Preserving and Restoring Behavioral Potential Within the Spinal Cord Using an Instrumental Training Paradigm

Eric D. Crown and James W. Grau

Department of Psychology, Texas A&M University, College Station, Texas 77843


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Crown, Eric D. and James W. Grau. Preserving and Restoring Behavioral Potential Within the Spinal Cord Using an Instrumental Training Paradigm. J. Neurophysiol. 86: 845-855, 2001. We have shown that spinal cord neurons can support a simple form of instrumental learning. In a typical experiment, rats are spinalized at the second thoracic vertebra (T2) and given shock to one hindleg. One group (master) receives shock whenever the leg is extended. This response-contingent shock causes an increase in response duration that decreases net shock exposure. This instrumental learning is not observed in yoked controls that receive the same amount of shock independent of leg position (noncontingent shock). Interestingly, rats that have received noncontingent shock also fail to learn when they are subsequently exposed to response-contingent shock on either the ipsilateral or contralateral leg. Just 6 min of noncontingent nociceptive stimulation, applied to the leg or tail, undermines behavioral potential for up to 48 h. The present experiments explore whether a behavioral therapy can prevent and/or reverse this deficit. In experiment 1, spinalized rats received 30 min of training with contingent shock, noncontingent shock, or nothing prior to noncontingent tailshock. They were then tested with contingent shock to the contralateral hindleg. Rats that had received noncontingent shock alone failed to learn. Prior exposure to contingent shock had an immunizing effect that prevented the deficit. Experiment 2 examined whether training with contingent shock after noncontingent shock exposure would restore behavioral potential. To facilitate performance during contingent shock training, subjects were given an intrathecal injection of the opioid antagonist naltrexone, a drug treatment that temporarily blocks the expression of the behavioral deficit. Twenty-four hours later subjects were tested with contingent shock on either the ipsilateral or contralateral leg. We found that naltrexone combined with contingent shock therapy restored spinal cord function. Naltrexone alone had no effect. The results suggest that noncontingent nociceptive stimulation can undermine behavioral potential after spinal cord injury and that instrumental training can help preserve, and protect, spinal cord function.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Prior studies have shown that the isolated mammalian spinal cord is quite plastic and that it can support a range of behavioral phenomena, including single stimulus learning [habituation and sensitization (Groves and Thompson 1970)], Pavlovian conditioning (Durkovic 1975; Durkovic and Damianopoulos 1986; Joynes and Grau 1996; Patterson et al. 1973), and instrumental learning (Chopin and Buerger 1976; Grau et al. 1996, 1998; Segal and Wolf 1994). Spinally transected (spinalized) animals can also be taught to step along a moving treadmill (Edgerton et al. 1997; Grillner and Dubuc 1988; Hodgson et al. 1994; Rossignol et al. 1999). Interestingly, a spinal cat that is stepping on a treadmill can learn to exhibit a stronger flexion response to minimize contact with an obstacle (Hodgson et al. 1994; Nakada et al. 1994), an observation that suggests that instrumental (response-reinforcer) learning can affect the locomotive system.

Our laboratory has been exploring the capacity for instrumental learning within the spinal cord using a procedure developed by Horridge (1962). The apparatus is illustrated in Fig. 1A. Spinalized rats rest comfortably in an opaque restraining tube. To prevent forelimb activity from introducing variability in hindleg position, a belt is used to stabilize the hindquarters. Shock is applied through intracutaneous electrodes at an intensity that elicits a vigorous (0.4-0.6 N) upward movement of the foot. To minimize lateral movements, the leg is stabilized using a piece of adhesive tape. Under these conditions, legshock elicits a flexion at the ankle joint that decreases the angle between the tibia and the foot. Because the tape maintains the tibia in a relatively fixed position, the angular displacement is translated into a change in tarsal joint angle. Tarsal joint angle can be monitored using a contact electrode that is taped to the plantar surface of the foot. Importantly, the contact electrode is electrically insulated from the subject. A saline solution is placed under the subject and the height of the solution is adjusted so that the contact electrode (at rest) is submerged 4 mm. Whenever the contact electrode touches the saline solution, it completes a circuit that is monitored by a computer. This simple apparatus allows us to vary the response criterion (solution height) and monitor a number of behavioral parameters (Fig. 1B), including response number, time in solution, and response duration.1



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Fig. 1. A: the apparatus used to apply legshock and monitor foot position. B, bottom: the behavioral measures monitored during instrumental training. The function depicts a snapshot of behavior during training and represents the location (in mm) of the contact electrode tip as a function of time (in seconds). The relative position of the foot is illustrated above the graph. The flexion counter is incremented each time the contact electrode leaves the solution. Average response duration is derived every 60 s using the following formula: response duration = (60 - time in solution)/(response number + 1). C: drawing that simulates foot position during training using the master-yoke paradigm. Master rats receive response-contingent shock. Yoked rats receive shock at the same time, and for the same duration, as the master rats but the shock occurs independent of foot position (noncontingent shock). Thus master rats receive shock whenever their foot reaches a particular position (defined by the response criterion) while yoked rats experience a variable relation between foot position and shock onset (top). Response-contingent shock causes a progressive increase in response duration in master subjects (bottom) that decreases net shock exposure. In the absence of a regular relation between leg position and shock onset, yoked rats fail to exhibit an increase in response duration.

