0022·3565/90/2521·0235$00.00/0 THE JOURNAL OP PHARMACOLOGY AND EXPEIUMENTAL THERAPEUTICS Vol. 252, No. 1 Copyri,bt Cll990 by The Americ:an Society for Pharmacology and Ezperimental Therapeutics Printed in U.SA.

Respiratory and Locomotor Stimulation by Low Doses of Dermorphin, a Mu1 Receptor-Mediated Effect1

PIRKKO PAAKKARI, ILARI PAAKKARI, ANNA-LEENA SIREN and GIORA FEUERSTEIN Depattment of Neurology (P.P., I.P., A.-L.S., G.F.), Uniformed Services University of the Health Sciences, Bethesda Maryland and Depattment of Pharmacology (G.F.), Smith Kilne Beecham, Philadelphia Pennsy/vania Accepted for publication September 29, 1989

ABSTRACT The selective opioid mu receptor agonist dermorphin increased dermorphin. The selective benzodiazepine antagonist flumazenil the locomotor activity of rats dose dependently at 10 to 100 (5 mgfkg), which has been shown previously to antagonize pmolfkg i.c.v. Respiratory rate, relative tidal volume and respi­ catalepsy and respiratory depression produced by relatively high ratory minute volume also increased unrelated to changes in doses of dermorphin, did not antagonize the respiratory or locomotor activity. Higher doses, on the other hand, produced locomotor stimulant effect of dermorphin. The data suggest that catalepsy and respiratory depression. Pretreatment of the rats mu,-opioid receptors are responsible for the low dose stimulant with the mu,-selective antagonist naloxonazine (10 mgfkg i.v.) effects of dermorphin on locomotor activity and respiration blocked the stimulant locomotor and respiratory effects of low whereas mu2 receptors mediate the respiratory depressant effect doses of dermorphin (1 0--1 00 pmolfkg), but potentiated the of dermorphin. respiratory depressant effect of a high dose (1 0 nmolfkg) of

Low doses of morphine or opioid peptides increase locomotor Furthermore, t.he role of mu1-opioid receptors in mediating the activity, whereas higher doses induce a cataleptic state, char­ stimulatory effect of dermorphin was examined in rats treated acterized by the absence of movement and muscular rigidity with mu, receptor antagonist naloxonazine (Hahn et al., 1982). (Tortella et al., 1978; Browne et al., 1979; Brady and Holtzman, In addition, because the cataleptic and ventilatory depressant 91981; Havemann et al., 1983; Locke and Holtzman, 1986). effect of dermorphin were antagonized by the benzodiazepine Locke and Holtzman (1986) suggested that the depressant antagonist flumazenil (Ro 15-1788) (Paakkari and Feuerstein, effect of opioids on locomotion might be mediated by mu-opioid 1988; Paakkari et al., 1988), we found it of interest to examine and stimulant effect by delta-opioid receptors. In accordance whether the benzodiazepine/GABAergic system is also associ­ with this concept, a potent and selective mu receptor agonist ated with dermorphin-induced locomotor and respiratory stim­ dermorphin (Rossi et al., 1986; Krumins, 1987) induced cata­ ulation. lepsy in the rat when administered i.c.v. at doses above 0.3 nmol/kg (Paakkari and Feuerstein, 1988). Methods The well known respiratory depressant effect of opioids (for Animals. Conscious male Sprague-dawley rats (250-330 g, Taconic review see Borison 1977) was also demonstrated for dermorphin Farms, Germantown, NY) were used. The animals received standard in conscious rats (Paakkari et al., 1988). However, small doses rat pellets and water ad libitum and were kept at 22•c and 12 hr/hr of opioids can also induce an increase in ventilatory frequency light dark cycle. (Hassen et al., 1982; Hurle et al., 1985) or mean instantaneous Procedure for i.c. v. and i. v. injections. Rats were anesthetized minute ventilation (Haddad et al., 1984; Schaeffer and Haddad, with an i.m. injection of ketamine (130 mg/kg) and acepromazine (1.3 1985). mg/kg). A stainless-steel guide cannula was inserted stereotaxically (Oavid Kopf Instruments, Tujunga, CA) through the skull into tbe The objective of the present study were to investigate right lateral brain ventricle (i.c.v.) and fixed with glue (Eastman 910 whether low doses of dermorphin would also induce locomotor adhesive). Coordinates for the lateral ventricle were: AP, -0.8 mm and and respiratory stimulation and, if so, would the respiratory L, 1.2 mm (1967). After the operation the rats were allowed to recover stimulation be secondary to the increase in locomotor activity. for at least 3 days in single cages, with food and water ad libitum. A group of rats was implanted also with PE-50 tubing, filled with hepa­ rinized ( 100 U /ml) saline, in the extemal jugular vein for naloxonazine Received for publication Aprill9, 1989. 1 Thia work waa supported by United States Army Medical Research and injections. The tubing was threads s.c. to the posterior neck, exited Development command protocol G39228. through a small incision, then cut to a lengtb of about 5 cm and sealed

