Dong-Yuan Cao

Local application of morphine suppresses glutamate-evoked activities of C and Aδ afferent fibers in rat hairy skin

Behavior studies have demonstrated that local application of morphine in peripheral tissues resulted in a significant antinociceptive effect, but there has been no electrophysiological evidence to support the peripheral mechanism of opioid antinociception. The purpose of the present study was to investigate whether local application of morphine suppressed the glutamate-evoked activities of C and Adelta primary afferent fibers in dorsal hairy skin of rat in vivo. The single unit activities of the C and Adelta afferent fibers were recorded by means of isolation of the fiber filaments from the dorsal cutaneous nerve branches, and the effects of glutamate and glutamate plus morphine injected into the receptive field on these activities were determined. The results revealed that most of the C and Adelta fibers were excited significantly by local injection of glutamate (0.3 mM), with the percentage being 81% (22/27, for C fibers) and 73% (36/49, for Adelta fibers), respectively. The glutamate-induced excitatory response was significantly suppressed by co-injection of morphine (1.0 mM). The mean discharge rates of C fibers and Adelta fibers decreased from 28.96 +/- 6.85, 28.99 +/- 3.79 impulses/min to 4.40 +/- 1.76, 2.72 +/- 0.71 impulses/min, respectively. The suppressing effect of morphine was reversed by pretreatment with opioid receptor antagonist naloxone (1.0 mM). These findings suggest that local application of morphine can suppress the glutamate-evoked activities of the fine fibers in rat hairy skin and thus provide an electrophysiological evidence for peripheral antinociception of opioids.

Relationship between Biomechanical Characteristics of Spinal Manipulation and Neural Responses in an Animal Model: Effect of Linear Control of Thrust Displacement versus Force, Thrust Amplitude, Thrust Duration, and Thrust Rate

High velocity low amplitude spinal manipulation (HVLA-SM) is used frequently to treat musculoskeletal complaints. Little is known about the interventions biomechanical characteristics that determine its clinical benefit. Using an animal preparation, we determined how neural activity from lumbar muscle spindles during a lumbar HVLA-SM is affected by the type of thrust control and by the thrusts amplitude, duration, and rate. A mechanical device was used to apply a linear increase in thrust displacement or force and to control thrust duration. Under displacement control, neural responses during the HVLA-SM increased in a fashion graded with thrust amplitude. Under force control neural responses were similar regardless of the thrust amplitude. Decreasing thrust durations at all thrust amplitudes except the smallest thrust displacement had an overall significant effect on increasing muscle spindle activity during the HVLA-SMs. Under force control, spindle responses specifically and significantly increased between thrust durations of 75 and 150 ms suggesting the presence of a threshold value. Thrust velocities greater than 20–30 mm/s and thrust rates greater than 300 N/s tended to maximize the spindle responses. This study provides a basis for considering biomechanical characteristics of an HVLA-SM that should be measured and reported in clinical efficacy studies to help define effective clinical dosages.

Effects of thrust amplitude and duration of high-velocity, low-amplitude spinal manipulation on lumbar muscle spindle responses to vertebral position and movement.

OBJECTIVE Mechanical characteristics of high-velocity, low-amplitude spinal manipulations (HVLA-SMs) can vary. Sustained changes in peripheral neuronal signaling due to altered load transmission to a sensory receptors local mechanical environment are often considered a mechanism contributing to the therapeutic effects of spinal manipulation. The purpose of this study was to determine whether variation in an HVLA-SMs thrust amplitude and duration alters the neural responsiveness of lumbar muscle spindles to either vertebral movement or position. METHODS Anesthetized cats (n = 112) received L6 HVLA-SMs delivered to the spinous process. Cats were divided into 6 cohorts depending upon the peak thrust force (25%, 55%, 85% body weight) or thrust displacement (1, 2, 3 mm) they received. Cats in each cohort received 8 thrust durations (0-250 milliseconds). Afferent discharge from 112 spindles was recorded in response to ramp and hold vertebral movement before and after the manipulation. Changes in mean instantaneous frequency (∆MIF) during the baseline period preceding the ramps (∆MIFresting), during ramp movement (∆MIFmovement), and with the vertebra held in the new position (∆MIFposition) were compared. RESULTS Thrust duration had a small but statistically significant effect on ∆MIFresting at all 6 thrust amplitudes compared with control (0-millisecond thrust duration). The lowest amplitude thrust displacement (1 mm) increased ∆MIFresting at all thrust durations. For all the other thrust displacements and forces, the direction of change in ∆MIFresting was not consistent, and the pattern of change was not systematically related to thrust duration. Regardless of thrust force, displacement, or duration, ∆MIFmovement and ∆MIFposition were not significantly different from control. CONCLUSION Relatively low-amplitude thrust displacements applied during an HVLA-SM produced sustained increases in the resting discharge of paraspinal muscle spindles regardless of the duration over which the thrust was applied. However, regardless of the HVLA-SMs thrust amplitude or duration, the responsiveness of paraspinal muscle spindles to vertebral movement and to a new vertebral position was not affected.

