Activation of serotonergic neurons in the raphe magnus is not necessary for morphine analgesia

A wealth of pharmacological and behavioral data suggests that spinally projecting serotonergic cells mediate opioid analgesia. A population of medullary neurons, located within raphe magnus (RM) and the neighboring reticular nuclei, contains serotonin and is the source of serotonin in the spinal dorsal horn. To test whether serotonergic neurons mediate opioid analgesia, morphine was administered during recordings from medullary cells that were physiologically characterized as serotonergic (5HTp) by their slow and steady discharge pattern in the lightly anesthetized rat. Selected 5HTp cells (n = 14) were intracellularly labeled, and all contained serotonin immunoreactivity. The discharge of most 5HTp cells was not affected by an analgesic dose of systemic morphine. In a minority of cases, 5HTp cells either increased or decreased their discharge after morphine administration. However, morphine altered the discharge of some 5HTp cells in the absence of producing analgesia and conversely did not alter the discharge of most 5HTp cells in cases in which analgesia occurred. RM cells with irregular discharge patterns and excitatory or inhibitory responses to noxious tail heat were classified as ON and OFF cells, respectively. All ON and OFF cells that were intracellularly labeled (n = 9) lacked serotonin immunoreactivity. All ON cells were inhibited, and most OFF cells were excited by systemic morphine. Because 5HTp cells do not consistently change their discharge during morphine analgesia, they are unlikely to mediate the analgesic effects of morphine. Instead, nonserotonergic cells are likely to mediate morphine analgesia in the anesthetized rat. In light of the sensitivity of morphine analgesia to manipulations of serotonin, serotonin release, although neither necessary nor sufficient for opioid analgesia, is proposed to facilitate the analgesic effects of nonserotonergic RM terminals in the spinal cord.     

Peripheral morphine analgesia: synergy with central sites and a target of morphine tolerance

Morphine injected s.c. in the tail is a potent analgesic in the tail-flick assay when the radiant heat source is focused directly over the injection site (ED50, 4.5 micrograms), but not if the radiant heat source is moved 1 cm proximally or distally to the injection site. Naloxone given systemically reverses this peripheral analgesia. Antisense oligodeoxynucleotides directed against exons 1 and 4 of MOR-1, a cloned mu opioid receptor, administered intrathecally (i.t.) block the local analgesic effect of morphine in the tail, indicating that the local response is mediated through mu receptors located on the terminals of sensory neurons from the dorsal root ganglia. Combinations of morphine given locally in the tail and spinally (i.t.) are synergistic. Spinal morphine also synergizes with systemic morphine in analgesia assays. Supraspinal morphine enhances systemic morphine analgesia, but less dramatically. We also examined tolerance on these analgesic systems by using a daily morphine injection paradigm which shifts the dose-response curve for systemic morphine approximately 2-fold after 5 days. In this paradigm, morphine's analgesic potency after either supraspinal or spinal administration alone does not change. However, the dose-response curve for local morphine in the tail is shifted by over 19-fold. The analgesic activity of the combination of supraspinal and systemic morphine is lowered approximately 2-fold and the combination of i.t. and systemic morphine by 12-fold. These studies confirm the presence of a peripheral mechanism for morphine analgesia mediated by mu receptors located on sensory neurons from the dorsal root ganglia, which is extremely sensitive to chronic morphine dosing.     

Inescapable shock-induced potentiation of morphine analgesia in rats: Sites of action.

Inescapable shock (IS) enhances analgesia to systemic morphine (MOR) 24 hr later. IS activates serotonin neurons in the dorsal raphe nucleus (DRN), rendering them hyperexcitable. These studies tested whether IS potentiates the analgesic effect of MOR microinjected in the DRN, as predicted by this hypothesis. To test site specificity, the effect of previous IS was examined on MOR microinjected lateral to the DRN and into 2 other sites that support MOR analgesia, the nucleus raphe magnus (NRM) and spinal cord. Twenty-four hours after IS, potentiated analgesia was observed after 0.5 μg MOR microinjected into, but not lateral to, the DRN. Potentiated analgesia was also observed after NRM (1.0 μg) and spinal cord (3.0 μg) MOR microinjections. These data suggest that IS-induced excitability changes within the DRN synergize with opiates microinjected in other analgesia areas and that this potentiates the responses to opiates 24 hr after IS. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Endogenous opioid peptides acting at mu-opioid receptors in the dorsal horn contribute to midbrain modulation of spinal nociceptive neurons

