to a similar degree in both the hyperpolarized and depolarized portions of the locomotor step cycle. This indicates that Vth reduction is a modulation of motoneurone membrane currents throughout the locomotor state rather than being related to the phasic synaptic input within the locomotor cycle. Potential ionic mechanisms of this locomotor‐state‐dependent increase in excitability were examined using three five‐compartment models of the motoneurone innervating slow, fast fatigue resistant and fast fatigable muscle fibres. Passive and active membrane conductances were set to produce input resistance, rheobase, afterhyperpolarization (AHP) and membrane time constant values similar to those measured in adult cat motoneurones in non‐locomoting conditions. The parameters of 10 membrane conductances were then individually altered in an attempt to replicate the hyperpolarization of Vth that occurs in decerebrate cats during fictive locomotion. The goal was to find conductance changes that could produce a greater than 3 mV hyperpolarization of Vth with only small changes in AP height (< 3 mV) and width (< 1.2 ms). Vth reduction without large changes in AP shape could be produced either by increasing fast sodium current or by reducing delayed rectifier potassium current. The most effective Vth reductions were achieved by either increasing the conductance of fast sodium channels or by hyperpolarizing the voltage dependency of their activation. These changes were particularly effective when localized to the initial segment. Reducing the conductance of delayed rectifier channels or depolarizing their activation produced similar but smaller changes in Vth. Changes in current underlying the AHP, the persistent Na+ current, three Ca2+ currents, the ‘h’ mixed cation current, the ‘A’ potassium current and the leak current were either ineffective in reducing Vth or also produced gross changes in the AP. It is suggested that the increased excitability of motoneurones during locomotion could be readily accomplished by hyperpolarizing the voltage dependency of fast sodium channels in the axon hillock by a hitherto unknown neuromodulatory action.

An intracellular study of perineal and hindlimb afferent inputs onto sphincter motoneurons in the decerebrate cat.

SummaryThe external urethral sphincter (EUS) and external anal sphincter (EAS) are striated muscles that function to maintain urinary and fecal continence respectively. This study examines the short-latency synaptic input from a variety of cutaneous perineal and muscle/cutaneous hindlimb afferents to the motoneurons innervating these muscles. Intracellular recordings from anti dromically identified EUS and EAS motoneurons provided records of the postsynaptic potentials (PSPs) produced by electrical stimulation of peripheral afferents in decerebrate or chloralose anesthetized cats. Excitatory postsynaptic potentials (EPSPs) were produced in most EUS and EAS motoneurons by stimulation of ipsilateral and contralateral sensory pudendal (SPud) and superficial perineal (SPeri) cutaneous nerves. The shortest cen tral latencies in the study (1.5 ms) suggest that there are disynaptic excitatory, in addition to tri-and oligosynap tic, connections within these reflex pathways. EPSPs mixed with longer latency inhibitory potentials (E/I PSPs) were observed in both motoneuron populations but were found more frequently in EAS motoneurons. These E/I PSPs were evoked more often from contralat eral afferents than from ipsilateral afferents. Cutaneous nerves innervating the hindlimb had weaker if any synaptic effects on sphincter motoneurons. Stimulation of ipsilateral hindlimb muscle nerves rarely produced PSPs in EUS motoneurons and had weak synaptic actions on EAS motoneurons. In 2 of 22 animals (both decerebrate), large inhibitory potentials predominated over early small EPSPs suggesting that inhibitory pathways from these afferents to sphincter motoneurons can be released under certain circumstances. The relation between the segmental afferents to EUS and EAS motoneurons and the neural circuitry influencing them during micturition and defecation are discussed.

Intraspinally mediated state-dependent enhancement of motoneurone excitability during fictive scratch in the adult decerebrate cat

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

Effects of electrical stimulation of the thoracic spinal cord on bladder and external urethral sphincter activity in the decerebrate cat.

SummaryElectrical stimulation of the spinal cord above the sacral segments was used to produce coordinated micturition in the paralysed decerebrate cat. Stimulation of the superficial aspect of the dorsolateral funiculus (DLF) within the lower thoracic (T9-T13) segments produced a bladder contraction coordinated with decreased activity in the external urethral sphincter (EUS) branch of the pudendal nerve during which time fluid was expelled. In addition, a similar response was observed with DLF stimulation at the boundary of the L5/L6 segments. At the second cervical spinal segment, however, stimulation of a more lateral and ventral portion of the superficial spinal white matter was the only effective site for producing micturition. The spinal cord-evoked response was comparable to the micturition evoked by electrical stimulation of the pontine micturition centre (PMC) within the brainstem. A bilateral lesion of the dorsal columns (DC) and the dorsolateral funiculi (DLF) at the lower thoracic levels abolished reflex micturition evoked by bladder distension. However stimulation rostral to the lesion, within the PMC or thoracic DLF, continued to produce coordinated bladder and sphincter response during voiding. Stimulation caudal to the lesion produced a decrease in pudendal nerve activity but did not produce a void or bladder pressure change. This reduction in pudendal nerve activity could be abolished with a second lesion of the superficial DLF caudal to the stimulation site. It was concluded that stimulation of the thoracic dorsolateral funiculus activates both ascending and descending fibres which can influence the bladder and/or sphincter muscles. The spinal cordevoked voiding was hypothesized to be due to activation of some portion of the ascending limb of the spinobulbospinal micturition reflex loop. The decreased activity produced by stimulation of the thoracic DLF caudal to a bilateral DC/DLF subtotal cord lesion may be mediated by fibres descending in the dorsolateral funiculus. The possibility that the spinal cord stimulation antidromically activated axons of neurons having segmental collaterals capable of influencing pudendal neural activity cannot be exclused at this time.