Mitigating aberrant Cdk5 activation alleviates mitochondrial defects and motor neuron disease symptoms in spinal muscular atrophy.

Spinal muscular atrophy (SMA), the top genetic cause of infant mortality, is characterized by motor neuron degeneration. Mechanisms underlying SMA pathogenesis remain largely unknown. Here, we report that the activity of cyclin-dependent kinase 5 (Cdk5) and the conversion of its activating subunit p35 to the more potent activator p25 are significantly up-regulated in mouse models and human induced pluripotent stem cell (iPSC) models of SMA. The increase of Cdk5 activity occurs before the onset of SMA phenotypes, suggesting that it may be an initiator of the disease. Importantly, aberrant Cdk5 activation causes mitochondrial defects and motor neuron degeneration, as the genetic knockout of p35 in an SMA mouse model rescues mitochondrial transport and fragmentation defects, and alleviates SMA phenotypes including motor neuron hyperexcitability, loss of excitatory synapses, neuromuscular junction denervation, and motor neuron degeneration. Inhibition of the Cdk5 signaling pathway reduces the degeneration of motor neurons derived from SMA mice and human SMA iPSCs. Altogether, our studies reveal a critical role for the aberrant activation of Cdk5 in SMA pathogenesis and suggest a potential target for therapeutic intervention.

Interference with glutamate antiporter system xc - enables post-hypoxic long-term potentiation in hippocampus.

Our group previously showed that genetic or pharmacological inhibition of the cystine/glutamate antiporter, system xc -, mitigates excitotoxicity after anoxia by increasing latency to anoxic depolarization, thus attenuating the ischaemic core. Hypoxia, however, which prevails in the ischaemic penumbra, is a condition where neurotransmission is altered, but excitotoxicity is not triggered. The present study employed mild hypoxia to further probe ischaemia-induced changes in neuronal responsiveness from wild-type and xCT KO (xCT-/-) mice. Synaptic transmission was monitored in hippocampal slices from both genotypes before, during and after a hypoxic episode. Although wild-type and xCT-/- slices showed equal suppression of synaptic transmission during hypoxia, mutant slices exhibited a persistent potentiation upon re-oxygenation, an effect we termed 'post-hypoxic long-term potentiation (LTP)'. Blocking synaptic suppression during hypoxia by antagonizing adenosine A1 receptors did not preclude post-hypoxic LTP. Further examination of the induction and expression mechanisms of this plasticity revealed that post-hypoxic LTP was driven by NMDA receptor activation, as well as increased calcium influx, with no change in paired-pulse facilitation. Hence, the observed phenomenon engaged similar mechanisms as classical LTP. This was a remarkable finding as theta-burst stimulation-induced LTP was equivalent between genotypes. Importantly, post-hypoxic LTP was generated in wild-type slices pretreated with system xc - inhibitor, S-4-carboxyphenylglycine, thereby confirming the antiporter's role in this phenomenon. Collectively, these data indicate that system xc - interference enables neuroplasticity in response to mild hypoxia, and, together with its regulation of cellular damage in the ischaemic core, suggest a role for the antiporter in post-ischaemic recovery of the penumbra.

A dynamic calcium-force relationship model for sag behavior in fast skeletal muscle.

In vitro studies using isolated or skinned muscle fibers suggest that the sigmoidal relationship between the intracellular calcium concentration and force production may depend upon muscle type and activity. The goal of this study was to investigate whether and how the calcium-force relationship changes during force production under physiological conditions of muscle excitation and length in fast skeletal muscles. A computational framework was developed to identify the dynamic variation in the calcium-force relationship during force generation over a full physiological range of stimulation frequencies and muscle lengths in cat gastrocnemius muscles. In contrast to the situation in slow muscles such as the soleus, the calcium concentration for the half-maximal force needed to drift rightward to reproduce the progressive force decline, or sag behavior, observed during unfused isometric contractions at the intermediate length under low-frequency stimulation (i.e., 20 Hz). The slope at the calcium concentration for the half-maximal force was required to drift upward for force enhancement during unfused isometric contractions at the intermediate length under high-frequency stimulation (i.e., 40 Hz). The slope variation in the calcium-force relationship played a crucial role in shaping sag behavior across different muscle lengths. The muscle model with dynamic variations in the calcium-force relationship also accounted for the length-force and velocity-force properties measured under full excitation. These results imply that the calcium sensitivity and cooperativity of force-inducing crossbridge formation between actin and myosin filaments may be operationally altered in accordance with the mode of neural excitation and muscle movement in intact fast muscles.

