Effects of morphine on: spontaneous, dorsal raphe, spinal tract of trigeminal nucleus, medial lemniscus and reticular lateral magnocellular evoked responses of hypothalamic units, in naive and morphine physically dependent rats

The spontaneous activity and the inputs to the medial basal hypothalamus (MBH) following dorsal raphe (DR), spinal tract of the trigeminal nerve (SpV), medial lemniscus (ML), reticular lateral magnocellular nucleus ( RLM ) and acoustic (Ac) stimulation and the effects of morphine and the opioid antagonist, naloxone, on these inputs, were investigated in morphine-naive and morphine-dependent animals. The observations were obtained in freely behaving animals previously implanted with permanent electrodes. The spontaneous activity of MBH neurons exhibits heterogenic spontaneous firing rates. This spontaneous activity is affected by acute and chronic morphine treatment. The MBH neuronal population exhibits neurophysiological patterns of tolerance of morphine dependence and withdrawal. The central input exerts a marked influence on MBH neurons in both naive and morphine-dependent animals. These inputs are modified by morphine challenge dose in both preparations, i.e., in morphine-naive and morphine-dependent animals, and are reversed by naloxone. The DR and Ac inputs affect the MBH neuronal activity differently from that observed following SpV, ML and RLM stimuli. The effects of morphine and naloxone on the DR and Ac input in morphine-naive and morphine-dependent animals differ from those observed following SpV, ML and RLM inputs. The MBH neurons exhibited a high percentage of convergence to Ac, DR, SpV, ML and RLM stimulation.     

Morphine and clonidine oral self-administration: a study in morphine dependent or abstinent rats

The ability of morphine and of clonidine to support self-administration has been evaluated in morphine dependent or abstinent rats, using an orally reinforced operant technique (F.R. 20). It was found that: Morphine drinking can function as a reinforcer of an operant response both in dependent and abstinent rats. The bitter taste of morphine becomes a secondary reinforcer for rats usually working for the alkaloid. Clonidine supports oral self-administration in morphine abstinent, but not in morphine dependent rats. The data are in line with clinical findings and give a further evidence that oral self administration in rats can be an useful model for the analysis of drug dependence.     

Studies on pain. Effects of morphine on a spinal nociceptive flexion reflex and related pain sensation in man

The nociceptive flexion reflex and the corresponding subjective pain score elicited by sural nerve stimulation were studied in 6 healthy volunteers. A significant correlation was found between the respective recruitment curves of the reflex and of the pain score as a function of stimulus intensity. Consequently, the reflex (Tr) and the pain (Tp) thresholds were found to be almost identical (mean: 10.6 and 10.3 mA, respectively). Similarly, the threshold of the maximal reflex response (Tmr) was very close to that of intolerable pain (Tip): 37.1 and 38.8 mA, respectively. These four parameters were studied before and after intravenous administration of morphine chlorhydrate (0.05, 0.1, 0.2 and 0.3 mg/kg) and subsequent administration of naloxone hydrochloride (0.02 mg/kg; i.v.). While 0.05 mg/kg morphine remained without any effect, higher doses produced an increase in the four thresholds (Tr, Tp, Tmr, Tip). Furthermore, a very significant linear relationship was found between the importance of the increase and the dose of morphine. Morphine also depressed in a dose-dependent fashion, the nociceptive reflexes elicited by a constant stimulation intensity (1.2-1.3 Tr). All these effects were immediately reversed by subsequent naloxone. During all the pharmacological situations, variations in Tr and Tp as well as in Tmr and Tip were found to be very significantly linearly related, indicating a close relationship between the effects of morphine on the nociceptive reflex and on the related pain sensation. These results suggest that, in our model involving a brief 'epicritic' nociceptive stimulus, the mechanisms of morphine-induced analgesia in man can be explained by a depressive effect on the nociceptive transmission directly at a spinal level.     

