Quaternary naloxone blocks morphine analgesia in spinal but not intact rats.

Quaternary derivatives of naloxone and other compounds are assumed not to enter the central nervous system following systemic administration. We report that i.p. naloxone methylbromide (5 mg/kg) completely reversed the antinociceptive effect of systemically administered morphine (6 mg/kg) in acutely spinalised rats, although it had no effect in the same animals prior to the transection. Naloxone hydrochloride was effective both before and after transection. Nuclear resonance spectra confirmed the purity of both compounds. These results suggest that acute spinal transection allows rapid entry of quaternary naloxone into the spinal cord. Quaternary compounds therefore may need to be used with caution in spinalised animals.     

mu- but not delta- and kappa-opioid receptor mediates the nucleus submedius interferon-alpha-evoked antinociception in the rat

Previous studies have indicated that interferon-alpha (IFN-alpha) can bind to opioid receptors and exerts an antinociceptive effect in both peripheral and central nervous systems. The current study investigated the antinociceptive effect of IFN-alpha unilaterally microinjected into the thalamic nucleus submedius (Sm) of rats on noxious thermal stimulus, and the roles of different subtypes of opioid receptors in mediating the Sm IFN-alpha-evoked antinociception. The results indicated that unilateral microinjection of IFN-alpha (4, 8, 16 pmol) into the Sm dose-dependently increased the hind paw withdrawal latency from the noxious heat stimulus, and this effect was reversed by pretreatment with non-selective opioid receptor antagonist naloxone (200 pmol) and specific mu-opioid receptor antagonist beta-FNA (1 nmol) into the same sites, whereas delta-opioid receptor antagonist ICI174,864 (1 nmol) and kappa-opioid receptor antagonist nor-BNI (1 nmol) failed to alter the effect of IFN-alpha. These results suggest that Sm is involved in IFN-alpha-evoked antinociception and mu- but not delta- and kappa-opioid receptor mediates the Sm IFN-alpha-evoked antinociception.     

Chronic osmotic stimulation reduces vasopressin but not synaptophysin content in rat neurohypophysis

The content of synaptophysin, a vesicular integral membrane protein of neurons and endocrine cells, and that of vasopressin was measured in neurohypophyses of rats during chronic osmotic stimulation. The animals received 2% NaCl in their drinking water for up to 4 days. Synaptophysin content of neurohypophyses was determined using quantitative immunoblotting, vasopressin content was measured by radioimmunoassay. Salt loading caused a decrease in the content of vasopressin to about 15% of that of control animals, whether expressed per neurohypophysis or relative to the total tissue protein. In contrast, no change was found in the synaptophysin content. Taken together with published evidence of changes in the relative numbers of the hormone-containing neurosecretory granules (NSGs) and the microvesicles (MVs) under the conditions of chronic osmotic stimulation, these results strongly indicate the surface density of synaptophysin on NSGs to be significantly lower than its surface density on MVs.     

Oral administration of IL-12 abrogates the induction but not the maintenance of oral tolerance

BACKGROUND: IL-12 is a Th1-inducing cytokine and known to be induced by some food factors. This function of such foods is expected to be applied for antiallergic materials. However, the influence of IL-12 on the immune responses has not been fully investigated. Oral tolerance is an immunologically unresponsive state induced by orally administered food antigens. Although a failure in the induction of oral tolerance would result in food allergy, the mechanisms for the induction and abrogation of oral tolerance have not been clarified. IL-12 induced by food factors may also affect the induction of oral tolerance. In this study we examined the effect of the oral administration of IL-12 on the induction and maintenance of oral tolerance. METHODS: BALB/c mice were fed beta-lactoglobulin with or without IL-12. After immunizing them with the antigen, the serum antibody titer of these mice was measured to evaluate the induction of oral tolerance. RESULTS: The induction of oral tolerance was prevented in the mice that had been simultaneously fed the antigen and IL-12. On the other hand IL-12 did not abrogate the already established oral tolerance when it was administered after feeding the antigen. We also found that oral tolerance could be induced normally in mice that had failed to induce such tolerance by simultaneous feeding of IL-12 and the antigen. CONCLUSIONS: The results indicate that IL-12 induced in the intestine by some food factors is involved in the regulation of oral tolerance.     

Tolerance to the antinociceptive effect of morphine microinjections into the ventral but not lateral-dorsal periaqueductal gray of the rat.