ABBAEVIAnON: DAMGO, o-Ala2-Me-Phe4-Giy-ol5-enkephalin. 235 236 Paakkari et al. Vol. 252 by heat. The rats received a single i.v. injection of naloxonazine (10 Results mg/kg, as the free base) or saline in a volume of 1 ml/kg 22 to 24 hr before dermorphin injections. Locomotor effects of dermorphin. Dermorphin was On the day of the experiment, a 7.5 mm long 30 g cannula was tested at doses of 3, 10, 30 and 100 pmol/kg. The three highest inserted into the ventricular space through the guide cannula. The doses increased locomotor activity, with 30 pmol/kg having the injection cannula was connected via polyethylene tubing to a Hamilton maximal effect (Fig. 1). lncreases in total (60 min) locomotor microliter syringe and the drug solution was injected over a period of activity produced by 100 pmol/kg of dermorphin did not reach 20 sec in a volume of 10 "1. Each rat received a single dermorphin statistical significance (P < .19). However, the increase in injection. The proper position of the i.c.v. cannula was ascertained at locomotor activity during the first 10 min after the injection the end of the experiment by inspection of the brain ventricles after was significant (fig. 2). an injection of methylene blue (10 "1). The spontaneaus locomotor activity of the resting ratswas Analgesia testing. Analgesia was measured by the tail-tlick test near zero during the 10 min before drug injections. (O'Amour and Smith, 1941). A cut-off time of 12 sec was used to avoid tissue damage. Tail-flick response times were represented as percentage Respiratory effects of dermorphin. Low doses of der­ of maximum possible effect (% of MPE) according to the equation: morphin (10 and 30 pmol/kg) increased the respiratory minute volume (fig. 3, lower panel). Respiration rate was increased % of MPE significantly after the dose of 30 pmol/kg (fig. 3, upper panel). = post drug response latency - predrug response latency x 100 Higher doses (3 and 10 nmol/kg) decreased both respiration cut-off time - predrug response latency rate and minute volume. The corresponding changes in relative Locomotor activity monitoring. Locomotor activity was moni­ tidal volume were 3 ± 6% (saline), 6 ± 10% (3 pmol/kg), 86 ± tored from 1 hr before until 2 hr after the drug administration. Digiscan 74% (10 pmol/kg), 179 ± 128% (30 pmol/kg), -3 ± 9% (100 Optical Digital Sensor Activity Monitors equipped with Datalogger pmol/kg), 23 ± 18 (3 nmol/kg) and -9 ± 7% (10 nmol/kg). The 8000 data collection devices (Omnitech Electronics Inc., Columbus, changes in relative tidal volume were not statistically signifi­ OH) were used to measure activity. The data was collected in counts cant. per 5 min. During the experiment the animals were placed in the Timecourse of locomotor and respiratory stimulation. Oxymax Plexiglas chambers (see Respiratory recording) set inside the The peak increase in locomotor activity was observed within activity monitors, which made the simultaneous recording of respira­ 15 min from the injection, whereas the respiratory stimulation tion and locomotor activity possible. was maximal 50 to 60 min after the injection (fig. 2). The effects Recording of respiration. Ventilation rate and relative ventilation of the dose of 100 pmol/kg at 10 min further shows that tidal volume were monitored by the Oxymax 85 system (Columbus Instruments, Columbus, OH). The recording system consists of two 1600 sealed Plexiglas test chambers and one reference chamber (internal 1400 ~lvent dimensions: 112 X 197 X 297 mm, volume: 6.551). Room air is pumped Dl.ll ...... 1200 !i:>:> 01<1 10 through each chamber at the constant rate of 2 liters/min. Recording .....-Vl c 1000 11111 OUJO of the respiration rate and relative tidal volume is based on the minute ::::l -01.1100 0 800 pressure changes in the chamber caused by the subject's breathing. u The tidal volume information is presented as a series of pulses whose '-" 600