The effect of morphine on responses of nucleus ventroposterolateralis neurons to colorectal distension in the rat

In 71 halothane-anesthetized rats, we characterized the responses of single neurons in the nucleus ventroposterolateralis (VPL) of the thalamus to a noxious visceral stimulus (colorectal balloon distension; CRD) and studied the effects of intravenous morphine on these responses using standard extracellular microelectrode recording techniques. One hundred nine neurons were isolated on the basis of spontaneous activity. Sixty-four (59%) responded to CRD, of which 52 (81 %) had excitatory and 12 (19%) had inhibitory responses. Neurons showed graded responses to graded CRD pressures (20-100 mmHg), with maximum excitation or inhibition occurring at 80 mmHg. Responses to noxious (pinch, heat) and innocuous (brush, tap) cutaneous stimuli were studied in 95 of the VPL neurons isolated. Eighty-three of these neurons (48 CRD responsive and 35 CRD nonresponsive) (87%) had cutaneous receptive fields, of which 96% were small and contralateral and 4% were large and contralateral or bilateral. Ninety-four percent of these neurons responded to both noxious and innocuous cutaneous stimulation, and 6% responded to only noxious stimulation. No neurons responded solely to innocuous stimulation. Cumulative doses of morphine (0.125, 0.25, 0.5, 1, and 2 mg/kg, i.v) produced statistically significant dose-dependent attenuation of neuronal responses to CRD. Naloxone (0.4 mg/ kg, i.v.) reversed the effects of morphine. Morphine and naloxone had no significant effects on spontaneous activity. These data support the involvement of VPL neurons in visceral nociception and are consistent with a role of VPL in sensory-discriminative aspects of nociception.     

Depressant and excitant effects of intraspinal microinjections of morphine and methionine-enkephalin in the cat

The effects of intraspinal microinjectins of morphine (10 microgram) and methionine-enkephalin (Met-enkephalin) (5 microgram) on the C-fiber and polysynaptic reflexes in the acute decerebrate low spinal cat were investigated. Microinjected into the dorsal horn, morphine and Met-enkephalin depressed the nociceptive C-fiber reflex (CFR) without altering the short latency polysynaptic reflex. Microinjected into the ventral horn, morphine and Met-enkephalin facilitated the C-fiber and polysynaptic reflexes. Pretreatment of the cats with intravenous naltrexone (2 mg/kg) antagonized the depressant effects produced by dorsal horn intraspinal microinjections of morphine and Met-enkephalin. The excitant effects of ventral horn microinjections of morphine were not antagonized by naltrexone (2 mg/kg). These results support a hypothesis that the analgesic effects of morphine at the spinal cord level are due to interactions with opiate receptors in the dorsal horn.     

Mutual potentiation of antinociceptive effects of morphine and clonidine on motor and sensory responses in rat spinal cord

Clonidine and morphine depress nociceptive reflex responses when given alone; when given in combination, the effect of each is potentiated by the other. The present study was designed to test if activity in ascending axons evoked by electrical stimulation of afferent C-fibers in the sural nerve of the rat also exhibits potentiation of the depressant effects of clonidine and morphine when both drugs are administered in combination by intrathecal (i.t.) injection to the lumbar spinal cord. For comparison, experiments were also carried out on the tail-flick response in rats. The results show that clonidine produced a dose-dependent inhibition of the tail-flick response (Ed50 20 micrograms); a combination of ineffective doses of clonidine (0.3 microgram) and morphine (2 micrograms) significantly inhibited the tail-flick response; clonidine (35 micrograms) reduced spontaneous, C-fiber-evoked and, due to co-activation, A delta-fibre-evoked activity in ascending axons; and clonidine at a threshold (0.3 microgram) or higher (3 micrograms) dose administered together with morphine at a dose (2 micrograms) that caused only a moderate inhibition produced a supra-additive effect in significantly depressing spontaneous. A delta- and C-fiber-evoked ascending activity. The dose-response curve of depression by morphine alone of C-fiber-evoked activity (ED50 8 micrograms) is significantly shifted by clonidine to the left (ED50 0.9 microgram). Naloxone (0.2 mg/kg) injected intravenously did not affect the inhibition of ascending activity caused by clonidine at the highest dose (35 micrograms), but it reduced the depressant effect of combined i.t. administration of clonidine and morphine. The potentiation of the antinociceptive effects of clonidine and morphine given in combination are possibly due to actions of the two drugs at different sites between the nociceptive afferents and the neurons sending their axons to the brain.     

