Actions of midazolam on excitatory transmission in dorsal horn neurons of adult rat spinal cord

BACKGROUND: Although intrathecal administration of midazolam, a water-soluble imidazobenzodiazepine derivative, has been found to produce analgesia, how it exerts this effect at the neuronal level in the spinal cord is not fully understood. METHODS: The effects of midazolam on electrically evoked and spontaneous excitatory transmission were examined in lamina II neurons of adult rat spinal cord slices using the whole cell patch clamp technique. RESULTS: Bath-applied midazolam (1 microm) diminished Adelta- and C-fiber evoked polysynaptic excitatory postsynaptic currents in both amplitude and integrated area. However, it affected neither Adelta- and C-fiber evoked monosynaptic excitatory postsynaptic currents in amplitude nor miniature excitatory postsynaptic currents in amplitude, frequency, and decay time constant. In the presence of a benzodiazepine receptor antagonist, flumazenil (5 microm), midazolam (1 microm) did not diminish Adelta-fiber evoked polysynaptic excitatory postsynaptic currents, suggesting that midazolam modulate the gamma-aminobutyric acid interneurons in the dorsal horn. CONCLUSIONS: Midazolam reduced excitatory synaptic transmission by acting on the gamma-aminobutyric acid type A/benzodiazepine receptor in interneurons, leading to a decrease in the excitability of spinal dorsal horn neurons. This may be a possible mechanism for the antinociception by midazolam in the spinal cord.     

Isoflurane reduces glutamatergic transmission in neurons in the spinal cord superficial dorsal horn: evidence for a presynaptic site of an analgesic action

The minimum alveolar concentration (MAC) of a volatile anesthetic defines anesthetic potency in terms of a suppressed motor response to a noxious stimulus. Therefore, the MAC of an anesthetic might in part reflect depression of motor neuron excitability. In the present study we evaluated the effect of isoflurane (ISO) on neurons in the substantia gelatinosa driven synaptically by putative nociceptive inputs in an in vitro spinal cord preparation of the rat. Whole-cell patch-clamp recordings were performed in neurons with their soma in the substantia gelatinosa of transverse rat spinal cord slices. We investigated the effect of ISO on excitatory postsynaptic currents (EPSC) evoked by dorsal root stimulation (eEPSC), spontaneous (sEPSC), and miniature (mEPSC) EPSC. ISO reversibly reduced the amplitude of eEPSC to 39% +/- 22% versus control. ISO decreased the frequency of sEPSC and mEPSC to 39% +/- 26% and 63% +/- 7%. Neither the amplitudes nor the kinetics of mEPSC and sEPSC were altered by ISO. We conclude that ISO depresses glutamatergic synaptic transmission of putative nociceptive primary-afferent inputs, presumably by reducing the release of the excitatory transmitter. This effect may contribute to an antinociceptive action of volatile anesthetics at the spinal cord level. IMPLICATIONS: The present electrophysiological in vitro experiments provide evidence that the volatile anesthetic isoflurane reduces excitatory transmitter release at the first site of synaptic integration of nociceptive inputs, the spinal cord superficial dorsal horn. This effect may contribute to the anesthetic action of volatile anesthetics at the spinal cord level.     

Uterine cervical distension induces cFos expression in deep dorsal horn neurons of the rat spinal cord

