Evidence That the Central Nervous System Can Induce a Modification at the Neuromuscular Junction That Contributes to the Maintenance of a Behavioral Response.

Neurons within the spinal cord are sensitive to environmental relations and can bring about a behavioral modification without input from the brain. For example, rats that have undergone a thoracic (T2) transection can learn to maintain a hind leg in a flexed position to minimize exposure to a noxious electrical stimulation (shock). Inactivating neurons within the spinal cord with lidocaine, or cutting communication between the spinal cord and the periphery (sciatic transection), eliminates the capacity to learn, which implies that it depends on spinal neurons. Here we show that these manipulations have no effect on the maintenance of the learned response, which implicates a peripheral process. EMG showed that learning augments the muscular response evoked by motoneuron output and that this effect survives a sciatic transection. Quantitative fluorescent imaging revealed that training brings about an increase in the area and intensity of ACh receptor labeling at the neuromuscular junction (NMJ). It is hypothesized that efferent motoneuron output, in conjunction with electrical stimulation of the tibialis anterior muscle, strengthens the connection at the NMJ in a Hebbian manner. Supporting this, paired stimulation of the efferent nerve and tibialis anterior generated an increase in flexion duration and augmented the evoked electrical response without input from the spinal cord. Evidence is presented that glutamatergic signaling contributes to plasticity at the NMJ. Labeling for vesicular glutamate transporter is evident at the motor endplate. Intramuscular application of an NMDAR antagonist blocked the acquisition/maintenance of the learned response and the strengthening of the evoked electrical response.SIGNIFICANCE STATEMENT The neuromuscular junction (NMJ) is designed to faithfully elicit a muscular contraction in response to neural input. From this perspective, encoding environmental relations (learning) and the maintenance of a behavioral modification over time (memory) are assumed to reflect only modifications upstream from the NMJ, within the CNS. The current results challenge this view. Rats were trained to maintain a hind leg in a flexed position to avoid noxious stimulation. As expected, treatments that inhibit activity within the CNS, or disrupt peripheral communication, prevented learning. These manipulations did not affect the maintenance of the acquired response. The results imply that a peripheral modification at the NMJ contributes to the maintenance of the learned response.

Acute theophylline exposure modulates breathing activity through a cervical contusion.

Cervical spinal contusion injuries are the most common form of spinal cord injury (>50%) observed in humans. These injuries can result in the impaired ability to breathe. In this study we examine the role of theophylline in the rescue of breathing behavior after a cervical spinal contusion. Previous research in the C2 hemisection model has shown that acute administration of theophylline can rescue phrenic nerve activity and diaphragmatic EMG on the side ipsilateral to injury. However, this effect is dependent on intact and uninjured pathways. In this study we utilized a cervical contusion injury model that more closely mimics the human condition. This injury model can determine the effectiveness of therapeutic interventions, in this case theophylline, on the isolated contused pathways of the spinal cord. Three weeks after a 150 kD C3/4 unilateral contusion subjects received a 15 mg/kg dose of theophylline prior to a contralateral C2 hemisection. Subjects that received theophylline were able to effectively utilize damaged pathways to breathe for up to 2 min, while subjects treated with saline were unable to support ventilation. Through these experiments, we demonstrate that theophylline can make injured pathways that mediate breathing more effective and therefore, suggest a potential therapeutic role in the critical time points immediately after injury.

Metaplasticity and behavior: how training and inflammation affect plastic potential within the spinal cord and recovery after injury.

Research has shown that spinal circuits have the capacity to adapt in response to training, nociceptive stimulation and peripheral inflammation. These changes in neural function are mediated by physiological and neurochemical systems analogous to those that support plasticity within the hippocampus (e.g., long-term potentiation and the NMDA receptor). As observed in the hippocampus, engaging spinal circuits can have a lasting impact on plastic potential, enabling or inhibiting the capacity to learn. These effects are related to the concept of metaplasticity. Behavioral paradigms are described that induce metaplastic effects within the spinal cord. Uncontrollable/unpredictable stimulation, and peripheral inflammation, induce a form of maladaptive plasticity that inhibits spinal learning. Conversely, exposure to controllable or predictable stimulation engages a form of adaptive plasticity that counters these maladaptive effects and enables learning. Adaptive plasticity is tied to an up-regulation of brain derived neurotrophic factor (BDNF). Maladaptive plasticity is linked to processes that involve kappa opioids, the metabotropic glutamate (mGlu) receptor, glia, and the cytokine tumor necrosis factor (TNF). Uncontrollable nociceptive stimulation also impairs recovery after a spinal contusion injury and fosters the development of pain (allodynia). These adverse effects are related to an up-regulation of TNF and a down-regulation of BDNF and its receptor (TrkB). In the absence of injury, brain systems quell the sensitization of spinal circuits through descending serotonergic fibers and the serotonin 1A (5HT 1A) receptor. This protective effect is blocked by surgical anesthesia. Disconnected from the brain, intracellular Cl(-) concentrations increase (due to a down-regulation of the cotransporter KCC2), which causes GABA to have an excitatory effect. It is suggested that BDNF has a restorative effect because it up-regulates KCC2 and re-establishes GABA-mediated inhibition.