Activation of neurons in the midbrain periaqueductal gray (PAG) inhibits spinal dorsal horn neurons and produces behavioral antinociception in animals and analgesia in humans. Although dorsal horn regions modulated by PAG activation contain all three opioid receptor classes (mu, delta, and kappa), as well as enkephalinergic interneurons and terminal fields, descending opioid-mediated inhibition of dorsal horn neurons has not been demonstrated. We examined the contribution of dorsal horn mu-opioid receptors to the PAG-elicited descending modulation of nociceptive transmission. Single-unit extracellular recordings were made from rat sacral dorsal horn neurons activated by noxious heating of the tail. Microinjections of bicuculline (BIC) in the ventrolateral PAG led to a 60-80% decrease in the neuronal responses to heat. At the same time, the responses of the same neurons to iontophoretically applied NMDA or kainic acid were not consistently inhibited. The inhibition of heat-evoked responses by PAG BIC was reversed by iontophoretic application of the selective mu-opioid receptor antagonists, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP). A similar effect was produced by naloxone; however, naloxone had an excitatory influence on dorsal horn neurons in the absence of PAG-evoked descending inhibition. This is the first demonstration that endogenous opioids acting via spinal mu-opioid receptors contribute to brain stem control of nociceptive spinal dorsal horn neurons. The inhibition appears to result in part from presynaptic inhibition of afferents to dorsal horn neurons.     

Intraoperative intravenous ketamine in combination with epidural analgesia: postoperative analgesia after renal surgery

We designed this double-blinded, randomized, controlled study to evaluate the effect of small-dose ketamine IV in combination with epidural morphine and bupivacaine on postoperative pain after renal surgery. An epidural catheter was inserted, and the administration of morphine and bupivacaine was started before surgery. Forty patients were assigned to one of two groups (ketamine or control). The ketamine group was administered a ketamine bolus and infusion during surgery. The median visual analog pain scale (VAS) scores at rest were significantly lower in the ketamine group during the first 6 h (P < 0.01). VAS pain scores on coughing were also significantly lower in the ketamine group (P < 0.01). Cumulative postoperative total analgesic consumption was less in the ketamine group on Days 1 and 2 (P < 0.001). The first analgesic demand time was shorter in the control group (9.2 +/- 11.5 min) than in the ketamine group (22.3 +/- 17.1 min) (P < 0.0001). The incidence of nausea and pruritus was more frequent in the control group (P < 0.05). In conclusion, postoperative analgesia was more effective when spinal cord and brain sensitization were blocked by a combination of epidural morphine/bupivacaine and IV ketamine. IMPLICATIONS: Renal nociception conducted multisegmentally by both the spinal nerves (T10 to L1) and the vagus nerve cannot be blocked by epidural analgesia alone. We demonstrated that IV ketamine had an improved analgesic or opioid-sparing effect when it was combined with epidural bupivacaine and morphine after renal surgery.     

Behaviorally functional opioid systems in infant rats: II. Evidence for pharmacological, physiological, and psychological mediation of pain and stress.

Two experiments, with 160 10-day-old Sprague-Dawley rat pups, examined the behavioral characteristics of the neonatal opioid system during distressful situations, using a modification of the hot-plate paw-lick test. Ss were analgesic to heat following intraperitoneal morphine (0.5 mg/kg). Subcutaneous naloxone (0.5 mg/kg) prevented the analgesia. Morphine analgesia was significantly greater in Ss group-isolated from the dam. Saline controls group-isolated from the dam exhibited longer latencies than their nest-housed siblings. Individual isolation for 5 min markedly increased paw-withdrawal latency, and this effect was naltrexone reversible. Analgesia was not seen when Ss were tested directly from the nest or when grouped with others for 5 min. It is suggested that the opioid systems for stress and pain are functional in 10-day-old rats and that short-term isolation from the dam is a probable natural stressor modulated by endogenous opioid release. (51 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Transient spinal ischemia in rat: characterization of spinal cord blood flow, extracellular amino acid release, and concurrent histopathological damage