120~------~ 240 c::J Solvwlt DU pmoi/Jct: DWnmol/11;: 200 rzll3 1Zl3 10 160 Q110 CSJ10 ~ -....._" 120 -30-100 > ::E 80

-10~------~ Fig. 5. lnfluence of naloxonazine (NAZ) and flumazenil (FLU) pretreat­ ments on the respiratory stimulant effect of dermorphin (DM, 30 pmol/ kg). ••p < .01 when compared to solvent; •p < .05 when compared to 2~r------~ DM; n = 6 in each group. 200 VR 1V w 1110 0 -...... ,~ 120 ~~ > 10 -20 ::::!: ~

..,...... 150 6--6 Dt.t 100 pmol/kg ·-· NAZ+DM 100 ~ 120 0-0 DM JO pmol/kg e-e NAZ+DM JO ::E 6 1600~------~ ~ 90 6-o- -6 1400 ':, 60 ~ 1200 ~ ~/0 "-o ---6""'~" 6 +J c 1000 gw JO .i.T-~ ~ •T """T0 ' ""' 6 ::J 0 800 < l 0 o••'/ • r~•~: -...._9 '-' 600 ~ . (''"•~.. : i . -JO • .& I --· ::s 400 • • • • 200 -60+---+--~----~--~---+----~--~ JO 01...--~-'--- 0 60 90 120 150 180 210 Fig. 4. lnfluence of naloxonazine (NAZ, 10 mgjkg i.p.) and flumazenil TIME (MIN) (FLU, 5 mgjkg i.p.) pretreatments on the locomotor effect of dermorphin Fig. 7. Analgesia induced by dermorphin (DM, 0.1 and 0.03 nmoljkg). (DM, 30 pmol/kg). •p < .05; ••p < .001 when compared to solvent, +p The filled symbols represent the effect of the same doses of DM in < .05 when compared to DM (30 pmol/kg; n = 6 in each group). naloxonazine (NAZ)-treated animals. •p < .05, n = 5 in all groups. MPE = maximum possible effect. respiratory and locomotor stimulation were unrelated to each loxonazine-treated ratswas given a respiratory depressant dose other, as the minute volume remained unchanged although of dermorphin, 10 nmol/kg. Respiratory depressionwas poten­ locomotor activity increased significantly (fig. 2). tiated by naloxonazine pretreatment (fig. 6). Effects of dermorpbin after pretreatment with nalox­ Analgesie effect of dermorphin. Dermorphin, 0.03 and onazine or fiumazenil. Naloxonazine (10 mg/kg i.v.) was 0.1 nmol/kg, induced analgesia, which was antagonized by given 20 to 24 hr before dermorphin. The dose of naloxonazine naloxonazine (fig. 7). and the time for pretreatment was chosen to obtain maximal mu -selectivity (Hahn et al., 1982; Ling et al., 1986). Naloxon­ 1 Discussion azine antagonized both the locomotor (fig. 4) and respiratory (fig. 5) stimulation produced by dermorphin, 30 pmol/kg, the We have shown previously that the central administration dose which had the maximal effect in the dose-response study. of the heptapeptide dermorphin produces catalepsy and respi­ Flumazenil (5 mg/kg i.p. 20 min before dermorphin) did not ratory depression at doses above 300 pmol/kg (Paakkari and significantly attenuate the locomotor and respiratory stimulant Feuerstein, 1988; Paakkari et al., 1988). This study disclosed effects of dermorphin (figs. 4 and 5). opposite effects of the same opioid, i.e., stimulation of locomo­ To confirm the selectivity of naloxonazine antagonism on tor activity and respiration when lower doses, 10 to 100 pmol/ dermorphin-induced respiratory stimulation, one group of na- kg, were given. 238 Peakkerf et el. Vol. 252 The high potency of dennorphin (up to 5000 times more and Goodman, 1985b), as weil as pharmacological studies with potent than morphine) described in analgesia studies and bioas­ mu1-antagonists and delta/mu1-agonists (Pasternak et al., 1980; says (Broccardo et al., 1981; Stevens and Yaksh, 1986), is Zhang and Pasternak, 1981, 1988; Bodnar et al., 1988) imply evident also when the effects of opioids on locomotion are that supraspinal opioid analgesia is mediated via mu1 receptors. compared: the doses of morphine that in previous