Contributions of glutamate receptors to the maintenance of mustard oil-induced hyperalgesia in spinalized rats.

N-methyl-d-aspartate (NMDA) receptors, which are widely distributed throughout the central nervous system, appear to play a critical role in several types of plasticity and long-term potentiation. In the pain system, increased sensitivity to somatosensory stimuli, known as hyperalgesia and allodynia, can arise from tissue damage or excessive C-fiber nociceptor activation. Previously, NMDA, non-NMDA ionotropic, and metabotropic glutamate receptors have been proposed to contribute to the sustained hyperalgesia following tissue injury or nociceptor activation. Although non-NMDA receptors appear to mediate both hyperalgesia and normal (nonhyperalgesic) responses and behavior, NMDA receptors have been reported to participate only in hyperalgesic responses. In contrast, other studies have implicated NMDA receptors in both hyperalgesic and normal responses. The aim of this study was to critically compare the effects of the glutamate receptor antagonists ketamine and 2-amino-5-phosphonovaleric acid (APV; NMDA receptor antagonists), 6,7-dinitroquinoxaline-2,3-dione (DNQX; non-NMDA ionotropic receptor antagonist), and 2-amino-3-phosphonopropionic acid (AP3; metabotropic receptor antagonist) on intra-articular mustard oil-induced facilitation of flexion withdrawal reflexes in spinalized rats. Our results showed that, as expected from previous studies, ketamine, APV, and DNQX dose-dependently inhibited the flexion withdrawal reflex evoked by C-fiber electrical stimulation of the sciatic nerve. Surprisingly, however, ketamine, APV, and DNQX also inhibited flexion withdrawal reflexes in normal (nonhyperalgesic) rats with similar ED50s. In contrast, AP3 had no effect in either hyperalgesic or normal rats. These results demonstrate that NMDA and non-NMDA ionotropic, but not metabotropic, glutamate receptors contribute without preference to both facilitated and normal flexion withdrawal reflexes evoked by high-intensity electrical stimulation in the spinalized rat. Thus, the apparent preference of NMDA receptors for hyperalgesic states seen in some previous studies on nociception, as well as in other model systems, may have arisen from differences in experimental paradigm, such as the intensity of sensory stimulation or excitability of the spinal cord, coupled with the voltage dependency of the NMDA conductance.

Intrinsic properties of deep dorsal horn neurons in the L6-S1 spinal cord of the intact rat.

1. Stable intracellular recordings were obtained from neurons (n = 62) in the L6-S1 deep dorsal horn of the spinal cord in pentobarbital-sodium-anesthetized, intact rats (n = 26). All neurons responded to natural mechanical stimuli and/or electrical stimulation of peripheral afferents. 2. Intracellular penetrations were maintained for 30 min-2 h. Action potentials occurred spontaneously in most neurons (n = 50) and could be evoked in the remainder (n = 12) by depolarizing current passage. Mean resting membrane potential was -60.9 mV, mean action potential height amplitude was 75.2 mV, mean half-width of the action potentials was 0.33 ms, mean input resistance was 38 M omega, and mean time constant was 9.1 ms. 3. Action potentials were followed by afterpotentials made up of at least three components; a fast afterhyperpolarization (fAHP), a slow afterhyperpolarization (sAHP), and an afterdepolarization (ADP). Most neurons (n = 40) exhibited all three afterpotentials, although some displayed only a fAHP and an ADP (n = 10) or a fAHP and a sAHP (n = 12). The durations and magnitudes of the afterpotentials varied widely among neurons. 4. Steady-state current-voltage relations were investigated in 14 neurons with depolarizing and hyperpolarizing current pulses. Of these 14 neurons, 5 exhibited inward rectification, 3 had outward rectification, and the remaining 6 showed a predominantly linear change of membrane potential to current injection. In addition, several neurons (n = 9) exhibited a postinhibitory rebound that was sometimes (n = 4) accompanied by a "sag" in voltage during the preceding hyperpolarizing current step. 5. Four patterns of spike frequency adaptation occurred during step depolarizing current passage. The firing of most neurons gradually decreased with a simple, approximately exponential time course (n = 21), in some neurons it decreased with both a fast and a slow time course (n = 8), in several it incremented in rate (n = 3), and one neuron showed a complex combination of multiple decrementing and incrementing adaptations. Time constants, magnitude of adaptation, and the slopes of the steady-state current-voltage relation varied widely. 6. Oscillations in membrane potential and firing rate occurred in three neurons. The oscillations arose from endogenous mechanisms in at least one neuron because manipulation of membrane potentials altered the frequency of oscillation; a depolarizing current increased the period of oscillation and eventually produced tonic firing, and a hyperpolarizing current increased the frequency of oscillation and eventually terminated firing. 7. The results demonstrate that neurons in the L6-S1 region of the dorsal horn exhibit a diversity of cellular mechanisms that may significantly modulate normal somatosensory and visceral input.