It has been previously shown that a reactive system, which is incapable of instrumental learning, can produce systematic changes in both response number and time in solution (Church and Lerner 1976; Grau et al. 1998). Instrumental learning can, however, be inferred from changes in response duration, and consequently we focus on this behavioral measure. Other behavioral indices (initial response force and duration, response number) are reported to evaluate the impact of our experimental manipulations on instrumental performance.

Elsewhere we have discussed the defining characteristics of instrumental learning (Grau 2000; Grau et al. 1998). These criteria are designed to establish whether a neural system is sensitive to response-reinforcer relations. To evaluate whether neurons within the spinal cord can meet these criteria, we introduce a response (foot position)-outcome (shock) contingency by applying shock to subjects in one group (master) whenever the tip of the contact electrode falls below the response criterion (Fig. 1C). We have shown that this training brings about an increase in response duration (illustrated in Fig. 1C, bottom) and that the critical outcome in this paradigm is shock onset (Grau et al. 1998). A critic could charge, however, that this increase in response duration reflects an unconditioned (unlearned) reaction that has nothing to do with the instrumental relation between foot position and shock onset. To address this possibility, a yoked control is included. Each yoked rat is experimentally coupled to a master subject and receives shock at exactly the same time, and for the same duration, as the master subject. But for the yoked rat, shock occurs independent of foot position; there is no contingency between foot position (the response) and shock onset (the outcome). At the start of training, the yoked rat will, by chance, sometimes receive shock when the foot is up and at other times when the foot is down. From the performance of the yoked rat, we can derive the consequence of exposure to the outcome alone, independent of foot position. Because only the master group exhibits an increase in response duration (Fig. 1C, bottom), we can infer that the response-outcome relation matters (Grau et al. 1996, 1998), a defining characteristic of instrumental learning. Manipulations of response-outcome contiguity lead to a similar conclusion (Grau et al. 1998).

To demonstrate that these behavioral differences reflect learning requires additional steps. In particular, we need to show that exposure to the response-outcome relation has a lasting effect on behavior and that the behavioral differences can be observed when subjects are tested under common conditions (Grau et al. 1998; Rescorla 1988). We have addressed these issues by testing master, yoked, and unshocked rats with response-contingent shock applied to either the same (ipsilateral) or opposite (contralateral) leg. We found that prior exposure to contingent shock (the master condition) facilitates learning relative to a group that was previously unshocked. Conversely, prior exposure to noncontingent shock (the yoked condition) undermines the capacity for instrumental learning. In both cases, prior training affects learning when contingent shock is applied to the contralateral leg. This transfer from one leg to the other suggests a common system, involving neurons within the spinal cord, must mediate both effects.

Further evidence for spinal mediation is provided by studies showing that transection of the sciatic nerve prevents both the acquisition of the instrumental response and the induction of the behavioral deficit (Ferguson et al. 1999). We have also demonstrated that the acquisition of the instrumental response is prevented by intrathecal application of either the local anesthetic lidocaine or the N-methyl-D-aspartate (NMDA) antagonist APV (Joynes et al. 1995). Similarly, the expression of the behavioral deficit can be prevented by the intrathecal application of either an opioid antagonist (Joynes and Grau 1998) or the GABA-A antagonist bicuculline (Ferguson et al. 2000). Together, these results suggest that both the acquisition of the instrumental response and the expression of the behavioral deficit depend on neurons within the spinal cord. Casually speaking, it is as if contingent shock enables behavioral potential within the spinal cord while noncontingent shock has a disabling effect on spinal plasticity.

We soon recognized that the loss of behavioral function associated with noncontingent shock exposure could have important clinical implications. For example, it could hurt the recovery of spinally mediated locomotive function (Edgerton et al. 1997; Grillner and Dubuc 1988; Hodgson et al. 1994; Rossignol et al. 1999; Wernig et al. 1995, 1998). It could also hurt the development of new functional connections from spared fibers or neuronal implants (Cheng et al. 1996).

For these reasons, additional studies were performed to characterize the behavioral deficit. We have shown that the deficit can also be produced using nociceptive stimuli applied to the tail through extracutaneous electrodes (Grau and Joynes 2001a,b; E. D. Crown, R. L. Joynes, A. R. Ferguson, and J. W. Grau, unpublished data). Moreover, just 6 min of intermittent tailshock induces a behavioral deficit that lasts up to 48 h, and a longer-lasting effect can be induced by increasing the duration of intermittent shock exposure. Parametric studies have shown that the behavioral deficit emerges at a shock intensity known to engage nociceptive mechanisms within the spinal cord. Indeed our inducing stimulus (constant current AC shock) is routinely used to study nociceptive mechanisms and both its functional consequences and psychophysical characteristics have been well characterized (King et al. 1996; Meagher et al. 1990, 2001; Terman et al. 1984; Watkins and Mayer 1986).

Given the potential clinical significance of the behavioral deficit, we sought procedures that could be used to prevent its development and restore behavioral potential after the deficit has been induced. In the present study, we show that instrumental training has a beneficial effect that can protect spinal neurons (experiment 1) and, in combination with a pharmacological manipulation, help restore behavioral potential after the deficit has been induced (experiment 2).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects

The subjects were male Sprague-Dawley rats (Rattus norvegicus) obtained from Harlan (Houston, TX). The rats were approximately 100-120 days old (400-480 g) and were individually housed with food and water continuously available. Rats were maintained on a 12-h light-dark cycle and were generally tested during the last 6 h of the light cycle. Each experiment used eight subjects per cell.

