Neuroscience Vol. 51, No. 2, pp. 347-355, 1992 Printed in Great Britain
0306-4522/92 $5.00 + 0.00 Pergamon Press Ltd IBRO
KAPPA-/MU-RECEPTOR INTERACTIONS IN THE OPIOID CONTROL OF THE IN VIVO RELEASE OF SUBSTANCE P-LIKE MATERIAL FROM THE RAT SPINAL CORD E. COLLIN,*A. MAUBORGNE,S. BOURGOIN, S. MANTELET,L. FERHAT, M. HAMON and F. CESSELIN INSERM U.288, Neurobiologie Cellulaire et Fonctionnelle, Facult6 de M6decine Piti6-Salp&ri6re, 91, boulevard de l'H6pital, 75634 Paris cedex 13, France Abstract--The possible involvement of # and x receptors in the opioid control of the spinal release of substance P-like material was assessed in vivo, in halothane-anaesthetized rats whose intrathecal space was continuously perfused with an artificial cerebrospinal fluid supplemented with various opioid receptor agonists and antagonists. Whereas the intrathecal perfusion with the # agonist DAGO (10 #M) significantly enhanced ( ~ + 50%) the spontaneous release of substance P-like material, that with the x agonist U 50488 H (10 #M) produced no change in the peptide outflow. The respective antagonists naloxone (10pM) for the p receptors and nor-binaltorphimine (10#M) for the x receptors did not affect the spontaneous release of substance P-like material, indicating that endogenous opioids acting at # and x receptors do not exert a tonic control on substance P-containing neurons in the spinal cord of halothane-anaesthetized rats. However, as expected from the involvement of p receptors, the stimulatory effect of DAGO on the peptide outflow could be prevented by naloxone but not norbinaltorphimine. Furthermore, instead of an increase with DAGO alone, a significant decrease in the spinal release of substance P-like material was observed upon the intrathecal perfusion with DAGO plus U 50488 H. Additional experiments with the respective # and x antagonists naloxone and nor-binaltorphimine demonstrated that this effect actually resulted from the simultaneous stimulation of # and x receptors. As previous studies showed that 6 agonists also reduce the release of substance P from primary afferent fibres, the present data could explain why non-selective opioid agonists such as morphine acting at 6, # and x receptors are especially potent to reduce the peptide release through additive inhibitory influences mediated by the stimulation of 6 receptors on the one hand and that of # plus x receptors on the other hand. Such additive inhibitory influences on substance P-containing fibres probably contribute to the potent analgesic action of non-specific opioids administered via the intrathecal route.
Substance P (SP) is one of the neuropeptides present in primary afferent fibres which is thought to play a role in the transmission of nociceptive signals from the periphery to the spinal cord. 15 Thus, a variety of stimuli which can activate small diameter primary afferent fibres such as noxious stimuli, 13,14,21,28,35,45high intensity stimulation of a peripheral nerve 19,5° and local application of capsaicin 1,17,37 can trigger the release of SP at the spinal level. Several lines of evidence indicate that SP-containing primary afferent fibres projecting within the dorsal horn of the spinal cord can be controlled by various neuroactive compounds. In particular, the analgesic action of opioids injected via the intrathecal route has been attributed, at least partly, to the capacity of these drugs to inhibit presynaptically the SP-containing primary afferent
fibres. 49 Indeed, autoradiographic studies have shown that opioid receptors are very probably located on these fibres. 3'11'29Furthermore, opioid receptor stimulation produces a marked reduction in the release of SP from the trigeminal nucleus 25 and the spinal cord.1,6,19,21,31,50
*To whom correspondence should be addressed: Abbreviations: ACSF, artificial cerebrospinal fluid; DAGO, TyrD-Ala-Gly-MePhe-Gly-ol; DPDPE; D-Pen 2, D-Pen 5enkephalin; DTLET, Tyr-D-Thr-Gly-Phe-Leu-Thr; PL017, MePhe 3, D-Pro4-morphiceptin; SP, substance P; SP-LM, substance P-like material; U 50488 H, 3,4-dichloro-Nmethyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzacetamide methansulphonate hydrate, trans-( +__). 347
A m o n g opioids, those stimulating selectively 6 receptors are especially potent to control SP-containing primary afferent fibres. Indeed, the 6 opioid agonists Tyr-D-Thr-Gly-Phe-Leu-Thr ( D T L E T ) s2 and D-Pen 2 (D-PenS-enkephalin; D P D P E ) 36 inhibit both the spontaneous l° and the evoked1'19'34'37release of SP from the rat spinal cord in vitro as well as in vivo. Furthermore, the selective blockade of 6 receptors by naltrindole 38 induces an enhancement of the spinal release of SP in the rat in vivo, suggesting that endogenous opioids acting at these receptors exert a tonic inhibitory control on SP-containing fibres.l° In contrast, the possible involvement of other opioid receptor subtypes in the control of SPcontaining primary afferent fibres is still a matter of debate. In the case of x receptors, most in vitro and in vivo investigations showed that their selective