Functional properties of spinal interneurons activated by muscular free nerve endings and their potential contributions to the clasp-knife reflex.

1. The goal of this study was to characterize the functional properties of spinal interneurons that are excited by muscular free nerve endings and to assess their contributions to the clasp-knife reflex. 2. The patterns of activity of 82 spinal interneurons that were excited by squeezing the Achilles tendon or manipulation of the muscle surfaces, preferential stimuli for muscular free nerve endings, were extracellularly recorded in lamina V-VII of the L5-S1 spinal cord in decerebrated and spinalized cats. 3. Interneurons were uniformly excited by increases in muscular length and force. Responses to muscle stretch exhibited gradual decay during maintained stretch, afterdischarge after stretch release, and adaptation to repeated stretch. Responses to isometric contraction induced by electrical stimulation of motor axons was also prolonged after contraction, but did not decay during maintained contraction. For similar increases in force, stretch evoked greater excitation than contraction, indicating that both stretch and contraction contributed to interneuronal activity. Overall, the time course and magnitude of the interneuronal responses to stretch and contraction paralleled the time course and magnitude of the clasp-knife reflex. 4. Interneurons were powerfully excited by muscular free nerve endings, which mediate the clasp-knife reflex, and by cutaneous receptors. Only occasionally were they excited by primary spindle or Golgi tendon organ afferents, which suggests that activation of muscular free nerve endings mediated the interneuronal responses to stretch and contraction. 5. Simultaneous recordings of interneuronal activity and the clasp-knife reflex revealed a broad correlation between interneuronal activity and clasp-knife inhibition. 6. Because the patterns of activity of free nerve ending-responsive interneurons during stretch and contraction were similar to the clasp-knife reflex, were closely correlated with clasp-knife inhibition during simultaneous interneuronal and reflex recordings, and were powerfully excited by muscular free nerve endings, it is likely that the interneurons described above contributed to the clasp-knife reflex. 7. In contrast, a small number (n = 16) of interneurons were recorded that were only weakly excited by muscular free nerve endings but strongly excited by group I afferents, exhibited less spontaneous and evoked activity, and had significantly different responses to stretch and contraction. These interneurons are less likely to have contributed to the clasp-knife reflex.

Respiratory-modulated and phrenic afferent-driven neurons in the cervical spinal cord (C4-C6) of the fluorocarbon-perfused guinea pig.

The potential contributions of cervical spinal interneurons to the neural control of respiration have been investigated by extracellularly recording the patterns of activity of neurons in the C4-C6 spinal cord during fictive respiration in the fluorocarbon-perfused, adult guinea pig. Two types of neurons were recorded: respiratory-modulated neurons, whose activity was modulated with respiration, and phrenic-driven neurons, which were excited by electrical stimulation of the phrenic nerve. Respiratory-modulated neurons (n = 20) could be divided into inspiratory, expiratory, and phase-spanning neurons, based on their patterns of activity during fictive respiration. Respiratory-modulated neurons showed varying dependencies on the type of breathing; during spontaneous augmented breaths, one-half exhibited patterns of activity that were significantly different to those seen during normal, fictive respiration, whereas the other half of the respiratory-modulated neurons showed similar patterns of activity during both normal and augmented breaths. Phrenic-driven neurons (n = 22) could be divided into short-latency (7-12 ms), moderate-latency (12-25 ms), and inhibited neurons, but were only occasionally and weakly modulated with respiration. The results suggest that respiratory-modulated C4-C6 spinal neurons may contribute to the neural control of respiration, with different subpopulations specialized for different types of respiratory tasks, and that phrenic-driven neurons may be interposed in sensory or reflex pathways, such as the spinothalamic tract or phrenic-to-phrenic inhibitory reflex.