Position Sensitivity of Feline Paraspinal Muscle Spindles to Vertebral Movement in the Lumbar Spine

Muscle spindles contribute to sensorimotor control by supplying feedback regarding muscle length and consequently information about joint position. While substantial study has been devoted to determining the position sensitivity of spindles in limb muscles, there appears to be no data on their sensitivity in the low back. We determined the relationship between lumbar paraspinal muscle spindle discharge and paraspinal muscle lengthening estimated from controlled cranialward movement of the L(6) vertebra in anesthetized cats. Ramp (0.4 mm/s) and hold displacements (0.2, 0.4, 0.6, 0.8, and 1.2 mm for 2.5 s) were applied at the L(6) spinous process. Position sensitivity was defined as the slope of the relationship between the estimated increase in muscle length and mean instantaneous frequency at each length. To enable comparisons with appendicular muscle spindles where joint angle was measured, we also calculated sensitivity in terms of the L(6) and L(7) intervertebral flexion angle (IVA). This angle was estimated from measurements of facet joint capsule strain (FJC) based on a previously established relationship between IVA and FJC strain in the cat lumbar vertebral column during lumbar flexion. Single-unit recordings were obtained from 12 muscle spindle afferents. Longissimus and multifidus muscles contained the receptive field of 10 and 2 afferents, respectively. Mean position sensitivity was 16.3 imp.s(-1).mm(-1) [10.6-22.1, 95% confidence interval (CI), P < 0.001]. Mean angular sensitivity was 5.2 imp.s(-1). degrees (-1) (2.6-8.0, P < 0.003). These slope estimates were more than 3.5 times greater compared with appendicular muscle spindles, and their CIs did not contain previous slope estimates for the sensitivity of appendicular spindles from the literature. Potential reasons for and the significance of the apparently high position sensitivity in the lumbar spine are discussed.

Sex differences in the responses of spinal wide-dynamic range neurons to subcutaneous formalin and in the effects of different frequencies of conditioning electrical stimulation.

The purpose of this study was to investigate sex-related differences in nociception elicited by s.c. injection of different concentrations (1-5%) of formalin. S.c. formalin-induced biphasic (early and late phases) persistent nociception was assessed by extracellularly recording the spontaneous activities of single spinal dorsal horn wide-dynamic range neurons in anesthetized male and female rats. The nociceptive responses of the dorsal horn wide-dynamic range neurons following s.c. injection of 5%, but not 1% and 2.5%, formalin in female rats were significantly stronger than the responses obtained in male rats. However, these concentration-dependent differences with respect to different sexes existed only in the late, but not the early, phase of formalin-induced nociception in intact, not spinal rats. The 5% formalin-induced late phase nociception in male rats was significantly depressed by 15 min of repeated conditioning electrical stimulation at a frequency of 5 Hz as well as 50 Hz during and after the period of conditioning electrical stimulation (intensity: 1 mA; pulse duration: 1 ms). In contrast, the inhibitory effect of 50 Hz conditioning electrical stimulation on the 5% formalin-elicited late phase response in female rats was markedly greater in magnitude and longer in duration than that of 5 Hz conditioning electrical stimulation. No significant depressive effects of 5 Hz conditioning electrical stimulation on formalin-induced nociception were found in female rats, indicating that the distinct effects of conditioning electrical stimulation at different frequencies are different in animals of opposite sexes. In conclusion, s.c. administration of different concentrations of formalin shows a distinct sex-related difference in its late tonic nociception of spinal nociceptive sensory neurons. Sex differences in formalin-induced tonic nociception are stimulus intensity dependent and related to the modulation from the supraspinal regions. S.c. formalin-induced late phase nociception in female rats is only sensitive to depression at a frequency of 50 Hz, but not 5 Hz, of conditioning electrical stimulation. This suggests that the involvement of the central mechanisms in the antinociceptive effects of conditioning electrical stimulation may be different at various frequencies of stimulation.