BACKGROUND: Uterine cervical distension underlies labor pain, yet its neurophysiology and pharmacology of inhibition remain unexplored. The authors examined uterine cervical distension-evoked cFos immunoreactivity in rat spinal cords, and the inhibitory effect of spinal cyclo-oxygenase inhibition on cFos expression. METHODS: Female rats were anesthetized with halothane, and pairs of metal rods were inserted in each cervical os through a mid-line laparotomy. A submaximal distension force (75 g) was applied for either 30 or 60 min, or, in control animals, no force was applied. Other animals received cervical lidocaine infiltration prior to uterine cervical distension. At the end of the experiments, the spinal cord at T12 to L2 levels was harvested and immunostained for cFos protein. Other animals received intrathecal ketorolac (0, 5, 25, and 50 microg; n = 5-6 for each group) prior to uterine cervical distension. RESULTS: Uterine cervical distension significantly increased cFos immunoreactivity in the spinal cord from T12 to L2, with most cFos expression in the deep dorsal and central canal regions. Surgical preparation alone without uterine cervical distension resulted in minimal cFos expression, primarily in the superficial dorsal horn. Uterine cervical distension-evoked cFos expression was prevented by prior infiltration of lidocaine into the cervix. Intrathecal ketorolac produced a dose-dependent inhibition of uterine cervical distension-induced cFos expression. CONCLUSION: The present study demonstrates that uterine cervical distension results in a similar pattern of spinal cord neuronal activation as seen with other noxious visceral stimuli. The inhibition of cFos expression by intrathecal ketorolac suggests that spinal cyclo-oxygenase plays a role in uterine cervical distension-induced nociception.     

Changes in properties of spinal dorsal horn neurons and their sensitivity to morphine after spinal cord injury in the rat

BACKGROUND: To gain a better understanding of spinal cord injury (SCI)-induced central neuropathic pain, the authors investigated changes in properties of spinal dorsal horn neurons located rostrally and caudally to the lesion and their sensitivity to morphine in rats after SCI. METHODS: The right spinal cord of Sprague-Dawley rats was hemisected at the level of L2. At 10 to 14 days after the SCI, when mechanical hyperalgesia/allodynia had fully developed, spontaneous activity and evoked responses to mechanical stimuli of wide-dynamic-range (WDR) and high-threshold neurons rostral and caudal to the lesion were recorded. Effects of cumulative doses of systemic (0.1-3 mg/kg) and spinal (0.1-5 microg) administration of morphine on spontaneous activity and evoked responses to the stimuli of the neurons were evaluated. RESULTS: Spontaneous activity significantly increased in WDR neurons both rostral and caudal to the SCI site, but high-frequency background discharges with burst patterns were only observed in neurons rostral to the SCI site. Significant increases in responses to the mechanical stimuli were seen both in WDR and high-threshold neurons located both rostrally and caudally to the lesion. The responses to nonnoxious and noxious stimuli were significantly greater in caudal WDR neurons than in rostral WDR neurons. In contrast, the responses to pinch stimuli were significantly higher in rostral high-threshold neurons than those in caudal high-threshold neurons. Systemically administered morphine had a greater effect on responses to nonnoxious and noxious stimuli of rostral WDR neurons than those of caudal WDR neurons. Spinally administered morphine significantly suppressed responses of WDR neurons in SCI animals to nonnoxious stimuli compared with those in sham-operated control animals. CONCLUSIONS: The findings suggest that changes in properties of spinal dorsal horn neurons after SCI are caused by different mechanisms, depending on the classification of the neurons and their segmental locations.     

Immunocytochemical study of parvalbumin,calbindin D-28k,and calretinin in the superficial dorsal horn of the rat spinal cord following unilateral hindpaw inflammation

The effect of noxious stimulation on the immunore-activity of the calcium-binding proteins parvalbumin (PV), calbindin-D-28k (CB) and calretinin (CR) was investigated in the superficial dorsal horn of lumbar levels L5-L3 of the rat spinal cord. Freund's adjuvant was injected unilaterally into the hindpaw to induce inflammation. Immunohistochemical techniques were utilized to investigate changes in the calcium-binding proteins 2h and 1, 2, 4, and 7 days after injection. At 24h after injection, a decrease in the intensity of fluorescence of PV-immunoreactive (IR) fibers was observed in the superficial layer (substantia gelatinosa) of the ipsilateral dorsal horn (L5-L3) in most animals. Comparatively fewer animals exhibited changes in the CB- and CR-IR fibers, except at the L3 level 2 days after, and at the L4 level 7 days after the hindpaw injection. After the peak response, at 24h in most animals, there was a decline in the number of responders at 2 days and no differences were noted at 4 days. However, at 7 days, there was again an increase in the number of animals revealing diminished fluorescence intensity in the ipsilateral substantia gelatinosa. Changes in immunoreactivity of calcium binding proteins in the interneurons of the superficial lumbar dorsal horn may reflect hyperactivity within these neurons following noxious stimulation.     