Differences in estimated persistent inward currents between ankle flexors and extensors in humans.

Persistent inward currents (PICs) are responsible for amplifying motoneuronal synaptic inputs and contribute to generating normal motoneuron activation. Delta-F (ΔF) is a well-established method that estimates PICs in humans indirectly from firing patterns of individual motor units. Traditionally, motor unit firing patterns are obtained by manually decomposing electromyography (EMG) signals recorded through intramuscular electrodes (iEMG). A previous iEMG study has shown that in humans the elbow extensors have higher ΔF than the elbow flexors. In this study, EMG signals were collected from the ankle extensors and flexors using high-density surface array electrodes during isometric sitting and standing at 10-30% maximum voluntary contraction. The signals were then decomposed into individual motor unit firings. We hypothesized that comparable to the upper limb, the lower limb extensor muscles (soleus) would have higher ΔF than the lower limb flexor muscles [tibialis anterior (TA)]. Contrary to our expectations, ΔF was higher in the TA than the soleus during sitting and standing despite the difference in cohort of participants and body positions. The TA also had significantly higher maximum discharge rate than the soleus while there was no difference in rate increase. When only the unit pairs with similar maximum discharge rates were compared, ∆F was still higher in the TA than the soleus. Future studies will focus on investigating the functional significance of the findings.NEW & NOTEWORTHY With the use of high-density surface array electrodes and convolutive blind source separation algorithm, thousands of motor units were decomposed from the soleus and tibialis anterior muscles. Persistent inward currents were estimated under seated and standing conditions via delta-F (∆F) calculation, and the results showed that unlike the upper limb, the flexor has higher ∆F than the extensor in the lower limb. Future studies will focus on functional significance of the findings.

Bursting interneurons in the deep dorsal horn develop increased excitability and sensitivity to serotonin after chronic spinal injury.

The loss of descending serotonin (5-HT) to the spinal cord contributes to muscle spasms in chronic spinal cord injury (SCI). Hyperexcitable motoneurons receive long-lasting excitatory postsynaptic potentials (EPSPs), which activate their persistent inward currents to drive muscle spasms. Deep dorsal horn (DDH) neurons with bursting behavior could be involved in triggering the EPSPs due to loss of inhibition in the chronically 5-HT-deprived spinal cord. Previously, in an acutely transected preparation, we found that bursting DDH neurons were affected by administration of the 5-HT1B/1D receptor agonist zolmitriptan, which suppressed their bursts, and by N-methyl-d-aspartate (NMDA), which enhanced their bursting behavior. Nonbursting DDH neurons were not influenced by these agents. In the present study, we investigate the firing characteristics of bursting DDH neurons following chronic spinal transection at T10 level in adult mice and examine the effects of replacing lost endogenous 5-HT with zolmitriptan. Terminal experiments using our in vitro preparation of the sacral cord were carried out ~10 wk postransection. Compared with the acute spinal stage of our previous study, DDH neurons in the chronic stage became more responsive to dorsal root stimulation, with burst duration doubling with chronic injury. The suppressive effects of zolmitriptan were stronger overall, but the facilitative effects of NMDA were weaker. In addition, the onset of DDH neuron activity preceded ventral root output and the firing rates of DDH interneurons correlated with the integrated long-lasting ventral root output. These results support a contribution of the bursting DDH neurons to muscle spasms following SCI and inhibition by 5-HT.NEW & NOTEWORTHY We investigate the firing characteristics of bursting deep dorsal horn (DDH) neurons following chronic spinal transection. DDH neurons in the chronic stage are different from those in the acute stage as noted by their increase in excitability overall and their differing responses serotonin (5-HT) and N-methyl-d-aspartate (NMDA) receptor agonists. Also, there is a strong relationship between DDH neuron activity and ventral root output. These results support a contribution of the bursting DDH neurons to muscle spasms following chronic spinal cord injury (SCI).

Robust and accurate decoding of motoneuron behaviour and prediction of the resulting force output.