Ethological analysis of scopolamine treatment or pretreatment in morphine dependent rats

Although scopolamine is currently used to treat morphine addiction in humans, its extensive actions on behaviors have not been systematically analyzed yet, and the underlying mechanisms of its effects still remain ambiguous. The present study was carried out to clarify the possible mechanisms by evaluating the effects of scopolamine pretreatment and treatment on naloxone-precipitated withdrawal signs and some of other general behaviors in morphine dependent rats. Our results showed that scopolamine pretreatment and treatment attenuated naloxone-precipitated withdrawal signs including jumping, writhing posture, weight loss, genital grooming, teeth-chattering, ptosis, diarrhea and irritability, except for wet dog shakes, while general behaviors such as water intake, urine volume and morphine excretion in urine were increased. Our findings suggest that scopolamine has significant actions in the treatment of opiate addiction, which might result from increasing morphine excretion from urine.

Involvement of peripheral ionotropic glutamate receptors in activation of cutaneous branches of spinal dorsal rami following antidromic electrical stimulation of adjacent afferent nerves in rats

The aim of the present study was to investigate the role of peripheral ionotropic glutamate receptors in the process of signal transmission between adjacent different peripheral sensory nerves. The T9 and T10 cutaneous branches of spinal dorsal rami were dissociated and cut proximally in pentobarbital anesthetized rats. Eighty-seven single afferents from T10 nerve filaments were recorded and characterized by assessing their spontaneous activities. Following 30 s antidromic electrical stimulation (intensity: 1 mA; duration: 0.5 ms; frequency: 20 Hz) of T9 cutaneous branches, the spontaneous activities of Abeta, Adelta and C fibers of T10 nerve were significantly enhanced from 2.00+/-0.34, 2.42+/-0.33, and 2.19+/-0.32 impulses/min to 4.31+/-0.58, 5.22+/-0.55, and 5.27+/-0.69 impulses/min, respectively (n=29 for each type, P<0.05). These enhanced spontaneous discharges of T10 nerve were significantly blocked by local treatment of its receptive field with either N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 or non-NMDA receptor antagonist DNQX (0.1 mM, 10 microl for each drug) (P<0.05). These results suggest that peripheral ionotropic glutamate receptors are involved in the activation of peripheral nerves following the antidromic stimulation of adjacent afferents from different spinal segments. We further provide the direct evidence that neurotransmitters released from adjacent peripheral nerves may also contribute to the occurrence of allodynia as well as secondary hyperalgesia during the pathological nociception.

Lengthening but not shortening history of paraspinal muscle spindles in the low back alters their dynamic sensitivity.

Proprioception is considered important for maintaining spinal stability and for controlling posture and movement in the low back. Previous studies demonstrate the presence of thixotropic properties in lumbar muscle spindles, wherein a vertebras positional history alters spindle responsiveness to position and movement. This study investigated whether a vertebras movement history affects the velocity sensitivity of paraspinal muscle spindles in the low back. Afferent activity from multifidus and longissimus muscle spindles was recorded in the L(6) dorsal root in 30 anesthetized cats. To alter movement history, a feedback-controlled motor attached to the L(6) spinous process held (conditioned for 4 s) the L(6) vertebra at an intermediate position or at positions that either lengthened or shortened the muscles. With the vertebra returned to the intermediate position, resting spindle discharge was measured over the next 0.5 s (static test) and then during a dynamic test consisting of ramp vertebral movement at four velocities (0.2, 0.5, 1.0, 2.0 mm/s). Spindle activity during the tests was measured relative to hold-intermediate. For both tests, hold-long decreased and hold-short increased muscle spindle responsiveness. For the static test position responsiveness was not different among the velocity protocols for either hold-long or hold-short (P = 0.42 and 0.24, respectively). During the dynamic test, hold-long conditioning significantly decreased [F((3,119)) = 7.99, P < 0.001] spindle responsiveness to increasing velocity. Mean velocity sensitivity was 4.44, 3.39, and 1.41 (impulses/s)/(mm/s) for the hold-short, hold-intermediate, and hold-long protocols, respectively. The nearly 2.5-fold decrease in velocity sensitivity following hold-long was significantly less than that for either hold-intermediate (P = 0.005) or hold-short conditioning (P < 0.001). Hold-short conditioning had little effect on velocity responses during the dynamic test [F((3,119)) = 0.23, P = 0.87]. In conclusion, only movement histories that stretch but not shorten muscle spindles alter their velocity sensitivity. In the low back, forward flexion and lateral bending postures would likely be the most provocative.

