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Reduction of NMDA-induced behaviour after acute and chronic administration of desipramine in mice
Neuropharmacology Vol. 32, No. 6, pp. 591-595, 1993 Printed in Great Britain. All rights reserved
Copyright
REDUCTION OF NMDA-INDUCED BEHAVIOUR ACUTE AND CHRONIC ADMINISTRATION DESIPRAMINE IN MICE
0
W28-3908/93 $6.00+ 0.00 1993 Pergamon Press Ltd
AFTER OF
N. MJELLEM, A. LUND and K. HOLE Department of Physiology, University of Bergen, Arstadveien 19, N-5009 Bergen, Norway (Accepted 14 December
1992)
Summary-The mechanisms of the antinociceptive effect of desipramine (DMI) are only partly known. It is generally accepted that excitatory amino acids act as neurotransmitters in primary nociceptive fibres and recent in vitro studies have shown an interaction between tricyclic antidepressants and the N-methyl-D-aspartic acid (NMDA) receptor complex. In this study, the modulatory effect of DMI on the biting and scratching behaviour induced by intrathecal (i.th.) administration of NMDA (0.25 nmol) was investigated. Desipramine was administered acutely, either intrathecally (0.7-35 pg) or intraperitoneally (i.p., 10 mg/kg), or chronically in the drinking water (0.15 g/l) for 3 weeks. The NMDA-induced behaviour
was significantly reduced both after acute and chronic administration of DMI. Several studies have shown a functional upregulation of the 5-HT,, receptor after chronic treatment with DMI. The activation of this receptor using the 5-HT,, agonist, %hydroxy-2-(di-n-propy1amino)tetralin hydrobromide (8-OH-DPAT), leads to a reduction in NMDA-induced behaviour. Using the 5-HT,, antagonist NAN-190 (lOpg, i.th.), the effect of chronic administration of DMI on the NMDA-induced behaviour was reversed. However, NAN-190 also increased NMDA-induced behaviour in the control group, suggesting that a tonic inhibition of this behaviour, mediated by the 5-HT,, receptor, may exist. These findings indicate that DMI may reduce glutaminergic transmission at the spinal NMDA receptor. As this receptor is central in spinal nociceptive transmission, this could be one mechanism for the antinociceptive effect of DMI. Key words-desipramine,
N-methyl-o-aspartate,
serotonin, nociception, spinal cord, mice.
Investigations of the antinociceptive effect of tricyclic antidepressants have given conflicting results (Ansuategui, Naharro and Feria, 1989; Fasmer, Post and Hole, 1987; France, 1987). Antinociception has been consistently reported after administration of desipramine (DMI); however, the mechanisms underlying this remain unclear (Lund, Tjolsen and Hole, 1989, 1990; Lund, Mjellem-Joly and Hole, 1992). Recent in vitro studies have demonstrated that tricyclic antidepressants may interact with the Nmethyl-D-aspartatic acid (NMDA) receptor complex (Rehavi and Schnitzer, 1991; Reynolds and Miller, 1988; Sernagor, Kuhn, Vyklicky and Mayer, 1989; White, Lovinger, Peoples and Weight, 1990). Tricyclic antidepressants have also been shown to protect mice from lethal intraperitoneal injections of NMDA (Leander, 1989). The excitatory amino acids are today considered to be neurotransmitters in primary afferent nociceptive neurones (Aanonsen and Wilcox, 1987, 1990; De Biassi and Rustioni, 1988; Dickenson and Sullivan, 1987; Gerber and Randic, 1989; Skilling, Smullin, Beitz and Larson, 1988). Therefore, whether the antinociceptive effect of DMI could be related to an ability to reduce glutaminergic transmission at the spinal NMDA receptor was investigated.
