Neurochemical Research, Vol. 28, No. 9, September 2003 (© 2003), pp. 1369–1373
Alterations in Brain Metabolism Induced by Chronic Morphine Treatment: NMR Studies in Rat CNS Sushil K. Sharma,3,4,* Kiran Yashpal,1,† Marian E. Fundytus,3,4 Françoise Sauriol,5,‡ James L. Henry,3,4 and Terence J. Coderre1,2,6 (Accepted March 25, 2003)
High-resolution (500 MHz) multiresonance/multinuclear proton (1H) nuclear magnetic resonance (NMR) spectroscopy was used to detect metabolic changes and cellular injury in the rat brain stem and spinal cord following chronic morphine treatment. Compensatory changes were observed in glycine, glutamate, and inositols in the brain stem, but not the spinal cord, of chronic morphinetreated rats. In spinal cord, increases were detected in lactate and N-acetyl-aspartate (NAA), suggesting that there is anaerobic glycolysis, plasma membrane damage, and altered pH preferentially in the spinal cord of chronic morphine-treated rats.
KEY WORDS: Opioids; nuclear magnetic resonance; spectroscopy; withdrawal; inositol.
channels (1) and increases inwardly rectifying potassium (K⫹) currents (2). Opioid receptors are also negatively coupled to adenylate cyclase and inhibit the production of cyclic adenosine monophosphate (c-AMP) (3). There is also evidence that opioid receptors are coupled to phosphatidylinositol (PI) hydrolysis (4), influence the production of inositol-1,4,5-trisphosphate (IP3) and protein kinase C (PKC), and enhance intracellular Ca2⫹ release (5) through a PI-dependent mechanism. Although the acute actions of opioids on various signal transduction mechanisms are well described, the effects of chronic opioid administration, which lead to the development of tolerance and dependence, are less well understood. Proposed mechanisms of opioid tolerance and dependence include: receptor downregulation (6), an uncoupling of receptors from G-proteins (7), and compensatory changes in membrane conductances or signal transduction pathways (8). The evidence for the former two proposals has been equivocal, thus the latter proposal has received much recent attention. Evidence suggests that there are compensatory changes in adenylate cyclase activity associated with chronic opioid treatment. Thus, although acute morphine treatment results in reductions in adenylate cyclase activity, these levels
INTRODUCTION Considerable evidence indicates that opioids produce multiple influences on signal transduction mechanisms in the central nervous system (CNS). Acute activation of opioid receptors inhibits voltage-gated calcium (Ca2⫹) 1
Department of Anesthesia, McGill University, Montreal, Quebec, Canada. 2 Departments of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada. 3 Department of Physiology, McGill University, Montreal, Quebec, Canada. 4 Department of Psychiatry, McGill University, Montreal, Quebec, Canada. 5 Department of Chemistry, McGill University, Montreal, Quebec, Canada. 6 Address reprint requests to: Terence J. Coderre, McGill University, McIntyre Medical Sciences Bldg., Room 1203, 3655 Dummond Street, Montreal, Quebec, Canada. H3G 1Y6. Tel: (514) 398-5773; Fax: (514) 398-8241; E-mail:
[email protected] *SKS’s current address: Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, University of Manitoba, Faculty of Medicine, 351 Taché Ave., Winnipeg, Manitoba, Canada, R2H 2A6. † KY and JLH’s current address: Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, N6A 5C1. ‡ FS’s current address: Department of Chemistry, Queen’s University, 90 Queen’s Crescent, Kingston, Ontario, Canada K7L 3N6.
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increase after chronic exposure, and exhibit greater increases (or an overshoot) after precipitation of morphine withdrawal (3,8). Although opioids have been found to influence PI hydrolysis (4), there is little evidence for compensatory changes in this system with chronic opioid administration. Furthermore, since chronic opioid treatment is known to enhance Ca2⫹ influx (5), the long-term consequences of increased intracellular Ca2⫹ may have serious implications for normal cell function and possibly produce metabolic changes or cellular injury. In the current study, we use high-resolution (500 MHz) multiresonance/multinuclear proton (1H) nuclear magnetic resonance (NMR) spectroscopy to assess the effects of morphine exposure on brain and spinal cord metabolism, neurotransmitters, and on various indicators of cellular injury. In particular, we wish to compare the effects of acute and chronic morphine exposure, as well as the precipitation of morphine withdrawal after chronic morphine treatment, to determine whether there are changes in brain or spinal cord metabolism that reflect compensatory processes that are initiated to mitigate cellular injury.