studies The mu1 receptor antagonist naloxonazine selectively antago­ (Brady and Holtzman, 1981) induced maximallocomotor acti­ nized morphine analgesia without affecting the hypoxemia and vation were 10,000-fold greater than the equipotent doses of hypercarbia produced by morphine (Ling et al., 1983, 1985), dermorphin in our study. Even the corresponding doses of the suggesting that the respiratory depressant effect of opioicls is specific mu agonist DAMGO were still 30 to 200-fold greater mediated by m~ receptors. Along this hypothesis, it can be than those of dermorphin in our study (Locke and Holtzman, speculated that the stimulatory effects of the selective mu­ 1987; Latimer et al., 1987). The outstanding potency of der­ agonist dermorphin on respiration are mediated via mu1 recep­ morphin, which has been shown to have high selectivity and tors. The aforementioned respiratory stimulation by small affinity for mu sites (about 3 times higher affinity than doses of mu-agonists or delta-agonists (Hassen et al., 1982; DAMGO; Krumins 1987), suggests that the locomotor activa­ Schaeffer and Haddad, 1985; Hurle et al., 1985), known to bind tion is mediated via mu receptors. to mu1 receptors with high affinity (W olozin and Pasternak, To our knowledge, the respiratory changes have not been 1981; Nishimura et aL, 1982; Lutz et al., 1985; Itzhak and measured in previous studies in which locomotor effects of Pasternak, 1987), could also be explained assuming that the opioids were investigated. It is conceivable that an increase in small doses would bind to high affinity mu1 receptors only, locomotor activity could subsequently Iead to an increase in leading to stimulation; at higher doses, the respiratory depres­ respiration. Indeed, low doses of dermorphin consistently in­ sant m~-effect would dominate. In our study dermorphin at creased respiratory rate and relative tidal volume. However, the dose of 100 pmol/kg did neither stimulate nor depress simultaneaus recording of respiration and locomotion revealed respiration; this Iack of change from base line (fig. 3) may be that Stimulation of respiration was unrelated to the increase in due to a balance between the respiratory stimulant and depres­ locomotor activity for the following reasons: first, the peak sant effect of this dose. stimulation in locomotion was observed 10 to 15 min after Stimulation of locomotor activity may similarly be a mu1 dermorphin injection, whereas the maximum effect on respi­ receptor effect, as dermorphin does not bind to delta receptors, rationwas reached 50 to 60 min after dermorphin administra­ formerly proposed to mediate the opioid-induced increase in tion; second, locomotor activity returned back to basal values locomotor activity (Locke and Holtzman, 1986). In the present at time of maximal respiratory stimulation; and third, the dose study naloxonazine did antagonize the respiratory and the of 100 pmol/kg of dermorphin increased locomotor activity locomotor stimulation by dermorphin, implying that these in­ significantly without respiratory stimulation. deed were mu1 receptor-mediated effects. Latimer et aL (1987) Centrally administered mu-opioid agonists induced in most also suggested recently that activation of dopamine neurons in studies a respiratory depression (Holaday, 1982; Pfeiffer et al., the ventral tegmental region of the rat brain by morphine and 1983; Pazos and Florez, 1983; Ward and Takemori, 1983; enkephalin analogs, and the subsequent increase in motor

