Effects of locus coeruleus lesions on morphine-induced antinociception

These studies were designed to examine the role of the norepinephrine-containing cells comprising the nucleus locus coeruleus (LC) in the mediation of pain perception and morphine-induced antinociception. Nociceptive threshold and morphine-induced analgesia were measured following monosodium-L-glutamate lesions of the LC and adjacent tegmentum (nucleus parabrachialis ventralis; PBV) at 17, 24 and 31 days after surgery. Nociceptive thresholds assessed by the tail flick and hot plate assays were not altered following lesions which included both the LC and PBV (Group 1) or by lesions of the PBV (Group 2) alone. Examination of lesion-induced effects on the capacity of morphine to induce analgesia revealed that damage which included both LC and PBV as well as that confined primarily to the PBV resulted in attenuation of analgesia induced by morphine. Those lesions which involved the LC altered norepinephrine content in the cortex, spinal cord and medial brain stem; however, no correlation could be demonstrated between the attenuation of morphine-induced analgesia and the changes in norepinephrine content of the brain regions examined. Thus, destruction of the LC does not appear to be responsible for the decreased effectiveness of morphine. The only region consistently damaged in both groups 1 and 2 was the ventral parabrachial nucleus. Therefore, we tentatively conclude that destruction of the PBV was responsible for the observed attenuation of morphine analgesia.     

Involvement of 5-hydroxytryptamine-containing neurons in antinociception produced by injection of morphine into nucleus raphe magnus or onto spinal cord

We studied whether antinociception produced by injection of morphine into the nucleus raphe magnus (NRM) or superfusion onto the spinal cord involved serotonergic neurons that descend from brainstem to spinal cord. Involvement of 5-hydroxytryptamine (5-HT)-containing neurons was determined by correlating morphine-induced analgesia with an increase in turnover of 5-HT and by determining if depletion of cord 5-HT with the neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT) could attenuate the antinociceptive effects of morphine. When injected directly into the NRM, 10 micrograms of morphine produced profound analgesia as measured by the paw-pressure technique, and significantly increased the turnover of 5-HT in both posterior medulla and spinal cord. Depletion of cord 5-HT to less than 10% of control concentrations attenuated the antinociceptive effect of morphine injected into the NRM. When various concentrations of morphine (1, 10 or 50 micrograms) were injected directly into the spinal subarachnoid space, a dose-dependent analgesia was observed. No change in 5-HT turnover in spinal cord was observed with any dose of morphine superfused onto the cord. In addition, depletion of cord 5-HT with 5,7-DHT did not alter the analgesic response to either 1 or 10 micrograms of intrathecal morphine. These results suggest that although 5-HT-containing neurons descending from brainstem into spinal cord are involved with analgesia produced by morphine injection into the NRM, they are not involved in the analgesia induced by applying morphine directly to the cord.     

Effect of morphine-induced cortical excitation on spinal sensory transmission

Bioelectrical responses evoked in the ventrolateral funiculus (VLF) of the spinal cord by electrical stimulation of the contralateral hind limb were studied following topical application of 1% morphine solution to the somatosensory SI area of the rat cerebral cortex. After morphine, a typical pattern was observed in the electrocorticogram, characterized by the appearance of rhythmic spiking activity. Time-related with each cortical spike, a significant reduction in the amplitude of VLF responses was observed. It is concluded that cortical excitation induced by morphine generates descending influences having the ability to inhibit spinal sensory transmission.     

Neonatal 6-hydroxydopamine destroys spinal cord noradrenergic axons from the locus coeruleus, but not those from lateral tegmental cell groups

Subcutaneous injection of 6-hydroxydopamine (6-OHDA) in neonatal rats results in sprouting of collateral axons in locus coeruleus (LC) and lateral tegmental noradrenergic neurons. It has been suggested that this sprouting represents maintenance of neuronal membrane area following "pruning" of axon terminals of long projections to cortex and cord. The chemical or surgical lesions of long axons used to produce "pruning" could also result in the loss of some parent cell bodies. We tested the hypothesis that long axon damage, rather than cell loss, is sufficient to produce collateral sprouting of proximal axons in noradrenergic neurons. With neonatal injections of 6-OHDA at doses which do not produce a loss of LC neurons, there is an 85% decrease in retrograde LC labeling following horseradish peroxidase or true blue injections into the spinal cord but no significant change in the numbers of retrogradely labeled neurons in other noradrenergic cell groups which also sprout collaterals. There is no change in the number of labeled LC neurons following cerebellar injections. In experiments using the fluorescent dyes diamidino yellow and true blue, the number and distribution of LC neurons labeled from spinal cord and cerebellum injections are similar to those in the horseradish peroxidase experiments. Doubly labeled neurons are found in the caudal two-thirds of LC in control rats, but as expected, rarely observed in 6-OHDA-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lesion in nucleus reticularis gigantocellularis: effect on the antinociception produced by micro-injection of morphine and focal electrical stimulation in the periaqueductal gray matter