348
E. COLLIN et al.
s t i m u l a t i o n by 3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzacetamide methan sulp h o n a t e hydrate, trans-( +_ ) ( U 50488 H ) 47 o r dynorp h i n did n o t exert any effect on the spinal release of S P , 1'19'21'34'37 However, C h a n g et al. 6 r e p o r t e d t h a t U 50488 H inhibits SP release from p r i m a r y sensory neurons. Similarly, contradictory data h a v e been reported regarding the m o d u l a t i o n o f spinal SP release by p opioid receptors. In vitro studies have s h o w n t h a t the selective /~ agonists T y r - D - A l a - G l y M e P h e - G l y - o l ( D A G O ) TM a n d M e P h e 3, D - P r o 4m o r p h i c e p t i n (PL017) 7 e n h a n c e the K +- or capsaicinevoked release o f SP f r o m rat spinal cord slices, 34'37 whereas A i m o n e a n d Y a k s h I f o u n d t h a t /~ opioid receptor stimulation by these drugs markedly inhibits the spinal release of SP evoked by intrathecal a d m i n i s t r a t i o n of capsaicin in anaesthetized rats. These c o n t r a d i c t o r y data led us to re-examine the possible effects o f / z a n d x receptor stimulation by D A G O a n d U 50488 H, respectively, o n the s p o n t a n e o u s release of SP from the rat spinal cord in vivo. In addition, since it has recently been reported t h a t the stimulation of one type o f opioid receptor c a n interact with t h a t o f a n o t h e r , 22 the possible effects o f the simultaneous stimulation of # a n d x receptors o n the spinal release of SP were also investigated.
EXPERIMENTAL PROCEDURES
Surgery Male Spragu~Dawley rats (Charles River, Saint-Aubinl~s-Elbeuf, France) weighing 320-380 g were kept under controlled environmental conditions (22 + I°C, 12 h alternate light,lark cycles, food and water ad libitum) for at least seven days before being used for the experiments. The surgical procedures were performed under deep anaesthesia (2% halothane in a nitrous oxide-oxygen mixture, 2:1). Following tracheal cannulation and insertion of a catheter in a jugular vein, the head was immobilized in a ventroflexed position using a Horsley~Clarke apparatus, the body lying on a sloping surface. The animal was artificially ventilated, the rate and the volume being adjusted with the use of a capnometer (Datex Normocap) to maintain a normal acid-base equilibrium. The heart rate was continuously monitored and the body temperature was kept constant (37 + 0.5°C) by means of a homeothermic blanket system. Special attention was paid to the peripheral vascularization by considering the colour of hindpaws and ears. In case of deterioration of any of these physiological parameters in the course of the intrathecal perfusion, the experiment was stopped immediately. Preparation of the animals for the intrathecal perfusion was essentially as described by Yaksh and Tyce. 5~ A transverse incision was made over the external occipital bump and on the midline overlying the cisterna magna. Muscles were drawn aside from the skull and atlas, and the occipitoatlantoidal membrane was carefully retracted from the cisterna dura. A small incision of the dura and the arachnoid was made over the obex. A nylon inflow catheter (0.50 mm i.d., 0.63 mm o.d.) was then carefully inserted and conveyed 85 mm into the subarachnoid space. An outflow catheter was inserted parallel to the former, with its extremity overlying the lower medulla.