Somatostatin inhibits activation of dorsal cutaneous primary afferents induced by antidromic stimulation of primary afferents from an adjacent thoracic segment in the rat

To investigate the effect of somatostatin on the cross-excitation between adjacent primary afferent terminals in the rats, we recorded single unit activity from distal cut ends of dorsal cutaneous branches of the T10 and T12 spinal nerves in response to antidromic stimulation of the distal cut end of the T11 dorsal root in the presence and absence of somatostatin and its receptor antagonist applied to the receptive field of the recorded nerve. Afferent fibers were classified based upon their conduction velocity. Mean mechanical thresholds decreased and spontaneous discharge rates increased significantly in C and Adelta but not Abeta fibers of the T10 and T12 spinal nerves in both male and female rats following antidromic electrical stimulation (ADES) of the dorsal root from adjacent spinal segment (DRASS) indicating cross-excitation of thin fiber afferents. The cross-excitation was not significantly different between male and female rats. Microinjection of somatostatin into the receptive field of recorded units inhibited the cross-excitation. This inhibitory effect, in turn, was reversed by the somatostation receptor antagonist cyclo-somatostatin (c-SOM). Application of c-SOM alone followed by ADES of DRASS significantly decreased the mechanical thresholds and increased the discharge rates of C and Adelta fibers, indicating that endogenous release of somatostatin plays a tonic inhibitory role on the cross-excitation between peripheral nerves. These results suggest that somatostatin could inhibit the cross-excitation involved in peripheral hyperalgesia and have a peripheral analgesic effect.

ELECTROPHYSIOLOGICAL EVIDENCE FOR THE INTERACTION OF SUBSTANCE P AND GLUTAMATE ON Aδ AND C AFFERENT FIBRE ACTIVITY IN RAT HAIRY SKIN

1 The purpose of the present study was to investigate whether there was a cooperative interaction between substance P (SP) and glutamate (GLU) administered subcutaneously on Aδ and C primary afferent fibre activity in dorsal hairy skin of the rat in vivo. The single unit activities of Aδ and C afferent fibres were recorded by isolation of fibre filaments from the dorsal cutaneous nerve branches and the effects of subcutaneous injections of low doses of SP, GLU and SP + GLU on activity were determined. 2 Sub‐threshold doses of SP (1 µmol/L, 10 µL) administered subcutaneously into the dorsal hairy skin had no effect on the afferent discharges of either Aδ or C units. 3 The afferent discharges of 35% (11/31) of Aδ fibres and 33% (6/18) of C fibres were increased by local injection of the submaximal doses of GLU (10 µmol/L, 10 µL) into the receptive fields. 4 The GLU‐induced excitatory response was significantly enhanced by coinjection of subthreshold doses of SP. The mean discharge rates of Aδ fibres and C fibres were increased from 5.84 ± 1.54 and 5.02 ± 2.65 impulses/min to 19.91 ± 4.35 and 17.58 ± 5.59 impulses/min, respectively, whereas the excitatory proportions of Aδ and C fibres were increased from 35 and 33% to 84 and 83%, respectively. The duration of the excitation for Aδ fibres and C fibres was also significantly increased after coinjection of SP + GLU compared with that observed when either substance was given alone. 5 The present study provides electrophysiological evidence for an interaction between receptors for SP and GLU on the fine fibres activities in rat hairy skin, which may be involved in the mechanisms of hyperalgesia.