Tolerance to the antinociceptive effect of morphine is mediated at least in part by morphine's action within the periaqueductal gray (PAG). The objective of the present study was to determine whether both ventral and lateral-dorsal PAG regions contribute to the development of tolerance. It was found that the antinociceptive efficacy of microinjecting morphine (5 microg/0.4 microl) into the ventral but not the lateral-dorsal PAG diminished with successive injections. Control experiments indicated that this decrease was caused by tolerance to morphine and was not a result of cell death caused by repeated microinjections or habituation from repeated behavioral testing. The finding of greater susceptibility of the ventral compared with the lateral-dorsal PAG to the development of tolerance adds to a growing literature distinguishing antinociception from these two regions.     

Lamina-specific effects of morphine and naloxone in dorsal horn of rat spinal cord in vitro.

1. Extracellular recordings were made from 95 dorsal horn neurons in a sagittal slice preparation of rat spinal cord. Neurons were synaptically activated by electrical stimulation of the dorsal root entry zone (n = 72) or driven by pressure ejection of L-glutamate into the substantia gelatinosa (SG; n = 23). For the majority of neurons low-intensity electrical stimuli evoked a burst of spikes with short latency (early firing). An increase in the stimulus intensity evoked the early firing followed by a characteristic prolonged period of activity (late firing). 2. The patterns of synaptically induced activity observed from neurons located in the SG (n = 45) and in deeper laminae (n = 27) were similar. Early and late firing of both SG and deep neurons was reduced by the nonspecific excitatory amino acid antagonist kynurenate. Raising [Mg2+] in the superfusate to 5 from 2.0 mM selectively reduced the late firing of both SG and deep neurons. These findings suggest that fibers present in the dorsal root zone make excitatory amino acid-mediated synapses with dorsal horn neurons. 3. The majority of deep neurons showed reduced responses to electrical stimuli in the presence of morphine, mimicking the findings reported in vivo. Naloxone reversed morphine inhibitions or, when applied to morphine-naive slices, caused modest increases in the responses of some deep neurons. 4. Most neurons located in the SG had their responses enhanced by morphine (late firing: 86.5 +/- 19.6%, mean +/- SE) and were inhibited by naloxone (-78.3 +/- 22.7%). Morphine-induced enhancements often persisted long after the morphine had washed out of the bath. Inhibitions by naloxone, whether pre- or postmorphine, were short lived; and responses generally returned to either control or morphine-enhanced levels on washout of the naloxone. 5. The gamma-aminobutyric acid (GABA) antagonist bicuculline, applied to GABA-naive slices, caused an increase in response of SG neurons while slightly depressing activity of deep neurons. A tonic release or presence of endogenous GABA, affecting neurons that are inhibited by exogenously applied GABA, may be responsible for the observed action of bicuculline. 6. Intracellular recordings were made from a further 32 dorsal horn neurons located in the translucent band of the spinal cord slices. Of 11 neurons examined, 5 showed increased input resistance (Rin) and were depolarized in response to morphine applied in the superfusate. One neuron showed no change in Rin or potential with morphine, whereas 5 of the 11 were hyperpolarized with decreased Rin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Estrogen reduces the excitability of the female rat medial amygdala afferents from the medial preoptic area but not those from the lateral septum

Electrical stimulation of the medial amygdala (AMY) elicited antidromic action potentials in neurons in the preoptic area (POA) and the lateral septum (LS) of 36 urethane-anesthetized ovariectomized female rats, which were either treated with estrogen o not treated. The extracellular potentials from the two sites showed similar characteristics, with the exception of the sensitivity to estrogen: they had latencies between 3 and 35 ms. Thresholds were as low as 100 A. The mean relative refractory period was 2.2 ms. The peak-to-peak amplitudes of the positive-negative biphasic potential ranged from 1.0 mV to 12.0 mV. Estrogen had site-specific effects on parameters of antidromic activation in the POA. Estrogen-treated rats had a significantly higher threshold (937 vs 664 A) and a longer refractory period (2.5 vs 2.1 ms) than the ovariectomized rats (P < 0.05 for each). The effects were absent in the LS. Selective cutting of the stria terminalis diminished the AMY-induced antidromic responses in the POA and LS. Electrical stimulation of the stria blocked the AMY-induced antidromic potentials by collision. Thus, estrogen-sensitive POA efferents as well as non-estrogen-sensitive LS efferents project to the AMY via the stria terminalis. Reductions in axonal excitability would inhibit neural conduction and transmission. Estrogen may therefore reduce the AMY inputs from the POA, without affecting those from the LS. Such alterations in the neural impulse flow may underlie estrogen-dependent neuroendocrine or behavioral regulation.     