The first part of the present study examined whether acute or chronic administration of DMI modulated the biting and scratching behaviour, earlier demonstrated after intrathecal (ith.) administration of NMDA (Mjellem-Joly, Lund, Berge and Hole, 1991; Mjellem, Lund, Eide, Storkson and Tjolsen, 1993). A functional upregulation of the 5-hydroxytryptamine (S-HT,,) receptor was previously demonstrated after chronic administration of DMI (Lund et al., 1992). Several studies have indicated that 5-hydroxytryptamine modifies the action of amino acids on central neurones (Gardette, Krupa and Crepel, 1987; Hicks, Krupa and Crtpel, 1989; Marshall and Xiong, 1991) and the present authors’ investigations have shown that administration of the selective S-HT,, receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (&OH-DPAT) reduced the biting and scratching behaviour induced after intrathecal administration of NMDA (Mjellem et al., 1993). The second part of the study therefore investigated whether blocking the spinal 5-HTIA receptors would reverse the effect of DMI on the NMDAinduced behaviour. Also, the possible effect of the S-HT,, receptor blocker on NMDA-induced nociceptive behaviour was investigated.
591
N.
592
MJELLEM
METHODS
ef
al.
Male albino mice (Born: NMRI. Born-mice, Ry, Denmark), weighing 20-25 g at the start of experiments, were used. The animals were housed in colony cages (16 mice in each). They were maintained in climate- and light-controlled rooms (12/12 hr dark/ light cycle with lights on at 07.00 hr) for at least 1 week prior to the experiments. Food and tap water were freely available for the control animals and food and tap water with DMI (0.15 mg/ml), were freely available to the groups treated chronically with DMI. The temperature in the testing room was 21-22°C during all experiments.
animal was returned to the cage and the amount of time spent biting and scratching during the first minute was scored by an observer, unaware of the pretreatment of the animal. In the acute experiments, DMI was either administered intraperitoneally (10 mg/kg), 90 min prior to intrathecal injection of NMDA, or intrathecally (0.7-35 pg), 5 min prior to administration of NMDA. In the experiments with chronic administration of DMI, the animals were drinking DMI dissolved in the drinking water for 3 weeks prior to the intrathecal injection of NMDA. In the experiments with chronic administration of DMI, all the animals were decapitated the day after testing and blood was collected for determination of serum concentrations.
Drugs
Statistical analysis
N-methyl-D-aspartate (NMDA) was purchased from Sigma Chemical Co., U.S.A., desipramine HCl from Ciba-Geigy, I-(2-methoxyphenyl)-4-[4-(2phthalimido)bytyl]piperazine hydrobromide (NAN190) and 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (&OH-DPAT) from Research Biochemicals Inc., U.S.A. The NMDA, NAN-190 and 8-OH-DPAT were dissolved in 0.9% saline. For chronic administration of the antidepressant, DMI was dissolved in the drinking waster in a concentration of 0.15 mg/ml. This was the only source of drinking water in the treatment group, yielding a dose of desipramine of approximately 1 mg/kg/ 24 hr. There was no significant difference in the drinking volume between groups drinking tap water and the groups receiving DMI, dissolved in the drinking water.
Comparison between two means were done by Student’s t-test and statistical significance was accepted at the 5% level (P ~0.05). Results are presented as mean + SEM.
Animals
RESULTS
A significant decrease in NMDA-induced biting and scratching was found when animals were pretreated with DMI. There was a dose-dependent decrease in NMDA-induced behaviour when DMI (0.7, 7, 35 pg) was preadministered intrathecally (Fig. 1). A significant reduction of NMDA-induced behaviour was also found both after preadministration of DMI (i.p., lOmg/kg) (PcO.05, Student’s t-test), compared to controls [Fig. 2(A)] or when DMI was given in the drinking water (0.15 g/l) for
Injection procedure
The technique of intrathecal injection was performed as described by Hylden and Wilcox (1980). A lumbar puncture was performed, using a 30-gauge needle, connected to a microsyringe with polyethylene tubing. A cutaneous incision (1 cm) was performed 1 hr before the injection. The needle was inserted between LS and L6. Puncture of the dura was reliably indicated by a flick of the tail. In previous experiments, injections of methylene blue dye had confirmed that the distribution of dye was consistently intradural when this injection procedure was used. The substances were administered intrathecally in a volume of 7 ~1. Veh
Testing protocols
Two hours before testing, the animals were placed individually in standard cages (30 x 12 x 13 cm), which served as observation chambers. The intrathecal injection of NMDA (0.25 nmol) produced a dose-dependent behaviour of caudally-directed biting and scratching that lasted approximately 60 set after the injection. After the intrathecal injection, the
0.7
7
3.5
DMI dose pg Fig. 1. The effect of pretreatment with desipramine (DMI) (0.7, 7, 35pg) on the biting and scratching behaviour, caused by intrathecal injection of NMDA (0.25 nmol). DMI was injected in a volume of 7 ~1, 5 min prior to the injection of NMDA. The time spent biting and scratching during the first 6Osec after injection of NMDA was recorded. (mean f SEM; n = 8-11). **P
Effects of desipramine on NMDA-induced
behaviour
593
At the end of the chronic experiment, the mean body weight of control animals was 40 f 1.2 g and of DMI-treated animals, 40 f 1.0 g. None of the animals seemed distressed or impaired in any way. The dose of DMI administered was approximately 1 mg/kg/24 hr. The serum concentration of DMI after 3 weeks of administration in the drinking water, was 240 + 77.8 nmol/l.
