Cannabinoid receptor involvement in stress-induced cocaine reinstatement: potential interaction with noradrenergic pathways.

This study examined the role of endocannabinoid signaling in stress-induced reinstatement of cocaine seeking and explored the interaction between noradrenergic and endocannabinergic systems in the process. A well-validated preclinical model for human relapse, the rodent conditioned place preference assay, was used. Cocaine-induced place preference was established in C57BL/6 mice using injections of 15 mg/kg cocaine. Following extinction of preference for the cocaine-paired environment, reinstatement of place preference was determined following 6 min of swim stress or cocaine injection (15 mg/kg, i.p.). The role of endocannabinoid signaling was studied using the cannabinoid antagonist AM-251 (3 mg/kg, i.p.). Another cohort of mice was tested for reinstatement following administration of the cannabinoid agonist CP 55,940 (10, 20, or 40 µg/kg, i.p.). The alpha-2 adrenergic antagonist BRL-44408 (5 mg/kg, i.p.) with or without CP 55,940 (20 µg/kg) was administered to a third group of mice. We found that: (1) AM-251 blocked forced swim-induced, but not cocaine-induced, reinstatement of cocaine-seeking behavior; (2) the cannabinoid agonist CP 55,940 did not reinstate cocaine-seeking behavior when administered alone but did synergize with a non-reinstating dose of the alpha-2 adrenergic antagonist BRL-44408 to cause reinstatement. These results are consistent with the hypothesis that stress exposure triggers the endogenous activation of CB1 receptors and that activation of the endocannabinoid system is required for the stress-induced relapse of the mice to cocaine seeking. Further, the data suggest that the endocannabinoid system interacts with noradrenergic mechanisms to influence stress-induced reinstatement of cocaine-seeking behavior.

Detection and mapping of quantitative trait loci that determine responsiveness of mice to nitrous oxide antinociception.

Exposure to 70% N(2)O evokes a robust antinociceptive effect in C57BL/6 (B6) but not in DBA/2 (D2) inbred mice. This study was conducted to identify quantitative trait loci (QTL) in the mouse genome that might determine responsiveness to N(2)O. Offspring from the F(2) generation bred from B6 and D2 progenitors exhibited a broad range of responsiveness to N(2)O antinociception as determined by the acetic acid-induced abdominal constriction test. QTL analysis was then used to dissect this continuous trait distribution into component loci, and to map them to broad chromosomal regions. To this end, 24 spleens were collected from each of the following four groups: male and female F(2) mice responding to 70% N(2)O in oxygen with 100% response (high-responders); and male and female F(2) mice responding with 0% response (low-responders). Genomic DNA was extracted from the spleens and genotyped with simple sequence length polymorphism MapPairs markers. Findings were combined with findings from the earlier QTL analysis from BXD recombinant inbred mice [Brain Res 725 (1996) 23]. Combined results revealed two significant QTL that influence responsiveness to nitrous oxide on proximal chromosome 2 and distal chromosome 5, and one suggestive QTL on midchromosome 18. The chromosome 2 QTL was evident only in males. A significant interaction was found between a locus on chromosome 6 and another on chromosome 13 with a substantial effect on N(2)O antinociception.

Increased mRNA expression for the alpha(1) subunit of the GABA(A) receptor following nitrous oxide exposure in mice.

The mechanisms by which nitrous oxide (N(2)O) produces physical dependence and withdrawal seizures are not well understood, but both N(2)O and ethanol exert some of their effects via the GABA(A) receptor and several lines of evidence indicate that withdrawal from N(2)O and ethanol may be produced through similar mechanisms. Expression levels of mRNA transcripts encoding several GABA(A) receptor subunits change with chronic ethanol exposure and, therefore, we hypothesized that N(2)O exposure would produce changes in mRNA expression for the alpha(1) subunit. Male, Swiss--Webster mice, 10--12 weeks of age, were exposed for 48 h to either room air or a 75%:25% N(2)O:O(2) environment. Brains were sectioned and mRNA for the alpha(1) subunit was detected by in situ hybridization using an 35S-labelled cRNA probe. N(2)O exposure produced a significant increase in expression levels of the alpha(1) subunit mRNA in the cingulate cortex, the CA1/2 region of the hippocampus, the dentate gyrus, the subiculum, the medial septum, and the ventral tegmental area. These results lend support to the hypothesis that N(2)O effects are produced, at least in part, through the GABA(A) receptor and that N(2)O produces these effects through actions in the cingulate cortex, hippocampus, ventral tegmental area and medial septum. These results are also further evidence that ethanol and N(2)O produce dependence and withdrawal through common mechanisms.