EXPERIMENTAL PROCEDURE Proton (1H) nuclear magnetic resonance (NMR) spectroscopy was used to assess changes in signal transduction pathways in rat CNS in response to chronic opioid treatment. (1H) NMR spectroscopy was used in order to determine the effects of morphine administration on neurotransmitters and their metabolites, signal transduction molecules, and indicators of metabolic disturbances or cellular injury. Assessments were made in tissues taken from rat CNS following in vivo administration of the prototypic opiate—morphine—either acutely, chronically, or chronically followed by acute administration of naloxone to precipitate withdrawal, and were compared with assessments in untreated control rats. We assessed both the specific spinal and supraspinal influences of acute and chronic morphine administration. To study supraspinal effects, the locus coeruleus (LC) and periaqueductal gray (PAG) regions were selected not only because of the critical roles they play in opiate analgesia and the development of opiate tolerance and dependence (9), but also because these areas are rich in opioid receptors (10). For similar reasons, the dorsal horn of the spinal cord was used to determine the spinal effects of morphine (11, 12). The experiments were performed with male Long Evans hooded rats (250–350 g) from Charles River, Quebec, under protocols approved by our Institutional Animal Care Committees. Four groups of five rats each were prepared as follows: (1) controls—untreated, (2) acute morphine treatment—a single 8 mg/kg SC dose of morphine, (3) chronic morphine treatment—SC infusion of 28.8 mg/day of morphine by Alzet® pump for 7 days (60 mg/ml morphine sulphate solution infused at a rate of 20 l/h), (4) precipitated withdrawal treatment—chronic morphine-treated rats were given an acute injection of naloxone (1 mg/kg, SC) on the 7th day of SC morphine infusion. Rats were sacrificed by decapitation 1 h after acute morphine treatment, on the 7th day of chronic morphine treatment,
or 10 min after naloxone injection for rats in the precipitated withdrawal group. Following treatment, each rat was decapitated and the dorsal horn of the spinal cord was isolated after removal of the spinal cord by pressure ejection with ice-cold saline. At the same time, the brain was removed from the skull, which was cooled on ice. Both spinal cord dorsal horn and the brain were snap frozen within 1 min of decapitation by immersion in isopentane cooled with dry ice. Subsequently, the locus coeruleus (LC) and periaqueductal gray (PAG) were isolated using stereotaxic coordinates from the atlas of Paxinos and Watson (13). For (1H) NMR spectra, tissues from the LC and PAG together, and the spinal cord dorsal horn, of 5 rats from each treatment group were processed. Two hundred fifty–milligram tissue samples were sonicated in 0.5 ml of 0.3 M perchloric acid buffer (pH 6.5) and centrifuged (11,200 rpm) for 20 min at 4°C. The pellets were further reconstituted in 0.5 ml of 0.3 M perchloric acid buffer to extract the remaining metabolites in the tissue. Pooled supernatant was treated with (1H) NMR buffer (1.5 M KOH, 0.3 M KCl, 0.1 M Na2PO4, pH 7.0) to precipitate potassium perchlorate, which was removed by further centrifugation at 11,200 rpm at 4°C. The pellet was used to estimate protein concentration using Bradford’s (14) method. The supernatant was lyophilized for 48 h and reconstituted in 1 ml of deuterium oxide containing 1 mM of internal standard trisilylproprionate (TSP) (Aldrich Techware, Milwaukee, MN). Chelex 100 (200–400 mesh, 4% solution) (BioRad, Hercules, CA) was used as an ion exchange resin for 48 h at 4°C to remove ions from the samples. The pD of the samples was adjusted to 7.5 using deuterated hydrochloric acid (DCl) or deuterated sodium hydroxide (NaOD). The samples were filtered through 0.2 m Gelman filters and placed in 5-mm-diameter NMR tubes (Sigma, St. Louis, MO). (1H) NMR spectra of all treatment groups were measured using a Varian (Unity NMR) 500 MH spectrometer with 128 transients of 5.3 each. After signal averaging, a fast Fourier transformation was performed to obtain the spectral peaks. Saturation factors were determined to analyze the concentration of substances in the aliphatic region of the proton spectra as described by Luyten et al. (15). Samples were spiked using Sigma Chemical compounds to estimate resonance assignments of morphine, its metabolite morphine 3-glucoronide, and naloxone. The spectral frequencies of these compounds did not overlap with those identified in the proton spectra of morphineor morphine ⫹ naloxone–treated rats. Reproducibility of the spectral analysis is indicated by the consistent appearance of spectral peaks across the four treatment groups for both brain stem and spinal cord samples, and alterations in peak heights were confirmed by spectral analysis in duplicate samples. For spectral simplification and cross correlation of the various spectral peaks, two-dimensional coherence spectroscopy (COSY) and stacked representations were also constructed for LC ⫹ PAG (data not shown); these additional measures fully supported findings of the one-dimensional (1H) NMR spectroscopic analysis.