Veh
DMI
Veh DMI
Fig. 2. The effect of pretreatment with DMI on the biting and scratching behaviour caused by intrathecal injection of NMDA (mean k SEM; n = 841). lP ~0.05 (Student’s ttest, compared to controls). (A) DMI (IOmg~g) injected intra~riton~ly (i.p.), 90 min prior to intrathecal injection of NMDA. (B) Chronic ad~~stmtion of DMI in the drinking water ‘(0.15 g/l) for 21 days, prior to intrathecal injection of NMDA.
3 weeks (P ~0.05, compared to animals drinking tap water) [Fig. 2(B)]. In animals having received DMI in the drinking water for 3 weeks, the intrathecal administration of the 5-HT,, antagonist, NAN-190 (10 pg), 5min prior to NMDA (ith.), reversed the DMIinduced reduction of NMDA-induced behaviour (P
A 0
VEH NAN-190
DMI
Control
Fig. 3.The effect of the S-HT,, antagonist NAN-190 (10 pg, ith.) on the reduction of NMDA-induced behaviour after chronic administration of DMI (mean f SEM; n = 8-10). *P
This study demonstrated that intrathecal administration of DMI induced a dose-dependant reduction in the biting and scratching behaviour, induced by intrathecal injection of NMDA. This effect was also found both after acute intraperitoneal and after chronic administration of DML These findings may reflect a direct interaction between DMI and the spinal NMDA receptor complex, as suggested by several in vitro studies, showing that tricyclic antidepressants bind to the NMDA receptor complex (Reynolds and Miller, 1988; Semagor, Kuhn, Vyklicky and Mayer, 1989; Sills and Loo, 1989). These studies have suggested that DMI binds either to the recognition site for Zn*+ (Reynolds and Miller, 1988) or to the phencyclidine binding site within the cation channel (Rehavi and Schnitzer, 1991; Reynolds and Rush, 1990; Sernagor et al., 1989; Sills and Loo, 1989). An inhibition of NMDAinduced lethalithy by DMI in mice (Leander, 1989) has also been reported. Contradicting these results is a report showing a potentiation of NMDAinduced depolarizations in a mouse cortical wedge preparation after DMI (Lancaster and Davies, 1991). Previous experiments have indicated an enhanced functional sensitivity of the 5-HT,, receptor after chronic administration of DMI (Lund er al., 1992), as well as an inhibitory action of the 5-HT,, agonist, 8-OH-DPAT on NMDA-induced behaviour (Mjellem et al., 1993). A possible mechanism of the reduction in NMDA-induced behaviour after DMI could then be through a functional enhancement of the spinal 5-HT,, receptors, as interactions between the serotonergic and glutaminergic transmitter systems have been suggested by several studies (Gardette, Krupa and Crepel, 1987; Hicks, Krupa and Crepel. 1989; Marshall and Xiong, 1991). Blocking the 5-HTrA receptor should then attenuate the observed effect on NMDA-induced behaviour. Indeed, when the S-HT,, receptor antagonist, NAN190 (Berendsen, Broekklamp and Van Delft, 1990; Rydelek-Fitzgerald, Teitler, Fletcher, Ismaiel and Glennon, 1990) was injected intrathecally, prior to NMDA, the effect of chronic administration of DMI on NMDA-induced behaviour was reversed. However, since pretreatment with NAN-190 also increased the biting and scratching behaviour after NMDA in the control animals, this precluded any definite concIusion as to whether blockade of 5-HT,, receptors may inhibit the effect of DML
