The genetics of pain and pain inhibition.

The present review summarizes the current state of knowledge about the genetics of pain-related phenomena and illustrates the scope and power of genetic approaches to the study of pain. We focus on work performed in our laboratories in Jastrzebiec, Poland; Portland, OR; and Los Angeles, which we feel demonstrates the continuing usefulness of classical genetic approaches, especially when used in combination with newly available molecular genetic techniques.

Opioid and nonopioid swim stress-induced analgesia: a parametric analysis in mice.

Environmental stress causes the activation of two types of endogenous pain inhibitory systems in animals: opioid analgesia is antagonized by opiate receptor blockers (e.g., naloxone and naltrexone), whereas analgesia produced by nonopioid systems is insensitive to such antagonism. A large literature documents that the parameters of the laboratory stressor will determine the neurochemical identity of the resultant analgesia. In rats, low severity stressors produce opioid analgesia and higher severity stressors produce nonopioid analgesia. A recent parametric analysis of swim stress-induced analgesia (SSIA) in the female Quackenbush mouse, however, observed the opposite pattern. The present study is a parametric analysis of SSIA using a range of swim temperatures (15-38 degrees C), swim durations (45 s to 7 min), and genetic models [male Swiss-Webster mice, and mice selectively bred from this outbred strain for high (HA), low (LA), or control SSIA]. We find that in nonselected mice low severity swims (i.e., warm temperature, short duration) produce naloxone-sensitive opioid SSIA, whereas high severity swims (i.e., cold temperature, long duration) produce nonopioid SSIA. This pattern is also seen in HA mice displaying very high analgesic magnitudes, but not in LA mice displaying minimal SSIA. In the selectively bred mice, analgesia and hypothermia from forced swimming are positively correlated, but can be dissociated both genetically and neurochemically. Furthermore, swimming in body temperature (38 degrees C) water produces analgesia without concommitant hypothermia, and the increased magnitude of 38 degrees C SSIA displayed by HA mice over control levels is entirely opioid.

1,3-Di-o-tolylguanidine (DTG) differentially affects acute and tonic formalin pain: antagonism by rimcazole.

The role of the sigma receptor in prolonged pain was examined by assessing the effects of 1,3,di-o-tolylguanidine (DTG), a selective sigma receptor ligand, on the formalin test in mice. Formalin injected subcutaneously into the hindpaw produces a biphasic pain response: an acute phase of short duration followed by a longer-lasting tonic phase. DTG (10 mg/kg, i.p.) potently reduced pain behavior in the acute phase but increased pain behavior in the tonic phase. Rimcazole (5 and 10 mg/kg, i.p.), a selective sigma receptor antagonist, blocked both the DTG-induced decrease and increase in pain behavior observed in the acute and tonic phases, respectively. These data support previous findings indicating a modulatory role for the sigma receptor in nociceptive processes, and suggest that this receptor differentially modulates acute vs. tonic pain.

Oligogenic determination of morphine analgesic magnitude: a genetic analysis of selectively bred mouse lines.

Two ongoing selective breeding projects have produced mice that display divergent analgesic responses to morphine. These two projects have selected for similar phenotypes: high and low levorphanol analgesia (HAR/LAR lines; Portland, OR) and high and low swim stress-induced analgesia (HA/LA lines; Jastrzebiec, Poland). Evidence suggests genetic commonalities between mice of the two projects. Using a Mendelian breeding protocol, we have recently found that one or two genetic loci predominantly determine the high morphine analgesia exhibited by HA mice. In the present study we demonstrate that the differential morphine analgesia (5 mg/kg, i.p.) displayed by HAR and LAR mice is similarly oligogenic, predominantly determined by two unlinked loci. A complementation analysis, in which the analgesic responses to morphine of the recessive homozygotes of each project (HAR and HA) were compared to those of their hybrid offspring (HAR x HA), revealed that different genetic loci have been fixed in each project. An intriguing bimodal distribution was observed in the HAR x HA population: Some HAR x HA hybrids displayed greater morphine analgesia than either HAR or HA mice, whereas others displayed minimal analgesia. LAR x LA hybrids displayed less analgesia than either LAR or LA mice. The analgesic responses of HAR x LA and LAR x HA mice were comparable to those of their low-line parents. These findings indicate not only that different loci were responsible for producing high morphine responders in each selection project but that these distinct loci can interact synergistically to produce "superhigh" and "superlow" responders.

The analgesic response to stress: genetic and gender considerations.