Extracellular concentrations of amino acids in halothane-anesthetized rats were measured using a microdialysis fiber inserted transversely through the dorsal spinal cord at the level of the lumbar enlargement in conjunction with HPLC and ultraviolet detection. After a 2-h washout and a 1-h control period, 20 min of reversible spinal cord ischemia was achieved by the inflation of a Fogarty F2 catheter passed through the femoral artery to the descending thoracic aorta. After 2 h of postischemic reperfusion, animals were transcardially perfused with saline followed by 10% formalin or 4% paraformaldehyde. The glutamate concentration in the dialysate was significantly elevated after 10 min of occlusion and returned to near-baseline during the first 30 min of reperfusion. Taurine was elevated significantly 0.5 h postocclusion and continued to increase throughout the 2 h of reperfusion. Glycine concentrations showed a tendency to be slightly above baseline during the reperfusion period. Glutamine concentrations modestly increased following 2 h of reperfusion. No significant changes in aspartate, asparagine, and serine were detected. In control animals no significant changes in any amino acids were detected. To assess the role of complete spinal ischemia on spinal glutamate release, studies were carried out using cardiac arrest. Twenty minutes after induction of cardiac arrest, the glutamate concentration was increased about 350-400%. In a separate group of animals, spinal cord blood flow (SCBF) and its response to decreased CO2 were measured using a laser probe implanted into the epidural space at the level of the L2 vertebral segment. SCBF decreased to 5-6% of the control during aortic occlusion. After reversible ischemia, marked hyperemia was seen for the first 15 min, followed by hypoperfusion at 60 min. Under control-preischemic conditions a decrease in arterial CO2 content caused a decrease in SCBF of about 25%. This autoregulatory response was almost completely absent when assessed 60 min after a 20-min interval of aortic occlusion. Histopathological analysis of spinal cord tissue from these animals demonstrated heavy neuronal argyrophilia affecting small and medium-sized neurons located predominantly in laminae III-V. These changes corresponded to signs of irreversible damage at the ultrastructural level. Occasionally, small areas of focal necrosis, located in the dorsolateral part of the dorsal horn and anterolateral part of the ventral horn, were found. The results are consistent with a role for glutamate in ischemically induced spinal cord damage and suggest that taurine elevation detected during the early reperfusion period may serve as an important indicator of irreversible spinal cord neuronal damage.     

Epidural morphine plus ketamine for upper abdominal surgery: improved analgesia from preincisional versus postincisional administration

Increased postoperative pain may be caused by central nervous system plasticity, which may be related to actions of N-methyl-D-aspartic acid (NMDA) receptors on neurons in the dorsal horn of the spinal cord. Opioids act mainly on presynaptic receptors and reduce neurotransmitter release, while ketamine antagonizes NMDA receptors and prevents wind-up and long-term potentiation. Thus, we postulated that central nervous system sensitization would be prevented more effectively by the preoperative use of these two drugs simultaneously, and the effect of preemptive analgesia would be demonstrated. Ketamine, 60 mg, and morphine, 2 mg, were injected epidurally through an indwelling catheter that was inserted at the T7-8 interspace in 60 ASA physical status class 1-2 patients. The drugs were injected before induction of anesthesia (Group 1; n = 30) or immediately after removal of a surgical specimen (Group 2; n = 30). An additional 2 mg of morphine was injected when the patients complained of resting pain. The analgesic effect was assessed by the time from first analgesic injection to second dose and the number of patients who needed supplemental injections. Complications were also noted. The duration of analgesia was longer (P < 0.01) in Group 1 (31.1 +/- 16.0 h) than in Group 2 (21.1 +/- 12.0 h), and the proportion of patients who needed supplemental injections was decreased (P < 0.05) in Group 1 (56.7%) compared with Group 2 (90.0%). The incidence of adverse effects was not different between the two groups. In conclusion, preoperative administration of morphine and ketamine is more effective in reducing postoperative pain than it is when given during the operation.     