瑞芬太尼和芬太尼对大鼠脊髓背角神经元NMDA受体通道电流的影响

目的 探讨瑞芬太尼和芬太尼对大鼠脊髓背角神经元NMDA受体通道电流的影响.方法 采用全细胞膜片钳技术记录NMDA受体通道电流.原代培养的E14SD大鼠脊髓背角神经元(DH细胞)30个,采用随机数字表法,将其分为3组(n=10):瑞芬太尼组(R组)、芬太尼组(F组)、对照组(C组).4 nmol/L瑞芬太尼(R组)、10 μmol/L芬太尼(F组)灌流DH细胞60 min后洗脱.于给药后即刻(T0)、药物作用15 min(T1)、30 min(T2)、45 min(T3)、60 min(T4)、洗脱后15 min(T5)、30 min(T6)时记录NMDA受体通道电流.结果 与C组比较,F组各时点NMDA受体通道峰电流差异无统计学意义,R组T0、T1时NMDA受体通道峰电流差异无统计学意义(P＞0.05),T2-T6时NMDA受体通道峰电流升高(P＜0.01);与T0时比较,R组T3-T6时NMDA受体通道峰电流升高(P＜0.01);与T5时比较,R组T2-T4时、T6时NMDA峰电流下降(P＜0.01).结论 瑞芬太尼可增强大鼠脊髓背角神经元NMDA受体功能,于洗脱后达峰效应,芬太尼无此作用.     

The effect of isoflurane on excitatory synaptic transmission in the rat hippocampus

The purpose of this investigation was to study the effect of isoflurane on excitatory synaptic transmission. Rat hippocampal slices maintained in vitro were used as a model. Isoflurane caused a dose-dependent reduction of the excitatory postsynaptic potential (EPSP); 1.5% isoflurane reduced the EPSP by 35 +/- 9% (mean +/- s.d.) and 3% by 57 +/- 11%. Neither spontaneous nor potassium-stimulated efflux of the glutamate analogue D-(3H)aspartate was changed, but the content of D-(3H)aspartate in slices loaded during isoflurane was reduced to 83 +/- 12% of control (P less than 0.05). The intracellularly recorded response to direct application of glutamate increased by 37 +/- 20% during isoflurane (3%) and 50 +/- 5% during halothane (2%). Isoflurane (3%) enhanced the response to the glutamate receptor agonist quisqualate by 44 +/- 19%, whereas the N-methyl-D-aspartate response was unchanged. Isoflurane enhanced the tetanic depression of the population spike. The present results suggest that isoflurane reduces excitatory synaptic transmission by a presynaptic mechanism.     

The effect of pentobarbital sodium on the dorsal horn of the spinal cord

The effect of intravenously administered pentobarbital sodium on the activity of single unit in Rexed lamina V of the transected feline lumbar spinal cord was studied using an extracellular microelectrode recording technique. Pentobarbital sodium 1.0 mg·kg⁻¹, 2.5 mg·kg⁻¹, and 5.0 mg·kg⁻¹ administered intravenously suppressed both the spontaneous and the evoked activity in Rexed lamina V cells, known to respond principally to noxious stimuli, in a dose-dependent manner. The maximum depression of cell activity occurred within 5 min after intravenous administration. The recovery of cell activity occurred within 70 min after intravenous administration of pentobarbital sodium. We conclude that pentobarbital sodium intravenously administered has a suppressive effect on single unit activity of cells in Rexed lamina V and probably has an analgesic effect. Its suppressive effect is dose-dependent.     