Morin-Surun et al., 1984), as did dermorphin in our previous activity, are mediated by mu receptors, perhaps the mu1-sub­ study (Paakkari et al., 1988). However, local injections of small type. doses (3-30 pmol) of the mu-opioid agonist DAMGO into the The analgesic effect of dermorphin was studied to character­ nucleus tractus solitarius increased respiratory frequency, ize further its mu1 and m~ receptor-mediated actions. Der­ whereas a high dose (300 pmol) bad a depressant effect (Hassen morphin-induced analgesia was antagonized by naloxonazine, et al., 1982). Furthermore, the mu agonists morphine (0.5 mg/ corroborating the theory that mu1 receptors are involved in this kg) and morphiceptin analog (Tyr-Pro-NMePhe-o-Pro-NH2, effect. After the bighest dose of dermorpbin the naloxonazine­ 2-125 ~o~g/kg) increased instantaneous minute ventilation in treated animals were also clearly less cataleptic than in control conscious dogs upon intracisternal administration (Haddad et group (ordinary catalepsy tests were, however, not performed aL, 1984; Schaeffer and Haddad, 1985). Also, the mu-agonist due to the recording of respiration), which is in concordance 2 4 5 o-Ala -Me-Phe -Met(O)ol -enkephalin (FK-33824) and the with the suggestion that catalepsy also is mediated via mu1 delta-agonist o-Ala2-o-Leu5-enkephalin increased respiratory receptors (Ling and Pasternak, 1982). frequency and decreased tidal volume when applied to the To ascertain the selectivity of naloxonazine antagonism ventral surface of the medulla oblongata of the anesthetized against mu1 receptors, we studied its influence on the respira­ cat, whereas when applied to the rostro-dorsal surface of the tory depressant effect of a higher dose of dermorphin. lnstead pons they depressed respiratory frequency and increased tidal of antagonism, there was a potentiation of respiratory depres­ volume (Hurle et al., 1985). sion. This is in accordance with the concept that m~ receptors

Pharmacological, biochemical and receptor binding studies mediate respiratory depression (Ling et al., 1985) and mu1 support the presence of two subtypes of mu receptors: the mu1 receptors respiratory Stimulation: when the mu1 receptors were receptor which binds both opiates and most enkephalins with blocked by naloxonazine, dermorphin's depressant effect on the similar very high affinities (Kd values < 1 nM), and the m~ available m~ receptors was unopposed and potentiated. receptor which binds morphine preferentially (Wolozin and We have sbown previously that the benzodiazepine antago­ Pasternak, 1981; Nishimura et aL, 1983; Lutz et al., 1985; nist flumazenil attenuates the cataleptic (Paakkari and Feuer­ Goodman and Pasternak, 1985). The autoradiographic distri­ stein, 1988) and respiratory depressant (Paakkari et al., 1988) bution of mu1-sites to brain areas involved in antinociception effect of dermorphin. lt also antagonizes the analgesic (Brady (Goodrnan and Pasternak, 1985; Moskowitz and Goodman, et al., 1984) and intestinal (Fioramonti et al., 1987) effects of 1985a), and the correlation between the density of mu1 -sites centrally given mu-agonist opioids. However, the locomotor and analgesic sensitivity in different mice strains (Moskowitz and respiratory stimulant effects of low doses of dermorphin 1990 Oploid Mu, Aeceptor EHeeta 239