AMPA receptor mediated behavioral plasticity in the isolated rat spinal cord.

Previous research has demonstrated that the spinal cord is capable of a simple form of instrumental learning. Spinally transected rats that receive shock to a hind leg in an extended position quickly learn to maintain the leg in a flexed position, reducing net shock exposure whenever that leg is flexed. Subjects that receive shock independent of leg position (uncontrollable shock) do not exhibit an increase in flexion duration and later fail to learn when tested with controllable shock (learning deficit). The present study examined the role of the ionotropic glutamate receptor α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in spinal learning. Intrathecal application of the AMPA receptor antagonist CNQX disrupted performance of a spinal instrumental learning in a dose dependent fashion (Experiment 1). CNQX also disrupted the maintenance of the instrumental response (Experiment 2) and blocked the induction of the learning deficit (Experiment 3). Intrathecal application of the agonist AMPA had a non-monotonic effect, producing a slight facilitation of performance at a low dose and disrupting learning at a high concentration (Experiment 4). Within the dose range tested, intrathecal application of AMPA did not have a long-term effect (Experiment 5). The results suggest that AMPA-mediated transmission plays an essential role in both instrumental learning and the induction of the learning deficit.

Maladaptive spinal plasticity opposes spinal learning and recovery in spinal cord injury.

Synaptic plasticity within the spinal cord has great potential to facilitate recovery of function after spinal cord injury (SCI). Spinal plasticity can be induced in an activity-dependent manner even without input from the brain after complete SCI. A mechanistic basis for these effects is provided by research demonstrating that spinal synapses have many of the same plasticity mechanisms that are known to underlie learning and memory in the brain. In addition, the lumbar spinal cord can sustain several forms of learning and memory, including limb-position training. However, not all spinal plasticity promotes recovery of function. Central sensitization of nociceptive (pain) pathways in the spinal cord may emerge in response to various noxious inputs, demonstrating that plasticity within the spinal cord may contribute to maladaptive pain states. In this review we discuss interactions between adaptive and maladaptive forms of activity-dependent plasticity in the spinal cord below the level of SCI. The literature demonstrates that activity-dependent plasticity within the spinal cord must be carefully tuned to promote adaptive spinal training. Prior work from our group has shown that stimulation that is delivered in a limb position-dependent manner or on a fixed interval can induce adaptive plasticity that promotes future spinal cord learning and reduces nociceptive hyper-reactivity. On the other hand, stimulation that is delivered in an unsynchronized fashion, such as randomized electrical stimulation or peripheral skin injuries, can generate maladaptive spinal plasticity that undermines future spinal cord learning, reduces recovery of locomotor function, and promotes nociceptive hyper-reactivity after SCI. We review these basic phenomena, how these findings relate to the broader spinal plasticity literature, discuss the cellular and molecular mechanisms, and finally discuss implications of these and other findings for improved rehabilitative therapies after SCI.

Impact of behavioral control on the processing of nociceptive stimulation.

How nociceptive signals are processed within the spinal cord, and whether these signals lead to behavioral signs of neuropathic pain, depends upon their relation to other events and behavior. Our work shows that these relations can have a lasting effect on spinal plasticity, inducing a form of learning that alters the effect of subsequent nociceptive stimuli. The capacity of lower spinal systems to adapt, in the absence of brain input, is examined in spinally transected rats that receive a nociceptive shock to the tibialis anterior muscle of one hind leg. If shock is delivered whenever the leg is extended (controllable stimulation), it induces an increase in flexion duration that minimizes net shock exposure. This learning is not observed in subjects that receive the same amount of shock independent of leg position (uncontrollable stimulation). These two forms of stimulation have a lasting, and divergent, effect on subsequent learning: controllable stimulation enables learning whereas uncontrollable stimulation disables it (learning deficit). Uncontrollable stimulation also enhances mechanical reactivity. We review evidence that training with controllable stimulation engages a brain-derived neurotrophic factor (BDNF)-dependent process that can both prevent and reverse the consequences of uncontrollable shock. We relate these effects to changes in BDNF protein and TrkB signaling. Controllable stimulation is also shown to counter the effects of peripheral inflammation (from intradermal capsaicin). A model is proposed that assumes nociceptive input is gated at an early sensory stage. This gate is sensitive to current environmental relations (between proprioceptive and nociceptive input), allowing stimulation to be classified as controllable or uncontrollable. We further propose that the status of this gate is affected by past experience and that a history of uncontrollable stimulation will promote the development of neuropathic pain.