KEY POINTS: The spinal alpha motoneuron is the only cell in the human CNS whose discharge can be routinely recorded in humans. We have reengineered motor unit collection and decomposition approaches, originally developed in humans, to measure the neural drive to muscle and estimate muscle force generation in the in vivo cat model. Experimental, computational, and predictive approaches are used to demonstrate the validity of this approach across a wide range of modes to activate the motor pool. The utility of this approach is shown through the ability to track individual motor units across trials, allowing for better predictions of muscle force than the electromyography signal, and providing insights in to the stereotypical discharge characteristics in response to synaptic activation of the motor pool. This approach now allows for a direct link between the intracellular data of single motoneurons, the discharge properties of motoneuron populations, and muscle force generation in the same preparation. ABSTRACT: The discharge of a spinal alpha motoneuron and the resulting contraction of its muscle fibres represents the functional quantum of the motor system. Recent advances in the recording and decomposition of the electromyographic signal allow for the identification of several tens of concurrently active motor units. These detailed population data provide the potential to achieve deep insights into the synaptic organization of motor commands. Yet most of our understanding of the synaptic input to motoneurons is derived from intracellular recordings in animal preparations. Thus, it is necessary to extend the new electrode and decomposition methods to recording of motor unit populations in these same preparations. To achieve this goal, we use high-density electrode arrays and decomposition techniques, analogous to those developed for humans, to record and decompose the activity of tens of concurrently active motor units in a hindlimb muscle in the in vivo cat. Our results showed that the decomposition method in this animal preparation was highly accurate, with conventional two-source validation providing rates of agreement equal to or superior to those found in humans. Multidimensional reconstruction of the motor unit action potential provides the ability to accurately track the same motor unit across multiple contractions. Additionally, correlational analyses demonstrate that the composite spike train provides better estimates of whole muscle force than conventional estimates obtained from the electromyographic signal. Lastly, stark differences are observed between the modes of activation, in particular tendon vibration produced quantal interspike intervals at integer multiples of the vibration period.

Acyloxyacyl hydrolase modulates pelvic pain severity.

Chronic pelvic pain causes significant patient morbidity and is a challenge to clinicians. Using a murine neurogenic cystitis model that recapitulates key aspects of interstitial cystitis/bladder pain syndrome (IC), we recently showed that pseudorabies virus (PRV) induces severe pelvic allodynia in BALB/c mice relative to C57BL/6 mice. Here, we report that a quantitative trait locus (QTL) analysis of PRV-induced allodynia in F2CxB progeny identified a polymorphism on chromosome 13, rs6314295 , significantly associated with allodynia (logarithm of odds = 3.11). The nearby gene encoding acyloxyacyl hydrolase ( Aoah) was induced in the sacral spinal cord of PRV-infected mice. AOAH-deficient mice exhibited increased vesicomotor reflex in response to bladder distension, consistent with spontaneous bladder hypersensitivity, and increased pelvic allodynia in neurogenic cystitis and postbacterial chronic pain models. AOAH deficiency resulted in greater bladder pathology and tumor necrosis factor production in PRV neurogenic cystitis, markers of increased bladder mast cell activation. AOAH immunoreactivity was detectable along the bladder-brain axis, including in brain sites previously correlated with human chronic pelvic pain. Finally, AOAH-deficient mice had significantly higher levels of bladder vascular endothelial growth factor, an emerging marker of chronic pelvic pain in humans. These findings indicate that AOAH modulates pelvic pain severity, suggesting that allelic variation in Aoah influences pelvic pain in IC.

Chronic electromyograms in treadmill running SOD1 mice reveal early changes in muscle activation.