Segmental reflex action in normal and decerebrate cats.

1. The objective of this study was to evaluate the action of the stretch reflex on the ankle extensor muscles of normal and decerebrate cats. 2. Experiments were performed on nine freely standing, unrestrained cats and repeated after decerebration at the premammillary level. The length, force, and electromyograph (EMG) of the soleus (SOL) and lateral gastrocnemius (LG) muscles were recorded with the use of implanted transducers and electrodes. 3. The left ankle joint was unexpectedly and reproducibly dorsiflexed by briefly stimulating the common peroneal (CP) nerve with electrodes within an implanted nerve cuff. The ensuing twitch contractions of the ankle dorsiflexor muscles stretched the ankle extensor muscles by 0.3-2.0 mm. Lidocaine was infused into another nerve cuff proximal to the stimulation site, to reversibly block the central propagation of evoked volleys in the CP nerve. 4. Reflex action before and after decerebration was measured from the responses to perturbations of similar amplitude and duration delivered at approximately matched background values of muscle length and force. In most cats the temperature of the hindlimb was monitored with an implanted thermistor and was restored to normal values with radiant heat after decerebration. 5. A stretch imposed on the tonically active ankle extensor muscles immediately caused a considerable rise in the force recorded from the triceps tendon. Within 30-40 ms the triceps force peaked, reaching a value 10-20 N greater than background, and then rapidly declined while the extensor muscles were still lengthening. The initial rise in force preceded any change in triceps EMG. It was attributed to the intrinsic viscoelasticity of the stretched muscles and tendons. After decerebration the magnitude and timing of the initial force peak did not change. 6. A short-latency reflex EMG burst was typically recorded from both the SOL and LG muscles, starting 11-17 ms after stimulus onset. After decerebration the area of the reflex EMG burst increased in all nine cats, typically by a factor of 2 or 3. 7. After decerebration a second, smaller increase in force was typically observed starting 60-80 ms after onset of stretch. This later force rise, interpreted to be of reflex origin, was rarely apparent in normal cats. 8. Decerebration introduced consistent modifications in postural behavior that were revealed by pushing down on the back of quietly standing cats. In normal cats, after brief pushes that stretched the ankle extensor muscles by 1-2 mm, the EMG, force, and length quickly stabilized near their initial values.(ABSTRACT TRUNCATED AT 400 WORDS)

Neural mechanisms underlying the clasp-knife reflex in the cat. I. Characteristics of the reflex.

1. The goal of this study was to characterize the clasp-knife reflex by the use of stretch and isometric contraction of ankle extensor and flexor muscles in decerebrated cats with bilateral dorsal hemisections of their spinal cords at segment T12. 2. Stretch of an extensor muscle evoked inhibition in both homonymous and synergistic extensor muscles. The similarities between homonymous and synergistic inhibition suggest that similar neural mechanisms were responsible. 3. Homonymous and synergistic clasp-knife inhibition showed several characteristic features: 1) inhibition was evoked only by large stretches that produced significant muscle force. Short stretches that did not produce large forces evoked only excitation; 2) the magnitude of clasp-knife inhibition increased with increasing initial motor output, as reflected in the level of rectified EMG; 3) the time course of reflex inhibition evoked by ramp-and-hold stretch was characterized by segmentation of EMG during ramp stretch, dynamic overshoot of inhibition at the end-of-ramp stretch, and slow but usually complete decay of inhibition during maintained stretch; 4) inhibition persisted beyond the termination of stretch, and 5) inhibition showed adaptation to repeated stretch. 4. Isometric contraction of the soleus or medial gastrocnemius, produced by electrical stimulation of the muscle nerve, also evoked powerful synergistic-reflex inhibition via similar mechanisms as stretch-evoked, clasp-knife inhibition. Stretch evoked a greater degree of inhibition than did contraction, indicating that receptors responsive to both stretch and contraction contribute to clasp-knife inhibition. 5. The reflex effects produced by stretching the soleus or medial gastrocnemius were not confined to the homonymous and close synergistic muscles. Extensor muscles were inhibited and flexor muscles were excited throughout the hindlimb, which paralleled the pattern of a flexion-withdrawal reflex evoked by cutaneous stimulation. 6. Stretch of a flexor muscle, the tibialis anterior, evoked the same spatial pattern and time course of reflex action as stretch of an extensor muscle--inhibition of extensor muscles and excitation of flexor muscles throughout the hindlimb, including homonymous excitation of the tibialis anterior. 7. We conclude that neither Golgi tendon organs nor secondary spindle afferents are likely to contribute significantly to clasp-knife inhibition because their responses to stretch and isometric contraction differ from the reflex actions evoked by stretch and contraction.(ABSTRACT TRUNCATED AT 400 WORDS)

