Pain and learning in a spinal system: contradictory outcomes from common origins.

The long-standing belief that the spinal cord serves merely as a conduit for information traveling to and from the brain is changing. Over the past decade, research has shown that the spinal cord is sensitive to response-outcome contingencies, demonstrating that spinal circuits have the capacity to modify behavior in response to differential environmental cues. If spinally transected rats are administered shock contingent on leg extension (controllable shock), they will maintain a flexion response that minimizes shock exposure. If, however, this contingency is broken, and shock is administered irrespective of limb position (uncontrollable shock), subjects cannot acquire the same flexion response. Interestingly, each of these treatments has a lasting effect on behavior; controllable shock enables future learning, while uncontrollable shock produces a long-lasting learning deficit. Here we suggest that the mechanisms underlying learning and the deficit may have evolved from machinery responsible for the spinal processing of noxious information. Experiments have shown that learning and the deficit require receptors and signaling cascades shown to be involved in central sensitization, including activation of NMDA and neurokinin receptors, as well as CaMKII. Further supporting this link between pain and learning, research has also shown that uncontrollable stimulation results in allodynia. Moreover, systemic inflammation and neonatal hindpaw injury each facilitate pain responding and undermine the ability of the spinal cord to support learning. These results suggest that the plasticity associated with learning and pain must be placed in a balance in order for adaptive outcomes to be observed.

A new measure of hindlimb stepping ability in neonatally spinalized rats.

One of the most widely used animal models for assessing recovery of locomotor functioning is the spinal rat. Although true differences in locomotor abilities of these animals are exhibited during treadmill testing, current measurement techniques often fail to detect them. The HiJK (Hillyer-Joynes Kinematics) scale was developed in an effort to distinguish more effectively between groups of spinal rats. Scale items were compiled after extensive review of the literature concerning development and analysis of rat locomotion and a thorough examination of the current tools. Treadmill tests for 137 Sprague-Dawley rats were taped and scored. The structure of the scale was tested with principle components and factor analysis, in which six of the eight items accounted for 59% of the variance, while all eight accounted for 78%. Validity tests demonstrate that HiJK is measuring locomotor performance accurately and powerfully. First, the HiJK scale correlates highly (>.8) with the widely used BBB scale and second, as shown with ANOVA, can distinguish between different groups of spinal rats. Reliability of the scale was also analyzed. Cronbach's alpha was shown to be .91, indicating considerable internal consistency. Additionally, inter-rater and intra-rater reliabilities were substantial, with correlations for most items reaching above .80. We believe that the HiJK scale will help researchers verify existing experimental differences, advance the field of spinal cord research, and, hopefully, lead to discovery of methods to enhance recovery of function.

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.

Local anesthetic treatment significantly attenuates acute pain responding but does not prevent the neonatal injury-induced reduction in adult spinal behavioral plasticity.

Recent findings indicate that neonatal injury results in decreased spinal plasticity in adult subjects (E. E. Young, K. M. Baumbauer, A. E. Elliot, & R. L. Joynes, 2007). Previous research has shown that acute manipulations of pain processing (i.e., administration of formalin, carrageenan, capsaicin) result in a loss of spinal behavioral plasticity (A. R. Ferguson, E. D. Crown, & J. W. Grau, 2006). Moreover, neonatal injury results in a lasting reduction in adult spinally mediated plasticity resembling the deficit seen following acute manipulations in adults (E. E. Young et al., 2007). The present study was designed to determine whether the effects of neonatal injury could be prevented by lidocaine administration during the initial healing period. Subjects (injured or uninjured) received lidocaine or saline on 1 of 4 administration schedules (preinjury only, postinjury only, for 24 hr postsurgery, or for 72 hr postsurgery). Results demonstrated that lidocaine administration did not prevent the hypersensitivity and reduced spinal plasticity associated with neonatal injury. This suggests that (a) the mechanisms underlying neonatal injury are independent of peripheral input in the initial healing period and (b) lidocaine is ineffective at preventing long-term spinal plasticity changes following neonatal injury.