The neonatal injury-induced spinal learning deficit in adult rats: central mechanisms.

Previous research has shown that small injuries early in development can alter adult pain reactivity and processing of stimuli presented to the side of injury. However, the mechanisms involved and extent of altered adult spinal function following neonatal injury remain unclear. The present experiments were designed to 1) determine whether the effects of neonatal injury affect processing contralateral to the injury and 2) evaluate the role of cells expressing the NK1 receptor, shown to be involved in central sensitization in adults, in the negative effects of neonatal injury. The present findings indicate that the effects of neonatal injury are primarily isolated to the injured hind limb and do not result in a bilateral alteration in adult spinal function. In addition, the effects of neonatal injury appear to be partially dependent on cells expressing the NK1 receptor as ablating these cells at the time of injury or in adulthood results in attenuation of the neonatal injury-induced spinal learning deficit.

Neurokinin receptors modulate the impact of uncontrollable stimulation on adaptive spinal plasticity.

Previous research has demonstrated that spinally transected rats can acquire a prolonged flexion response to prevent the delivery of shock. However, rats that receive shock irrespective of leg position cannot learn to maintain the same response. The present experiments examined the role of neurokinin receptors in this learning deficit. Results demonstrated that neurokinin (NK1 and NK2) antagonists blocked the induction of the learning deficit, whereas NK agonists induced a learning deficit. The study found that NK agonist administration did not substitute for uncontrollable shock exposure. Finally, administration of an NK1 agonist prior to uncontrollable shock prevented the induction of the deficit. These results provide additional evidence that engaging nociceptive plasticity undermines the capability of spinal neurons to support adaptive changes.

Administration of a Ca-super(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor prevents the learning deficit observed in spinal rats after noncontingent shock administration.

Research has shown that spinal rats given shock to the hind leg when it is in an extended position (contingent shock) will learn to maintain a flexion response. However, subjects that experience shock irrespective of leg position (noncontingent shock) do not exhibit this learning. The current studies examined the role of Ca-super(2+)/calmodulin-dependent protein kinase II (CaMKII) in this learning deficit. Subjects were given intrathecal injections of CaMKII inhibitor solution or artificial cerebrospinal fluid (aCSF) 15 min prior to and immediately or 4 hr following noncontingent shock training. Results demonstrate that the CaMKII inhibitor successfully reversed the learning deficit when injected prior to and immediately following training. These results indicate the importance of CaMKII in the learning deficit present in spinal animals trained with noncontingent shock.

Intrathecal administration of neurokinin 1 and neurokinin 2 receptor antagonists undermines the savings effect in spinal rats seen in an instrumental learning paradigm.

Research has demonstrated that the isolated spinal cord is capable of modifying its behavior in response to changes in environmental stimuli. Previous studies have shown that rats with complete thoracic spinal transections can learn to maintain a flexion response when shock delivery is paired with leg position. The current experiments examined whether neurokinin (NK) 1 and 2 receptors are involved in the acquisition and retention of this prolonged flexion response. Results demonstrated that L-703,606 (NK1 antagonist) facilitated response acquisition, whereas MEN-10,376 (NK2 antagonist) hindered acquisition. Furthermore, pretraining administration of either antagonist undermined subjects' ability to reacquire the prolonged flexion response during testing. These results demonstrate the importance of NK receptors in spinally mediated behavioral plasticity.

Intrathecal infusions of anisomycin impact the learning deficit but not the learning effect observed in spinal rats that have received instrumental training.

Previous research has shown that spinally transected rats will learn to maintain a flexion response when administered shock contingent upon leg position. In short, a contingency is arranged between shock delivery and leg extension so that Master rats exhibit an increase in flexion duration that lasts throughout the training session. Furthermore, when Master rats are later tested they reacquire the flexion response in fewer trials, indicative of some savings. As a control, a second group of spinal rats (Yoked rats) are given shock irrespective of leg position (noncontingent shock). These animals fail to show the same increase in leg flexion duration. Interestingly, when Yoked rats are later tested with a shock contingency in place, they still fail to learn (learning deficit). The present experiments were designed to determine whether both forms of instrumental learning in spinal animals require de novo protein synthesis. As such, we administered various doses of anisomycin intrathecally prior to training. Additionally, spinal rats were trained and tested either immediately or 24 h after test. We found that only the highest dose of anisomycin (125 microg/microl) had an effect in Yoked animals that were tested 24 h after training. Specifically, the highest dose of anisomycin reversed the learning deficit in those animals. Moreover, anisomycin had a similar effect when administered prior to training and immediately following training, but not 6 h after training. Finally, the results demonstrated that the observed effect of anisomycin was not due to state-dependency.