Effects of serotonin and morphine on spontaneous and evoked firing of nociceptive neurons in the trigeminal spinal nucleus of rats

Spontaneously firing neurons that were responsive to noxious face pinch or noxious heat were studied in the trigeminal spinal nucleus of the rat brain. These eurons responded with either an increase or decrease in firing rate. In these neurons serotonin (5-hydroxytryptamine; 5-HT) apparently acts through two mechanisms to attenuate the response to a noxious stimulus. One mechanism is mimicked by morphine; these two drugs block the response to the noxious stimuli without having a consistent effect on spontaneous firing. The effects of the two drugs were somewhat selective depending on the noxious stimulus used and the effect of the noxious stimulus; morphine and 5-HT were more effective in blocking the increase in firing rate evoked by the face pinch but 5-HT and morphine were more effective in blocking the decrease in firing rate evoked by the noxious heat stimulus. Interestingly, the direction of the response to a particular noxious stimulus frequently predicted whether or not both morphine and 5-HT would act on the same or different neurons. A second mechanism by which 5-HT, but not morphine, acted was to change the spontaneous firing in a direction opposite that evoked by the noxious stimulus. This type of effect apparently modulated the response to a noxious stimulus by changing the spontaneous firing rate such that a noxious stimulus had to be more intense before it could significantly alter the neuronal firing in the opposite direction. Morphine occasionally produced a change in firing pattern in neurons; this effect remains to be documented more extensively.     

Effects of pain, morphine and naloxone on the duration of animal hypnosis

Previous research has shown that animal hypnosis (tonic immobility) in the rabbit may be elicited in a condition of prolonged nociceptive stimulation. These experiments show that long-lasting irritative pain, produced within 15 min of formalin injection, potentiates the duration of hypnosis. Morphine, in the absence of painful stimuli, also potentiates hypnosis duration and this effect is antagonized by naloxone. Naloxone reduces hypnosis duration, but only at high doses (15 mg/kg). In a condition of irritative pain, the potentiation of hypnosis duration is abolished by naloxone (5 mg/kg). Hypnosis response is abolished in 6 out of 7 morphine-tolerant rabbits, but prolonged pain restores the response. The hypothesis that an opioid mechanism may be activated during animal hypnosis is discussed.     

Effects of footshock stress and morphine on natural killer lymphocytes in rats: studies of tolerance and cross-tolerance

Exposure to a form of footshock stress known to cause opioid-mediated analgesia suppresses the cytotoxic activity of natural killer (NK) cells in rats. This suppression is blocked by the opioid antagonist, naltrexone and is mimicked by morphine administration, suggesting mediation by opioid receptors. Supporting this hypothesis, we now report that the morphine-induced suppression of NK activity shows tolerance after 14 daily injections. The NK-suppressive effect of stress, however, shows neither tolerance with repetition nor cross-tolerance in morphine-tolerant rats.     

In situ-produced 7-chlorokynurenate has different effects on evoked responses in rats with limbic epilepsy in comparison to naive controls

PURPOSE: Uncontrolled epilepsy remains a significant health concern and requires new approaches to therapy. N-methyl-d-aspartate (NMDA) receptor blockade has been considered, but the adverse cognitive and behavioral effects of conventional NMDA-receptor antagonists have prevented the development of clinically useful compounds. An alternative approach may be the blockade of the glycine coagonist ("glycine(B)") site of the NMDA receptor. METHODS: As a first step in the exploration of this approach, we examined the effect of 4-chloro-kynurenine (4-Cl-KYN), which is converted by astrocytes to the potent NMDA glycine-site antagonist 7-chloro-kynurenic acid (7-Cl-KYNA), on the in vivo epileptiform evoked potentials in the CA1 region of rats with chronic limbic epilepsy (CLE). 4-Cl-KYN (100 mg/kg) was administered intraperitoneally to naive and epileptic rats. Evoked potentials were induced in area CA1 of the hippocampus by electrical stimulation of the midline region of the thalamus. Simultaneous microdialysis was performed in the contralateral hippocampus to determine the extracellular levels of 7-Cl-KYNA over the course of the experiment. RESULTS: Administration of 4-Cl-KYN caused a significant reduction in the amplitude of the population spike and in the number of population spikes in epileptic animals (p < 0.01) but had no effect on the evoked response in naive rats. In contrast, 4-Cl-KYN significantly altered the paired response in naive animals (p < 0.01), but had no significant effect on this parameter in epileptic animals. The levels of 7-Cl-KYNA measured achieved known pharmacologically effective concentrations and paralleled the observed physiological effects. CONCLUSIONS: The use of glial cells for the neosynthesis and local delivery of neuroactive compounds may be a viable strategy for the treatment of limbic epilepsy. These results also underscore the unique pharmacology of neurons in epilepsy.     