Nitrous oxide antinociception in BXD recombinant inbred mouse strains and identification of quantitative trait loci.

Among inbred mouse strains, DBA/2 mice are unique because of their poor responsiveness to nitrous oxide (N2O) antinociception. As a first step towards identifying candidate genes involved in determining antinociceptive responsiveness to N2O, male mice from the DBA/2 strain, the more responsive C57BL/6 strain, their B6D2F1 offspring, and 22 BXD recombinant inbred (RI) strains derived from DBA/2 and C57BL/6 mice were exposed to N2O and evaluated using the acetic acid abdominal constriction test. When exposed to 70% N2O, C57BL/6, DBA/2 and B6D2F1 mice exhibited antinociceptive responses of 78, 22 and 55%, respectively. The BXD RI strains demonstrated varying degrees of responsiveness to N2O. Cluster analysis revealed one cluster of 16 strains approximating the C57BL/6 progenitor (61.9-100% antinociceptive response to 70% N2O) and another of six strains around the DBA/2 progenitor (9.1-40% antinociceptive response to 70% N2O). The robust strain differences permitted screening the strain means with 1492 marker loci previously mapped in BXD RI strains. Using a QTL analysis specifically tailored to existing mouse RI strains, we found associations at the 0.01 level on seven chromosomes with the most promising marker loci being Il2ra, Hbb, Hmg1rs7 and Gsl5 on chromosomes 2, 7, 16 and 19, respectively (P < 0.002).

Strain-dependent variability in nitrous oxide withdrawal seizure frequency.

Exposure to nitrous oxide produces handling induced convulsions following withdrawal in mice. Since strain differences in responsiveness to the antinociceptive potency of N2O have been found, we examined whether there were differences in susceptibility to N2O withdrawal seizures. Significant differences were found between mouse strains, varying between 100% and 0% of mice exhibiting withdrawal seizures. There was a lack of correlation between the sensitivity of the mouse strains to N2O-induced analgesia and N2O withdrawal seizures, suggesting different mechanisms of action.

Strain-dependent differences in responsiveness of mice to nitrous oxide (N2O) antinociception.

N2O antinociception was assessed in eight inbred and two outbred mouse strains. Results indicated the following order of responsiveness among the 10 strains: A/J (most sensitive), C57BL/6ByJ, C57BL/6J, BALB/cByJ, C3H/HeJ, Swiss-Webster, CXBK/ByJ, ICR, CBA/J and DBA/2J (least sensitive). These results demonstrate significant strain-dependent differences in antinociceptive responsiveness to N2O. The weak antinociceptive response to N2O in the DBA/2J strain, which is sensitive to morphine and U-50, 488H, indicates some underlying neurobiological difference in the DBA/2J mouse that imparts resistance to N2O. The responsiveness of CXBK/ByJ mice to N2O indicates that mu-opioid receptors may not play an important role in N2O antinociception in mice.

Benzodiazepine receptor mediation of behavioral effects of nitrous oxide in mice.

Nitrous oxide produces behavioral effects, the underlying mechanism of which is not known. In the mouse staircase test, exposure to nitrous oxide caused a reduction in rearing activity, an effect similar to that produced by benzodiazepines in this paradigm, when its opioid action on locomotion is blocked by naloxone. In this study, we tested whether effects of nitrous oxide might be mediated by benzodiazepine receptors, using chlordiazepoxide as a control. The abilities of nitrous oxide and chlordiazepoxide to reduce rearing were significantly attenuated in mice pretreated with the benzodiazepine receptor blocker flumazenil or rendered tolerant to benzodiazepines. These findings suggest an involvement of benzodiazepine receptors in mediation of certain behavioral effects of nitrous oxide.

Increased apomorphine-induced hypothermia precedes development of hypertension in SHRs.

Apomorphine produced a greater hypothermic response in spontaneously hypertensive rats (SHRs) than in normotensive Wistar-Kyoto rats (WKYs). Experiments were conducted in SHRs and WKYs of three age groups to determine whether the increased hypothermic responsiveness to apomorphine occurs prior to the development of hypertension. The mean systolic blood pressures (SBPs) of SHRs and WKYs were comparable at 4-6 weeks of age. The mean SBP of SHRs were significantly greater than that of WKYs at both 8-10 and 12-15 weeks of age. Yet SHRs responded to apomorphine with significantly greater hypothermia than WKYs at all three ages. These findings indicate that the hyperresponsiveness of SHRs to apomorphine-induced hypothermia precedes the development of hypertension. This sequence of events is consistent with the hypothesis that central DA systems play a role in development of hypertension in SHRs.