Intrathecal perfusion and treatments Following the surgical procedure, rats were paralysed by a slow i.v. infusion of gallamine triethiodide, and the level of halothane was lowered to 0.5% for the remainder of the experiment. The intrathecal space was then perfused with an artificial cerebrospinal fluid (ACSF, in mM: NaC1 126.5; NaHCO 3 27.5; KC1 2.4; KH2PO 4 0.5; CaC12 1.1; MgC12 0.85; Na2SO 4 0.5; glucose 5.9, adjusted to pH 7.3 by bubbling with an O2-CO2 mixture, 95 : 5, v/v) maintained at 37°C at the output of the inflow catheter. The flow rate was 0.1 ml/min. Thirty minutes after the beginning of the perfusion, fractions (1.5ml corresponding to 15 min) were collected at 0°C, and immediately divided into three aliquots (0.5ml) which were stored frozen at - 3 0 ° C until the measurement of their SP-like material (SP-LM) contents. Usually, three sets of three fractions were collected before (fractions 1-3), during (fractions 4 6) and after (fractions 7-9) the addition of a given compound to the perfusing fluid. When two or three compounds were used, antagonists of opioid receptors (naloxone, nor-binaltorphimine) and/or U 50488 H were added 15 min before and during the whole treatment (fractions 4-7) with the kt opioid receptor agonist (DAGO, fractions 5 7). Radioimmunoassay of substance P As previously described, 32,33 SP-LM was radioimmunoassayed directly in 0.5-ml aliquots of the collected perfusate fractions using monoradioiodinated [~zsI]Tyr8-SP as the tracer. The detection limit of the assay was 0.25 pg of SP per sample, and half displacement of [~z~I]Tyr8-SP bound to antibodies was obtained with 20 pg of the peptide. Each time a compound was added to the ACSF, a complete standard curve was drawn from radioimmunoassays of 0.25-100pg of SP performed in the presence of this compound at the same concentration as that used for the perfusion experiments. The antiserum used was directed toward the carboxy-terminal moiety of the SP molecule. The C-terminal fragments up to SP(5-11) showed 100% cross-reactivity [the shortest C-terminal fragment recognized was SP(7-11) with 25% crossreactivity], whereas SP(1 ll)-COOH and N-terminal fragments did not show any cross-reactivity (i.e. less than 0.01% as compared to 100% for SP). Neurokinins A and B were practically devoid of any immunoreactivity (0.4% and 0.03%, respectively; see Ref. 33 for details). In all cases, SP-LM content was expressed as SP equivalents, i.e. in pg of SP producing the same displacement of bound [125I]Tyr8-SP under standard radioimmunoassay conditions. Calculations In order to determine the significance of the effect produced by a given treatment, the mean value of SP-LM content in the three fractions preceding the treatment was calculated in each experiment and used as the control value (100%). SP-LM content in each successive fraction was then expressed as a percentage of this control value. Means ___S.E.M. of such data obtained in seven to 15 rats were calculated. Statistical analyses were carried out using ANOVA followed by Fischer PLSD test. When the P value was higher than 0.05, the difference was considered to be non-significant. As discussed previously, 5 such calculations were possible because the spontaneous release of SP-LM remained fairly constant throughout the experiment under control conditions. ~° Chemicals [l:5I]Tyr8-SP (2000 Ci/mmol) was from New England Nuclear (Boston, MA, U.S.A.) Other compounds were: DAGO (Bachem, Bubendorf, Switzerland), U 50488 H
~/p opioid control of spinal SP release (Upjohn, Kalamazoo, MI, U.S.A.), naloxone (ENDO, New York, U.S.A.), nor-binaltorphimine (Res. Biochem. Inc., Natick, MA, U.S.A.). All the SP fragments used for characterizing the antiserum were from Sigma (St Louis, MO, U.S.A.).