The interaction of tetrahydroisoquinoline derivatives with antinociceptive action of morphine and oxotremorine in mice

Summary. To extend our earlier data on synergistic action of tetrahydroisoquinolines and morphine, we have investigated the analgesic effects of 1,2,3,4-tetrahydroisoquinoline (TIQ) and its 1-methyl-(1-MeTIQ) and N-methyl (N-MeTIQ) analogs on analgesia induced by morphine and oxotremorine. 1-MeTIQ and N-MeTIQ induced a moderate, delayed and prolonged analgesic action measured in the tail-flick test in CD-1 mice; 1-MeTIQ and TIQ prolonged the opiate (morphine, 2.5mg/kg ip) analgesia while TIQ and N-TIQ potentiated cholinergic (oxotremorine, 0.02mg/kg ip) analgesia. The involvement of the opioid and noradrenergic systems in this effect is discussed.Received April 14, 2003; accepted July 9, 2003 Published online September 15, 2003     

Augmentation of LTP induced by Primed–Bursts tetanic stimulation in hippocampal CA1 area of morphine dependent rats

The effects of chronic morphine administration on the development of Long-term potentiation (LTP) were investigated at the Schaffer collateral-CA1 pyramidal cell synapses of the rat hippocampal slices using primed-bursts tetanic stimulation. Significant enhancement of orthodromic population spike (OPS) was found for all stimulus intensities after tetanic stimulation. OPS enhancement was greatest when tested with low to mid-range stimulus intensities (25 and 50 μA). There was also significant decrease in OPS delay. These responses were similar in slices from both control and morphine dependent rats. At all delivered stimulus intensities, the amount of LTP of OPS in slices from dependent rats was larger than that of control slices. However, these differences in LTP of OPS were significant at low stimulus intensities. These findings suggest that chronic morphine administration had induced changes in CA1 neurocircuitry which modulated synaptic plasticity during high frequency stimulation and appeared as augmented LTP.     

Accelerated release and production of orphanin FQ in brain of chronic morphine tolerant rats

Orphanin FQ has been shown to possess anti-opioid activity at supraspinal level. Our previous work revealed that chronic morphine tolerance could be reversed by intracerebroventricular (i.c.v.) injection of OFQ IgG to rats. In this study, we used radioimmunoassay (RIA) to assess the changes of Orphanin FQ immunoreactivity (OFQ-ir) in cerebroventricular perfusate, periaqueductal gray (PAG) and amygdala of rats made tolerance to morphine (10-60 mg/kg, s.c., t.i.d., for 5 days). The results indicated that: (1) In rats administrated with morphine for 3 and 5 days, the content of OFQ-ir in cerebroventricular perfusate increased by 25% and 52% over the NS control group. (2) The content of OFQ-ir in PAG of rats receiving 1d, 3d and 5d injections of morphine showed an increase of 17%, 48% and 81% respectively over NS group. (3) The content of OFQ-ir in amygdala of rats given 3d and 5d of morphine showed a 36% and 55% increase compared with corresponding control group. It is suggested that continuous use of high doses of morphine accelerated the release and biosynthesis of OFQ in rat brain to antagonize the effect of opioids, which may play a role in the development of morphine tolerance, and that brain OFQ may serve as a delayed negative feedback control on opioid analgesia.     

Dose effects of morphine on the spontaneous unit activity recorded from the thalamus,hypothalamus, septum,hippocampus, reticular formation,central gray,and caudate nucleus

Spontaneous activity was recorded from 652 units in 8 subcortical structures of unanesthetized rats. Recordings were obtained in central gray, mesencephalic reticular formation, parafasciculus thalami, caudate nucleus, anterior and ventro- medial hypothalamus, lateral septum, and dorsal hippocampus. Eighty recordings were obtained from untreated animals and 80 from saline-injected controls, none of which showed any significant changes of unit activity during the 4- 5-hr observation period. The effect of morphine, given in 5 incremental doses from 0.5 to 30.0 mg/kg ip, was followed in 492 units. Morphine enhanced or depressed spontaneous discharge rates, or caused biphasic effects, ie enhancement alternating with depression and vice versa. Naloxone induced increase in firing after either effect of morphine, or reduced spontaneous activity after morphineinduced increases. However, when morphine reduced neuronal discharges, naloxone never caused further depression. In 86 units, morphine at any dosage failed to alter neuronal activity, but in 54 of these units naloxone nevertheless induced alterations in firing rates. The pattern of responses to morphine differed between all 8 brain regions examined and was characteristic for each individual structure. This is the first systematic study describing the dose-response characteristics of morphine in 8 brain sites recorded simultaneously. Furthermore, it utilized freely behaving animals without the interference of anesthetics, which are themselves known to interact with opiates. The variety of response patterns seen supports the neuro pharmacological evidence for multiple opiate receptors or multiple sites of opiate action.     