Neural mechanisms underlying the clasp-knife reflex in the cat. II. Stretch-sensitive muscular-free nerve endings.

1. The goal of this study was to determine the contribution of muscular free nerve endings to the clasp-knife reflex by comparing their response properties and reflex actions to the clasp-knife reflex. 2. The responses of single muscle afferents were examined in anesthetized cats using stretch and isometric contraction of ankle extensor muscles identical to those that evoked clasp-knife inhibition in decerebrated and dorsal spinal-hemisectioned cats. 3. Fifty-three stretch-sensitive mechanoreceptor afferents were identified as free nerve ending afferents based on their conduction velocities, location within the muscle, uniformity of response, and dissimilarity to other muscle proprioceptors. The afferent conduction velocities were in both the group III (56%) and group II (44%) range, including five fast-conducting group II afferents (greater than 55 m/s). 4. The stretch response of stretch-sensitive, free nerve endings (SSFNEs) showed several characteristic features: 1) afferents were excited only by large stretches that produced significant passive force; 2) afferent activity began after a brief delay and exhibited segmentation of discharge during ramp stretch, a maximum at the end of ramp stretch, and rapid and complete decay during static stretch, and 3) afferent response adapted to repeated stretches. These properties match those of clasp-knife inhibition described in the companion paper, except that the SSFNE segmentation and maximum were more pronounced and their decay during maintained stretch was more rapid. 5. Isometric contraction produced by electrical stimulation of the muscle nerve, which induced force-evoked inhibition in decerebrated and dorsal hemisectioned cats, also consistently excited SSFNEs. Stretch evoked greater excitation than contraction, indicating that both length and force contribute to SSFNE activity. 6. Stimulation of free nerve endings by squeezing the achilles tendon in cats exhibiting the clasp-knife reflex evoked powerful, homonymous inhibition and a flexion-withdrawal pattern of reflex action--that is, inhibition of extensor and excitation of flexor muscles throughout the hindlimb, which parallels the spatial divergence of the clasp-knife reflex. 7. Intrathecal application of capsaicin, which preferentially blocks the reflex actions of small afferent fibers, blocked clasp-knife inhibition in decerebrated, dorsal hemisectioned cats. 8. The similarities between the reflex actions and response properties of SSFNEs and the properties of the clasp-knife reflex suggest that SSFNEs mediate clasp-knife inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)

Force-sensitive interneurons in the spinal cord of the cat.

The input-output properties of interneurons mediating spinal reflexes were investigated by extracellularly recording the response of interneurons to excitation from muscle receptors in the ankle extensor muscles of decerebrated, spinal cats. A population ofinterneurons in the intermediate region ofthe spinal cord is potently excited by increases in muscle force. Unlike the discharge of Golgi tendon organs, which accurately encodes moment-to-moment variations in the force of a single muscle, the discharge of these interneurons depends in a dynamic and usually nonlinear way on the force in several muscles. Powerful input from unidentified mechanoreceptors in muscle, presumably free nerve endings, is at least partly responsible for these properties. These force-sensitive interneurons are more likely to mediate clasp knife-type inhibition than simple negative force feedback.