Systemic naloxone does not affect pain-related behaviour in the formalin test in rat

The effects of systemic naloxone on pain behaviour in rats were examined in modified formalin test. Dilute formalin was subcutaneously injected in the plantar surface of the hindpaw, after which pain was rated according to behavioural criteria. Fifteen minutes after formalin injection, naloxone in various doses (10 micrograms/kg, 100 micrograms/kg, 300 micrograms/kg, 600 micrograms/kg, 800 micrograms/kg, 2000 micrograms/kg) or saline was intraperitoneally injected. Pain behaviour in naloxone injected and saline injected rats was not different. The findings are discussed in relation to previous reports about naloxone effects on different types of pain.     

Time-dependent effects of oral morphine on autotomy following brachial nerve section in the rat.

In this study, the effects of morphine in drinking water on autotomy in male F344 rats having undergone brachial nerve sections were assessed during a 3-week period. Morphine was self-administered orally in tap water by the rats. Rats administered morphine just after the nerve section or 1 week after the nerve section showed a significant increase in the severity of autotomy when compared to the rats administered distilled water. Morphine had no effect on autotomy when it was administered 2 weeks after the nerve section. These results suggested that the effect of oral morphine on autotomy changes in a time-dependent manner.     

Evidence for the involvement of the mu but not delta opioid receptor subtype in the synergistic interaction between opioid and alpha 2 adrenergic antinociception in the rat spinal cord.

The interaction between the spinal antinociceptive effects of selective mu or delta opioid agonists morphine and DSTBULET (Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr), respectively, and the selective alpha 2 adrenergic agonist dexmedetomidine was examined on convergent dorsal horn neuronal responses in the intact anaesthetized rat. The coadministration of intrathecal morphine (0.5 microgram, 2.5 micrograms) and dexmedetomidine (0.5 microgram) produced a greater than additive inhibition of C fibre-evoked responses. Inhibitions were reversed by either the opioid antagonist naloxone or the alpha 2 adrenergic antagonist atipamezole. The coadministration of intrathecal DSTBULET (1 microgram, 2.5 micrograms) and dexmedetomidine did not result in a supra-additive inhibition of C fibre-evoked responses. The results suggest that mu rather than delta opioid receptors are involved in the synergism of spinal opioid and alpha 2 adrenergic antinociception.     

Selective modulation of the cardiovascular response but not the antinociception evoked from the dorsal PAG,by 5-HT in the ventrolateral medulla

In lightly ananesthetised rats, electrical stimulation in the dorsal part of the periaqueductal grey matter (dPAG) produced an increase in the latency of the tail flick response accompanied by an increase in blood pressure and heart rate. Microinjection of 40-50nmol 5-HT bilaterally into the rostral part of nucleus paragigantocellularis lateralis (PGL) at the level of the facial nucleus attenuated the cardiovascular response to stimulation of the dPAG but had no effect on the analgesia. It is suggested that cardiovascular control neurones, but not the pain control neurones in the rostral part of PGL are subject to inhibitory serotonergic modulation.     

Inhibition of acoustic startle in the rat by a footshock prestimulus: effects of morphine and naloxone

In a series of three experiments, we investigated the ability of footshock to inhibit the rat's acoustic startle response. Based on the mean thresholds obtained from a flinch-jump test, 8 rats were tested in a startle inhibition procedure with prestimulus intensities of 0.1, 0.2, 0.4, and 0.8 mA. Subjects were presented with a series of startle-eliciting noise bursts, preceded by a 300-ms footshock of one of the four intensities. Control trials consisting of the noise alone were also included. Startle amplitude was measured during the 100 ms immediately following the noise onset. Footshock reliably inhibited startle at all intensities; inhibition was near maximum at 0.2 mA. Morphine administration reliably interfered with the inhibition at all intensities, and this was reversed by naloxone administration. These data suggest that morphine may have a more general effect than is currently believed.     

Cholinomimetics, but not morphine, increase antinociceptive behavior from pontine reticular regions regulating rapid-eye-movement sleep