A new, highly selective CCK-B receptor radioligand ([3H][N-methyl-Nle28,31]CCK26-33): evidence for CCK-B receptor heterogeneity.

[N-methyl-Nle28,31]CCK26-33 (SNF 8702) is a nonsulfated cholecystokinin octapeptide analog that is highly selective for cholecystokinin-B (CCK-B) receptors. Inhibition studies using [125I] Bolton-Hunter-labeled CCK-8 show that SNF 8702 has over 4,000-fold greater affinity for CCK receptors in guinea pig cortex relative to those in guinea pig pancreas. SNF 8702 was tritium-labeled to a specific activity of 23.7 Ci/mmol and its binding properties characterized for guinea pig brain membrane preparations. [3H]SNF 8702 binds to a single site with high affinity (Kd = 0.69-0.90 nM) in guinea pig cortex, cerebellum, hippocampus and pons-medulla. Of these four tissues, the highest receptor density was measured in the cortex (86 fmol/mg of protein) and the lowest in the pons-medulla (22 fmol/mg of protein). In contrast to findings of single-site binding in some brain regions, evidence for CCK-B receptor heterogeneity is observed under other conditions. [3H]SNF 8702 binding to membranes prepared from whole guinea pig brain shows biphasic association kinetics at a concentration of 2.0 nM consistent with the presence of binding site heterogeneity. Binding site heterogeneity is consistently observed for [3H]SNF 8702 binding to guinea pig whole brain membranes in saturation studies where a high-affinity site (Kd = 0.31 nM) is distinguished from a low-affinity site (Kd = 3.3 nM). Binding site heterogeneity is also observed for the midbrain-thalamic region. CCK-B receptor heterogeneity is suggested by the effect of the guanyl nucleotide analogue, guanylyl-imidodiphosphate (Gpp(NH)p), on [3H]SNF 8702 binding to CCK-B receptors in the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation between rat brain and mouse vas deferens delta opioid receptors.

Certain enkephalin analogues, including those which contain the conformationally restricted amino acid E-(2R,3S)-cyclopropylphenylalanine [2R,3S)-delta E Phe), have been shown to have high affinity for brain delta opioid receptors but are much less active in mouse vas deferens bioassays. To investigate whether there are differences between delta opioid receptors in brain and mouse was deferens, the ability of a selective delta opioid compound, [D-Pen2,pCl-Phe4,D-Pen5]enkephalin (pCl-DPDPE), and [D-Ala2,(2R,3S)-delta E Phe4,Leu5]enkephalin methyl ester (CP-OMe), to inhibit [3H]pCl-DPDPE binding in both rat brain and mouse vas deferens were measured. pCl-DPDPE recognized brain and mouse vas deferens binding sites with equal affinity, however, CP-OMe showed 33 fold lower affinity in mouse vas deferens compared to brain. This suggests that mouse vas deferens delta opioid receptors may be distinct from brain delta opioid receptors.

Mediation of nitrous oxide analgesia in mice by spinal and supraspinal kappa-opioid receptors.

Exposure to nitrous oxide produced concentration-dependent analgesia in the mouse abdominal constriction test. Intracerebroventricular or intrathecal pretreatment with naltrexone or nor-binaltorphimine significantly reduced nitrous oxide analgesia. However, similar pretreatment with beta-funaltrexamine had no appreciable effect. These findings suggest that nitrous oxide analgesia involves spinal and supraspinal kappa-opioid receptors.

A high affinity, highly selective ligand for the delta opioid receptor: [3H]-[D-Pen2, pCl-Phe4, d-Pen5]enkephalin.