Surgery

All rats were anesthetized with pentobarbital (50 mg/kg) before being shaved both on the back and on the hindlimbs. A spinal transection was made at the second thoracic vertebra (T2) as described in Grau et al. (1998). Briefly, the tissue in front of T2 was cleared away, and the cord was transected by cauterization. The exposed cord was then covered with oxidized cellulose (Oxycel, Parke-Davis), and the wound was closed with Michel clips. Rats were maintained in a temperature-controlled environment (approximately 25.5°C) during recovery and testing. To prevent injury to the hindlimbs during recovery, the rat's rear legs were maintained in a normal flexed position by a piece of porous tape [Orthaletic, 1.3 cm (width)] that was gently wrapped once around the rat's body.

Subjects in experiment 2 had intrathecal cannulae lowered into the lumbar region of the spinal cord following the procedure of Yaksh and Rudy (1976). After the spinal cord was transected, a segment of polyurethane tubing (25 cm; PE-10) fitted with a 0.23-cm (diameter) stainless steel wire (SWGX-090, Small Parts) was inserted 9 cm down the spinal cord. The tubing was inserted into the subarachnoid space, between the dura and the white matter, so as to lie on the dorsal surface of the cord. The exposed end of the tubing was secured to the adjacent tissue using an adhesive (Superglue). The wire was then pulled from the tubing and the wound caudal to the exposed length of tubing was closed using Michel clips.

During recovery, hydration was maintained with supplemental injections of saline, and the rat's bladders were expressed at regular intervals. At the end of behavioral testing, subjects were killed with pentobarbital (100 mg/kg).

Transections were confirmed by inspecting the cord during the operation, observing the behavior of the subjects after they have recovered to insure that they exhibited paralysis below the level of the forepaws and did not vocalize to leg or tail shock, and examining the spinal cord postmortem in a randomly selected subset of the subjects.

Apparatus

Instrumental training was conducted while spinal rats were loosely restrained in tubes [23.5 cm (length) × 8 cm ID; see Fig. 1A]. Two slots [5.6 cm (l) × 1.8 cm (w)] were cut 4 cm apart, 1.5 cm from the end of the tube, allowing both hind legs to hang freely. Legshock was applied by attaching one lead from a BRS/LVE shock generator (Model SG-903) to a stainless steel wire inserted through the skin over the tibia 1.5 cm from the tarsals. The other lead was attached to a 2.5-cm stainless steel pin that was inserted 0.4 cm into the tibialis anterior muscle 1.7 cm above the other electrode.

Leg position was monitored using a contact electrode constructed from a 7-cm 0.018-in stainless steel rod that was taped to the foot. The portion of the rod that contacted the rat was electrically insulated from the foot with a 2.5-cm section of heat shrink tubing. A fine wire [0.01 sq. mm (36 AWG)] was attached to the end of the rod. This wire (20 cm) extended from the rear of the foot and was connected to a digital input monitored by a Macintosh computer. The insulated rod was taped to the plantar surface of the rat's foot using approximately 8-cm of porous tape (Orthaletic, 1.3 cm) with the end positioned directly in front of the plantar protuberance. A plastic rectangular dish [11.5 (w) × 19 (l) × 5 (d)] containing a NaCl solution was placed approximately 7.5 cm below the restraining tube. A drop of detergent was added to the solution to reduce surface tension. A ground wire was connected to a 1-mm stainless steel rod that was placed in the solution. When the contact electrode attached to the rat's paw touched the solution, it completed the circuit monitored by the computer, delivering shock to the tibialis anterior muscle. The state of this circuit was sampled at a rate of 30 times/s.

Flexion force was measured by attaching a monofilament plastic line ("4-lb test" Stren, Dupont) to the rat's foot immediately behind the plantar protuberance. The 40-cm length of line was passed through an eyelet attached to the apparatus directly under the paw, 16 cm beneath the base of the tube. The end of the line was attached to a strain gauge that was fastened to a ringstand. After the line was connected to the rat's paw, the ringstand was positioned so that the line was taut, just barely engaging the gauge. The strain gauge has been calibrated by determining the relationship between voltage and force in Newtons.

A 660-V transformer was employed to generate the tail shocks. These shocks were administered through electrodes constructed from a modified fuse clip that was covered with electrode paste and taped to the rat's tail approximately 15 cm from the tip. A computer was used to control the onset and offset of tailshock. The shocks were 80 ms in duration and occurred on a variable time schedule with a mean of 2 s (range: 0.2-3.8 s).