RESULTS The most obvious changes between the various treatment groups in the (1H) NMR spectra for the LC ⫹ PAG regions were obtained in the resonances for glycine, which acts as an inhibitory transmitter, and inositols, intracellular metabolites linked to various metabotropic receptors (Fig. 1A). Specifically, in acute
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Fig. 1. A, (1H) NMR spectra of excised rat brain stem (LC ⫹ PAG) for untreated control rats and rats treated either acutely or chronically with morphine, or for chronic morphine-treated rats given acute treatment with naloxone. B, (1H)NMR spectra of excised rat spinal cord dorsal horn for untreated control rats and rats treated either acutely or chronically with morphine, or for chronic morphine-treated rats given acute treatment with naloxone. GABA, gamma-aminobutyric acid; GPC, glycophosphocholine; NAA, N-acetylaspartate; PC, phosphocholine; Pcr, phosphocreatine.
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morphine-treated rats there was a dramatic decrease in glycine and inositols compared to untreated controls. Furthermore, in chronic morphine-treated rats, glycine and inositol levels were increased slightly, relative to the acute morphine treatment, suggesting that a compensatory mechanism may be triggered by chronic treatment. A large increase in glycine and inositols over control levels, and particularly over the acute treatment levels, in the precipitated-withdrawal group suggests there is an overcompensation in these substances associated with morphine withdrawal. Glutamate and glutamine levels varied in the same pattern as did glycine and inositols, although the spectral peaks were initially lower and the changes less dramatic. In contrast, the pattern of changes of glycine, inositols, and glutamate were very different in the dorsal horn of the spinal cord (Fig. 1B). Glycine and inositols were relatively unchanged in acute and chronic morphinetreated rats, although they were increased in the precipitated withdrawal group. Glutamate and glutamine resonances, however, did not differ between any of the groups. Thus, unlike the LC ⫹ PAG regions, there was not the same clear pattern of compensatory changes in the signal transduction molecules or glutamate in the dorsal horn of the spinal cord. Further NMR measures indicated that in the LC and PAG regions there was no change between the untreated and morphine-treated groups in the spectral resonances for N-acetylaspartate (NAA, an intracellular marker of cell injury) (16) and only a slight decrease in lactate (an indicator of anaerobic glycolysis) (17). These findings suggest that the morphine treatment did not produce measurable metabolic changes or cellular injury (Fig. 1A). In contrast, in the dorsal horn NAA and lactate (at 1.3 ppm) levels were increased in acute and chronic morphine-treated rats, but were reduced relative to chronic morphine-treated rats in the precipitated withdrawal group (Fig. 1B). This suggests that unlike in the LC ⫹ PAG regions, in the spinal cord dorsal horn region there are signs of metabolic disturbances and cellular injury associated with morphine administration.
study, however, provides the first direct evidence for compensatory changes in the levels of glutamate and glutamate-linked metabolites (inositols) in brain stem with chronic opioid treatment. Using (1H) NMR spectroscopy we have shown changes in these molecules reflecting a decrease with acute morphine treatment, a compensatory increase with chronic morphine treatment, and a dramatic increase, or overcompensation during naloxone-precipitated withdrawal. The increase in inositols could be derived from inositols phospholipids (related to metabotropic and iontropic glutamate receptor activation, as well as other G protein–coupled receptors) or from myoinositol (found mostly in glia, and may suggest gliosis [20]). However, the parallel compensatory changes observed with glutamate and inositols after chronic morphine treatment suggest an interaction between these two molecules may contribute to morphine dependence and withdrawal. Importantly, these compensatory changes appear to occur only in the brain regions studied (LC ⫹ PAG), but not in the spinal cord. There also were differences in indicators of metabolic changes and cellular injury that suggest major differences between the effects of chronic morphine on brain stem vs. spinal cord neurons. Specifically, in the dorsal horn of the spinal cord, chronic morphine treatment was associated with increased indicators of glycolysis and altered pH (increased lactate) as well as cellular damage (increased NAA). In the LC ⫹ PAG regions there were no changes in lactate or NAA after chronic morphine treatment; thus it is proposed that brain-sparing mechanisms, associated with compensatory changes in glutamate-linked metabolites, may play a protective role in neurons within these regions.
DISCUSSION
REFERENCES
The development of morphine tolerance or dependence has been linked conceptually to glutamate and glutamate-linked metabolites, because NMDA receptor and metabotropic glutamate receptor antagonists (18), as well as inhibitors of intracellular Ca2⫹ release and protein kinases (19), inhibit the development of morphine tolerance and/or dependence. The present
ACKNOWLEDGMENTS This work was supported by Canadian Institutes of Health Research (CIHR) grants to TJC and JLH, and an NIH grant to JLH. TJC is a CIHR Investigator.
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