Binding characteristics of a new, conformationally constrained, halogenated enkephalin analogue, [3H]-[D-penicillamine2, pCl-Phe4, D-penicillamine5]enkephalin ([3H]pCl-DPDPE), were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C determined a dissociation constant (Kd) of 328 +/- 27.pM and a receptor density (Bmax) of 87.2 +/- 4.2 fmol/mg protein. Kinetic studies demonstrated biphasic association for [3H]pCl-DPDPE, with association rate constants of 5.05 x 10(8) +/- 2.5 x 10(8) and 0.147 +/- 10(8) +/- 0.014 x 10(8) M-1 min-1. Dissociation was monophasic with a dissociation rate constant of 2.96 x 10(-3) +/- 0.25 x 10(-3) min-1. The average Kd values determined by these kinetic studies were 8.4 +/- 2.7 pM and 201 +/- 4 pM. Competitive inhibition studies demonstrated that [3H]pCl-DPDPE has excellent selectively for the delta opioid receptor. [3H]pCl-DPDPE binding was inhibited by low concentrations of ligands selective for delta opioid receptor relative to the concentrations required by ligands selective for mu and kappa sites. These data show that [3H]pCl-DPDPE is a highly selective, high affinity ligand which should be useful in characterizing the delta opioid receptor.

The effect of venous blood stream cooling on survival of bacterially infected rabbits.

The effect of physical cooling on the mortality rate of rabbits infected with Pasteurella multocida was investigated. Rabbits were cooled for 48 hours after bacterial injection by passing cold fluid through small hollow metal cuffs which had been surgically implanted around the abdominal vena cavae of rabbits. The average body temperatures of the rabbits during the 24-hour period after the intravenous injection of live Pasteurella multocida was 40.92 +/- 0.20 degrees C in control rabbits and 38.98 +/- 0.71 degrees C in cooled rabbits. 90% of physically cooled rabbits survived compared with 46% of control rabbits 48 hours after bacterial injection, suggesting that thermoregulatory effector mechanisms involved in cold defense may enhance survival.

Rearing and step climbing behavior of X-linked hypophosphatemic (Hyp) mice.

Hyp mice are a model for X-linked hypophosphatemia, the most common form of vitamin D-resistant rickets. Previous reports indicate alterations in the nervous system of these mice. The present study examined rearing and step climbing behavior in Hyp mice in the staircase test. We found that male Hyp mice have large reductions in both the number of rears and steps climbed when compared to normal male mice. When compared to normal female mice, female Hyp mice have a small reduction in the number of steps climbed but no significant reduction in the number of rears. However, they have greater rearing and climbing activity than male Hyp mice. These reductions in activity are not correlated with the severity of bone disease. Gonadectomy had no effect on activity in male mice but reduced activity comparably in both Hyp and normal female mice. We conclude that Hyp mice have altered behavior in the staircase test and that the magnitude and type of the alteration is influenced by the sex of the animal.

Increased metabolic rate in X-linked hypophosphatemic mice.

Hyp mice are a model for human X-linked hypophosphatemia, the most common form of vitamin D-resistant rickets. It has previously been observed that Hyp mice have a greater food consumption per gram body weight than do normal mice. This led to the search for some alteration in metabolism in Hyp mice. We found that oxygen consumption was significantly higher in Hyp mice than in normal C57BL/6J mice and this was accompanied by an increased percentage of cardiac output being delivered to organs of heat production (liver and skeletal muscle), to the skin, and to bone and a decreased percentage to the gastrointestinal tract of Hyp mice. The increased oxygen consumption in Hyp mice was not associated with increased plasma free T4 levels and was not affected by alterations in plasma phosphate produced by a low phosphate diet. The cause of the increased oxygen consumption is not known, and the role that this change and reported changes in distribution of cardiac output may play in the development of X-linked hypophosphatemia is also unknown. Study of the cardiovascular and thermoregulatory systems in Hyp mice should help increase understanding of the underlying mechanisms of this disease.

Narcotic antagonist-induced hypotension in the spontaneously hypertensive rat.

Intravenous naloxone or naltrexone produced transient, dose-related reductions in the mean arterial pressure (MAP) and heart rate (HR) of urethane-anesthetized spontaneously hypertensive rats (SHRs). Yet these same doses of narcotic antagonists reduced HR but not MAP of normotensive Wistar-Kyoto rats (WKYs). Such effects were not observed upon administration to SHRs of increasing doses of methylnaltrexone, which possesses no central activity. (+)-Naloxone, which does not block opiate receptors, reduced HR but not MAP of both SHRs and WKYs. These findings indicate that SHRs and WKYs differ in their MAP and HR responses to narcotic antagonists. The high doses required for effect plus the brevity of the responses suggest that these drug effects are perhaps not mu-opiate receptor-mediated; however, the methylnaltrexone and (+)-naloxone findings clearly implicate a central specificity of action. We conclude that narcotic antagonist-induced changes in MAP and HR in SHRs are possibly specific and central in origin yet not mediated by mu-opiate receptors.