N. MJELLEMetal.
594
In one group of mice, DMI was administered chronically in the drinking water (Lund, MjellemJoly and Hole, 1991; Lund et al., 1992), both to avoid the stress of repeated injections and to obtain a continuous treatment, not assured by injections once or twice daily (Eschalier, Fialip, Varoquaux, Makambila, Marty and Bastide, 1988; Fialip, Marty, Makambila, Civiale and Eschalier, 1988). The serum concentrations obtained by this dose and mode of administration seemed to be compatible to those reported in humans and in rats (Eschalier, 1990; Lund et al., 1991, 1992). The results presented here indicate that administration of DMI reduced glutaminergic transmission at the spinal NMDA receptor. This could represent an interaction between DMI and the spinal NMDA receptors, which would be in accordance with results from several in vitro receptor binding studies. As the activation of the NMDA receptor complex is central in nociceptive transmission in the spinal cord, this interaction may be another mechanism of the antinociceptive effect of DMI.
relevance in a study of the influence of clomipramine on morphine analgesia in mice. J. Pharmac. Exp. Ther. 248: 747-751.
France R. D. (1987) The future for antidepressants: Treatment of pain. Psychopathology 20: Suppl. 1, 99-113. Gardette R., Krupa M. and Crepe.1 F. (1987) Differential effects of serotonin on the spontaneous discharge and on the excitatory amino acid-induced responses of deep cerebellar nuclei neurons in rat cerebellar slices. Neuroscience 23: 49 l-500.
Gerber G. and Randic M. (1989) Excitatory amino acidmediated components of synaptically evoked input from dorsal roots to deep dorsal horn neurons in the rat spinal cord slice. Neurosn’. Left. 106: 211-219. Hicks T. P.. Kruua _ M. and Crtoel F. (19891 Selective effects of serotonin upon excitatory amino acid-induced depolarizations of Purkinje cells in cerebellar slices from young rats. Brain Res. 492: 371-376. Hylden J. L. K. and Wilcox G. L. (1989) Intrathecal morphine in mice: a new technique. Eur. J. Pharmac. 67: 313-316.
Lancaster J. M. methyhmipramine mouse cortical
and Davies J. A. (1991) Despotentiates NMDA responses in a slice preparation. Neuroreport 2:
66M8.
Leander J. D. (1989) Tricyclic antidepressants block N-methyl-o-aspartic acid-induced lethalithy in mice. Br. J. Pharmac. %: 256258.
Lund A, Mjeilem-Joly N. and Hole K. (1991) Chronic administration of desipramine and zimelidine changes the behavioural response in the formalin test in rats.
REFERENCES
Aanonsen L. M. and Wilcox G. L. (1987)Nociceptive action of excitatory amino acids in the mouse: effects of spinally administered opioids, phencyclidine and sigma agonists. J. Pharmac. Exp. Ther. 243: %19.
Aanonsen L. M., Lei S. and Wilcox G. L. (1990) Excitatory amino acid receptors and nociceptive neurotransmission in rat spinal cord. Pain 41: 309-321. Ansuategui M., Naharro L and Feria M. (1989) Noradrenergic and opioidergic influences on the antinociceptive effect of clomipramine in the formahn test of rats. Psychopharmacology 98: 93-96. Berendsen H. H. G., Broekklamp C. L. E. and Van Delft A. M. L. (1990) Antagonism of I-OH-DPATinduced behaviour in rats. Eur. J. Pharmac. 187: 97-103.
De Biasi S. and Rustioni A. (1988) Glutamate and substance P coexist in primary afferent terminals in the superficial laminae of spinal cord. Proc. Natn. Acad. Sci. U.S.A. 85: 7820-7824.
Dickenson A. H. and Sullivan A. F. (1987) Evidence for a role of the NMDA receptor in the frequency dependent potentiation of deep rat dorsal horn nociceptive neurones following C-fibre stimulation. Neuropharmacology 26: 1235-1238.