KEY POINTS: The present study demonstrates that electromyograms (EMGs) obtained during locomotor activity in mice were effective for identification of early physiological markers of amyotrophic lateral sclerosis (ALS). These measures could be used to evaluate therapeutic intervention strategies in animal models of ALS. Several parameters of locomotor activity were shifted early in the disease time course in SOD1G93A mice, especially when the treadmill was inclined, including intermuscular phase, burst skew and amplitude of the locomotor bursts. The results of the present study indicate that early compensatory changes may be taking place within the neural network controlling locomotor activity, including spinal interneurons. Locomotor EMGs could have potential use as a clinical diagnostic tool. ABSTRACT: To improve our understanding of early disease mechanisms and to identify reliable biomarkers of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease, we measured electromyogram (EMG) activity in hind limb muscles of SOD1G93A mice. By contrast to clinical diagnostic measures using EMGs, which are performed on quiescent patients, we monitored activity during treadmill running aiming to detect presymptomatic changes in motor patterning. Chronic EMG electrodes were implanted into vastus lateralis, biceps femoris posterior, lateral gastrocnemius and tibialis anterior in mice from postnatal day 55 to 100 and the results obtained were assessed using linear mixed models. We evaluated differences in parameters related to EMG amplitude (peak and area) and timing (phase and skew, a measure of burst shape) when animals ran on level and inclined treadmills. There were significant changes in both the timing of activity and the amplitude of EMG bursts in SOD1G93A mice. Significant differences between wild-type and SOD1G93A mice were mainly observed when animals locomoted on inclined treadmills. All muscles had significant effects of mutation that were independent of age. These novel results indicate (i) locomotor EMG activity might be an early measure of disease onset; (ii) alterations in locomotor patterning may reflect changes in neuronal drive and compensation at the network level including altered activity of spinal interneurons; and (iii) the increased power output necessary on an inclined treadmill was important in revealing altered activity in SOD1G93A mice.

Synaptic control of the shape of the motoneuron pool input-output function.

Although motoneurons have often been considered to be fairly linear transducers of synaptic input, recent evidence suggests that strong persistent inward currents (PICs) in motoneurons allow neuromodulatory and inhibitory synaptic inputs to induce large nonlinearities in the relation between the level of excitatory input and motor output. To try to estimate the possible extent of this nonlinearity, we developed a pool of model motoneurons designed to replicate the characteristics of motoneuron input-output properties measured in medial gastrocnemius motoneurons in the decerebrate cat with voltage-clamp and current-clamp techniques. We drove the model pool with a range of synaptic inputs consisting of various mixtures of excitation, inhibition, and neuromodulation. We then looked at the relation between excitatory drive and total pool output. Our results revealed that the PICs not only enhance gain but also induce a strong nonlinearity in the relation between the average firing rate of the motoneuron pool and the level of excitatory input. The relation between the total simulated force output and input was somewhat more linear because of higher force outputs in later-recruited units. We also found that the nonlinearity can be increased by increasing neuromodulatory input and/or balanced inhibitory input and minimized by a reciprocal, push-pull pattern of inhibition. We consider the possibility that a flexible input-output function may allow motor output to be tuned to match the widely varying demands of the normal motor repertoire.NEW & NOTEWORTHY Motoneuron activity is generally considered to reflect the level of excitatory drive. However, the activation of voltage-dependent intrinsic conductances can distort the relation between excitatory drive and the total output of a pool of motoneurons. Using a pool of realistic motoneuron models, we show that pool output can be a highly nonlinear function of synaptic input but linearity can be achieved through adjusting the time course of excitatory and inhibitory synaptic inputs.

The potential for understanding the synaptic organization of human motor commands via the firing patterns of motoneurons.

Motoneurons are unique in being the only neurons in the CNS whose firing patterns can be easily recorded in human subjects. This is because of the one-to-one relationship between the motoneuron and muscle cell behavior. It has long been appreciated that the connection of motoneurons to their muscle fibers allows their action potentials to be amplified and recorded, but only recently has it become possible to simultaneously record the firing pattern of many motoneurons via array electrodes placed on the skin. These firing patterns contain detailed information about the synaptic organization of motor commands to the motoneurons. This review focuses on parameters in these firing patterns that are directly linked to specific features of this organization. It is now well established that motor commands consist of three components, excitation, inhibition, and neuromodulation; the importance of the third component has become increasingly evident. Firing parameters linked to each of the three components are discussed, along with consideration of potential limitations in their utility for understanding the underlying organization of motor commands. Future work based on realistic computer simulations of motoneurons may allow quantitative "reverse engineering" of human motoneuron firing patterns to provide good estimates of the relative amplitudes and temporal patterns of all three components of motor commands.

PICs in motoneurons do not scale with the size of the animal: a possible mechanism for faster speed of muscle contraction in smaller species.