Behavioral procedures

The behavioral procedures were initiated approximately 24 h after surgery. Before the rats were placed in the restraining tubes, their rear legs were shaved and marked for placement of the shock leads. The wire electrode was then inserted over the tibia at the distal mark and the rats were placed in the restraining tubes. Next, the contact electrode used to monitor leg position was taped to the paw. To minimize lateral movements of the tibia and fibula, a 20-cm piece of porous tape (Orthaletic, 1.3 cm) was wrapped around the ankle and taped to a bar extending across the apparatus directly under the front panel of the restraining tube. Next one lead from the shock generator was attached to the stainless steel wire inserted over the tibia. The shock generator was set to deliver a 0.1-mA shock, and the region over the second mark was probed to find a site that elicited a vigorous flexion response. The pin was then inserted perpendicular to the body into the tibialis anterior muscle. After the line connected to the strain gauge was placed over the rat's paw, we verified that a single intense (1.6 mA) test shock (0.3 s) elicited a flexion response of at least 0.8 N. Shock intensity was then adjusted so that a 0.3-s shock produced a flexion force of 0.4 N. The plastic line was then removed. Finally, three 0.15-s legshocks were administered, spaced about 1 s apart, to establish the tarsus' resting position, and the height of the solution was adjusted so that the tip of the rod lay 4 mm below the surface.

EXPERIMENT 1. Forty-eight rats were divided into six groups (n = 8) for experiment 1. After receiving a spinal transection, rats were placed in the tubes. In phase 1, shock electrodes were attached to their leg, and the shock intensity needed to induce a 0.4 N change in flexion force was set. A third of the subjects (master) was exposed to 30 min of training during which they received legshock for contacting the underlying salt solution (response-contingent shock). Another group (yoked) was yoked to these subjects and received the same amount of legshock independent of leg position (noncontingent shock). The last third served as the unshocked controls (see Fig. 2). Immediately following master-yoke training or restraint, tail electrodes were attached, and half of the animals in each condition were given 6 min of noncontingent tailshock while the other half remained unshocked (phase 2). The tail electrodes were then removed, and all rats were tested under common conditions during a 30-min interval in which response-contingent legshock was applied to the previously untreated (contralateral) leg. Prior to testing, both flexion force (0.4 N) and contact electrode depth (4 mm) were equated across subjects.

EXPERIMENT 2. Forty-eight rats were used in experiment 2. Following spinal transection, rats were placed in the tubes and tail electrodes were attached. In phase 1, 40 subjects were given noncontingent tailshock as described in the preceding text. The remaining subjects were prepared in the same fashion but were unshocked (see Fig. 2). Prior to phase 2 treatment (master, yoke, or nothing), all rats received naltrexone treatment. A Hamilton syringe containing 1 µl of naltrexone (7 µg/µl) was inserted into the exposed end of the catheter, and the drug was administered over a period of 30 s, followed by a 20-µl saline flush over a period of 3 min. Previous research in our laboratory has shown that this drug treatment can temporarily eliminate the behavioral deficit observed after noncontingent shock (Joynes and Grau 1998). Once the shock electrodes were attached to the rat's leg and the shock intensity necessary to induce a 0.4 N change in flexion force had been established, 16 of the previously shocked rats were given contingent legshock (shocked right-arrow master) while another 16 received the same amount of shock independent of leg position (shocked right-arrow yoked). The remaining 16 subjects (8 shocked and 8 unshocked) remained unshocked during the phase 2 treatment, yielding the two additional experimental conditions (shocked right-arrow unshocked and unshocked right-arrow unshocked). Subjects were tested 24 h later, after the drug had cleared the system (Joynes and Grau 1998). Rats were returned to the tubes, and flexion force and electrode depth were equated both across subjects and test leg. The capacity for instrumental learning was then assessed by exposing the rats to 30 min of response-contingent legshock. Half of the subjects (n = 8) in the shocked right-arrow master and shocked right-arrow yoked groups were tested with contingent shock applied to the previously treated (ipsilateral) leg while the remaining subjects were tested on the untreated (contralateral) leg. For rats that did not receive master-yoke training (unshocked right-arrow unshocked, shocked right-arrow unshocked), both legs were unshocked during the phase 2 treatment. For these subjects, the assignment of contralateral versus ipsilateral was arbitrary, and there was no functional difference between the two conditions. Half of the subjects in these conditions were tested with contingent shock applied to the left hind leg while the remaining half were tested with contingent shock applied to the right hind leg. For the purpose of the data analyses, half of the rats in each of these conditions were randomly assigned to the contralateral condition and the remaining half were assigned to the ipsilateral condition.

For both experiments, noncontingent tailshock was applied using a program that emulated the master-yoke shock schedule. This program administered 80-ms shocks on a variable time schedule that ranged from 0.2 to 3.8 s (mean = 2 s). We have shown that this shock schedule produces a behavioral deficit that lasts 24-48 h (Grau and Crown 2000).

Behavioral measures

Three behavioral measures were used to monitor performance: time in solution, response number, and response duration (see Grau et al. 1998) (Fig. 2). The computer recorded when the contact electrode touched the underlying solution (time in solution). Whenever the electrode left the solution, the number of responses was increased by 1 (response number). To obtain a measure of performance over time, we divided the session into 30, 1-min time bins.



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Fig. 2. The experimental designs for experiments 1 (top) and 2 (bottom). Each experiment involved 2 pretreatment phases and a test phase. In experiment 1, spinalized rats received contingent legshock (master), noncontingent legshock (yoked), or no shock (unshocked) prior to noncontingent tailshock or no shock. In experiment 2, the order of treatment was reversed. Following noncontingent tailshock or an equivalent period of restraint, subjects were trained with contingent legshock (master), experienced an equivalent amount of noncontingent legshock (yoked), or remained unshocked. Immediately before this training episode, rats were given an intrathecal injection of naltrexone. In both experiments, rats were tested under common conditions with 30 min of response-contingent legshock. In experiment 1, contingent shock was applied to the untreated (contralateral) leg. In experiment 2, half of the subjects were tested on the contralateral leg while the remaining subjects were tested on the pretreated (ipsilateral) leg. Testing was delayed for 24 h in experiment 2 to allow the drug to clear the system.