Eschaher A. (1990) Antidepressants and pain management. In: Serotonin and Pain (Besson J.-M.. Ed.)., .vn. 305-325. Excerpta Medica, Amsterdam. Eschalier A., Fiahp J., Varoquaux O., Makambila M.-C., Marty H. and Bastide P. (1988) Pharmacokinetic patterns of repeated administration of antidepressants in animals. I. Implications for antinociceptive action of clomipramine in mice. J. Pharmac. Exp. Ther. US: 963-968.
Fasmer 0. B., Post C. and Hole K. (1987) Changes nociception after acute and chronic administration zimelidine: Different effects in the formalin test and substance P behavioural assay. Neuropharmacology
in of the 26:
309-312.
Fialip J., Marty H., Makambila M.-C., Civiale M.-A. and Eschalier A. (1989) Pharmacokinetic patterns of repeated administration of antidepressants in animals. II Their
Neuropharmacology 301 481487.
Lund A., Mjellem-Joly N. and Hole K. (1992) Desipramine, administered chronically, influences 5-hydroxytryptarnine,,-receptors, as measured by behavioural tests and receptor binding in rats. Neuropharmacology 31: 25-32.
Lund A., Tjelsen A. and Hole K. (1989) Desipramine in small doses induces antinociception in the increasing temperature hot-plate test, but not in the tail-flick test. Neuropharmacology 28: 1169-l 173. Lund A., Tjelsen A. and Hole K. (1990) Antinociceptive effect of intrathecally administered desipramine and zimelidine in rats. Neuropharmacology 29: 8 19-823. Marshall K. C. and Xiong H. (1991) Modulation of amino acid neurotransmitter actions by other neurotransmitters: some examples. Can. J. Physiol. Pharmac. 69: 1115-1122.
Mjellem-Joly N., Lund A., Berge O.-G. and Hole K. (1991) Potentiation of a behavioural response in mice by spinal coadministration of substance P and excitatory amino acid agonists. Neurosci. Letf. 133: 121-124.
Mjellem N., Lund A., Eide P. K., Stsrkson R. and Tjelsen A. (1993) The role of spinal 5-HT,, and 5-HT,, receptors in nociceptive transmission and in the modulation of N-methyl-o-aspartate induced behaviour. Neuroreport. In press. Rehavi M. and Schnitzer E. (1991) Long term desipramine or chlorpromazine treatment and rat brain N-methylo-aspartate receptor. J. Bas. Clin. Phys. Pharm. 2: 111-121.
Reynolds I. J. and Miller R. J. (1988) Tricyclic antidepressants block N-methyl-o-aspartate receptors: similarities to the action of zinc. J. Pharmac. 95: 95-102. Reynolds I. J. and Rush E. A. (1990) Role of lipid solubility in the interaction of drugs with the N-methyl-o-aspartate receptor. Synapse 5: 71-76. Rydelek-Fitzgerald L., Teitler M., Fletcher P. W., Ismaiei A. M. and Glennon R. A. (1990) NAN-190: agonist and antagonist interactions with brain 5-HT,, receptors. Brain Res. 532: 191-196.
Effects of desipramine on NMDA-induced
behaviour
595
Semagor E., Kuhn D., Vyklicky L. and Mayer M. L. (1989) Open channel block of NMDA 51:
127-132. White G., Lovinger D. M., Peoples R. W. and Weight F. F. (1990) Inhibition of N-methyl-D-aspartate activated ion current by desmethylimipramine. Brain Res. 537: 337-339.