The effect of bilateral electrolytic lesions in the nucleus reticularis giganto-cellularis (NGC) on the antinociceptive efficacy of morphine and electrical stimulation applied in the periaqueductal central gray matter (PAG) was investigated. Antinociception, evaluated by standard hot plate and tail-flick analgesiometric tests, was reliably produced by morphine (5 microgram) and focal electrical stimulation (40-200 micro A) administered in the PAG of rats via chronic indwelling cannula/electrode assemblies. Subsequent to the initial antinociceptive testing, bilateral electrolytic lesions were introduced in the NGC and the antinociceptive efficacy of morphine and stimulation in the PAG was again evaluated. Lesions in the NGC prevented the expression of the antinociception produced by the microinjection of morphine in the PAG whereas the antinociception resulting from electrical stimulation in the PAG was unaffected. Further, lesions in the NGC did not alter baseline (control) nociceptive thresholds in either analgesiometric test. These results provide additional support for involvement of the NGC in morphine-induced antinociception and, in addition, suggest that the NGC is not essential to a tonically-active inhibitory system or to the antinociception produced by focal electrical stimulation in the PAG.     

Microinjection of morphine into nucleus reticularis paragigantocellularis of the rat: Suppression of noxious-evoked activity of nucleus raphe magnus neurons

Microinjection of 1 microgram of morphine into nucleus reticularis paragigantocellularis (Pgc) of anesthetized rats depressed both noxious-evoked and spontaneous activity of nociresponsive neurons in the nucleus raphe magnus (NRM). This effect was naloxone-reversible, and was not observed after control injections dorsal to Pgc. The percent change in spontaneous firing was significantly greater than the percent change in pinch-evoked firing. This reduction in NRM neuronal discharge may contribute to the antinociceptive effects produced by microinjection of morphine into Pgc.     

Microinjection of morphine into nucleus reticularis paragigantocellularis of the rat suppresses spontaneous activity of nucleus raphe magnus neurons

Female rats were neonatally treated with estradiol-17 beta-benzoate or the long-acting dibenzoate esters of the isomeric catecholestrogens, 2-hydroxyestradiol-17 beta and 4-hydroxyestradiol-17 beta. Estrogen benzoates were administered subcutaneously from day 1 to 5 of life at doses of 0.05, 0.10, 0.50 and 1.00 micrograms/day. All rats were ovariectomized as adults and, 4 weeks later, the luteinizing hormone (LH) response to progesterone (2.5 mg) was tested after priming with estradiol-17 beta-benzoate (20 micrograms). At a dose of 0.5 micrograms/day, estradiol-17 beta-benzoate and 4-hydroxyestradiol-17 beta-dibenzoate were equally effective in neonatally defeminizing the LH surge mechanism. In contrast, up to a dose of 1.00 micrograms/day, 2-hydroxyestradiol-17 beta-dibenzoate did not interfere with the LH response in adult life. In the pituitary gland and uterus of the neonatally defeminized rats estrogen responsiveness of cytosolic progestin receptor induction was unimpaired. Moreover, in the uterus of these rats nuclear translocation of cytosolic progestin receptors was intact.     

Comparison of the effects of ventral medullary lesions on systemic and microinjection morphine analgesia

The effects of electrolytic lesions of the nucleus raphe magnus (NRM), nucleus reticularis paragigantocellularis (PGC) and nucleus raphe alatus (NRA) on analgesia elicited in the rat from systemic morphine and morphine microinjection into the periaqueductal gray (PAG) were evaluated using the tail flick test. No consistent change in baseline pain sensitivity was observed following lesions of the NRM, PGC or NRA. To determine the effect of ventral medullary lesions on systemic porphine analgesia, pain sensitivity was assessed prior to and 40 min after 6 mg/kg morphine administration (i.p.) at 2 days preceding lesioning and 5, 12 and 19 days post-lesion. NRM and PGC lesions produced only slight reductions in analgesia at 5 days after surgery. It was observed that large NRM, large PGC, and NRA lesions significantly attenuated analgesia evaluated at 12 days post-lesion. Smaller lesions confined within the NRM or PGC were reliably less effective than the larger lesions in reducing analgesia. In a subsequent study, 5 μg morphine in 0.5 μl saline was microinjected into the ventral PAG at the level of the dorsal raphe. Identical testing procedures were used and the analgesia was assessed at 2 days before lesioning and 5 and 12 days post-lesion. In contrast to the previous study, large NRM lesions abolished analgesia as early as 5 days following lesioning. Small NRM lesions were less effective and PGC lesions were generally ineffective in attenuating analgesia induced by morphine microinjection. We conclude that the NRA may act as a functional unit in the mediation of systemic morphine analgesia. In contrast, analgesia elicited from intracerebral (PAG) morphine microinjection is mediated via the NRM.     