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.

Neonatal hind-paw injury disrupts acquisition of an instrumental response in adult spinal rats.

The present study was designed to evaluate the impact of neonatal injury on adult spinal plasticity in rats. Subjects were randomly assigned to 1 of 4 experimental conditions: (a) hind-paw injury at Postnatal Day (PD) 2, (b) hind-paw injury at PD 5, (c) anesthesia exposure only on PD 2, or (d) anesthesia exposure only on PD 5. Subjects receiving a unilateral neonatal hind-paw injury showed decreased mechanical threshold (hyperalgesia) on the previously injured hind paw throughout development. This decrease in threshold survived spinal transection (at T2) at 12 weeks of age. Injured subjects also showed significant impairment in a spinal instrumental learning task performed by the previously injured hind paw. This disruption of learning indicates a disruption of spinal plasticity that may be due to induction of long-term changes in nociceptive processing within the spinal cord.

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.

Lipopolysaccharide induces a spinal learning deficit that is blocked by IL-1 receptor antagonism.

Previous studies have shown that spinal neurons are capable of supporting a form of instrumental conditioning. Subjects receiving a spinal transection will learn to maintain a flexion response after exposure to shock contingent on leg position. In contrast, subjects receiving shock irrespective of leg position will not show increased flexion duration. Activation of the immune system has deleterious effects on learning in intact animals, but the impact of immune system activation on learning spinal animals is not known. We found that a large dose of i.p. LPS (1.0mg/kg) significantly disrupted the acquisition of the instrumental flexion response. The LPS-induced learning deficit was not prevented by preexposure to contingent shock (i.e. immunization) (Experiment 2). Co-administration of the iNOS inhibitor L-NIL (0.1, 1.0 and 10.0 microg/microL) failed to block the deficit (Experiment 3). Co-administration of an IL-1 receptor antagonist (r-metHuIL-1ra [10.0, 30.0 and 100.0 microg/microL) prevented the LPS-induced learning deficit when given in a dose of 100.0 microg/microL(i.t.) only (Experiment 4). Findings indicate a role for spinal IL-1 in the decreased plasticity following LPS administration.

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.

Instrumental learning within the spinal cord: VI. The NMDA receptor antagonist, AP5, disrupts the acquisition and maintenance of an acquired flexion response.

Prior studies have shown that circuits within the spinal cord can support a simple form of instrumental learning. Spinally transected rats are given shock to one hind leg whenever the leg is extended. This response-outcome contingency causes an increase in flexion duration. The present experiments examine whether the NMDA receptor is involved in the acquisition and maintenance of this instrumental response. Experiment 1 showed that the NMDA receptor antagonist 2-amino-5-phosphonovalerate acid (AP5) reduces instrumental responding in a dose-dependent fashion. Experiment 2 showed that AP5 given after training eliminates the increase in flexion duration. The results implicate the NMDA receptor in the acquisition and maintenance of spinally mediated instrumental behavior.

Instrumental learning within the spinal cord: III. Prior exposure to noncontingent shock induces a behavioral deficit that is blocked by an opioid antagonist.

Spinally transected rats given legshock whenever one hindleg is extended learn to maintain a flexion response that decreases net shock exposure. Prior exposure to response-independent (noncontingent) shock prevents learning. This behavioral deficit was eliminated by systemic administration of the nonselective opioid antagonist naltrexone (Experiment 1). The deficit was also blocked by intrathecal (i.t.) naltrexone at a dose of 7 microg/microl (Experiment 2). Noncontingent shock undermined behavioral potential for 24 h (Experiment 3). The expression of the deficit was blocked by naltrexone (7 microg/microl, i.t.) given prior to testing. The same dose prior to initial shock exposure had no effect. Administration of an antagonist that acts on the kappa opioid receptor (nor-BNI) restored learning (Experiment 4). Equal molar concentrations of antagonists that act on the micro (CTOP) or delta (naltrindole) receptor had no effect.