The brain contains neuronal circuits, activation of which by electrical stimulation or environmental stress causes analgesia. Both opioid and non-opioid forms of stimulus-induced analgesia exist, and are anatomically differentiated. Several transmitters have been postulated for non-opioid stimulus-induced analgesia, N-methyl-D-aspartic acid being a particularly likely candidate. In mice there are marked gender differences in the underlying neurochemical medication of stress-induced analgesia, the development of which is sensitive to the hormonal environment during early post-natal development and which changes with age in both sexes. Mice can be bred for a high or low analgesic response to stress and there is evidence that this is determined by a single gene. Operative pain, as a stressor, inhibits natural killer (NK) cell activity and influences the propensity to develop metastases when mice are inoculated with an experimental tumour after abdominal surgery. This can be influenced by peri-operative morphine in analgesic doses.

Antinociception following 1,3,-di-o-tolylguanidine, a selective sigma receptor ligand.

The role of sigma receptors in antinociceptive processes remains equivocal, because previous sigma drugs also bind to PCP/NMDA and opiate receptors. The present study examined the antinociceptive effects of the high-affinity, sigma-selective ligand 1,3-di-o-tolylguanidine (DTG; 10, 15, and 20 mg/kg, IP) on tail-withdrawal latencies in mice. DTG produced significant but short-lived increases in withdrawal latencies at all dose levels. DTG also produced hypothermia, but this effect was dissociable from antinociception. The highly selective sigma ligand rimcazole (10 and 25 mg/kg, IP) antagonized DTG antinociception in a dose-dependent manner. The opiate antagonist naloxone and the PCP/NMDA antagonist MK-801 were, however, without effect. Haloperidol, which also binds to sigma receptors, increased withdrawal latencies but did not alter DTG antinociception. These data implicate sigma receptors as the site of DTG antinociception, and more generally support the distinction between sigma, opiate, and PCP/NMDA receptors.

The role of LGL/NK cells in surgery-induced promotion of metastasis and its attenuation by morphine.

Painful stress such as surgery has been shown both to suppress immune function and to promote metastasis, although the degree to which alterations in immunity underlies the tumor-enhancing effects of surgery remains unclear. We recently reported that an experimental laparotomy results in a twofold increase in the number of lung metastases following iv injection of MADB106 tumor cells, a natural killer (NK)-sensitive mammary adenocarcinoma cell line, syngeneic to the Fischer 344 rats we studied. Further, the administration of an analgesic dose of morphine prevented these metastatic-enhancing effects of surgery. The aim of the present study was to investigate the role of NK cells in both the metastatic-enhancing effects of surgery and the attenuation of these effects by morphine. Using a simple 2 x 2 experimental design (surgery with anesthesia vs anesthesia only, and morphine vs vehicle), we found that surgery resulted in a decrease in both whole blood NK cytotoxic activity and number of circulating LGL/NK cells assessed 4 h postoperatively. In a second experiment involving an 18-h lung clearance assay, we used the mAb 3.2.3 to deplete rats of LGL/NK cells with the following rationale: if LGL/NK cells are necessary to mediate an event, then in their absence, that event should not occur. Normal and LGL/NK-depleted animals were assigned to the same four experimental groups, and radiolabeled MADB106 tumor cells were injected iv 4 h after surgery. In normal animals, there was a significant interaction between surgery and morphine such that morphine attenuated the surgery-induced increase in tumor cell retention without affecting tumor cell retention in the anesthesia groups. In the LGL/NK-depleted animals, however, although the tumor-enhancing effects of surgery remained evident, morphine did not mitigate this outcome. These results suggest that: (a) both LGL/NK cell activity and other factors independent of LGL/NK cells play a role in the surgery-induced increase in tumor cell retention; and (b) LGL/NK cells play a critical role in morphine's attenuating effects on this outcome. Finally, these results reinforce concern about the pathogenic consequences of unrelieved pain.

Mu-opiate receptor binding is up-regulated in mice selectively bred for high stress-induced analgesia.

Pain perception and sensitivity to opiate analgesics strongly depend on genotype. Mice selectively bred for high (HA) and low (LA) swim stress-induced analgesia display markedly divergent morphine analgesia, a difference that appears to be determined by one or at the most two major genes. In an attempt to provide candidate genes mediating the supranormal analgesia displayed by HA mice, we performed mu-opiate receptor binding on 27th generation HA, LA, and control (C) mice using [3H]naloxone. HA mice were found to have significantly higher whole-brain receptor density (Bmax) than LA mice in whole brain homogenates; no significant difference in affinity (Kd) was observed. Quantitative autoradiography confirmed the line difference in whole-brain receptor binding. In the medial thalamus, a brain area implicated in ascending pathways of pain inhibition, HA mice were found to display significantly higher [3H]naloxone binding than C mice (a 64% increase) and LA mice (a 128% increase). No significant line differences were observed in any other brain locus. Thalamic mu receptors may therefore play an important role in a central 'volume control' mechanism of pain inhibition, and underlie individual differences in the responses of mice to opiate analgesic drugs.