Increases in protein kinase C gamma immunoreactivity in the spinal cord of rats associated with tolerance to the analgesic effects of morphine

Our previous studies have indicated a critical role of protein kinase C (PKC) in intracellular mechanisms of tolerance to morphine analgesia. In the present experiments, we examined (1) the cellular distribution of a PKC isoform (PKC gamma) in the spinal cord dorsal horn of rats associated with morphine tolerance by utilizing an immunocytochemical method and (2) the effects of the N-methyl-D-aspartate receptor antagonist MK-801 on tolerance-associated PKC gamma changes. In association with the development of tolerance to morphine analgesia induced by once daily intrathecal administration of 10 micrograms morphine for eight days, PKC gamma immunoreactivity was clearly increased in the spinal cord dorsal horn of these same rats. Within the spinal cord dorsal horn of morphine tolerant rats, there were significantly more PKC gamma immunostained neurons in laminae I-II than in laminae III-IV and V-VI. Such PKC gamma immunostaining was observed primarily in neuronal somata indicating a postsynaptic site of PKC gamma increases. Moreover, both the development of morphine tolerance and the increase in PKC gamma immunoreactivity were prevented by co-administration of morphine with 10 nmol MK-801 between Day 2 and Day 7 of the eight day treatment schedule. In contrast, PKC gamma immunoreactivity was not increased in rats receiving a single i.t. administration of 10 micrograms morphine on Day 8, nor did repeated treatment with 10 nmol MK-801 alone change baseline levels of PKC gamma immunoreactivity. These results provide further evidence for the involvement of PKC in NMDA receptor-mediated mechanisms of morphine tolerance.     

Erythrocyte Na/K ATPase activity and diabetes: relationship with C-peptide level

The purpose of this study is to clarify the volume effect of epidural saline injection 20 min after spinal anesthesia. Thirty patients undergoing combined spinal and epidural anesthesia for orthopedic surgery were randomly divided into two groups: a control group (n = 15) and a saline group (n = 15). In the control group, 2% lidocaine 3 ml with 0.4% tetracaine was injected into the subarachnoid space from L 4-5 interspace using Durasafe (Becton Dickinson, USA) and saline was not injected into the epidural space. In the saline group, saline 10 ml was injected through an epidural catheter 20 min after spinal anesthesia. The levels of analgesia 20 min after spinal anesthesia were not significantly different between the groups. However, the levels of analgesia 3, 5, 10, 40 and 100 min after epidural saline injection in the saline group were significantly higher than those in the control group (P < 0.05). The highest analgesic level was obtained 10 min after epidural saline injection and reached to T 4.3 +/- 1.1. In conclusion, epidural saline injection increases the analgesic level 20 min after spinal anesthesia because of the volume effect.     

Quality of center day care and attunement between parents and caregivers: center day care in cross-national perspective

The efferent projections from the periaqueductal gray matter (PAG) to the parabrachial nucleus (PB) were studied in the rat following microinjections of the anterograde axonal tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) into restricted regions of the PAG. The dorsomedial and dorsolateral PAG columns project almost exclusively to the superior lateral PB subnucleus, whereas the lateral and ventrolateral PAG columns project to five lateral PB sites: dorsal lateral subnucleus, medial and lateral crescent areas (which flank the dorsal lateral PB subnucleus), central lateral subnucleus (rostral portion), and superior lateral subnucleus. The PAG region lying near the cerebral aqueduct projects to five lateral PB sites: external lateral subnucleus (inner subdivision), medial and lateral crescent areas, central lateral subnucleus (rostral portion), and dorsal lateral subnucleus. The internal lateral PB subnucleus, which projects exclusively to the intralaminar thalamic nuclei, and the K?lliker-Fuse nucleus were not innervated by the PAG. The PAG selectively innervates individual PB subnuclei that may be part of the spino-parachio-forebrain pathway. All PAG columns, including the aqueductal region, project to the superior lateral PB subnucleus, a presumed nociceptive relay site that receives inputs from multiple spinal cord regions (laminae I, V, and VIII) and projects to the ventromedial and retrochiasmatic hypothalamic areas-two regions that have been implicated in complex goal-directed behavior (e.g., food intake and reproductive function). Earlier studies demonstrated that the dorsal lateral and external lateral PB subnuclei (inner division) receive overlapping inputs from the superficial dorsal horn (laminae I and II) and the nucleus tractus solitarius, and both PB subnuclei send projections to limbic forebrain areas (e.g., hypothalamus, preoptic region, amygdala). Because the PAG projects to both of these PB subnuclei, this projection system possibly functions as a behavioral state-dependent filter system that modulates ascending nociceptive and/or visceral information as it is relayed through the PB to forebrain sites.     