TABLE 1 Leu•-enkephalin injections into the nucleua accumbena on the motility of rats. Putative receptcn lnvolved in verlous etfects of dermorphln Life Sei. 33: Supp. I, 627-630, 1983. NAZ, naloxonazine; FLU, ftumazeplne; BZ/GASA, benzodiazepine--y-aminobutyric HOLADAV, J. W.: Cardioreapiratory effects of mu and delta opiate agoniats ecid (GABA) receptor complex. following third or fourth ventricular injectiona. Peptides 3: 1023-1029, 1982. HURLE, M. A., MEDIA VILLA, A. AND FLOREZ, J.: Differential reapiratory patterns Antagonz8d by induced by opioida applied to the ventral medullary and donal pontine surfaces Effect Dole Receplm NAZ FLU of cats. Neuropharmacology 24: 597-606, 1985. ITZHAK, Y. AND PABTERNAK, G. W.: Interaction of [D-Ser, Leu1 J-enkephalin­ pmdf/cg Thr• (DSLET), a relatively selective delta-ligand, with mu opioid binding i.c.v. 1 sites. Life Sei 40: 307-311, 1987. Respiratory >10 Yes No Mu, KRUMINS, S. A.: Characterization of dermorphin binding to membranes of rat Stimulation brain and heart. Neuropeptides 9: 93-102, 1987. Locomotor >10 Yes No Mu 1 LATIMER, L. G., DUFFV, P. AND KALIVAS, P. W.: Mu opioid receptor involvement Stimulation in enkephalin activation of dopamine neurons in the ventral tegmental area. Mu,; Analgesia >30 Yes Yes BZ/GABA J. Pharmacol. E:.:p. Ther. 241: 328-337, 1987. Mu,; Catalepsy >300 Yes Yes BZ/GABA LING, G. S. AND PASTERNAK, G. W.: Morphine catalepsy in the rat: lnvolvement Respiratory >1000 No Yes Mu2; BZ/GABA of mu 1 (high affinity) opioid binding sites. Neuroaci. Lett. 32: 193-196, 1982. depression LING, G. S. F., SIMANTOV, R., CLARK, J. A. AND PASTERNAK, G. W.: Nalo:.:ona· zine actions in uivo. Eur. J. Pharmacol. 129: 33-38, 1986. were not antagonized by flumazenil. Hypothetically, this can LING, G. S. F., SPIEGEL, K., LOCKHART S. H. AND PASTERNAK, G. W .: Separation be explained by postulating that very low concentrations of of opioid analgesia from respiratory depreuion: Evidence for different receptor mechanisma. J. Pharmacol. Exp. Ther. 232: 149-155, 1985. dermorphin would bind to high-affinity, naloxonazine-sensitive LING, G. S. F., SPIEGEL, K., NISHIMURA, S. L. AND PASTERNAK, G. W.: mu. receptors only; at higher analgesic, respiratory depressant Diasociation of morphine's analgeaic and reapiratory depreasant actions. Eur. and cataleptic doses it would interact with benzodiazepine/ J. Pharmacol. 88: 487-488, 1983. GABAergic pathways (table 1), suggested to modulate various LOCKE, K. W. AND HOLTZMAN, S. G.: Behavioral effects of opioid peptides effects of opioids (Cooper et al., 1983; Naughton et al., 1985; aelective for mu or delta receptors. Locomotor activity in nondependent and morphine-dependent rats. J. Pharmacol. E:.:p. Ther. 238: 997-1003, 1986. Zambotti et al., 1987; Moreau and Pieri, 1988). LUTZ, R. A., CRUCIANI, R. A., MUNSON, P. J. AND RODBARD, D.: Mu,: A very high affinity subtype of enkephalin binding aites in rat brain. Life Sei. 36: Aclmowleclgmenta 2233-2238, 1985. The experimenta reported herein were conducted according to the principles MOREAU, J.-L. AND PIERI, L.: Effects of an intrathecally adminiatered benzodi· aet forth in the Guide for Care and Uae of Labaratory Animals, Institute of azepine receptor agonist, ant.agonist and inverse agoniat on morphine-induced Laboretory Anima! Medicine, National Research Council, Department of Health, Education and Welfare Publication No. (NIH) 85-23, 1985. We wish to thank inhibition of a spinal nociceptive refle:.:. Br. J. Pharmacol. 93: 964-968, 1988. Dr. Jamea E. Barrett for his uaeful commenta during this study. MORIN-SURUN, M.-P., BUODINOT, E., GACEL, G., CHAMPAGNAT, J., ROQUES, 8. P. AND DENAVIT-SAUBIE, M.: Different effecta of" and delta opiate agonists Referencee on respiration. Eur. J. Phannacol. 98: 235-240, 1984. MOSKOWITZ, A. S. AND GOODMAN, R. R.: Autoradiographie diatribution of mu BODNAR, R. J., WILLIAMS, C. L., LEE, S. J. AND PASTERNAK, G. W.: Role of 1 and mu, opioid binding in the mouae centrat nervoua syatem. Brain Res. 380: mu -opiate recepton in supraspinal opiate analgesia: A microinjection study. 1 117-129, 1985a. Brain Res. 447: 25-:U, 1988. MOSKOWITZ, A. S. AND GOODMAN, R. R.: Autoradiographie analyais of mu" mu BORJSON, H. L.: Centrat nervoua reapiratory depreaaanta: Narcotic analgesics. 2 Pharmacol. Ther. Part B Gen. Syst. Pharmacol. 3: 227-237, 1977. and delta opioid binding in the centrat nervoua ayatem of C57BL/6BY and BRADV, L. S. AND HOLTZMAN, S. G.: Effecta of intraventricular morphine and CXBK (opioid receptor-deficient) mice. Brain Res. 380: 108-116, 1985b. enkephalins on locomotor activity in nondependent, morphine-dependent and NAUGHTON, N., HOFFMAN, W. E., LARSCHEID, P., COOK, J. M., ALBRECHT, R. poet-dependent rata. J. Pharmacol. Ezp. Ther. 218: 613-620, 1981. F. 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