RESULTS
In the absence of drugs, perfusion of the intrathecal space with A C S F at a flow rate of 0.1 ml/min yielded a constant spontaneous outflow of SP-LM for at least 135 min (i.e. nine fractions), after an initial washing period of 30 min after recovery from surgery. The mean SP-LM content in fractions collected under such control conditions was equal to 5.01 _+ 0.41 pg SP equivalents (mean + S.E.M., n = 12), which corresponded to a rate of 0.33 + 0.03pg/min for the spontaneous release of SP-LM from the whole spinal cord in halothane-anaesthetized rats. Neither 1 0 # M U 50488 H nor 1 0 # M nor-binaltorphimine (a selective x opioid receptor antagonist 4~)
349
altered the spontaneous release of SP-LM, as shown in Fig. 1A and B. By contrast, the addition of D A G O (10 # M ) to the perfusing A C S F led to a significant increase in SP-LM outflow (Fig. 2A). Indeed, although the SP-LM content (133___ 15% of the control value) of the first fraction (nb 4) collected in the presence of the # agonist did not significantly differ from the control value, those of the two following fractions (nbs 5 and 6, 151 + 18% and 1 4 7 + 13%, respectively) were significantly higher (P
0306-4522/92 $5.00 + 0.00 Pergamon Press Ltd IBRO
KAPPA-/MU-RECEPTOR INTERACTIONS IN THE OPIOID CONTROL OF THE IN VIVO RELEASE OF SUBSTANCE P-LIKE MATERIAL FROM THE RAT SPINAL CORD E. COLLIN,*A. MAUBORGNE,S. BOURGOIN, S. MANTELET,L. FERHAT, M. HAMON and F. CESSELIN INSERM U.288, Neurobiologie Cellulaire et Fonctionnelle, Facult6 de M6decine Piti6-Salp&ri6re, 91, boulevard de l'H6pital, 75634 Paris cedex 13, France Abstract--The possible involvement of # and x receptors in the opioid control of the spinal release of substance P-like material was assessed in vivo, in halothane-anaesthetized rats whose intrathecal space was continuously perfused with an artificial cerebrospinal fluid supplemented with various opioid receptor agonists and antagonists. Whereas the intrathecal perfusion with the # agonist DAGO (10 #M) significantly enhanced ( ~ + 50%) the spontaneous release of substance P-like material, that with the x agonist U 50488 H (10 #M) produced no change in the peptide outflow. The respective antagonists naloxone (10pM) for the p receptors and nor-binaltorphimine (10#M) for the x receptors did not affect the spontaneous release of substance P-like material, indicating that endogenous opioids acting at # and x receptors do not exert a tonic control on substance P-containing neurons in the spinal cord of halothane-anaesthetized rats. However, as expected from the involvement of p receptors, the stimulatory effect of DAGO on the peptide outflow could be prevented by naloxone but not norbinaltorphimine. Furthermore, instead of an increase with DAGO alone, a significant decrease in the spinal release of substance P-like material was observed upon the intrathecal perfusion with DAGO plus U 50488 H. Additional experiments with the respective # and x antagonists naloxone and nor-binaltorphimine demonstrated that this effect actually resulted from the simultaneous stimulation of # and x receptors. As previous studies showed that 6 agonists also reduce the release of substance P from primary afferent fibres, the present data could explain why non-selective opioid agonists such as morphine acting at 6, # and x receptors are especially potent to reduce the peptide release through additive inhibitory influences mediated by the stimulation of 6 receptors on the one hand and that of # plus x receptors on the other hand. Such additive inhibitory influences on substance P-containing fibres probably contribute to the potent analgesic action of non-specific opioids administered via the intrathecal route.
Substance P (SP) is one of the neuropeptides present in primary afferent fibres which is thought to play a role in the transmission of nociceptive signals from the periphery to the spinal cord. 15 Thus, a variety of stimuli which can activate small diameter primary afferent fibres such as noxious stimuli, 13,14,21,28,35,45high intensity stimulation of a peripheral nerve 19,5° and local application of capsaicin 1,17,37 can trigger the release of SP at the spinal level. Several lines of evidence indicate that SP-containing primary afferent fibres projecting within the dorsal horn of the spinal cord can be controlled by various neuroactive compounds. In particular, the analgesic action of opioids injected via the intrathecal route has been attributed, at least partly, to the capacity of these drugs to inhibit presynaptically the SP-containing primary afferent
fibres. 49 Indeed, autoradiographic studies have shown that opioid receptors are very probably located on these fibres. 3'11'29Furthermore, opioid receptor stimulation produces a marked reduction in the release of SP from the trigeminal nucleus 25 and the spinal cord.1,6,19,21,31,50
*To whom correspondence should be addressed: Abbreviations: ACSF, artificial cerebrospinal fluid; DAGO, TyrD-Ala-Gly-MePhe-Gly-ol; DPDPE; D-Pen 2, D-Pen 5enkephalin; DTLET, Tyr-D-Thr-Gly-Phe-Leu-Thr; PL017, MePhe 3, D-Pro4-morphiceptin; SP, substance P; SP-LM, substance P-like material; U 50488 H, 3,4-dichloro-Nmethyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzacetamide methansulphonate hydrate, trans-( +__). 347
A m o n g opioids, those stimulating selectively 6 receptors are especially potent to control SP-containing primary afferent fibres. Indeed, the 6 opioid agonists Tyr-D-Thr-Gly-Phe-Leu-Thr ( D T L E T ) s2 and D-Pen 2 (D-PenS-enkephalin; D P D P E ) 36 inhibit both the spontaneous l° and the evoked1'19'34'37release of SP from the rat spinal cord in vitro as well as in vivo. Furthermore, the selective blockade of 6 receptors by naltrindole 38 induces an enhancement of the spinal release of SP in the rat in vivo, suggesting that endogenous opioids acting at these receptors exert a tonic inhibitory control on SP-containing fibres.l° In contrast, the possible involvement of other opioid receptor subtypes in the control of SPcontaining primary afferent fibres is still a matter of debate. In the case of x receptors, most in vitro and in vivo investigations showed that their selective
