Joseph R. Holtman

Characterization of the antinociceptive effect of oxycodone in male and female rats

A number of investigators have shown that sex plays an important role in the analgesic effects of opioids. Typically, the antinociceptive responsiveness to mu opioid agonists such as morphine is greater in male than in female rats. The effect of sex on kappa opioid analgesia is less known. The present study was conducted to examine sex-related differences in responsiveness to oxycodone (putative kappa/mu opioid agonist). This information is important since oxycodone is widely used clinically for treatment of pain. The present results indicated that oxycodone had a greater antinociceptive response in female rats compared to male rats. This sex specific responsiveness to oxycodone, however, was lost with chronic administration. The greater antinociception in female rats was even more prominent with U50,488H (selective kappa agonist). Further, low (subanalgesic) doses of oxycodone and U50,488H enhanced the sensitivity to pain (hyperalgesia) to a greater extent in male than in female rats. This is in contrast to the previously shown greater hyperalgesic effect of subanalgesic doses of the mu opioid agonist, morphine, in female than in male rats. The present findings suggest that sexual dimorphism in the effect of opioids is related to the opioid receptors on which they predominately act.

Ultrastructural evidence for serotonin-immunoreactive terminals contacting phrenic motoneurons in the cat

The innervation of the phrenic motor nucleus in the cat by serotonin-containing neurons has been studied using retrograde tracing combined with immunohistochemistry at the electron microscope level. It was found that phrenic motoneuron cell bodies and dendrites are contacted by serotonin-immunoreactive synaptic terminals. This finding suggests that the activity of phrenic motoneurons is directly affected by serotonergic neurons.

Sex-related differences in the enhancement of morphine antinociception by NMDA receptor antagonists in rats.

The effect of the N-methyl-D-aspartate (NMDA) receptor antagonists dextromethorphan (DEX), ketamine (KET), and MK-801 on morphine (MOR)-induced antinociception has been investigated in male and female rats. DEX (7.5, 15, and 30 mg/kg), KET (0.75, 1.5, and 3 mg/kg), and MK-801 (0.075, 0.15, and 0.3 mg/kg) dose-dependently enhanced MOR-induced (3 mg/kg) analgesia in female rats. DEX and KET enhanced the peak effect, whereas MK-801 increased both magnitude and duration of analgesia. DEX also enhanced MOR-induced analgesia in male rats. However, the interaction was of less magnitude in male compared with female rats. The effects of KET and MK-801 on MOR-induced analgesia were negligible in male rats. A 3-mg/kg dose of MOR given alone produced greater analgesia in male than in female rats, but in the presence of NMDA antagonists, MOR elicited similar analgesic responses in both sexes.

Morphine tolerance in male and female rats

Several studies indicate greater sensitivity to morphine (MOR) analgesia in male compared to female rats under the acute dosing condition. The present study investigated whether the same sex difference in sensitivity persists in MOR-tolerant rats. MOR was administered chronically (7 mg/kg twice daily) until tolerance developed in each rat. Tolerant rats were treated randomly with higher graded doses of MOR (10-25 mg/kg). Analgesia (tail-flick test) and spontaneous motor activity (total locomotion) were measured. The present data confirmed previous studies showing a greater sensitivity to acute MOR in male than in female rats. However, the sex differences seen in MOR sensitivity were abolished in tolerant rats. The rate of acquisition of tolerance was similar in male and female rats. The analgesic response was not affected by motor depression.

Interaction between morphine and norketamine enantiomers in rodent models of nociception.

Ketamine, one of a few clinically-available N-Methyl-D-aspartate (NMDA)-receptor antagonists, is known to improve the analgesic efficacy of opioids in humans and rodents. However, the use of ketamine in combination with opioids is mainly restricted to the perioperative setting, due to severe psychotomimetic, sedative and motor side effects. Recent data from our laboratory demonstrated that a major metabolite of ketamine, norketamine, in particular the S(+) enantiomer, had a better antinociception/side effects profile than ketamine in rats. It is unknown if norketamine affects opioid antinociception. In the present study, morphine (a low dose) was combined with S(+)- and R(-)-norketamine (sub-antinociceptive doses) and characterized utilizing rodent models of pain including: thermal nociception (the tail-flick test), peripheral neuropathy (chronic constriction nerve injury) and tonic inflammatory pain (the formalin test). The data showed that: 1) Norketamine enhanced morphine antinociception and blocked tolerance to this effect; 2) Norketamine potentiated morphine effectiveness in the alleviation of symptoms resulting from injury to nerve (mechanical hyperalgesia, tactile allodynia) and peripheral tissue (formalin-induced nociceptive behavior); 3) S(+)-norketamine was more potent than R(-)-norketamine; 4) Antinociception was not confounded by significant side effects. Morphine-S(+)-norketamine combination drug therapy may prove clinically useful for the alleviation of acute and chronic pain of differing etiology.