We have previously shown how instrumental learning can be distinguished from a reactive system that is insensitive to response-outcome [reinforcer] relations (Grau et al. 1998). A key difference concerns response duration; only the instrumental account anticipates that contingent shock will produce a progressive increase in response duration at the tarsal joint. Response duration was derived from time in solution and response number using the following equation: response durationi = (60 s - time in solutioni)/(Response numberi + 1) where i was the current time bin. To address the possibility that differences in response duration during testing reflect a loss of responding in the previously shocked rats, we also present response number.

Statistics

The results were analyzed using an ANOVA. Post hoc comparisons were made using Duncan's new multiple range test. In all cases, a criterion of P < 0.05 was used to judge statistical significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experiment 1: behavioral immunization

Experiment 1 examined whether prior exposure to contingent shock has a protective effect on behavioral plasticity, immunizing the spinal cord against the deleterious effects of subsequent noncontingent nociceptive stimulation.

TRAINING. Prior to training in phase 1, the shock intensity needed to induce a flexion force of 0.4 N ranged from 0.55 ± 0.07 (SE) mA to 0.60 ± 0.05 mA. The initial response durations ranged from 0.20 ± 0.03 s to 0.25 ± 0.002 s. Independent ANOVAs confirmed that the groups did not differ prior to training, both Fs < 1.0, P > 0.05.

During training, spinalized rats exposed to contingent legshock (master rats) learned, exhibiting a progressive increase in response duration (Fig. 3, top). Rats given noncontingent legshock (yoked) failed to learn. An ANOVA revealed a significant main effect of training condition, F(2, 42) = 1729.57, P < 0.0001. There was also a significant main effect of time and a significant training condition × time interaction, both Fs > 13.80, P < 0.0001, indicating that, over the course of the 30-min training session, master rats spent progressively more time with their leg in the flexed position relative to both the yoked and unshocked controls.



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Fig. 3. Performance over time during the training phase (1) for subjects in experiment 1. The change in response duration (top) and response number (bottom) observed over the 30-min training period for subjects given contingent shock (master, ), noncontingent shock (yoked, ), or nothing (unshocked, triangle ). The error bars indicate the SE.

Master and yoked rats made more flexion responses during training than rats that remained unshocked (Fig. 3, bottom). An ANOVA confirmed a significant main effect of training condition, F(2, 42) = 12.02, P < 0.0001. The main effect of time and the training condition × time interaction were also significant, both Fs > 10.11, P < 0.0001. Post hoc comparisons of the group means showed that both master and yoked rats made significantly more contacts with the salt solution than rats in the unshocked group, both Ps < 0.05. No other differences were significant, P > 0.05.

TESTING. Subjects were tested by applying contingent shock to the contralateral leg. Prior to testing, there were no differences in either the shock intensity required to elicit a flexion force of 0.4 N (range: 0.58 ± 0.10 mA to 0.85 ± 0.10 mA) or initial response duration (range: 0.18 ± 0.04 s to 0.33 ± 0.14 s), both Fs < 2.38, P > 0.05.

Rats that were unshocked in phases 1 and 2 exhibited a progressive increase in response duration when tested with response-contingent shock (Fig. 4). Preexposure to noncontingent shock disrupted this learning. Prior training with contingent shock (master) facilitated acquisition and prevented the behavioral deficit. An ANOVA performed on the response duration data yielded a significant main effect of initial training condition (master, yoked, or unshocked) and tailshock (unshocked or shocked), as well as a significant training condition × tailshock interaction, all Fs > 97.38, P < 0.0001. There was also a significant main effect of time as well as separate training condition × time and tailshock × time interactions that were qualified by a training condition × tailshock × time three-way interaction, all Fs > 3.12, P < 0.0001. These interactions indicate that the change in response duration observed over time depended on both phase 1 and 2 treatment condition. Post hoc comparisons confirmed that during the 30-min testing session, the master groups (master right-arrow unshocked, master right-arrow shocked), and the subjects that previously received no shock (unshocked right-arrow unshocked), exhibited longer response durations relative to both yoked groups (yoked right-arrow unshocked, yoked right-arrow shocked) and the group that received noncontingent shock in phase 2 (unshocked right-arrow shocked), all Ps < 0.05. No other differences were significant, P > 0.05. 



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Fig. 4. Response duration (top) and response number (bottom) over the 30-min period of testing in experiment 1. Rats had previously received contingent legshock (master, left), noncontingent legshock (yoked, middle), or nothing (unshocked, right), followed by either noncontingent tailshock (shocked, ) or nothing (unshocked, ). The error bars indicate the SE.

Subjects that failed to learn tended to exhibit a greater number of flexion responses (Fig. 4, bottom). An ANOVA performed on these data revealed a significant main effect of legshock, F(2, 42) = 8.48, P < 0.05, as well as a significant legshock × tailshock interaction, F(2, 42) = 5.60, P < 0.05. The main effect of tailshock, however, was not significant, F(1, 42) = 2.18, P > 0.05. There was also a significant main effect of time and a significant time × legshock interaction, both Fs > 1.36, P < 0.05. Post hoc analyses revealed that rats pretrained with contingent shock (master right-arrow unshocked, master right-arrow shocked) and the unshocked controls (unshocked right-arrow unshocked) made significantly fewer responses than subjects that received noncontingent shock (yoked right-arrow unshocked, yoked right-arrow shocked, or unshocked right-arrow shocked), all Ps < 0.05. No other differences were significant, P > 0.05.