Effects of ontogeny on performance of rats in a novel object-recognition task.

The current experiment investigated ontogenetic forgetting on a novel object-recognition task similar to that of Besheer and Bevins. 18-day-old pups (n = 49) and adult (n = 29) rats were tested at two retention intervals (1 min. or 120 min.). By employing exclusion criteria which demanded minimum amounts of object exploration at training and test, the performance of 18-day-old pups but not that of adults was significantly impaired at 120 min. relative to 1 min. Analysis indicated that the ontogeny of the learning and memory measured in novel object recognition follows a developmental trend similar to that of other forms of learning, with older animals remembering more and thus performing better than younger animals. Unfortunately, given the extreme variability inherent to the task and large N necessary to achieve significance, the use of this task in studies of learning, memory, and development is discouraged.

Instrumental learning within the spinal cord: V. Evidence the behavioral deficit observed after noncontingent nociceptive stimulation reflects an intraspinal modification.

Spinally transected rats given leg shock whenever one hindlimb is extended learn to maintain the leg in a flexed position, which minimizes net shock exposure. Yoked rats, that receive an equal amount of shock independent of leg position (noncontingent shock), do not exhibit an increase in flexion duration. Yoked rats also fail to learn when response contingent shock is applied to the previously shocked leg, a behavioral deficit that resembles learned helplessness. This deficit could reflect either a peripheral (e.g. muscle fatigue) or central effect. Experiment 1 showed that spinalized rats given noncontingent shock to one hind limb fail to learn when response-contingent shock is applied to the contralateral leg. Experiment 2 demonstrated that blocking the afferent input to the spinal cord, by cutting the sciatic nerve, blocked the development of the deficit. Experiment 3 found that intrathecal lidocaine has a protective effect and prevents the deficit. These findings suggest that noncontingent nociceptive stimulation induces an intraspinal modification that undermines behavioral potential.

Instrumental learning within the spinal cord: IV. Induction and retention of the behavioral deficit observed after noncontingent shock.

Spinalized rats given shock whenever 1 hind leg is extended learn to maintain that leg in a flexed position, a simple form of instrumental learning. Rats given shock independent of leg position do not exhibit an increase in flexion duration. Experiment 1 showed that 6 min of intermittent legshock can produce this deficit. Intermittent tailshock undermines learning (Experiments 2-3), and this effect lasts at least 2 days (Experiment 4). Exposure to continuous shock did not induce a deficit (Experiment 5) but did induce antinociception (Experiment 6). Intermittent shock did not induce antinociception (Experiment 6). Experiment 7 addressed an alternative interpretation of the results, and Experiment 8 showed that presenting a continuous tailshock while intermittent legshock is applied can prevent the deficit.

Instrumental learning within the spinal cord. II. Evidence for central mediation.

Rats spinally transected at the second thoracic vertebra can learn to maintain their leg in a flexed position if they receive legshock for extending the limb. These rats display an increase in the duration of a flexion response that minimizes net shock exposure. The current set of experiments was designed to determine whether the acquisition of this behavioral response is mediated by the neurons of the spinal cord (i.e., is centrally mediated) or reflects a peripheral modification (e.g., a change in muscle tension). Experiment 1 found that preventing information from reaching the spinal cord by severing the sciatic nerve blocked the acquisition of this behavioral response. Spinalized rats also failed to learn if the spinal cord was anesthetized with lidocaine during exposure to response-contingent shock (Experiment 2). Experiment 3 demonstrated that prior exposure to response-contingent shock on one hindleg facilitated acquisition of the response when subjects were later tested on the opposite leg. These findings suggest that acquisition of the instrumental response depends on neurons within the spinal cord.