Noradrenergic and serotonergic involvement in brief shock-induced analgesia in rats.

Performed 4 experiments to investigate the effects of different techniques causing noradrenergic and serotonergic depletions in the brain and spinal cord on brief shock-induced analgesia (BSIA). Newborn pups were administered N-2-choloroethyl-N-ethyl-2-bromobenzylamine systemically and 6-hydroxydopamine (6-OHDA) administered either systemically or directly into the locus ceruleus region, or intrathecally into the lumbar subarachnoidal space, caused notable and consistent attenuations of analgesia. Treatments reduced noradrenaline concentrations in the spinal cord drastically. A potentiation of BSIA was caused by the administration of p-chlorophenyl-alanine, whereas administration of 5,7-dihydroxytryptamine, into the nucleus raphe magnus or intrathecally into the subarachnoidal space, produced attenuation of the analgesic effect. Biochemical analyses revealed marked 5-hydroxytryptamine (5-HT) depletions in the spinal cord. Findings are discussed with regard to the role of spinal noradrenaline and 5-HT involvement in BSIA and in reactions to stressful events. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A physiologic assessment of intrathecal amitriptyline in sheep

BACKGROUND: Intrathecal injection of amitriptyline enhances antinociception from intravenous morphine and reduces neuropathic pain behavior in animals. This study represents part of a preclinical assessment of intrathecal amitriptyline to determine its safety for use in humans. METHODS: Low thoracic intrathecal, femoral, and pulmonary arterial catheters were inserted in 18 adult ewes, followed 96 h later by intrathecal injection of saline or 5 mg amitriptyline and by determination of spinal cord blood flow, hemodynamic variables, behavioral changes, cerebrospinal fluid concentrations of catecholamines and amitriptyline, and spinal tissue concentrations of amitriptyline. In six other ewes, low thoracic intrathecal and femoral arterial catheters were inserted and blood pressure and heart rate were measured after intrathecal injection of saline or 0.25, 1, or 5 mg amitriptyline. Four other ewes received cervical intrathecal injection of 5 and 10 mg amitriptyline, and antinociception was determined. RESULTS: Thoracic intrathecal injection of amitriptyline produced dose-dependent sedation but did not significantly affect spinal cord blood flow or hemodynamic variables. Spinal cord tissue concentrations of amitriptyline were 100 times greater in tissue near the tip of the thoracic intrathecal catheter compared with cervical cord tissue. Cerebrospinal fluid concentrations of catecholamines did not significantly change after amitriptyline was administered. Cervical intrathecal injection of 5 mg amitriptyline produced mild antinociception, whereas 10 mg produced intense sedation and, in one sheep, seizures and death. CONCLUSIONS: Although other preclinical toxicity studies are necessary before introducing intrathecal amitriptyline for use in humans, this study did not reveal dangerous changes in blood pressure or spinal cord blood flow from this agent.     

Analgesic response to stress is reduced in perinatally undernourished rats

Stress-induced analgesia was evaluated in adult rats submitted early in life to a protein deprivation schedule. Rats were undernourished with a hypoproteic diet containing 80 g casein/kg diet from d 14 of gestation until 50 days of age. Rats were thereafter fed a balanced nonpurified diet until 140 days of age, when they were exposed to two stressors: forced swimming and acute restraint, after which the analgesic response was evaluated. In addition, the analgesic response induced by different morphine doses was determined in another group of rats. Basal latency was not different in deprived and control rats. Undernourished rats presented a significantly lower analgesic response in both stress situations. However, when the analgesic response induced by different morphine doses (1, 2, 4 and 8 mg/kg, s.c.) was assessed, a significantly higher response occurred in undernourished rats compared to control rats. This lower stress-induced analgesia in undernourished rats may account for the behavioral alterations attributed to early undernutrition.     