Sleep disruption is a significant problem associated with the subjective experience of pain. Both rapid-eye-movement (REM) sleep and nociception are modulated by cholinergic neurotransmission, and this study tested the hypothesis that antinociceptive behavior can be evoked cholinergically from medial pontine reticular formation (mPRF) regions known to regulate REM sleep. The foregoing hypothesis was investigated by quantifying the effect of mPRF drug administration on tail flick latency (TFL) of cat during polygraphically defined sleep/wake states. The mPRF was microinjected with 0.25 ml saline, carbachol (4.0 microg), neostigmine (6.7 microg), or morphine sulfate (14.7 microg), and TFL measures were obtained in response to radiant heat. During wakefulness TFL (% increase) was not increased by morphine or saline, but was significantly increased by mPRF administration of carbachol (42.4%) and neostigmine (35.2%). Cortical somatosensory potentials (SSEPs) were reliably evoked by tail stimulation before and after mPRF microinjections of carbachol. The results show for the first time that mPRF administration of cholinomimetics significantly increased TFL. During NREM sleep and REM sleep, TFL was significantly increased compared to waking TFL (110% and 321%, respectively). The finding of sleep-dependent alterations in TFL demonstrates that mPRF regions known to regulate REM sleep can modulate supraspinal cholinergic antinociceptive behavior.     

Osteoprotegerin reduces the loss of periarticular bone mass in primary and secondary spongiosa but does not influence inflammation in rat antigen-induced arthritis

Objective: To assess the effect of osteoprotegerin (OPG) on joint swelling, synovial inflammation and cartilage destruction, periarticular and axial bone volume, and bone turnover in rat antigen-induced arthritis (AIA). Design: Rats were treated with OPG (3 mg/kg/day) at regular intervals from day 1 to day 20 of AIA. Disease activity was evaluated by measurement of joint swelling as well as, joint inflammation and destruction by histology. Bone volume and cellular turnover parameters of secondary spongiosa of the right tibia head and the third lumbar vertebra were evaluated by histomorphometry. Periarticular bone volume of the primary spongiosa at the right tibia head was measured by linear scanning. The findings were compared with those of PBS-treated AIA and healthy animals. Result: OPG treatment did not reduce joint swelling or histological signs of inflammation. Cartilage destruction was reduced. However, this effect did not reach statistical significance . In the secondary spongiosa OPG treatment reduced the loss of periarticular bone volume. However, the latter did not reach the level of healthy controls. OPG treatment significantly reduced parameters of bone formation and bone resorption. In the primary spongiosa, OPG-treatment led to a higher amount of mineralized tissue and a greater number of trabeculae compared to PBS-treated animals with AIA or healthy controls. In the axial skeleton, OPG treatment reduced bone formation and bone resorption parameters compared to healthy animals. This treatment had no influence on bone volume. Conclusions: In periarticular bone of AIA rats, OPG treatment reduced the loss of bone volume and decreased the bone turnover, thus preventing periarticular bone destruction. OPG treatment had no influence on inflammatory process or on cartilage destruction. Received 2 June 2005; returned for revision 26 July 2005; returned for final revision 9 August 2005; accepted by M. Parnham 24 September 2005 Presented in part at the 66. Annual Meeting of the American College of Rheumatology, New Orleans, U.S.A., October 2002, and at the 25. Annual Meeting of the American Society of Bone and Mineral Research, Minneapolis, USA, September 2003 Supported by grants from the Thuringian Ministry of Science, Research and Art (B307-01025, B378-01017), the Interdisciplinary Center for Clinical Research (IZKF) Jena, and the Deutsche Forschungsgemeinschaft (Br 1372/5-1) Osteoprotegerin was generously provided by Amgen (Thousand Oaks, CA, USA). Drs. Neumann and Oelzner contributed equally to this work.     

Acute noxious stimulation modifies morphine effect in serotonergic but not dopaminergic midbrain areas

It is poorly understood if and how pain may modify the effect of opioids on neural systems that contribute to reward and addictive behavior. We hypothesized that the activation of ascending dopaminergic and serotonergic nuclei by morphine is modified by the presence of noxious stimulation. Immunohistochemical double-labeling technique with Fos was used to examine if an intraplantar formalin injection, an acute noxious input, changed the effect of morphine on dopaminergic neurons of the ventral tegmental area (VTA), and serotonergic neurons of the dorsal raphe nucleus (DR). Four groups of rats were analyzed: (1) control injected with normal saline s.c., (2) rats treated with formalin into the hind paw 30 min after normal saline injection, (3) rats injected with morphine sulfate s.c., and (4) rats treated with formalin into the hind paw 30 min after morphine injection (morphine/formalin). Following morphine injection, there was an increase in the number of dopaminergic neurons in the VTA with Fos immunolabeling. However, noxious stimulation did not detectably change morphine's effect on Fos expression in VTA dopamine neurons. In contrast, the number of serotonergic neurons containing Fos was increased in the morphine/formalin group compared to all other groups and this effect was topographically selective for the dorsal area of the DR at mid rostro–caudal levels. Therefore, morphine's activation of the VTA, which is associated with motivated behavior and reward seeking, appears similar in the context of pain. However, activation of the ascending serotonin system, which influences mood and has the capacity to modify reward pathways, appears different. In addition, these findings reveal interactions between nociceptive signaling and opioids that contrasts with the notion that opioids simply block access of nociceptive signaling to supraspinal structures.     