Copyright
REDUCTION OF NMDA-INDUCED BEHAVIOUR ACUTE AND CHRONIC ADMINISTRATION DESIPRAMINE IN MICE
0
W28-3908/93 $6.00+ 0.00 1993 Pergamon Press Ltd
AFTER OF
N. MJELLEM, A. LUND and K. HOLE Department of Physiology, University of Bergen, Arstadveien 19, N-5009 Bergen, Norway (Accepted 14 December
1992)
Summary-The mechanisms of the antinociceptive effect of desipramine (DMI) are only partly known. It is generally accepted that excitatory amino acids act as neurotransmitters in primary nociceptive fibres and recent in vitro studies have shown an interaction between tricyclic antidepressants and the N-methyl-D-aspartic acid (NMDA) receptor complex. In this study, the modulatory effect of DMI on the biting and scratching behaviour induced by intrathecal (i.th.) administration of NMDA (0.25 nmol) was investigated. Desipramine was administered acutely, either intrathecally (0.7-35 pg) or intraperitoneally (i.p., 10 mg/kg), or chronically in the drinking water (0.15 g/l) for 3 weeks. The NMDA-induced behaviour
was significantly reduced both after acute and chronic administration of DMI. Several studies have shown a functional upregulation of the 5-HT,, receptor after chronic treatment with DMI. The activation of this receptor using the 5-HT,, agonist, %hydroxy-2-(di-n-propy1amino)tetralin hydrobromide (8-OH-DPAT), leads to a reduction in NMDA-induced behaviour. Using the 5-HT,, antagonist NAN-190 (lOpg, i.th.), the effect of chronic administration of DMI on the NMDA-induced behaviour was reversed. However, NAN-190 also increased NMDA-induced behaviour in the control group, suggesting that a tonic inhibition of this behaviour, mediated by the 5-HT,, receptor, may exist. These findings indicate that DMI may reduce glutaminergic transmission at the spinal NMDA receptor. As this receptor is central in spinal nociceptive transmission, this could be one mechanism for the antinociceptive effect of DMI. Key words-desipramine,
N-methyl-o-aspartate,
serotonin, nociception, spinal cord, mice.
Investigations of the antinociceptive effect of tricyclic antidepressants have given conflicting results (Ansuategui, Naharro and Feria, 1989; Fasmer, Post and Hole, 1987; France, 1987). Antinociception has been consistently reported after administration of desipramine (DMI); however, the mechanisms underlying this remain unclear (Lund, Tjolsen and Hole, 1989, 1990; Lund, Mjellem-Joly and Hole, 1992). Recent in vitro studies have demonstrated that tricyclic antidepressants may interact with the Nmethyl-D-aspartatic acid (NMDA) receptor complex (Rehavi and Schnitzer, 1991; Reynolds and Miller, 1988; Sernagor, Kuhn, Vyklicky and Mayer, 1989; White, Lovinger, Peoples and Weight, 1990). Tricyclic antidepressants have also been shown to protect mice from lethal intraperitoneal injections of NMDA (Leander, 1989). The excitatory amino acids are today considered to be neurotransmitters in primary afferent nociceptive neurones (Aanonsen and Wilcox, 1987, 1990; De Biassi and Rustioni, 1988; Dickenson and Sullivan, 1987; Gerber and Randic, 1989; Skilling, Smullin, Beitz and Larson, 1988). Therefore, whether the antinociceptive effect of DMI could be related to an ability to reduce glutaminergic transmission at the spinal NMDA receptor was investigated.
The first part of the present study examined whether acute or chronic administration of DMI modulated the biting and scratching behaviour, earlier demonstrated after intrathecal (ith.) administration of NMDA (Mjellem-Joly, Lund, Berge and Hole, 1991; Mjellem, Lund, Eide, Storkson and Tjolsen, 1993). A functional upregulation of the 5-hydroxytryptamine (S-HT,,) receptor was previously demonstrated after chronic administration of DMI (Lund et al., 1992). Several studies have indicated that 5-hydroxytryptamine modifies the action of amino acids on central neurones (Gardette, Krupa and Crepel, 1987; Hicks, Krupa and Crtpel, 1989; Marshall and Xiong, 1991) and the present authors’ investigations have shown that administration of the selective S-HT,, receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (&OH-DPAT) reduced the biting and scratching behaviour induced after intrathecal administration of NMDA (Mjellem et al., 1993). The second part of the study therefore investigated whether blocking the spinal 5-HTIA receptors would reverse the effect of DMI on the NMDAinduced behaviour. Also, the possible effect of the S-HT,, receptor blocker on NMDA-induced nociceptive behaviour was investigated.
591
N.
592
MJELLEM
METHODS
ef
al.