Morphine fails to produce tolerance when administered in the presence of formalin pain in rats.

The present study examined the development of tolerance to morphine analgesia under conditions in which morphine was administered in the presence or absence of pain induced by subcutaneous injection of 50 microliters of 2.5% formalin into the hind paw of rats. Animals were injected with morphine (25 mg/kg, i.p.) or saline for 3 consecutive days either in the presence of pain (10 min after formalin injection) or in the absence of pain (6 h prior to formalin injection). On the 4th day, tolerance to the analgesic effect of test doses of morphine (6 or 10 mg/kg) was assessed in the formalin and tail-flick tests, respectively. Significant tolerance in both tests was observed in animals receiving morphine in the absence of pain during the tolerance induction period, but not in animals receiving morphine in the presence of pain.

NMDA receptor antagonists, MK-801 and ACEA-1011, prevent the development of tonic pain following subcutaneous formalin.

Subcutaneous injection of formalin produces a biphasic pain response: an early, transient phase followed by a late tonic phase. The present study examined the involvement of the N-methyl-D-aspartic acid (NMDA) receptor in the development of the late pain produced following subcutaneous injection of formalin into the hind paw in mice. Blockade of the NMDA receptor by its non-competitive antagonist, MK-801, prior to formalin injection, but not after, reduced pain during the late phase. Similarly, blockade of the NMDA receptor allosteric site by the novel glycine site antagonist, ACEA-1011, also reduced the pain response in the late phase. These results suggest that the development of the late phase of formalin pain is due to NMDA-mediated activity during the early phase.

Levorphanol and swim stress-induced analgesia in selectively bred mice: evidence for genetic commonalities.

Two independent selective breeding programs have developed divergent lines of mice expressing either high and low swim stress-induced analgesia (HA/LA lines; Jastrzebiec, Poland) or high and low levorphanol analgesia (HAR/LAR lines; Portland, OR). In the present study, mice from both programs were tested for both levorphanol analgesia (2 mg/kg) and an opioid-mediated swim stress-induced analgesia (3 min swimming in 32 degrees C water) in the hot-plate test. Mice selected for high and low levorphanol analgesia displayed high and low swim stress-induced analgesia, respectively; mice selected for high and low swim stress-induced analgesia displayed high and low levorphanol analgesia, respectively. This pattern of correlated responses suggests a high degree of common genetic determination in opiate and swim stress-induced analgesia. These findings also suggest that individual differences in analgesic responsiveness to opiate drugs result from genetically determined individual differences in endogenous pain inhibitory mechanisms.

Antagonism of the non-opioid component of ethanol-induced analgesia by the NMDA receptor antagonist MK-801.

Recent evidence from our laboratory suggests that the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (dizocilpine) selectively antagonizes non-opioid (i.e. naloxone-insensitive) mechanisms of stress-induced analgesia in mice. For example, we have recently demonstrated that a low dose of MK-801 (0.075 mg/kg, i.p.) antagonizes the non-opioid component of a mixed opioid/non-opioid swim stress-induced analgesia (SSIA) resulting from forced swimming for 3 min in 20 degrees C water. Since ethanol-induced analgesia (EIA) has been found to be only partially attenuated by naloxone, we hypothesized that MK-801 would similarly block the non-opioid component of EIA. The effects of MK-801 and of the opioid receptor antagonist naloxone (10 mg/kg, i.p.) on analgesia produced by ethanol (2.5 g/kg in 20% vol/vol, i.p.) were studied in control mice and in mice selectively bred for high (HA) or low (LA) SSIA. HA mice showed significantly more, and LA mice significantly less, EIA than controls. Naloxone and MK-801 significantly attenuated EIA in control and HA mice, and in these lines the combined administration of both antagonists blocked EIA completely. In LA mice, which displayed very little EIA, naloxone but not MK-801 reversed EIA completely. These findings provide additional evidence for the role of the NMDA receptor in non-opioid mechanisms of analgesia. The finding that mice selectively bred for high and low SSIA, also display high and low EIA suggests common mediation of the effects of stress and ethanol on antinociceptive processes.

The specific N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 blocks U-50,488, but not morphine antinociception.