SUMMARY. In phase 1, spinalized rats exposed to contingent shock exhibited a progressive increase in response duration indicative of learning. Yoked rats that received an equivalent amount of shock failed to learn. Yoked rats also failed to learn when they were subsequently tested with contingent shock applied to the contralateral leg (test phase). Six min of noncontingent tailshock (phase 2) also produced a robust behavioral deficit. This deficit was not observed in rats that had previously received training with contingent shock, which suggests that contingent shock training has a protective effect on spinal plasticity.

Experiment 2: behavioral therapy

Experiment 2 evaluated whether exposure to response-contingent shock after noncontingent tailshock has a therapeutic effect that restores behavioral potential. An obvious difficulty with testing this hypothesis is that the initial exposure to noncontingent shock will make subjects incapable of learning and ruin the capacity for behavioral therapy. To examine whether response-contingent shock has a beneficial therapeutic effect, we need a manipulation that can temporarily block the expression of the behavioral deficit. Recent work suggests that this may be accomplished by microinjecting the opioid antagonist naltrexone into the spinal cord (Joynes and Grau 1998, 2001). Although this pharmacological treatment has no effect on the induction of the behavioral deficit, it blocks the expression of the deficit and thereby temporarily restores the capacity for instrumental learning. The present experiment examines whether instrumental training in the presence of naltrexone has a long-term therapeutic effect that restores behavioral potential.

TRAINING. Prior to training in phase 2, the shock intensity required to induce a flexion force of 0.4 N ranged from 0.50 ± 0.03 (SE) mA to 0.64 ± 0.07 mA. The initial response durations ranged from 0.14 ± 0.014 s to 0.25 ± 0.06 s. Independent ANOVAs confirmed that the groups did not differ prior to training, both Fs < 1.44, P > 0.05.

Naltrexone-treated spinalized rats displayed a progressive increase in response duration during training with contingent shock (Fig. 5, top). An ANOVA revealed a significant main effect of group, F(3, 44) = 371.91, P < 0.0001. There was also a significant main effect of time as well as a significant group × time interaction, both Fs > 4.99, P < 0.0001, confirming that over the course of the training session, master rats spent significantly longer amounts of time with their legs in a flexed position relative to rats in the yoked and unshocked conditions.



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Fig. 5. Response duration (top) and response number (bottom) for rats that had previously received noncontingent tailshock (shocked,  and black-triangle) or nothing (unshocked,  and triangle ) followed by an injection of naltrexone. Shocked rats then received 30 min of training with contingent legshock (master), noncontingent legshock (yoked), or remained unshocked (unshocked). The error bars indicate the SE.

During the training period, rats given contingent (master) or noncontingent (yoked) shock made more flexion responses than the unshocked controls (Fig. 5, bottom). An ANOVA found a significant main effect of group, F(3, 44) = 2.95, P < 0.05. There was also a significant main effect of time and a significant group × time interaction, both Fs > 2.08, P < 0.0001.

TESTING. Subjects were tested with contingent shock to either the same (ipsilateral) or opposite (contralateral) leg shocked previously in phase 2. Prior to testing, there were no significant differences in either the shock intensity needed to induce a 0.4 N flexion force (range: 0.63 ± 0.07 to 0.70 ± 0.02 mA) or initial response duration (range: 0.14 ± 0.01 to 0.22 ± 0.04 s), all Fs < 1.30, P > 0.05.

As usual, previously unshocked rats (unshocked right-arrow unshocked) exhibited a progressive increase in response duration when tested with response-contingent shock (Fig. 6, top). Subjects that received noncontingent tailshock followed by naltrexone treatment alone (shocked right-arrow unshocked) failed to learn. So too did subjects given two sessions of noncontingent shock (shocked right-arrow yoked). Training with contingent shock after noncontingent shock exposure (shocked right-arrow master) attenuated the behavioral deficit and this was true irrespective of whether subjects were tested on the ipsilateral or contralateral leg. An ANOVA confirmed that similar results were obtained across test legs, all Fs < 1.00, P > 0.05, and consequently we collapsed the data across this variable (Fig. 6, right). The subsequent ANOVA yielded a significant main effect for group (shocked right-arrow master, shocked right-arrow yoked, shocked right-arrow unshocked, or unshocked right-arrow unshocked), time, and a group × time interaction, all Fs > 2.30, P < 0.05. Post hoc tests confirmed that shocked right-arrow master rats displayed significantly longer response durations than the other shocked groups (shocked right-arrow yoked, shocked right-arrow unshocked), all Ps < 0.05. Unshocked right-arrow unshocked rats had significantly longer response durations than all the other groups, all Ps < 0.05. No other differences were statistically significant, P > 0.05. 