Modification of morphine analgesia and tolerance by flumazenil in male and female rats

This study assessed the effect of the central benzodiazepine receptor antagonist, 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester (flumazenil), on morphine-induced analgesia, locomotor effects, and development of tolerance in rats. The thermally evoked pain (tail flick) response was determined after acute and chronic intraperitoneal (i.p.) administration of morphine and flumazenil, alone and in combination. In acute studies, flumazenil induced weak analgesia unrelated to dose and sex, whereas morphine-induced analgesia was dependent on both dose and sex (male>female). Flumazenil dose-dependently enhanced the analgesic effect of morphine in female but not in male rats. Isobolographic analysis suggested synergism between flumazenil and morphine in female rats, but antagonism in male rats. Flumazenil-induced locomotor changes (alone and with morphine) were related to sex but not dose. Chronic coadministration of flumazenil with morphine enhanced analgesia and attenuated tolerance development in female rats. The findings suggest a possible role for flumazenil as an adjunct with opioids in acute and chronic pain therapy.

Substance P immunoreactive projections to the ventral respiratory group in the rat

Retrograde tracing (rhodamine beads) combined with immunohistochemistry was used to determine the origin of neurons containing substance P that project to the rostral ventral respiratory group in the rat. Double-labeled neurons (rhodamine beads plus substance P immunoreactivity) were found in the midline caudal raphe nuclei (raphe obscurus, raphe pallidus, and raphe magnus) and in the ventrolateral medulla in the parapyramidal region. The findings of this study suggest that substance P-containing neurons in the caudal raphe nuclei and parapyramidal region project to inspiratory neurons in the rostral ventral respiratory group and may therefore influence their activity.

Effect of activation of 5-HT1A receptors at the ventral medulla on phrenic nerve activity

The purpose of this study was to determine the effects of the 5-HT1A receptor agonist, 8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide (8-OH-DPAT), on respiratory activity (phrenic nerve activity) following application to the ventral medullary surface in the cat. In addition, in order to determine if the action of 8-OH-DPAT was localized to structures at the ventral medulla, we examined the distribution of [3H]8-OH-DPAT to other brain regions and to the peripheral circulation. 8-OH-DPAT (0.0625-8 micrograms) produced a dose-related increase in respiratory rate when applied at either the intermediate or caudal areas on the ventral surface of the medulla. The maximal change in respiratory rate was 9 +/- 2 and 8 +/- 1 breaths/min at the intermediate and caudal areas, respectively. Integrated phrenic nerve amplitude was not significantly affected at these sites except at the 8 micrograms dose where it was decreased. No change in phrenic nerve activity was observed with 8-OH-DPAT application at the rostral area. [3H]8-OH-DPAT was not found to distribute to other brain regions or to the peripheral circulation following application to the ventral medullary surface. The results of this study suggest that 8-OH-DPAT causes changes in respiratory activity, primarily respiratory rate, by acting on neuronal structures at the ventral surface of the medulla.

Regulation of respiratory motor outflow to the larynx and diaphragm by GABA receptors

Effects of intracisternal administration of the gamma-aminobutyric acid receptor agonist, muscimol, on recurrent laryngeal and phrenic nerve activities were assessed in the cat. Muscimol (0.1-2.4 micrograms) produced a dose-related inhibition of the respiratory activity in both of these nerves. The inhibition was seen as a decrease in the amplitude of nerve activity with no significant change in respiratory rate. The ED50 values for muscimol and its 95% confidence interval for inhibition of the inspiratory activity in the recurrent laryngeal and phrenic nerves were 0.22 micrograms (0.17-0.29) and 0.27 micrograms (0.19-0.38), respectively. The potency of muscimol for inhibition of these nerve activities was not significantly different. Expiratory activity in the recurrent laryngeal nerve was completely inhibited prior to either the inspiratory activity in this nerve or in the phrenic nerve. Intracisternal administration of the gamma-aminobutyric acid receptor antagonist, bicuculline, rapidly reversed the inhibition in both nerves. Reversal times for inhibition of the inspiratory activities in the recurrent laryngeal and phrenic nerves were not significantly different. Return of inspiratory activity in each nerve always preceded the return of expiratory activity in the recurrent laryngeal nerve. These results demonstrate that CNS gamma-aminobutyric acid receptors are involved in the regulation of respiratory motor outflow to the larynx as well as the diaphragm.

The effects of diazepam dependence and withdrawal on morphine-induced antinociception and changes in locomotion in male and female rats.

Male and female rats were exposed for 3 weeks to diazepam (DZ)-filled or empty capsules (CTR) prior to the daily administration of morphine (MOR, 5 mg/kg, IP) for 5 days. Thereafter, capsules were removed and 48 h later MOR was injected for the next 5 days. The tail-flick latency (TFL) was measured prior to and 15, 30, and 60 min after MOR assessed analgesia. Locomotion (LOC) was determined before and 15 min after injection. Prior to MOR injection (baseline), male rats were more sensitive to the thermal stimulus and were less active than female rats. Daily MOR injections neither affected the baseline TFL nor LOC. Regardless of gender, MOR produced greater analgesia in DZ-dependent and withdrawn rats than in CTR. MOR analgesia was greater in DZ-dependent male than in female rats. Gender differences in MOR analgesia were not of statistical significance in DZ-withdrawn rats. The first dose of MOR produced more depression of LOC in DZ-dependent female than in male rats. Across the time of MOR injections, female DZ-dependent and withdrawn rats were less active than CTR. LOC increased with repeated administration of MOR in all groups of rats. In summary, DZ dependence and withdrawal enhanced MOR analgesia in rats of both sexes. Regardless of chronic treatment, MOR produced more analgesia and less depression of LOC in male than in female rats. It is suggested that a decrease in the function of the GABAergic system plays a role in alteration of MOR analgesia.