Cardiorespiratory and spinal cord blood flow effects of intrathecal neostigmine methylsulfate, clonidine, and their combination in sheep

BACKGROUND: Intrathecal neostigmine may produce analgesia by itself and may enhance analgesia from spinal clonidine. Before clinical trials, the spinal cord blood flow effects of these drugs alone and in combination should be examined in animals. METHODS: Conscious, nonpregnant ewes with indwelling vascular and thoracic spinal catheters received intrathecal injection of 0.2 or 2 mg neostigmine, 0.2 mg clonidine, or 2 mg neostigmine plus 0.2 mg clonidine. Mean systemic and pulmonary arterial and central venous pressures, heart rate, and cardiac output were monitored, arterial blood was sampled for blood gas tensions and pH, and spinal cord blood flow was determined by colored microsphere injection before and at 15, 60, and 240 min after spinal study drug injection. RESULTS: Neostigmine alone did not affect cardiorespiratory variables or spinal cord blood flow. Intrathecal clonidine alone decreased systemic arterial and central venous pressures, whereas these effects were not observed with addition of neostigmine. Clonidine or neostigmine alone or the combination of clonidine and neostigmine did not affect spinal cord blood flow. CONCLUSIONS: Intrathecal neostigmine alone or in combination with clonidine does not reduce spinal cord blood flow, an important preclinical toxicity issue. These results provide additional support for initial clinical trials of intrathecal neostigmine for analgesia.     

The antinociceptive effects of branched-chain amino acids: evidence for their ability to potentiate morphine analgesia

The effect of branched-chain amino acids (BCAA) on pain threshold was studied in rats. Nociception was induced by the hot-plate analgesia meter, a method measuring supraspinally organized pain responses. After a single intravenous injection of BCAA (320 mg/kg), the percent change in latency time to the pain response significantly increased by 19% in 60 min, and by 22% in 75 min (p < 0.005), as compared to an injection of an equal volume of a standard concentration of an amino acid solution or physiological saline. Subsequently, we studied the interaction of BCAA with opioid-type analgesia. In combination with intravenously injected morphine (3 mg/kg), BCAA significantly potentiated and prolonged the action of morphine using the hot-plate test. From 5 min after morphine injection, the latencies to a pain response were markedly higher with the combination of BCAA and morphine (+80% and +89% at 5 min after morphine injection, if BCAA was administered 45 or 60 min prior to morphine injection, respectively) when compared with the effect of morphine alone (+13% at 5 min; p < 0.005). BCAA demonstrated analgesic effects, which, in combination with morphine, potentiated and prolonged the antinociceptive action of morphine. BCAA may represent a new adjunct treatment modality for acute and chronic pain, and give us further insight into the mechanisms of pain control.     

Ability of periaqueductal gray subdivisions and adjacent loci to elicit analgesia and ability of naloxone to reverse analgesia.

This study is an investigation of the effects of stimulation of regions within and adjacent to the periaqueductal gray (PAG) matter. Eighty-five rats were implanted with 1 monopolar stimulating electrode into 1 of 5 loci. Potency of analgesia was evaluated by relative increases in tailflick latencies after brain stimulation, and threshold current intensity was used to elicit analgesia. The ability of naloxone to reverse the stimulation-induced analgesia was also evaluated. Results replicate the previous finding of differential naloxone reversibility of ventral vs. dorsal PAG sites, but they do not support a regional distinction in the potency of analgesia induced. The results suggest that dorsal PAG sites are involved in a separate nonopiate pain-inhibitory system, whereas ventral sites are involved in an opiate system. These systems, however, do not respect the cytoarchitectural boundaries of the PAG because sites adjacent to the PAG elicit similar effects with a corresponding dorsal-ventral distinction. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Thyroid hormones differentially affect sarcoplasmic reticulum function in rat atria and ventricles