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Fig. 6. Response duration (top) and response number (bottom) for rats that had previously received noncontingent tailshock (shocked,  and black-triangle) or nothing (unshocked,  and triangle ) followed by training with contingent legshock (master), noncontingent legshock (yoked), or nothing (unshocked). Half of the subjects were tested on the pretreated (ipsilateral, left) leg while the remaining rats were tested on the opposite (contralateral, middle) leg. Right: the combined results (collapsed across test leg). The error bars indicate the SE.

Rats that had previously received noncontingent shock (shocked right-arrow unshocked, shocked right-arrow yoked) continued to respond throughout the test session and exhibited a greater number of flexion responses (Fig. 6, bottom). Again similar results were obtained irrespective of whether contingent shock was applied to the ipsilateral or contralateral leg, all Fs < 1.00, P > 0.05. Consequently, we collapsed the data across this variable (Fig. 6, right). The subsequent ANOVA revealed a significant main effect of group and a group × time interaction, both Fs > 1.66, P < 0.01. The main effect of time was not significant, F(29, 1218) = 1.26, P > 0.05. Post hoc tests confirmed that rats that had received only noncontingent shock (shocked right-arrow yoked, shocked right-arrow unshocked) made more contacts with the salt solution than both the shocked right-arrow master rats and the unshocked right-arrow unshocked rats, all Ps < 0.05. No other differences approached significance, P > 0.05.

SUMMARY. As previously reported (Joynes and Grau 1998, 2001), intrathecal naltrexone blocked the expression of the behavioral deficit, allowing previously shocked rats to acquire the instrumental response. More importantly, this instrumental training had a long-term therapeutic effect that naltrexone alone did not, reducing the adverse consequences of noncontingent nociceptive stimulation. This benefit of behavioral training was observed 24 h after drug treatment and was evident on both the pretreated and opposite leg. The fact that the therapeutic effect extends to the contralateral leg suggests that the benefits of behavioral training do not reflect a peripheral modification. Rather it appears that instrumental training reverses a centrally mediated process that undermines behavioral potential within the spinal cord. Rats that received the drug in combination with either nothing (shocked right-arrow unshocked) or noncontingent shock (shocked right-arrow yoked) exhibited the usual behavioral deficit. This suggests that the drug treatment alone was not sufficient---the pharmacological manipulation was only effective when combined with behavioral training.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experiment 1 confirmed that exposure to response-contingent legshock produces a progressive increase in response duration (Grau et al. 1998). This learning was not observed in subjects exposed to an equivalent amount of noncontingent legshock. Exposure to noncontingent legshock did, however, have a long-term impact on behavioral potential---it eliminated the capacity to learn when subjects were subsequently tested with contingent shock. This behavioral deficit does not appear to reflect a peripheral modification because learning was disrupted when contingent shock was applied to the contralateral leg. Learning was also disrupted by 6 min of noncontingent shock applied to the tail. Importantly, rats that had received noncontingent shock failed to learn even though prior shock treatment had no effect on the shock intensity required to elicit a flexion response or initial response duration. Indeed rats that failed to learn generally responded throughout the test period and exhibited the highest number of flexion responses. Despite continued exposure to the response-reinforcer contingency, they never exhibited an adaptive increase in response duration. Thus prior exposure to noncontingent shock undermines the capacity for instrumental behavior. The induction of this behavioral deficit was blocked by prior exposure to response-contingent shock. It appears that contingent shock treatment engages a neurobiological mechanism that has a protective effect and that helps preserve behavioral potential.

Experiment 2 examined whether behavioral training after noncontingent shock could attenuate the behavioral deficit. To enable learning after noncontingent shock exposure, rats were pretreated with naltrexone. As previously reported, this drug treatment temporarily restored the capacity to learn (Joynes and Grau 1998, 2001). In the absence of behavioral training, this restorative effect waned within 24 h, as the drug cleared the animal's system. But when naltrexone treatment was combined with instrumental training, the behavioral deficit appeared to be permanently ameliorated. Together our findings suggest that instrumental training engages a process that counters the behavioral deficit, protecting the spinal cord from the deleterious effects of subsequent noncontingent events (experiment 1) and restoring behavioral potential after the degradative mechanism has been engaged (experiment 2).

Those familiar with the learned-helplessness literature will recognize the formal similarity of these findings to the results obtained in intact subjects using supraspinally mediated test procedures. In intact rats, exposure to noncontingent tailshock produces a behavioral deficit that undermines learning and motivation in a variety of behavioral tasks (Maier and Seligman 1976; Overmier and Seligman 1967; Seligman and Maier 1967). Rats that experience the same amount of shock, but have control over its termination, do not exhibit either a learning or performance deficit. Like the deficit observed in spinalized rats, noncontingent stimulation in intact animals produces a deficit that decays over the course of 48 h and is attenuated by administration of an opioid antagonist (Hyson et al. 1982; Maier and Minor 1993; Maier et al. 1980). Moreover, prior training with contingent shock in intact rats has an immunizing effect that prevents learned helplessness and training with contingent shock after noncontingent shock exposure has a therapeutic effect that reduces the behavioral deficit (Seligman et al. 1968, 1975). Early theorists suggested that these effects might be related to a cognition of no control (Maier and Jackson 1979; Maier and Seligman 1976). The present findings suggest an alternative view---that noncontingent nociceptive stimulation can have a degradative effect on behavioral function independent of cognition. The induction and maintenance of this unconditioned response to nociceptive stimulation appears to be modulated by behavioral variables and this relation appears to hold at multiple levels within the nervous system. These observations suggest that the rules explicated by helplessness theory summarize some basic principles of neuronal organization that may be inherent to any system capable of instrumental learning (Eisenstein and Carlson 1997).