348
E. COLLIN et al.
s t i m u l a t i o n by 3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzacetamide methan sulp h o n a t e hydrate, trans-( +_ ) ( U 50488 H ) 47 o r dynorp h i n did n o t exert any effect on the spinal release of S P , 1'19'21'34'37 However, C h a n g et al. 6 r e p o r t e d t h a t U 50488 H inhibits SP release from p r i m a r y sensory neurons. Similarly, contradictory data h a v e been reported regarding the m o d u l a t i o n o f spinal SP release by p opioid receptors. In vitro studies have s h o w n t h a t the selective /~ agonists T y r - D - A l a - G l y M e P h e - G l y - o l ( D A G O ) TM a n d M e P h e 3, D - P r o 4m o r p h i c e p t i n (PL017) 7 e n h a n c e the K +- or capsaicinevoked release o f SP f r o m rat spinal cord slices, 34'37 whereas A i m o n e a n d Y a k s h I f o u n d t h a t /~ opioid receptor stimulation by these drugs markedly inhibits the spinal release of SP evoked by intrathecal a d m i n i s t r a t i o n of capsaicin in anaesthetized rats. These c o n t r a d i c t o r y data led us to re-examine the possible effects o f / z a n d x receptor stimulation by D A G O a n d U 50488 H, respectively, o n the s p o n t a n e o u s release of SP from the rat spinal cord in vivo. In addition, since it has recently been reported t h a t the stimulation of one type o f opioid receptor c a n interact with t h a t o f a n o t h e r , 22 the possible effects o f the simultaneous stimulation of # a n d x receptors o n the spinal release of SP were also investigated.
EXPERIMENTAL PROCEDURES
Surgery Male Spragu~Dawley rats (Charles River, Saint-Aubinl~s-Elbeuf, France) weighing 320-380 g were kept under controlled environmental conditions (22 + I°C, 12 h alternate light,lark cycles, food and water ad libitum) for at least seven days before being used for the experiments. The surgical procedures were performed under deep anaesthesia (2% halothane in a nitrous oxide-oxygen mixture, 2:1). Following tracheal cannulation and insertion of a catheter in a jugular vein, the head was immobilized in a ventroflexed position using a Horsley~Clarke apparatus, the body lying on a sloping surface. The animal was artificially ventilated, the rate and the volume being adjusted with the use of a capnometer (Datex Normocap) to maintain a normal acid-base equilibrium. The heart rate was continuously monitored and the body temperature was kept constant (37 + 0.5°C) by means of a homeothermic blanket system. Special attention was paid to the peripheral vascularization by considering the colour of hindpaws and ears. In case of deterioration of any of these physiological parameters in the course of the intrathecal perfusion, the experiment was stopped immediately. Preparation of the animals for the intrathecal perfusion was essentially as described by Yaksh and Tyce. 5~ A transverse incision was made over the external occipital bump and on the midline overlying the cisterna magna. Muscles were drawn aside from the skull and atlas, and the occipitoatlantoidal membrane was carefully retracted from the cisterna dura. A small incision of the dura and the arachnoid was made over the obex. A nylon inflow catheter (0.50 mm i.d., 0.63 mm o.d.) was then carefully inserted and conveyed 85 mm into the subarachnoid space. An outflow catheter was inserted parallel to the former, with its extremity overlying the lower medulla.