Spinal pathways mediating coeruleospinal antinociception in the rat

In a previous study, we showed in rats that axons of some locus coeruleus/subcoeruleus (LC/SC) neurons involved in coeruleospinal modulation of nociception descend through the ipsilateral side of the spinal cord and cross the midline at spinal segmental levels. The present study was designed to investigate a possible spinal pathway of these descending axons from the LC/SC. Extracellular recordings were made from the left dorsal horn with a carbon filament electrode (4-6 M(omega)). To block impulses from the LC/SC which descend through spinal pathways ipsilateral to the recording sites, a hemisection of the spinal cord ipsilateral to the recording sites was performed at the C2 level with fine forceps in all rats tested. In these rats, responses of dorsal horn neurons to noxious heat (53 degrees C) applied to receptive fields were inhibited during electrical stimulation (100 microA, 100 Hz, 0.1 ms pulses) of the LC/SC. The transection of the dorsolateral funiculus contralateral to the recording sites did not affect LC/SC stimulation-produced inhibition. Following transection of the ventrolateral funiculus (VLF) contralateral to the recording sites, LC/SC stimulation failed to inhibit heat-evoked responses. These results suggest that interruption of descending inhibition from the LC/SC produced by the VLF transections is due to the blockage of axons descending in the ventrolateral quadrant of the spinal cord, but not in the dorsolateral quadrant.     

Estradiol reduces seizure-induced hippocampal injury in ovariectomized female but not in male rats

Estrogens protect ovariectomized rats from hippocampal injury induced by kainic acid-induced status epilepticus (SE). We compared the effects of 17beta-estradiol in adult male and ovariectomized female rats subjected to lithium-pilocarpine-induced SE. Rats received subcutaneous injections of 17beta-estradiol (2 microg/rat) or oil once daily for four consecutive days. SE was induced 20 h following the second injection and terminated 3 h later. The extent of silver-stained CA3 and CA1 hippocampal neurons was evaluated 2 days after SE. 17beta-Estradiol did not alter the onset of first clonus in ovariectomized rats but accelerated it in males. 17beta-Estradiol reduced the argyrophilic neurons in the CA1 and CA3-C sectors of ovariectomized rats. In males, estradiol increased the total damage score. These findings suggest that the effects of estradiol on seizure threshold and damage may be altered by sex-related differences in the hormonal environment.     

Isolation reduces contextual but not auditory-cue fear conditioning: a role for endogenous opioids.

Isolation for several hours after fear conditioning reduces contextual but not auditory-cue fear conditioning (J. W. Rudy, 1996). This isolation effect is reversed by both, centrally and peripherally acting opioid receptor antagonists. As in isolation, systemically administered morphine given immediately after conditioning also reduces contextual fear conditioning. Morphine's effect is also reversed by both centrally and peripherally acting opioid receptor antagonists. Exposure to the conditioning context has been shown to eliminate the effect of isolation on contextual fear conditioning (J. W. Rudy, 1996). Context preexposure also eliminated the effect of morphine on contextual fear conditioning. These results imply that opioids released in the periphery play an important role in producing the isolation effect and that they do so by disrupting the postconditioning memory consolidation processes.     

Oral administration of unmodified colonic but not small intestinal antigens protects rats from hapten-induced colitis

Colonic administration of a hapten, 2,4,6-trinitrobenzene sulphonic acid (TNBS) has been shown to induce colitis in rats. We are using this model to investigate the role of colonic antigens in the immunopathology. In this study, we show that colitis can be suppressed by oral administration of haptenized colonic antigens prior to the TNBS enema. Moreover, our data suggest that haptenization of the colonic antigens is not essential because oral feeding of non haptenized colonic antigens too protects rats from TNBS-induced colitis. Thus, unmodified colonic antigens may be involved in the induction of oral tolerance, and possibly in the pathogenesis in this model of colitis. Further, we show that the protective immunity or oral tolerance induced by non haptenized colonic antigens can be passively transferred to na?ve rats by mesenteric T lymphocytes. Interestingly, oral feeding of small intestinal antigens, haptenized and non haptenized, does not protect rats from colitis, suggesting a specific role for colonic antigens. These data underscore the usefulness of this rat model in the identification of pathogenic antigens in colitis and in the development of therapeutic strategies based on oral tolerance.