The majority of studies on the electrical properties of neurons are carried out in rodents, and in particular in mice. However, the minute size of this animal compared with humans potentially limits the relevance of the resulting insights. To be able to extrapolate results obtained in a small animal such as a rodent, one needs to have proper knowledge of the rules governing how electrical properties of neurons scale with the size of the animal. Generally speaking, electrical resistances of neurons increase as cell size decreases, and thus maintenance of equal depolarization across cells of different sizes requires the underlying currents to decrease in proportion to the size decrease. Thus it would generally be expected that voltage-sensitive currents are smaller in smaller animals. In this study, we used in vivo preparations to record electrical properties of spinal motoneurons in deeply anesthetized adult mice and cats. We found that PICs do not scale with size, but instead are constant in their amplitudes across these species. This constancy, coupled with the threefold differences in electrical resistances, means that PICs contribute a threefold larger depolarization in the mouse than in the cat. As a consequence, motoneuronal firing rate sharply increases as animal size decreases. These differences in firing rates are likely essential in allowing different species to control muscles with widely different contraction speeds (smaller animals have faster muscle fibers). Thus from our results we have identified a possible new mechanism for how electrical properties are tuned to match mechanical properties within the motor output system.NEW & NOTEWORTHY The small size of the mouse warrants concern over whether the properties of their neurons are a scaled version of those in larger animals or instead have unique features. Comparison of spinal motoneurons in mice to cats showed unique features. Firing rates in the mouse were much higher, in large part due to relatively larger persistent inward currents. These differences likely reflect adaptations for controlling much faster muscle fibers in mouse than cat.

Firing characteristics of deep dorsal horn neurons after acute spinal transection during administration of agonists for 5-HT1B/1D and NMDA receptors.

Spinal cord injury (SCI) results in a loss of serotonin (5-HT) to the spinal cord and a loss of inhibition to deep dorsal horn (DDH) neurons, which produces an exaggerated excitatory drive to motoneurons. The mechanism of this excitatory drive could involve the DDH neurons triggering long excitatory postsynaptic potentials in motoneurons, which may ultimately drive muscle spasms. Modifying the activity of DDH neurons with drugs such as NMDA or the 5-HT1B/1D receptor agonist zolmitriptan could have a large effect on motoneuron activity and, therefore, on muscle spasms. In this study, we characterize the firing properties of DDH neurons after acute spinal transection in adult mice during administration of zolmitriptan and NMDA, using the in vitro sacral cord preparation and extracellular electrophysiology. DDH neurons can be categorized into three major types with distinct evoked and spontaneous firing characteristics: burst (bursting), simple (single spiking), and tonic (spontaneously tonic firing) neurons. The burst neurons likely contribute to muscle spasm mechanisms because of their bursting behavior. Only the burst neurons show significant changes in their firing characteristics during zolmitriptan and NMDA administration. Zolmitriptan suppresses the burst neurons by reducing their evoked spikes, burst duration, and spontaneous firing rate. Conversely, NMDA facilitates them by enhancing their burst duration and spontaneous firing rate. These results suggest that zolmitriptan may exert its antispastic effect on the burst neurons via activation of 5-HT1B/1D receptors, whereas activation of NMDA receptors may facilitate the burst neurons in contributing to muscle spasm mechanisms following SCI.

Serotonin affects movement gain control in the spinal cord.

A fundamental challenge for the nervous system is to encode signals spanning many orders of magnitude with neurons of limited bandwidth. To meet this challenge, perceptual systems use gain control. However, whether the motor system uses an analogous mechanism is essentially unknown. Neuromodulators, such as serotonin, are prime candidates for gain control signals during force production. Serotonergic neurons project diffusely to motor pools, and, therefore, force production by one muscle should change the gain of others. Here we present behavioral and pharmaceutical evidence that serotonin modulates the input-output gain of motoneurons in humans. By selectively changing the efficacy of serotonin with drugs, we systematically modulated the amplitude of spinal reflexes. More importantly, force production in different limbs interacts systematically, as predicted by a spinal gain control mechanism. Psychophysics and pharmacology suggest that the motor system adopts gain control mechanisms, and serotonin is a primary driver for their implementation in force production.

Intrinsic excitability of human motoneurons in biceps brachii versus triceps brachii.