Intrathecal perfusion and treatments Following the surgical procedure, rats were paralysed by a slow i.v. infusion of gallamine triethiodide, and the level of halothane was lowered to 0.5% for the remainder of the experiment. The intrathecal space was then perfused with an artificial cerebrospinal fluid (ACSF, in mM: NaC1 126.5; NaHCO 3 27.5; KC1 2.4; KH2PO 4 0.5; CaC12 1.1; MgC12 0.85; Na2SO 4 0.5; glucose 5.9, adjusted to pH 7.3 by bubbling with an O2-CO2 mixture, 95 : 5, v/v) maintained at 37°C at the output of the inflow catheter. The flow rate was 0.1 ml/min. Thirty minutes after the beginning of the perfusion, fractions (1.5ml corresponding to 15 min) were collected at 0°C, and immediately divided into three aliquots (0.5ml) which were stored frozen at - 3 0 ° C until the measurement of their SP-like material (SP-LM) contents. Usually, three sets of three fractions were collected before (fractions 1-3), during (fractions 4 6) and after (fractions 7-9) the addition of a given compound to the perfusing fluid. When two or three compounds were used, antagonists of opioid receptors (naloxone, nor-binaltorphimine) and/or U 50488 H were added 15 min before and during the whole treatment (fractions 4-7) with the kt opioid receptor agonist (DAGO, fractions 5 7). Radioimmunoassay of substance P As previously described, 32,33 SP-LM was radioimmunoassayed directly in 0.5-ml aliquots of the collected perfusate fractions using monoradioiodinated [~zsI]Tyr8-SP as the tracer. The detection limit of the assay was 0.25 pg of SP per sample, and half displacement of [~z~I]Tyr8-SP bound to antibodies was obtained with 20 pg of the peptide. Each time a compound was added to the ACSF, a complete standard curve was drawn from radioimmunoassays of 0.25-100pg of SP performed in the presence of this compound at the same concentration as that used for the perfusion experiments. The antiserum used was directed toward the carboxy-terminal moiety of the SP molecule. The C-terminal fragments up to SP(5-11) showed 100% cross-reactivity [the shortest C-terminal fragment recognized was SP(7-11) with 25% crossreactivity], whereas SP(1 ll)-COOH and N-terminal fragments did not show any cross-reactivity (i.e. less than 0.01% as compared to 100% for SP). Neurokinins A and B were practically devoid of any immunoreactivity (0.4% and 0.03%, respectively; see Ref. 33 for details). In all cases, SP-LM content was expressed as SP equivalents, i.e. in pg of SP producing the same displacement of bound [125I]Tyr8-SP under standard radioimmunoassay conditions. Calculations In order to determine the significance of the effect produced by a given treatment, the mean value of SP-LM content in the three fractions preceding the treatment was calculated in each experiment and used as the control value (100%). SP-LM content in each successive fraction was then expressed as a percentage of this control value. Means ___S.E.M. of such data obtained in seven to 15 rats were calculated. Statistical analyses were carried out using ANOVA followed by Fischer PLSD test. When the P value was higher than 0.05, the difference was considered to be non-significant. As discussed previously, 5 such calculations were possible because the spontaneous release of SP-LM remained fairly constant throughout the experiment under control conditions. ~° Chemicals [l:5I]Tyr8-SP (2000 Ci/mmol) was from New England Nuclear (Boston, MA, U.S.A.) Other compounds were: DAGO (Bachem, Bubendorf, Switzerland), U 50488 H
~/p opioid control of spinal SP release (Upjohn, Kalamazoo, MI, U.S.A.), naloxone (ENDO, New York, U.S.A.), nor-binaltorphimine (Res. Biochem. Inc., Natick, MA, U.S.A.). All the SP fragments used for characterizing the antiserum were from Sigma (St Louis, MO, U.S.A.).
RESULTS
In the absence of drugs, perfusion of the intrathecal space with A C S F at a flow rate of 0.1 ml/min yielded a constant spontaneous outflow of SP-LM for at least 135 min (i.e. nine fractions), after an initial washing period of 30 min after recovery from surgery. The mean SP-LM content in fractions collected under such control conditions was equal to 5.01 _+ 0.41 pg SP equivalents (mean + S.E.M., n = 12), which corresponded to a rate of 0.33 + 0.03pg/min for the spontaneous release of SP-LM from the whole spinal cord in halothane-anaesthetized rats. Neither 1 0 # M U 50488 H nor 1 0 # M nor-binaltorphimine (a selective x opioid receptor antagonist 4~)
349
altered the spontaneous release of SP-LM, as shown in Fig. 1A and B. By contrast, the addition of D A G O (10 # M ) to the perfusing A C S F led to a significant increase in SP-LM outflow (Fig. 2A). Indeed, although the SP-LM content (133___ 15% of the control value) of the first fraction (nb 4) collected in the presence of the # agonist did not significantly differ from the control value, those of the two following fractions (nbs 5 and 6, 151 + 18% and 1 4 7 + 13%, respectively) were significantly higher (P