Localization of endogenous morphine-like compounds in the mouse spinal cord

Morphine, codeine, morphine-6-glucuronide, and morphine-3-glucuronide are synthesized de novo in mammalian cells and in the central nervous system. Knowledge on endogenous morphine-like compound distribution in the adult mouse brain has been recently improved, and new hypotheses have been suggested about the potential implications in brain physiology. Endogenous morphine-like compounds have been shown to be synthesized in the spinal cord, but their localization is unknown. Here we describe the distribution of endogenous morphine-like compounds (morphine and/or its glucuronides and/or codeine) in the adult mouse spinal cord using a well-validated antibody. By using different microscopy approaches, we found the presence of morphine, codeine, or morphine glucuronides in γ-aminobutyric acid (GABA)-ergic neurons and astrocytes of the spinal cord. Whereas GABAergic neurons containing endogenous morphine-like compounds were located primarily in the ventral horn, astrocytes that were labeled for morphine-like compounds were found throughout the gray matter and the white matter. Our study demonstrates the possibility that endogenous morphine-like compounds in the central nervous system have other functions beyond their analgesic functions.     

Reduction of fetal rat spinal cord volume following maternal morphine injection

Previous studies have shown behavioral changes in pre- and postnatal rats exposed to morphine before and during gestation. The present study has attempted to discover morphological correlates of these changes in the fetal rat spinal cord. Pregnant Wistar rats were injected subcutaneously on days 12–18 of gestation with 5 mg/kg of morphine 4 times daily. Non-injected, saline-injected and pair-fed controls were prepared for comparison. On day 18 of gestation the fetuses were perfused and the volume of the first thoracic spinal cord segment was measured using planimetric measurements of histological sections. The following volumes were measured within the segment: hemisection, gray matter, white matter, dorsal horn, ventral horn and length. All volumes were reduced by 20% in morphine-exposed fetuses and 10% in pair-fed fetuses. The length of the spinal cord segment was not different from controls. In addition, body weights were not reduced in either the morphine-exposed or pair-fed fetuses. This is the first study of rats exposed prenatally to morphine, exhibiting a decrease in nervous system volume without an accompanying decrease in body weight.     

Increased norepinephrine and uric acid levels in the spinal cord of arthritic rats

The purpose of the present study was to investigate whether the level of norepinephrine and its rate of disappearance after decarboxylase inhibition were modified in the spinal cord of a chronic pain model: the arthritic rat. Chromatographic studies allowing the simultaneous determination of norepinephrine and uric acid by means of HPLC with electrochemical detection are described. The norepinephrine and uric acid levels in the spinal cord were higher in arthritic rats than in normal rats. In addition the rate of disappearance of the amine was increased in the dorsal part of the cord in arthritic rats. These results agree with previous reports suggesting an activating effect of nociceptive stimuli on descending noradrenergic systems. They also indicate that studies on purine metabolism in the CNS in inflammatory and/or pain processes will be of interest.     

Spinal vs supraspinal actions of morphine on cat spinal cord multireceptive neurons

To examine whether morphine elicits a supraspinal mediated spinal inhibition of nociceptive transmission, several investigators have compared the effects of morphine on nociceptive transmission in animals with the spinal cord intact vs transected or cold-blocked. The results have been conflicting, possibly due to different methods of analysis. For example, some investigators have found i.v. administered morphine produces a greater percentage decrease in nociceptive transmission when the spinal cord is intact compared to the transected state. Therefore, they concluded that morphine elicits a supraspinal-mediated inhibition. Conversely, others have reported that the increase in noxious stimulus-evoked responses of dorsal horn neurons upon cold blocking the spinal cord was reduced by i.v. morphine. They therefore concluded that morphine decreases descending inhibition. We tested the effects of i.v. morphine on spinal cord multireceptive neurons in the presence and absence of descending inhibition. Using the above methods of analysis, our results were found to be consistent with their findings which indicate that the method of analysis used is critical to the interpretation reached. To determine how these calculations would be affected by a depressant effect on the spinal cord neurons only, we performed similar experiments iontophoresing gamma-aminobutyric acid (GABA) onto these dorsal horn neurons. The similarity between the morphine and GABA data suggests that the effects of systemically administered morphine on multireceptive dorsal horn neurons can be adequately explained by a spinal cord site of action.     