The effect of the specific NMDA receptor antagonist MK-801 on antinociception produced by the kappa opiate receptor agonist U-50,488 and the mu receptor agonist morphine was assessed using the tail-flick test in rats. MK-801 (0.05 and 0.1 mg/kg) antagonized antinociception induced by all three doses of U-50,488 (7.5, 15 and 30 mg/kg), and potentiated antinociception induced by the lower (1 mg/kg) but not higher (5 mg/kg) dose of morphine. Naloxone at a dose of 1.0 but not 0.1 mg/kg blocked U-50,488 antinociception, indicating that MK-801 affects opiate antinociception. The present results are the first to suggest a critical role for the NMDA receptor in opiate antinociception involving the kappa receptor.

N-methyl-D-aspartic acid (NMDA) receptor antagonist MK-801 blocks non-opioid stress-induced analgesia. II. Comparison across three swim-stress paradigms in selectively bred mice.

The effects of the specific N-methyl-D-aspartic acid (NMDA) receptor antagonist MK-801 (dizocilpine, 0.075 mg/kg, i.p.) on swim-stress-induced analgesia (SSIA) were studied in control (C) mice and in mice selectively bred for high (HA) or low (LA) SSIA. In three consecutive experiments, animals were subjected to forced swimming at water temperature of 20 degrees C, 32 degrees C and 15 degrees C and the resulting analgesia (hot-plate test) was found to be mixed opioid/non-opioid, opioid and non-opioid, respectively, as a function of the degree of antagonism by naloxone (10 mg/kg, i.p.). The major finding of this study is that MK-801 attenuated 15 degrees C SSIA, against which naloxone was ineffective, but had no effect on 32 degrees C SSIA, which naloxone blocked completely. A combination of naloxone and MK-801 significantly attenuated 20 degrees C SSIA in C and HA mice and in HA mice this attenuation was significantly larger than that produced by either drug alone. Morphine analgesia (10 mg/kg, i.p.) was unaffected by MK-801. It is concluded that low doses of MK-801 selectively block non-opioid mechanisms of SSIA.

The NMDA receptor antagonist MK-801 prevents long-lasting non-associative morphine tolerance in the rat.

Several studies have demonstrated that the N-methyl-D-aspartate (NMDA) antagonist MK-801 attenuates the development of morphine tolerance and withdrawal. These studies employed repeated morphine injections to induce tolerance, a procedure in which learning has been suggested to play a significant role in tolerance development. MK-801 has been reported to block some types of learning, and it is unclear, therefore, whether the effect of MK-801 on tolerance development is due to its antagonism of associative (learning) or non-associative factors. Moreover, previous studies have tested the effects of MK-801 on morphine tolerance only up to 48 h after its induction; yet morphine tolerance can persist for many months, and it is not known whether MK-801 can block long-lasting tolerance. In the present study, therefore, we adopted a model of morphine tolerance in which the involvement of learning is minimized by using a single injection of morphine in a sustained-release preparation, and we tested tolerance for up to 56 days. In the first experiment, simultaneously administering MK-801 (0.2 mg/kg, s.c.) and morphine (60 mg/kg, s.c.), each in a sustained-release preparation, abolished tolerance that lasted at least 12 days. Analgesia was measured in the hot-plate test following a test dose of morphine (15 mg/kg, i.p.). In the second experiment, delivering MK-801 and morphine as before, the duration of morphine-induced catalepsy and analgesia was prolonged. Nevertheless, 24 h later one symptom of naloxone-precipitated withdrawal was significantly attenuated in these same animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethanol increases tumor progression in rats: possible involvement of natural killer cells.

The effects of acute and chronic ethanol administration on tumor progression and metastasis were studied in rat models of leukemia and breast cancer, respectively. Acute administration of 1.5-3.5 g of ethanol/kg body weight significantly reduced survival of rats injected with CRNK-16 leukemia cells in a dose-related manner. Acute administration of 2.5-3.5 g of ethanol/kg body weight, one hour before tumor inoculation, or chronic consumption of liquid diet containing ethanol for two weeks before and three weeks after tumor inoculation, significantly increased the number of lung metastases of MADB106 mammary adenocarcinoma. The ethanol-induced increase in the number of metastases was not correlated with plasma levels of corticosterone and was not altered by the opiate antagonist naltrexone. Incubation of spleen cells in vitro in the presence of ethanol, at concentrations comparable to those measured in the blood of ethanol-treated rats, significantly suppressed natural killer (NK) cell activity against MADB106 cells in a standard chromium-release assay and decreased the binding of effector to MADB106 tumor cells. However, neither acute nor chronic ethanol administration in vivo altered splenic NK activity against this tumor in the same in vitro assay, in which the ethanol would have been washed away. These results suggest that, in the presence of ethanol, tumor progression is facilitated. The possibility that this facilitation is related to ethanol-induced impairment of the normal tumoricidal interaction between NK and tumor cells is discussed.