Male albino mice (Born: NMRI. Born-mice, Ry, Denmark), weighing 20-25 g at the start of experiments, were used. The animals were housed in colony cages (16 mice in each). They were maintained in climate- and light-controlled rooms (12/12 hr dark/ light cycle with lights on at 07.00 hr) for at least 1 week prior to the experiments. Food and tap water were freely available for the control animals and food and tap water with DMI (0.15 mg/ml), were freely available to the groups treated chronically with DMI. The temperature in the testing room was 21-22°C during all experiments.
animal was returned to the cage and the amount of time spent biting and scratching during the first minute was scored by an observer, unaware of the pretreatment of the animal. In the acute experiments, DMI was either administered intraperitoneally (10 mg/kg), 90 min prior to intrathecal injection of NMDA, or intrathecally (0.7-35 pg), 5 min prior to administration of NMDA. In the experiments with chronic administration of DMI, the animals were drinking DMI dissolved in the drinking water for 3 weeks prior to the intrathecal injection of NMDA. In the experiments with chronic administration of DMI, all the animals were decapitated the day after testing and blood was collected for determination of serum concentrations.
Drugs
Statistical analysis
N-methyl-D-aspartate (NMDA) was purchased from Sigma Chemical Co., U.S.A., desipramine HCl from Ciba-Geigy, I-(2-methoxyphenyl)-4-[4-(2phthalimido)bytyl]piperazine hydrobromide (NAN190) and 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (&OH-DPAT) from Research Biochemicals Inc., U.S.A. The NMDA, NAN-190 and 8-OH-DPAT were dissolved in 0.9% saline. For chronic administration of the antidepressant, DMI was dissolved in the drinking waster in a concentration of 0.15 mg/ml. This was the only source of drinking water in the treatment group, yielding a dose of desipramine of approximately 1 mg/kg/ 24 hr. There was no significant difference in the drinking volume between groups drinking tap water and the groups receiving DMI, dissolved in the drinking water.
Comparison between two means were done by Student’s t-test and statistical significance was accepted at the 5% level (P ~0.05). Results are presented as mean + SEM.
Animals
RESULTS
A significant decrease in NMDA-induced biting and scratching was found when animals were pretreated with DMI. There was a dose-dependent decrease in NMDA-induced behaviour when DMI (0.7, 7, 35 pg) was preadministered intrathecally (Fig. 1). A significant reduction of NMDA-induced behaviour was also found both after preadministration of DMI (i.p., lOmg/kg) (PcO.05, Student’s t-test), compared to controls [Fig. 2(A)] or when DMI was given in the drinking water (0.15 g/l) for
Injection procedure
The technique of intrathecal injection was performed as described by Hylden and Wilcox (1980). A lumbar puncture was performed, using a 30-gauge needle, connected to a microsyringe with polyethylene tubing. A cutaneous incision (1 cm) was performed 1 hr before the injection. The needle was inserted between LS and L6. Puncture of the dura was reliably indicated by a flick of the tail. In previous experiments, injections of methylene blue dye had confirmed that the distribution of dye was consistently intradural when this injection procedure was used. The substances were administered intrathecally in a volume of 7 ~1. Veh
Testing protocols
Two hours before testing, the animals were placed individually in standard cages (30 x 12 x 13 cm), which served as observation chambers. The intrathecal injection of NMDA (0.25 nmol) produced a dose-dependent behaviour of caudally-directed biting and scratching that lasted approximately 60 set after the injection. After the intrathecal injection, the
0.7
7
3.5
DMI dose pg Fig. 1. The effect of pretreatment with desipramine (DMI) (0.7, 7, 35pg) on the biting and scratching behaviour, caused by intrathecal injection of NMDA (0.25 nmol). DMI was injected in a volume of 7 ~1, 5 min prior to the injection of NMDA. The time spent biting and scratching during the first 6Osec after injection of NMDA was recorded. (mean f SEM; n = 8-11). **P
Effects of desipramine on NMDA-induced
behaviour
593
At the end of the chronic experiment, the mean body weight of control animals was 40 f 1.2 g and of DMI-treated animals, 40 f 1.0 g. None of the animals seemed distressed or impaired in any way. The dose of DMI administered was approximately 1 mg/kg/24 hr. The serum concentration of DMI after 3 weeks of administration in the drinking water, was 240 + 77.8 nmol/l.