Effect of methylprednisolone on motor function and spinal cord blood flow after spinal cord compression in rats

The effect of methylprednisolone (MP) on neurologic recovery and spinal cord blood flow (SCBF) was investigated up to 4 days after a spinal cord compression injury in rats. The injury was produced at midthoracic level by applying a load of 35 g on a 2.2 x 5.0 mm compression plate for 5 min, which resulted in transient paraparesis. MP was given as a bolus dose of 30 mg/kg i.v. 60 min after injury (n = 20) and controls were given saline (n = 10). The motor performance was assessed daily as the capacity angle on the inclined plane and SCBF was measured by 14C-iodoantipyrine autoradiography on Days 1 or 4. On Day 1 the capacity angle was reduced from about 63 degrees preoperatively to 33 +/- 2 degrees (mean +/- SEM) in the control group and to 50 +/- 1 degrees in the group treated with MP (p less than 0.05). Thereafter there was a slight improvement in both groups, but the difference persisted throughout the observation period. On Day 4 both gray and white matter SCBF was better preserved in MP-treated animals than in the control group (59 +/- 4 versus 49 +/- 3 ml/min/100 g tissue for gray matter and 13.6 +/- 0.6 versus 10.7 +/- 0.8 ml/min/100 g tissue for white matter). Posttraumatic treatment with MP, thus, improved both the neurologic recovery during the first 4 days and SCBF as measured on Day 4. It is speculated that the effect of MP is at least partly exerted on the vascular bed.     

Cerebellar norepinephrine depletion and impaired acquisition of specific locomotor tasks in rats

Previous work in our laboratory has shown that norepinephrine (NE)-depleted rats manifested impaired acquisition of a locomotor task as measured in a new rod runway paradigm. This paradigm involved the initial training of water-deprived rats on an equally spaced regular rod arrangement (REG), and subsequent testing, after intracisternal 6-hydroxydopamine (6-OHDA; 3 X 25 micrograms/microliter free base) infusion, on a more difficult irregular rod arrangement (IRR). These NE-depleted animals manifested impaired acquisition of the task as measured by running times (RT, 25 trials/day) over a 4 day post-infusion test period (IRR). In this present study, this same REG/IRR paradigm was employed in combination with a localized 6-OHDA lesion of the coeruleo-cerebellar pathway. A bilateral infusion of 6-OHDA (8 micrograms/2 microliters) induced cerebellar noradrenergic deafferentation (26% of controls) and produced a significant impairment of 4 day post-infusion RT. Thus, the coeruleo-cerebellar-lesioned rats demonstrated acquisitional impairment when tested on the new locomotor task (IRR). Moreover, the degree of impaired acquisitional, but not initial post-infusion motor performance, was found to correlate directly with the degree of cerebellar noradrenergic deafferentation. Furthermore, these rats showed no arousal, motivational or general cognitive learning deficits since no significant differences were observed in runway intertrial interval times, open field behavior, or in reversal of a T-maze position habit. Thus, cerebellar NE appears to be strongly associated with the adaptive ability to coordinate and choreograph the movements necessary to perform in this locomotor task.     

Separate involvement of the spinal noradrenergic and serotonergic systems in morphine analgesia: the differences in mechanical and thermal algesic tests

Experiments using 3 analgesic tests, the tail-pinch, hot-plate and tail-flick methods, were done to evaluate the roles of the spinal noradrenergic and serotonergic systems in the production of morphine analgesia in rats. To deplete noradrenaline or serotonin in the spinal cord, 6-hydroxydopamine or 5,6-dihydroxytryptamine was given intrathecally. 6-Hydroxydopamine suppressed the antinociceptive effects of morphine injected systemically or intracerebrally (into the nuclei reticularis gigantocellularis and paragigantocellularis or into the periaqueductal gray matter) in the tail-pinch test, but not significantly in the hot-plate and tail-flick tests. Conversely, 5,6-dihydroxytryptamine suppressed the antinociceptive effects of systemically given morphine in the hot-plate test, but not significantly in the tail-pinch and the tail-flick tests. The results not only provide further evidence for the involvement of the descending inhibitory systems in morphine antinociception, but also show that the extent of participation of the spinal noradrenergic and serotonergic systems in the effects of morphine has to be carefully assessed as different analgesic tests (tail-pinch, tail-flick and hot-plate) yield different results.     

Effect of a spinal cord photolesion injury on catalase

Ischemic injury to the spinal cord results in cell and tissue damage. Oxygen free radicals have been implicated in post-ischemic cell injury and death while free radical scavengers like superoxide dismutase and catalase are associated with an amelioration of ischemic injury. Measurement of catalase enzyme activity or protein in ischemic tissue presents mechanical problems due to extensive tissue destruction. Therefore, we looked at the effects of a photochemical lesion (which reproduces ischemic injury) on the levels of catalase mRNA in the spinal cord tissues of rodents under various experimental conditions. A significant depletion in the levels of catalase mRNA was observed in the spinal cord tissues of rats that received a severe lesion and were sacrificed 6 days post-lesion, while levels of catalase mRNA in the spinal cord tissues of similarly lesioned rats sacrificed 14 days post-lesion showed a return to control values.