Sex and strain differences in morphine-induced temperature effects in WKYs and SHRs.

Male and female normotensive Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) all responded to morphine treatment with biphasic dose-response curves, exhibiting hyperthermia at low doses and hypothermia at higher doses. However the direction and magnitude of temperature changes induced by different doses of morphine varied significantly depending upon the sex and strain (WKY vs. SHR) of the test animals. Both WKY and SHR males responded with little change in temperature at 1.0 mg/kg and hyperthermia at 5.0 mg/kg. Hypothermia appeared in SHR males at 10 mg/kg, while hypothermia in WKY males was not seen until 20 mg/kg was administered. Both WKY and SHR females demonstrated a greater sensitivity than their male counterparts to the thermotropic effects of morphine, exhibiting hyperthermia at 1.0 mg/kg, which was greater than the hyperthermia exhibited by male rats, and progressively greater hypothermia at 2.0, 5.0 and 10 mg/kg. SHR females demonstrated hypothermia at lower doses of morphine than did WKY females but were otherwise not different. These findings indicate that (1) morphine-induced temperature effects in SHRs and WKYs are dependent upon dose; (2) SHRs seem more sensitive than WKYs to the hypothermic effects of morphine; and (3) female rats seem more sensitive than male rats to the thermotropic effects of morphine in general.

Influence of chronic naloxone treatment on development of hypertension in the spontaneously hypertensive rat.

Chronic administration of naloxone by means of miniosmotic pumps retarded the development of hypertension in young spontaneously hypertensive rats (SHRs) in a dose-related manner. Abrupt termination of naloxone treatment resulted in acceleration in the rate of the blood pressure increase, while increasing the naloxone concentration further slowed the development of hypertension in SHRs. The heart rates of SHRs undergoing chronic naloxone treatment were generally lower than those of control SHRs. Naloxone had no influence upon the mean systolic blood pressures or heart rates of normotensive Wistar-Kyoto control rats. These findings indicate that chronic naloxone treatment can alter the development of hypertension in the SHR.

Effects of antipyresis on bacterial numbers in infected rabbits.

A previous investigation demonstrated that infusion of an antipyretic drug into the preoptic anterior hypothalamus (PO/AH) of rabbits reduced the fever usually seen during the initial stages of infection. This was followed by an increased fever and an increased mortality rate [32]. The work reported here investigated the hypothesis that the increased mortality was the result of decreased killing and/or increased multiplication of bacteria during the initial, attenuated phase of the febrile course in the antipyretic-treated rabbits. Rabbits were injected intravenously with Pasteurella multocida and either sodium salicylate or a control solution was infused directly into the PO/AH. Infusion of sodium salicylate reduced the mean fever 4 hours after injection of bacteria from 2.07 +/- 0.28 degrees C (S.E.M.) to 0.62 +/- 0.43 degrees C. Rabbits with reduced fevers had decreased blood leucocyte counts and greater numbers of bacteria in lung and liver samples. No differences were seen in reticuloendothelial clearance of carbon, hematocrit, or intracellular viability of bacteria when antipyretics were administered. This increase in bacterial numbers corresponds well to the increased mortality found in previous studies in animals with reduced fevers.

Antipyresis: its effect on mortality rate of bacterially infected rabbits.

The effect of an antipyretic drug administered directly into the preoptic-anterior hypothalamus was measured in order to investigate the role of fever on mortality of bacterially infected mammals. New Zealand white rabbits (Oryctolagus cuniculus) were injected intravenously with Pasteurella multocida and either sodium salicylate or a control solution was infused directly into the preoptic-anterior hypothalamus. Both groups developed fevers, but the fever of the rabbits infused with the antipyretic was reduced by 50% during the initial stage of infection. Hypothalamic sodium salicylate infusions produced a lower average fever than control infusions over an initial 5 hour period of infection, reducing average 5 hour fevers from 1.56 degrees C to 0.72 degrees C. All of the infected rabbits infused with sodium salicylate died whereas only 29% of the infected control rabbits died. Rabbits receiving sodium salicylate alone did not die. The increased mortality could possibly be the result of a fulminating infection caused by rapidly multiplying bacteria during the initial, attenuated phase of the febrile course in the salicylate-treated rabbits.