Yue Dai

Mechanisms underlying the early phase of spike frequency adaptation in mouse spinal motoneurones

Spike frequency adaptation (SFA) is a fundamental property of repetitive firing in motoneurones (MNs). Early SFA (occurring over several hundred milliseconds) is thought to be important in the initiation of muscular contraction. To date the mechanisms underlying SFA in spinal MNs remain unclear. In the present study, we used both whole‐cell patch‐clamp recordings of MNs in lumbar spinal cord slices prepared from motor functionally mature mice and computer modelling of spinal MNs to investigate the mechanisms underlying SFA. Pharmacological blocking agents applied during whole‐cell recordings in current‐clamp mode demonstrated that the medium AHP conductance (apamin), BK‐type Ca2+‐dependent K+ channels (iberiotoxin), voltage‐activated Ca2+ channels (CdCl2), M‐current (linopirdine) and persistent Na+ currents (riluzole) are all unnecessary for SFA. Measurements of Na+ channel availability including action potential amplitude, action potential threshold and maximum depolarization rate of the action potential were found to correlate with instantaneous firing frequency suggesting that the availability of fast, inactivating Na+ channels is involved in SFA. Characterization of this Na+ conductance in voltage‐clamp mode demonstrated that it undergoes slow inactivation with a time course similar to that of SFA. When experimentally measured parameters for the fast, inactivating Na+ conductance (including slow inactivation) were incorporated into a MN model, SFA could be faithfully reproduced. The removal of slow inactivation from this model was sufficient to remove SFA. These data indicate that slow inactivation of the fast, inactivating Na+ conductance is likely to be the key mechanism underlying early SFA in spinal MNs.

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.

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.

The upright posture improves plantar stepping and alters responses to serotonergic drugs in spinal rats

•  Locomotor training of rats held in an upright posture has been used recently to restore locomotion after spinal cord injury. Our results show that the upright posture alone improves locomotor recovery in spinal rats. •  This improvement is reversed by the removal of cutaneous afferent feedback from the paw, showing that sensory feedback from the foot facilitates the spinal central pattern generator (CPG) for locomotion. •  5‐HT2 and 5‐HT1A/7 agonists improve locomotion in the horizontal posture but can impair locomotion in the upright posture, suggesting that a proper balance of afferent feedback from the foot and 5‐HT receptor activation is necessary for optimal locomotor recovery. •  Our results provide new insights into the organization of the CPG for locomotion and the evolution of hominid bipedalism. The potent effects of cutaneous afferents from the paw revealed here must be taken into account in the design of strategies to restore locomotion after spinal cord injury.

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.

Staircase currents in motoneurons: insight into the spatial arrangement of calcium channels in the dendritic tree.

In spinal motoneurons, activation of dendritically located depolarizing conductances can lead to amplification of synaptic inputs and the production of plateau potentials. Immunohistochemical and computational studies have implicated dendritic CaV1.3 channels in this amplification and suggest that CaV1.3 channels in spinal motoneurons may be organized in clusters in the dendritic tree. Our goal was to provide physiological evidence for the presence of multiple discrete clusters of voltage-gated calcium channels in spinal motoneurons and to explore the spatial arrangement of these clusters in the dendritic tree. We recorded voltage-gated calcium currents from spinal motoneurons in slices of mature mouse spinal cords. We demonstrate that single somatic voltage-clamp steps can elicit multiple inward currents with varying delays to onset, resulting in a current with a “staircase”-like appearance. Recordings from cultured dorsal root ganglion cells at different stages of neurite development provide evidence that these currents arise from the unclamped portions of the dendritic tree. Finally, both voltage- and current-clamp data were used to constrain computer models of a motoneuron. The resultant simulations impose two conditions on the spatial distribution of CaV channels in motoneuron dendrites: one of asymmetry relative to the soma and another of spatial separation between clusters of CaV channels. We propose that this compartmentalization would provide motoneurons with the ability to process multiple sources of input in parallel and integrate this processed information to produce appropriate trains of action potentials for the intended motor behavior.

Electrophysiological and Pharmacological Properties of Locomotor Activity-Related Neurons in cfos-EGFP Mice

Although locomotion is known to be generated by networks of spinal neurons, knowledge of the properties of these neurons is limited. Using neonatal transgenic mice that express enhanced green fluorescent protein (EGFP) driven by the c-fos promoter, we visualized EGFP-positive neurons in spinal cord slices from animals that were subjected to a locomotor task or drug cocktail [N-methyl-D-aspartate, serotonin (5-HT), dopamine, and acetylcholine (ACh)]. The activity-dependent expression of EGFP was also induced in dorsal root ganglion neurons with electrical stimulation of the neurons. Following 60-90 min of swimming, whole cell patch-clamp recordings were made from EGFP+ neurons in laminae VII, VIII, and X from slices of segments T(12) to L(4). The EGFP+ neurons (n = 55) could be classified into three types based on their responses to depolarizing step currents: single spike, phasic firing, and tonic firing. Membrane properties observed in these neurons include hyperpolarization-activated inward currents (29/55), postinhibitory rebound (11/55), and persistent-inward currents (31/55). Bath application of 10-40 microM 5-HT and/or ACh increased neuronal excitability or output with hyperpolarization of voltage threshold and changes in membrane potential. 5-HT also increased input resistance, reduced the afterhyperpolarization (AHP), and induced membrane oscillations, whereas ACh reduced the input resistance and increased the AHP. In this study, we demonstrate a new way of identifying neurons active in locomotion. Our results suggest that the EGFP+ neurons are a heterogeneous population of interneurons. The actions of 5-HT and ACh on these neurons provide insights into the neuronal properties modulated by these transmitters for generation of locomotion.