The intrinsic excitability of spinal motoneurons is mediated in part by the presence of persistent inward currents (PICs), which amplify synaptic input and promote self-sustained firing. Studies using animal models have shown that PICs are greater in extensor motoneurons over flexor motoneurons, but this difference has not yet been demonstrated in humans. The primary objective of this study was to determine whether a similar difference exists in humans by recording from motor units in biceps and triceps brachii during isometric contractions. We compared firing rate profiles of pairs of motor units, in which the firing rate of the lower-threshold "control" unit was used as an indicator of common drive to the higher-threshold "test" unit. The estimated contribution of the PIC was calculated as the difference in firing rate of the control unit at recruitment versus derecruitment of the test unit, a value known as the delta-F (ΔF). We found that ΔF values were significantly higher in triceps brachii (5.4 ± 0.9 imp/s) compared with biceps brachii (3.0 ± 1.4 imp/s; P < 0.001). This difference was still present even after controlling for saturation in firing rate of the control unit, rate modulation of the control unit, and differences in recruitment time between test and control units, which are known to contribute to ΔF variability. We conclude that human elbow flexor and extensor motor units exhibit differences in intrinsic excitability, contributing to different neural motor control strategies between muscle groups.

Analyzing Modeled Torque Profiles to Understand Scale-Dependent Active Muscle Responses in the Hip Joint.

Animal locomotion is influenced by a combination of constituent joint torques (e.g., due to limb inertia and passive viscoelasticity), which determine the necessary muscular response to move the limb. Across animal size-scales, the relative contributions of these constituent joint torques affect the muscular response in different ways. We used a multi-muscle biomechanical model to analyze how passive torque components change due to an animal's size-scale during locomotion. By changing the size-scale of the model, we characterized emergent muscular responses at the hip as a result of the changing constituent torque profile. Specifically, we found that activation phases between extensor and flexor torques to be opposite between small and large sizes for the same kinematic motion. These results suggest general principles of how animal size affects neural control strategies. Our modeled torque profiles show a strong agreement with documented hindlimb torque during locomotion and can provide insights into the neural organization and muscle activation behavior of animals whose motion has not been extensively documented.

Multimodal parameter spaces of a complex multi-channel neuron model.

One of the most common types of models that helps us to understand neuron behavior is based on the Hodgkin-Huxley ion channel formulation (HH model). A major challenge with inferring parameters in HH models is non-uniqueness: many different sets of ion channel parameter values produce similar outputs for the same input stimulus. Such phenomena result in an objective function that exhibits multiple modes (i.e., multiple local minima). This non-uniqueness of local optimality poses challenges for parameter estimation with many algorithmic optimization techniques. HH models additionally have severe non-linearities resulting in further challenges for inferring parameters in an algorithmic fashion. To address these challenges with a tractable method in high-dimensional parameter spaces, we propose using a particular Markov chain Monte Carlo (MCMC) algorithm, which has the advantage of inferring parameters in a Bayesian framework. The Bayesian approach is designed to be suitable for multimodal solutions to inverse problems. We introduce and demonstrate the method using a three-channel HH model. We then focus on the inference of nine parameters in an eight-channel HH model, which we analyze in detail. We explore how the MCMC algorithm can uncover complex relationships between inferred parameters using five injected current levels. The MCMC method provides as a result a nine-dimensional posterior distribution, which we analyze visually with solution maps or landscapes of the possible parameter sets. The visualized solution maps show new complex structures of the multimodal posteriors, and they allow for selection of locally and globally optimal value sets, and they visually expose parameter sensitivities and regions of higher model robustness. We envision these solution maps as enabling experimentalists to improve the design of future experiments, increase scientific productivity and improve on model structure and ideation when the MCMC algorithm is applied to experimental data.

Motoneuron excitability and muscle spasms are regulated by 5-HT2B and 5-HT2C receptor activity.