We have recently reported that the short-acting anesthetic and analgesic drug midazolam can produce analgesia and decrease morphine tolerance and dependence in the rat by interacting with the opioid system. This study was designed to investigate the effect of midazolam, morphine, and both together on met-enkephalin levels in the rat. Male Sprague-Dawley rats were divided into four groups: (1) saline-saline; (2) saline-morphine; (3) midazolam-saline, and (4) midazolam-morphine groups. First, a saline or midazolam injection was given intraperitoneally and after 30 min a second injection of saline or morphine was given subcutaneously once daily for 11 days. Animals were sacrificed on the 11th day 60 min after the last injection to measure met-enkephalin by radioimmunoassay. Morphine tolerant animals showed a significant increase in met-enkephalin levels in the cortex (137%) and midbrain (89%), and a significant decrease in met-enkephalin levels in the pituitary (74%), cerebellum (34%) and medulla (72%). Midazolam treated animals showed a significant decrease in met-enkephalin levels in the pituitary (63%), cortex (39%), medulla (58%), kidneys (36%), heart (36%) and adrenals (43%), and a significant increase in met-enkephalin levels in the striatum (54%) and pons (51%). When morphine and midazolam were injected together, midazolam antagonized the increase in met-enkephalin levels in cortex and midbrain region and the decrease in met-enkephalin level in the medulla region observed in morphine tolerant animals. These results indicate that morphine tolerance and dependence is associated with changes in the concentration of met-enkephalin in the brain. Midazolam may inhibit morphine tolerance and dependence by reversing some of the changes induced in met-enkephalin levels in brain by morphine in morphine tolerant and dependent animals.     

Nociceptive c-fos expression in supraspinal areas in avoidance of descending suppression at the spinal relay station

The number and distribution of Fos-like-immunoreactive neurons in different supraspinal brain areas induced by formalin injection into one hindpaw was estimated in rats with transected dorsal half of the spinal cord at the thoracic level in an attempt to avoid most of the descending modulatory actions. The results showed that: (i) after spinal lesion, the peripheral noxious inputs, going up mainly through the ventral spinal cord, elicited a more widespread and densely located Fos-like-immunoreactive neurons in subcortical areas, many of them showed no Fos expression when noxious stimulation was given in rats with intact spinal cord; (ii) at the same time, a small number of subcortical areas, such as the lateral ventroposterior thalamic nucleus and dorsal raphe nucleus, exhibited no significant increase of nociceptive Fos-like immunoreactive neurons after spinal lesion as compared to that with intact spinal cord; and (iii) there appeared a prominent expansion of cortical areas with densely located Fos-like-immunoreactive neurons in spinal-lesioned rats as compared with the limited labelled areas in the control group with intact spinal cord. These results indicate that: (i) in avoiding the spinally descending modulatory mechanisms, more widespread supraspinal and cortical neurons will be recruited and activated in response to the noxious stimulation; and (ii) the descending systems exert differential actions on the spinal targets which project nociceptive signals to different supraspinal regions. The implication of these facts is discussed.     

Analgesic potency of mu and kappa opioids after systemic administration in amphibians

The relative analgesic potency of 11 opioid agents was assessed by using the acetic acid test in amphibians. Systemic administration of the mu agonists, fentanyl, levorphanol, methadone, morphine, meperidine and codeine; the partial mu agonist, buprenorphine; and the kappa agonists nalorphine, bremazocine, U50488 and CI-977 was made by s.c. injection into the dorsal lymph sac of the Northern grass frog, Rana pipiens. All agents produced a dose-dependent and long-lasting analgesia which persisted for at least 4 hr. The analgesic effects of single doses of each agent were significantly blocked or reduced by pretreatment with naltrexone. Systemic opioids produced log dose-response curves which yielded ED50 values ranging from 1.4 nmol/g for fentanyl to 320.9 nmol/g for nalorphine. Comparison of ED50 values gave a rank order of analgesic potency = fentanyl > CI-977 > levorphanol > U50488 > methadone > bremazocine > morphine > buprenorphine > meperidine > codeine > nalorphine. The relative analgesic potency of mu opioids in amphibians was significantly correlated with relative analgesic potency of these same agents obtained on the mouse writhing and hot plate tests. These data suggest that the amphibian model may serve as an adjunct or alternative model for the testing of opioid agents. Furthermore, given the inactivity of kappa opioids on rodent hot plate and tail-flick tests, the acetic acid test in amphibians may be especially well-suited for the assessment of opioid analgesia after administration of kappa-selective opioids.