Requirement of neuronal connexin36 in pathways mediating presynaptic inhibition of primary afferents in functionally mature mouse spinal cord

•  Reflexes evoked by sensory information from muscles and skin play an important role in controlling muscle activity during movement. •  The strength of these reflexes is regulated in part by presynaptic inhibition, a process controlling the release of chemical transmitters from sensory fibres terminating on spinal neurones. •  Our study is the first to show that electrical synapses among spinal neurones in young animals are essential for normal operation of processes that presynaptically regulate synaptic transmission between large diameter sensory fibres and spinal cord neurones. •  Transgenic mice lacking connexin36, a protein that mediates electrical communication via gap junctions between neurones, suffer a severe impairment of presynaptic inhibition and a similar impairment can be produced in normal mice with drugs that disrupt gap junction function. •  The wide distribution of connexin36 in the spinal cord suggests that neuronal gap junctions also play a role in other physiological processes.

Contributions of the input signal and prior activation history to the discharge behaviour of rat motoneurones

The principal computational operation of neurones is the transformation of synaptic inputs into spike train outputs. The probability of spike occurrence in neurones is determined by the time course and magnitude of the total current reaching the spike initiation zone. The features of this current that are most effective in evoking spikes can be determined by injecting a Gaussian current waveform into a neurone and using spike‐triggered reverse correlation to calculate the average current trajectory (ACT) preceding spikes. The time course of this ACT (and the related first‐order Wiener kernel) provides a general description of a neurones response to dynamic stimuli. In many different neurones, the ACT is characterized by a shallow hyperpolarizing trough followed by a more rapid depolarizing peak immediately preceding the spike. The hyperpolarizing phase is thought to reflect an enhancement of excitability by partial removal of sodium inactivation. Alternatively, this feature could simply reflect the fact that interspike intervals that are longer than average can only occur when the current is lower than average toward the end of the interspike interval. Thus, the ACT calculated for the entire spike train displays an attenuated version of the hyperpolarizing trough associated with the long interspike intervals. This alternative explanation for the characteristic shape of the ACT implies that it depends upon the time since the previous spike, i.e. the ACT reflects both previous stimulus history and previous discharge history. The present study presents results based on recordings of noise‐driven discharge in rat hypoglossal motoneurones that support this alternative explanation. First, we show that the hyperpolarizing trough is larger in ACTs calculated from spikes preceded by long interspike intervals, and minimal or absent in those based on short interspike intervals. Second, we show that the trough is present for ACTs calculated from the discharge of a threshold‐crossing neurone model with a postspike afterhyperpolarization (AHP), but absent from those calculated from the discharge of a model without an AHP. We show that it is possible to represent noise‐driven discharge using a two‐component linear model that predicts discharge probability based on the sum of a feedback kernel and a stimulus kernel. The feedback kernel reflects the influence of prior discharge mediated by the AHP, and it increases in amplitude when AHP amplitude is increased by pharmacological manipulations. Finally, we show that the predictions of this model are virtually identical to those based on the first‐order Wiener kernel. This suggests that the Wiener kernels derived from standard white‐noise analysis of noise‐driven discharge in neurones actually reflect the effects of both stimulus and discharge history.

Multiple Effects of Serotonin and Acetylcholine on Hyperpolarization-Activated Inward Current in Locomotor Activity-Related Neurons in Cfos-EGFP Mice

Hyperpolarization-activated inward current (I(h)) has been shown to be involved in production of bursting during various forms of rhythmic activity. However, details of I(h) in spinal interneurons related to locomotion remain unknown. Using Cfos-EGFP transgenic mice (P6-P12) we are able to target the spinal interneurons activated by locomotion. Following a locomotor task, whole cell patch-clamp recordings were obtained from ventral EGFP+ neurons in spinal cord slices (T(13)-L(4), 200-250 microm). I(h) was found in 51% of EGFP+ neurons (n = 149) with almost even distribution in lamina VII (51%), VIII (47%), and X (55%). I(h) could be blocked by ZD7288 (10-20 microM) or cesium (1-1.5 mM) but was insensitive to barium (2-2.5 mM). I(h) activated at -80.1 +/- 9.2 mV with half-maximal activation -95.5 +/- 13.3 mV, activation rate 10.0 +/- 3.2 mV, time constant 745 +/- 501 ms, maximal conductance 1.0 +/- 0.7 nS, and reversal potential -34.3 +/- 3.6 mV. 5-HT (15-20 microM) and ACh (20-30 microM) produced variable effects on I(h). 5-HT increased I(h) in 43% of EGFP+ neurons (n = 37), decreased I(h) in 24%, and had no effect on I(h) in 33% of the neurons. ACh decreased I(h) in 67% of EGFP+ neurons (n = 18) with unchanged I(h) in 33% of the neurons. This study characterizes the I(h) in locomotor-related interneurons and is the first to demonstrate the variable effects of 5-HT and ACh on I(h) in rodent spinal interneurons. The finding of 5-HT and ACh-induced reduction of I(h) in EGFP+ neurons suggests a novel mechanism that the motor system could use to limit the participation of certain neurons in locomotion.