Expression of hyperpolarization-activated and cyclic nucleotide-gated cation channel subunit 2 in axon terminals of peptidergic nociceptive primary sensory neurons in the superficial spinal dorsal horn of rats

Hyperpolarization-activated cyclic nucleotide-gated cation channel proteins (HCN1-4), which are potentially able to modulate membrane excitability, are abundantly expressed by neurons in spinal dorsal root ganglia (DRG). In the present experiment, we investigated whether HCN2 protein is confined exclusively to the perikarya of DRG neurons or is transported from the somata to the central axons of DRG neurons that terminate in the spinal dorsal horn. Using immunohistochemical methods, we have demonstrated that laminae I-IIo of the superficial spinal dorsal horn of the adult rat spinal cord show a strong punctate immunoreactivity for HCN2. Dorsal rhizotomy resulted in a complete loss of immunostaining in the dorsal horn, suggesting that HCN2 is confined to axon terminals of primary afferents. In double labelling immunohistochemical studies, we have also shown that HCN2 widely co-localizes with calcitonin gene-related peptide, but is almost completely segregated from isolectin-B4 binding, indicating that HCN2 is primarily expressed in peptidergic nociceptive primary afferents. The expression of HCN2 in central terminals of peptidergic primary afferents was also verified with electron microscopy. Utilizing the pre-embedding nanogold method, we found that HCN2 is largely confined to axon terminals with dense-core vesicles. Within these terminals, some of the silver grains marking the accurate location of HCN2 molecules were associated with the cell membrane, and others were scattered in the axoplasm. Within the cell membrane, HCN2 was found almost exclusively in extrasynaptic locations. The results suggest that HCN2 may contribute to the modulation of membrane excitability of nociceptive primary afferent terminals in the spinal dorsal horn.     

The analgesic effects of morphine, but not those of the enkephalinase inhibitor thiorphan, are enhanced in arthritic rats

The effects of various i.v. doses of morphine (0.1, 0.3 and 1 mg/kg) and of thiorphan, an inhibitor of enkephalinase (0.7, 2.5, 5, 10 and 15 mg/kg), were studied upon the vocalization threshold to foot pressure in normal rats and rats with Freund's adjuvant-induced arthritis. The vocalization threshold in arthritic rats was, before any injections, significantly lower than in normal rats (mean pressure threshold for vocalization: 115.2g ± 14.7 (n= 152) for arthritic rats vs 182.5g ± 21.3 for normal rats (n= 152). The various doses of morphine in raising the vocalization threshold were more efficient in arthritic than in normal rats (maximum vocalization threshold (% of control) following 1mg/kg morphine= 225.70 ± 10.21 in arthritic rats vs 140.75 ± 6.87 in normal rats, n= 9 in each case). This effect was dose-dependent, and in every case, naloxone-reversible. Injected at doses of 5–15 mg/kg, thiorphan increased the vocalization threshold (maximum= 223.91% ± 11.96 in arthritic rats vs 223.30% ± 5.93 in normal rats for 15 mg/kg i.v., n= 9 for each group). This effect was not greater in arthritic than in normal rats. The dose of 2.5 mg/kg of thiorphan was insufficient. Administered at 0.7 mg/kg, thiorphan significantly decreased the vocalization threshold in the arthritic rats only. These effects of thiorphan were all naloxone-reversible using doses of naloxone which were one-hundredth of those of thiorphan.     

Differential responses of serotonergic and non-serotonergic neurons in nucleus raphe magnus to systemic morphine in rats

Forty-eight raphe-spinal units in nucleus raphe magnus were identified by antidromic stimulation and further classified into serotonergic and non-serotonergic populations according to their conduction velocity and spontaneous discharge rate. Following morphine administration (5 mg/kg, i.p.) they showed different responses: excitatory, depressive or non-responsive. It was found that 6 of 7 nonresponsive, 3 of 20 depression-responsive and only 1 of 21 excitation-responsive units proved to be serotonergic neurons, indicating that the involvement of serotonergic neurons in morphine analgesia is probably insignificant. It is suggested that the raphe-spinal fiber systems, both excitatory and inhibitory are originated mainly from the non-serotonergic neurons.