Injury to dorsal root ganglia alters innervation of spinal cord dorsal horn lamina involved in nociception

STUDY DESIGN: A study of the relation between the development of mechanical allodynia and the reorganization of primary afferent terminals in the sensory lamina of the rat spinal cord dorsal horn after partial dorsal root ganglion injury in rats. OBJECTIVES: To investigate the pathologic mechanisms of mechanical allodynia after partial dorsal root ganglion injury. SUMMARY OF BACKGROUND DATA: After experimental peripheral nerve injury causing neuropathic pain, myelinated afferent fibers sprout into lamina II of the dorsal horn. This lamina is associated with nociceptive-specific neurons that generally are not stimulated by myelinated fiber input from mechanical receptors. These morphologic changes are suggested to have significance in the pathogenesis of chronic mechanical allodynia, although it is not known whether this kind of morphologic change occurs after dorsal root ganglion injury. METHODS: After partial dorsal root ganglion crush injury, the mechanical force causing footpad withdrawal was measured with von Frey hairs, and myelinated primary afferents were labeled with cholera toxin B subunit horseradish peroxidase, a selective myelinated fiber tracer that identifies transganglionic synapses. RESULTS: After partial dorsal root ganglion injury, mechanical allodynia developed in the corresponding footpad within 3 days and persisted throughout the experimental period. At 2 and 4 weeks after the injury, B subunit horseradish peroxidase-positive fibers, presumably myelinated afferents, were observed to be sprouting into lamina II of the dorsal horn on the injured side, but not on the contralateral control side. CONCLUSIONS: Morphologic change in spinal cord dorsal horn lamina II occurs after partial dorsal root ganglion injury. This change may have significance in the pathogenesis of chronic mechanical allodynia after partial dorsal root ganglion injury.     

Functional mu opioid receptors are reduced in the spinal cord dorsal horn of diabetic rats

BACKGROUND: The mechanisms of decreased spinal analgesic potency of morphine in neuropathic pain are not fully known. Agonist-stimulated [35S]GTPgammaS receptor autoradiography has been used to measure receptor activation of G proteins in vitro. Using this technique, we determined changes in the functional mu opioid receptors in the spinal dorsal horn in diabetic rats. METHODS: Rats were rendered diabetic with an intraperitoneal injection of streptozotocin. The lumbar spinal cord was obtained from age-matched normal and diabetic rats 4 weeks after streptozotocin treatment. [D-Ala2,N-MePhe4,Gly5-ol]-enkephalin (DAMGO, 10 microm)-stimulated [35S]GTPgammaS binding was performed in both tissue sections and isolated membranes. RESULTS: The DAMGO-stimulated [35S]GTPgammaS binding in the spinal dorsal horn was significantly reduced (approximately 37%) in diabetic rats compared with normal rats. However, [35S]GTPgammaS bindings in the spinal dorsal horn stimulated by other G protein-coupled receptor agonists, including [D-Pen2,D-Pen5]-enkephalin, R(-)N6-(2-phenylisopropyl)-adenosine, and WIN-55212, were not significantly altered in diabetic rats. The basal [35S]GTPgammaS binding in the spinal dorsal horn was slightly (approximately 13%) but significantly increased in diabetic rats. Western blot analysis revealed no significant difference in the expression of the alpha subunits of G(i) and G(o) proteins in the dorsal spinal cord between normal and diabetic rats. CONCLUSIONS: These data suggest that the functional mu opioid receptors in the spinal cord dorsal horn of diabetic rats are reduced. The impaired functional mu opioid receptors in the spinal cord may constitute one of the mechanisms underlying the reduced spinal analgesic effect of mu opioids in diabetic neuropathic pain.     