We have shown that a period of noncontingent shock engages a process that undermines behavioral potential within the spinal cord. At the neurochemical level, the behavioral deficit observed after noncontingent shock depends on an agent that acts on the kappa opioid receptor (Joynes and Grau 2001). In addition, the GABA antagonist bicuculline eliminates the deficit, suggesting an increase in GABAergic inhibition contributes to the deficit (Ferguson et al. 2000). These neurochemical systems could undermine the capacity for learning by attenuating NMDA-mediated excitation, an essential process in both Pavlovian and instrumental conditioning within the spinal cord (Durkovic and Prokowich 1998; Joynes et al. 1995). We have also begun to further characterize the stimulus conditions that produce the behavioral deficit. We first isolated the essential shock conditions, relating our stimulus parameters to prior studies of nociceptive modulation (Grau et al. 1996; Meagher et al. 1993). We found that the deficit does not emerge until shock intensity is increased to a relatively intense level, far more intense than the shock parameters needed to elicit vigorous withdrawal and escape behaviors in experimental animals and reports of intense pain in humans (Crown et al., unpublished data). Indeed a similar intensity is needed to engage antinociceptive mechanisms within the spinal cord. The behavioral deficit does not, however, appear to be mediated by the same process that generally inhibits nociceptive reflexes, for antinociception and the behavioral deficit are affected in opposite ways by the temporal spacing of the shock stimuli (Crown et al., unpublished data).

Other studies have explored whether the behavioral deficit can be induced by naturalistic stimuli that may accompany a spinal cord injury (Ferguson et al. 2001). We noted that there were a number of interesting parallels between the biochemical cascade that mediates the behavioral deficit and the consequences of peripheral inflammation. Inflammation is known to sensitize mechanical reactivity through a process that depends on the expression of the kappa opioid dynorphin-A and enhanced GABAergic inhibition (Dickenson 1996; Dray et al. 1994; Dubner and Ruda 1994; Stanfa and Dickenson 1993; Stanfa et al. 1994). Likewise exposure to noncontingent shock (for a duration sufficient to induce the behavioral deficit) enhances tactile reactivity and undermines instrumental learning through a kappa opioid-mediated process that can be blocked by administration of the GABA antagonist bicuculline (Ferguson et al. 2000). Experimentally inflammation can be induced by a number of chemical irritants injected subcutaneously into the hindpaw, including formalin and carrageenan (Honor et al. 1999). We have tested the impact of carrageenan on instrumental learning and have found that carrageenan disrupts instrumental behavior during the period of inflammation (Ferguson et al. 2001). This finding suggests that naturalistic stimuli not only sensitize spinal nociceptive pathways but also undermine spinal plasticity. Clinically, this finding is especially important, given that between 30 and 90% of human patients suffering from spinal cord injury report experiencing pathological pain, one potential marker of central sensitization (Coderre et al. 1993; Yezierski 1996). The biochemical alterations that accompany spinal cord trauma and sensitize nociceptive reactivity could undermine behavioral potential within the spinal cord and thereby hurt the recovery of locomotor function (Wernig et al. 1995, 1998). Supporting this, we have determined that exposure to the same shock schedules that induce the behavioral deficit in spinalized rats also retards the recovery of locomotor function in rats that have received a contusion injury (Grau et al. 2001).

Our studies have examined the functional properties that govern learning within the spinal cord (Ferguson et al. 2000; Grau and Crown 2000; Grau et al. 1996, 1998; Crown et al., unpublished observations). We have shown that these systems are sensitive to response-outcome relations and, in this way, meet the behavioral criteria for instrumental learning (Grau et al. 1996, 1998). We have further shown that experience with a response-outcome relation can have a general effect on spinal cord function with noncontingent events undermining behavioral potential while contingent relations have a protective/restorative effect (Ferguson et al. 2000; Grau and Crown 2000; Crown et al., unpublished results). Further studies are needed to determine whether these functional relations affect the recovery of function after spinal cord injury. Our work also lays the foundation for more detailed analyses of the underlying neurophysiological/neurochemical systems; analyses that could shed further light on the mechanisms that maintain plasticity within the spinal cord and encode response-outcome relations. By detailing when learning occurs, how long it lasts, the neurochemical systems involved, and its functional properties, our research program provides a system in which to understand the underlying components and a framework through which these findings can be related to clinically relevant training procedures.


    ACKNOWLEDGMENTS

The authors thank A. Sieve, A. Ferguson, S. Washburn, G. Garcia, and B. Patton for comments on a previous version of this manuscript.

This work was funded by National Institute of Mental Health Grant MH-60157 to J. W. Grau.


    FOOTNOTES

Address for reprint requests: E. D. Crown (E-mail: edc5078{at}neo.tamu.edu).

1 More precise information about foot angle can be obtained using a capacitative position device (Eisenstein and Carlson 1994). While such a device has some experimental advantages (e.g., allowing us to change the response criterion on-line), the essential features of the behavioral paradigm remain the same: a response criterion must be set (translating the analog signal into a dichotomous response) and response duration remains the primary measure of learning.

Received 12 December 2000; accepted in final form 27 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society




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