Veh
DMI
Veh DMI
Fig. 2. The effect of pretreatment with DMI on the biting and scratching behaviour caused by intrathecal injection of NMDA (mean k SEM; n = 841). lP ~0.05 (Student’s ttest, compared to controls). (A) DMI (IOmg~g) injected intra~riton~ly (i.p.), 90 min prior to intrathecal injection of NMDA. (B) Chronic ad~~stmtion of DMI in the drinking water ‘(0.15 g/l) for 21 days, prior to intrathecal injection of NMDA.
3 weeks (P ~0.05, compared to animals drinking tap water) [Fig. 2(B)]. In animals having received DMI in the drinking water for 3 weeks, the intrathecal administration of the 5-HT,, antagonist, NAN-190 (10 pg), 5min prior to NMDA (ith.), reversed the DMIinduced reduction of NMDA-induced behaviour (P
A 0
VEH NAN-190
DMI
Control
Fig. 3.The effect of the S-HT,, antagonist NAN-190 (10 pg, ith.) on the reduction of NMDA-induced behaviour after chronic administration of DMI (mean f SEM; n = 8-10). *P
This study demonstrated that intrathecal administration of DMI induced a dose-dependant reduction in the biting and scratching behaviour, induced by intrathecal injection of NMDA. This effect was also found both after acute intraperitoneal and after chronic administration of DML These findings may reflect a direct interaction between DMI and the spinal NMDA receptor complex, as suggested by several in vitro studies, showing that tricyclic antidepressants bind to the NMDA receptor complex (Reynolds and Miller, 1988; Semagor, Kuhn, Vyklicky and Mayer, 1989; Sills and Loo, 1989). These studies have suggested that DMI binds either to the recognition site for Zn*+ (Reynolds and Miller, 1988) or to the phencyclidine binding site within the cation channel (Rehavi and Schnitzer, 1991; Reynolds and Rush, 1990; Sernagor et al., 1989; Sills and Loo, 1989). An inhibition of NMDAinduced lethalithy by DMI in mice (Leander, 1989) has also been reported. Contradicting these results is a report showing a potentiation of NMDAinduced depolarizations in a mouse cortical wedge preparation after DMI (Lancaster and Davies, 1991). Previous experiments have indicated an enhanced functional sensitivity of the 5-HT,, receptor after chronic administration of DMI (Lund er al., 1992), as well as an inhibitory action of the 5-HT,, agonist, 8-OH-DPAT on NMDA-induced behaviour (Mjellem et al., 1993). A possible mechanism of the reduction in NMDA-induced behaviour after DMI could then be through a functional enhancement of the spinal 5-HT,, receptors, as interactions between the serotonergic and glutaminergic transmitter systems have been suggested by several studies (Gardette, Krupa and Crepel, 1987; Hicks, Krupa and Crepel. 1989; Marshall and Xiong, 1991). Blocking the 5-HTrA receptor should then attenuate the observed effect on NMDA-induced behaviour. Indeed, when the S-HT,, receptor antagonist, NAN190 (Berendsen, Broekklamp and Van Delft, 1990; Rydelek-Fitzgerald, Teitler, Fletcher, Ismaiel and Glennon, 1990) was injected intrathecally, prior to NMDA, the effect of chronic administration of DMI on NMDA-induced behaviour was reversed. However, since pretreatment with NAN-190 also increased the biting and scratching behaviour after NMDA in the control animals, this precluded any definite concIusion as to whether blockade of 5-HT,, receptors may inhibit the effect of DML
N. MJELLEMetal.
594
In one group of mice, DMI was administered chronically in the drinking water (Lund, MjellemJoly and Hole, 1991; Lund et al., 1992), both to avoid the stress of repeated injections and to obtain a continuous treatment, not assured by injections once or twice daily (Eschalier, Fialip, Varoquaux, Makambila, Marty and Bastide, 1988; Fialip, Marty, Makambila, Civiale and Eschalier, 1988). The serum concentrations obtained by this dose and mode of administration seemed to be compatible to those reported in humans and in rats (Eschalier, 1990; Lund et al., 1991, 1992). The results presented here indicate that administration of DMI reduced glutaminergic transmission at the spinal NMDA receptor. This could represent an interaction between DMI and the spinal NMDA receptors, which would be in accordance with results from several in vitro receptor binding studies. As the activation of the NMDA receptor complex is central in nociceptive transmission in the spinal cord, this interaction may be another mechanism of the antinociceptive effect of DMI.
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