Licking and c-fos expression in the dorsal horn of the spinal cord after the formalin test

We investigated whether c-fos expression in the dorsal horn is affected by licking in the formalin test. Thirty adult Sprague-Dawley rats were divided into 5 groups of 6 rats each: a free condition control (Free Cont) group, formalin test under free condition (Free F-test) group, scrub stimulation under free condition (Free Scrub) group, restrained condition control (Restricted Cont) group, and formalin test under restrained condition (Restricted F-test) group. Animals in the three free condition groups and two restricted groups were put in a clear plastic chamber and a restraining chamber, respectively. Ten percent formalin was injected into the left rear paw in the Free and Restricted F-test groups. Animals in the Free Scrub group were scrubbed on the left rear paw with a wet cotton swab. The Free Cont, Restricted Cont, and Free Scrub groups showed little c-fos expression. The number of c-fos positive cells in the ipsilateral surface dorsal horn of the Restricted F-test group was significantly less than that of the Free F-test group (P < 0.05). The results indicated that the licking action increased c-fos expression of the lumbar dorsal horn in the formalin test.     

Halothane modulates NMDA and non-NMDA excitatory synaptic transmission in rat cortical neurons

Purpose Although general anesthetics may decrease neuronal excitation, their detailed effects on spontaneous excitatory postsynaptic currents (EPSCs) remain controversial. We investigated and compared the effects of halothane on N-methyl-d-asparate (NMDA) and non-NMDA receptor-mediated postsynaptic currents.Methods Spontaneous synaptic currents were recorded by the patch clamp technique in cultured rat cortical neurons. They were isolated by specific pharmacological blocking agents and their electrophysiologic properties were examined.Results The frequency of NMDA EPSCs was preferentially decreased as compared with that of non-NMDA EPSCs at halothane 1.2mM. The total net charge of EPSCs mediated by NMDA and non-NMDA receptors was depressed to 56% ± 6% (mean ± SD) and 71% ± 7% of control by halothane 0.6mM, and to 11% ± 9% and 59% ± 11% of control by halothane 1.2mM, respectively.Conclusion These results show that halothane causes decrease of excitatory synaptic activity, with NMDA EPSCs being more sensitive than non-NMDA EPSCs.     

神经损伤诱发脊髓WDR神经元功能活动变化

在坐骨神经损伤的大鼠病理模型上，研究神经损伤诱发脊髓背角与痛觉传递相关的广动力学性（Wide-dynamic－range，WDR）神经元电活动的改变。实验结果显示，神经损伤后，损伤区远心端的电刺激诱发WDR神经元的早成分电活动的放电数目显著低于近心端和动物健侧（P＜0．01），而晚成分放电反应无减弱。提示WDR神经元通过它对外周神经元传入反应的编码特性而参与对伤害性刺激的定位和定性。     

Actions of norepinephrine and isoflurane on inhibitory synaptic transmission in adult rat spinal cord substantia gelatinosa neurons

Volatile inhaled anesthetics and nitrous oxide (N2O) are often used together in clinical practice to produce analgesia. Because the analgesic effect of N2O is, at least in part, mediated by norepinephrine (NE) release in the spinal cord, we examined the interaction between isoflurane (ISO) and NE in the adult rat spinal cord with respect to central nociceptive information processing. The effects of clinically relevant concentrations of ISO (1 MAC) and NE (20 microM) on spontaneous inhibitory transmission in substantia gelatinosa (SG) neurons were examined using the blind whole-cell patch-clamp method. ISO prolonged the decay time and increased the total charge transfer of spontaneous inhibitory postsynaptic currents. NE increased the frequency and mean amplitude of inhibitory postsynaptic currents and the charge transfer as well. Coapplication of both drugs led to an additive increase of the charge transfer and frequent temporal summation of inhibitory postsynaptic currents. We conclude that both ISO and NE enhance the inhibitory synaptic transmission in the rat SG neurons and their interaction is additive, suggesting that ISO may add to the analgesic action of N2O at the spinal cord dorsal horn level.