Immediately after spinal cord injury (SCI), a devastating paralysis results from the loss of brain stem and cortical innervation of spinal neurons that control movement, including a loss of serotonergic (5-HT) innervation of motoneurons. Over time, motoneurons recover from denervation and function autonomously, exhibiting large persistent calcium currents (Ca PICs) that both help with functional recovery and contribute to uncontrolled muscle spasms. Here we systematically evaluated which 5-HT receptor subtypes influence PICs and spasms after injury. Spasms were quantified by recording the long-lasting reflexes (LLRs) on ventral roots in response to dorsal root stimulation, in the chronic spinal rat, in vitro. Ca PICs were quantified by intracellular recording in synaptically isolated motoneurons. Application of agonists selective to 5-HT(2B) and 5-HT(2C) receptors (including BW723C86) significantly increased the LLRs and associated Ca PICs, whereas application of agonists to 5-HT(1), 5-HT(2A), 5-HT(3), or 5-HT(4/5/6/7) receptors (e.g., 8-OH-DPAT) did not. The 5-HT(2) receptor agonist-induced increases in LLRs were dose dependent, with doses for 50% effects (EC(50)) highly correlated with published doses for agonist receptor binding (K(i)) at 5-HT(2B) and 5-HT(2C) receptors. Application of selective antagonists to 5-HT(2B) (e.g., RS127445) and 5-HT(2C) (SB242084) receptors inhibited the agonist-induced increase in LLR. However, antagonists that are known to specifically be neutral antagonists at 5-HT(2B/C) receptors (e.g., RS127445) had no effect when given by themselves, indicating that these receptors were not activated by residual 5-HT in the spinal cord. In contrast, inverse agonists (such as SB206553) that block constitutive activity at 5-HT(2B) or 5-HT(2C) receptors markedly reduced the LLRs, indicating the presence of constitutive activity in these receptors. 5-HT(2B) or 5-HT(2C) receptors were confirmed to be on motoneurons by immunolabeling. In summary, 5-HT(2B) and 5-HT(2C) receptors on motoneurons become constitutively active after injury and ultimately contribute to recovery of motoneuron function and emergence of spasms.

Time Course of Alterations in Adult Spinal Motoneuron Properties in the SOD1(G93A) Mouse Model of ALS.

Although amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease, motoneuron electrical properties are already altered during embryonic development. Motoneurons must therefore exhibit a remarkable capacity for homeostatic regulation to maintain a normal motor output for most of the life of the patient. In the present article, we demonstrate how maintaining homeostasis could come at a very high cost. We studied the excitability of spinal motoneurons from young adult SOD1(G93A) mice to end-stage. Initially, homeostasis is highly successful in maintaining their overall excitability. This initial success, however, is achieved by pushing some cells far above the normal range of passive and active conductances. As the disease progresses, both passive and active conductances shrink below normal values in the surviving cells. This shrinkage may thus promote survival, implying the previously large values contribute to degeneration. These results support the hypothesis that motoneuronal homeostasis may be "hypervigilant" in ALS and a source of accumulating stress.

The Involvement of CaV1.3 Channels in Prolonged Root Reflexes and Its Potential as a Therapeutic Target in Spinal Cord Injury.

Spinal cord injury (SCI) results in not only the loss of voluntary muscle control, but also in the presence of involuntary movement or spasms. These spasms post-SCI involve hyperexcitability in the spinal motor system. Hyperactive motor commands post SCI result from enhanced excitatory postsynaptic potentials (EPSPs) and persistent inward currents in voltage-gated L-type calcium channels (LTCCs), which are reflected in evoked root reflexes with different timings. To further understand the contributions of these cellular mechanisms and to explore the involvement of LTCC subtypes in SCI-induced hyperexcitability, we measured root reflexes with ventral root recordings and motoneuron activities with intracellular recordings in an in vitro preparation using a mouse model of chronic SCI (cSCI). Specifically, we explored the effects of 1-(3-chlorophenethyl)-3-cyclopentylpyrimidine-2,4,6-(1H,3H,5H)-trione (CPT), a selective negative allosteric modulator of CaV1.3 LTCCs. Our results suggest a hyperexcitability in the spinal motor system in these SCI mice. Bath application of CPT displayed slow onset but dose-dependent inhibition of the root reflexes with the strongest effect on LLRs. However, the inhibitory effect of CPT is less potent in cSCI mice than in acute SCI (aSCI) mice, suggesting changes either in composition of CaV1.3 or other cellular mechanisms in cSCI mice. For intracellular recordings, the intrinsic plateau potentials, was observed in more motoneurons in cSCI mice than in aSCI mice. CPT inhibited the plateau potentials and reduced motoneuron firings evoked by intracellular current injection. These results suggest that the LLR is an important target and that CPT has potential in the therapy of SCI-induced muscle spasms.