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FMRFamide, a putative endogenous opiate antagonist: Evidence from suppression of defeat-induced analgesia and feeding in mice
Neuropeptides 6:
485-494,
1985
FMRFAMIDE, A PUTATIVE ENDOGFNOUS OPIATE ANTAGONIST: EVIDENCE FROM SUPPRESSION OF DEFEAT-INDUCED ANALGESIA AND FEEDING IN MICE Martin Kavaliers and Maurice Hirst Department of Zoology and Department of Pharmacology and Toxicology, University of Western Ontario, London, Ontario, Canada N6A 5B7 [reprints to MK] ABSTRACT Social conflict and defeat in mice leads to an activation of endogenous opiate systems. The effects of intracerebroventricular administration of the peptide FMRFamide (Phe-Met-Arg-Phe-NH*) and the opiate antagonist naloxone, on agrressive encounters, defeat-induced analgesia and defeat-induced feeding were examined in male mice. Both substances reduced the number of bites required to cause defeat in subordinate mice during aggressive encounters, as well as suppressing the subsequent defeat-induced analgesia. Administration of FMRFamide or naloxone also reduced defeat-induced feeding. These results indicate that FMRFamide (or FMRFamide-like neuropeptides) may function as endogenous opioid antagonists. INTRODUCTION The peptide Phe-Met-Arg-Phe-NH2 (FMRFamide) has been isolated from the ganglia of the Venus clam, Macrocallista nimbosa (1) and found to evoke a variety of cardiac and neuronal actions in invertebrates (l-3). FMRFamide-like immunoreactivity was subsequently demonstrated to have wide distribution in the central nervous system of mammals, with the highest concentrations occurring in the spinal cord and hypothalamus (4,5). This broad distribution led to the suggestion that FMRFamide, or FMRFamide-like peptides, may have regulatory roles in the mammalian central nervous system, which involve autonomic and endocrine regulation, as well as pain modulation (5). This speculation received support with the findings that iontophoretically applied FMRFamide has excitatory effects on mammalian neurons (6). it was demonstrated recently that both FMRFamide and an immunoreactive FMRFamide-like peptide purified from bovine brain could antagonize morphine-induced analgesia in rats (7). In addition, Tang et al. (7) showed that intrathecal or intracerebroventricular (i.c.v.)injections of antibody directed against FMRFamide induced long lasting analgesia and decreased tolerance to morphine-induced analgesia. These investigations suggested that FMRFamide, or related peptides in the 485
spinal cord, may affect pain threshold by functioning as endogenous opiate antagonists. Subsequently, Kavaliers -et al. (8) showed that i.c.v. injections of FMRFamide suppressed both morphine- and food deprivation-induced feeding in the mouse, in a manner analogous to that obtained after treatment with the opiate antagonist, naloxone. These data provide further support for the suggestion that either FMRFamide, or FMRFamide-like peptides, may function as physiological opiate antagonists. There is substantial evidence that after exposure to either physically or psychologically stressful situations endogenous opioid activity is increased in mammals (9-12). In both wild and laboratorybred mice, intraspecific aggressive interactions can result in the display of a specific defeat posture in the vanquished individual (12-14). This behavior has been considered to represent a generalized natural biological response to the stress of social confrontation (14). Defeat has been shown to result in activation of endogenous opiate systems (12), and the display of naloxone senstive analgesic and ingestive responses (14). In the present study we describe the effects of FMRFamide on the analgesic, behavioral and ingestive responses of mice subjected to the stress of social conflict and defeat. We show that FMRFamide can suppress these endogenous opiate activated responses, in a manner analogous to that observed with naloxone, and consistent with the proposal that FMRFamide, or related peptides, function as endogenous opiate antagonists. MATERIALS AND METHODS Animals Small (2-3 months of age, 25-30 gm) and large (6-8 months of age, 40-45 gm) male CF-1 mice (Charles River, Quebec) were used in these studies. The small mice were housed in groups of five, while the larger animals were held individually. All animals were kept under a 12 hour light: 12 hour dark cycle (LD 12:12, L 0700-1900 h, 20 VW/cm') at 22 f 1". Food (Purina mouse chow 5015) and water were available -ad libitum. Experimental Procedures Social Conflict and Nociceptive Responses. At mid-photophase groups of ten different small mice received either intraperitoneal (i.p.) injections of naloxone hydrochloride (1.0 mg/kg, Endo Laboratories, NJ) prepared in saline (10 ml/kg), saline vehicle (10 ml/kg), or i.c.v. injections of 0.01, 0.10, 1.0 and 10 ug (0.17, 1.7, 17 and 170 nmoles, respectively) of FMRFamide (Peninsula Laboratories, CA) dissolved in saline. Using the procedure of Haley and McCormick (15), mice received i.c.v. injections of FMRFamide in 2.0 ~1 (1.0 1.11in each side of the brain) or saline alone which served as a control. The i.c.v. injection interrupted the activity of the animals for approximately 2-3 min, after which they resumed apparently normal behaviors and activity. In order to gain an indication of the regions of the ventricular system to which the drugs penetrated, dilute India ink (2.0 ~1, 1.0 ~1 per side) was injected into over-anesthestized mice. The brains were dissected and
486
the ink was observed
to have penetrated
the ventricular
system.
Thirty minutes after i.p. or i.c.v. injection, a small mouse was placed in the home cage of a larger mouse. The number of bites received by the smaller mouse during this "resident-intruder" pairing was Once recorded until the intruder mouse displayed a full defeat posture. defeat was reached the encounter was terminated. Defeat was characterized by an upright position, limp forepaws, upward-angled head and retracted ears. The same large, aggressive resident mice were used in all of the studies. Results of previous investigations showed that the repeated aponistic interactions had no effects on the aggressive behavior of the larger resident mouse directed towards the small intruder (14). Each smell intruder mouse was used only once. Immediately following the aggressive encounter the nociceptive thresholds of the small intruder mice were assessed using a hot-plate Individual mice were placed onto the heated surface (50 + technique. 0.5'C) and the latency to a paw-licking response was recorded; after which the mouse was quickly removed from the surface and returned to the home cage. Response latencies of groups of ten other mice given saline (10 ml/kg i.p., 2.0 ~1, i.c.v.) but without experience of any social conflict were also determined. In feeding determinations, mice were Social Conflict and Food Intake. housed individually in elevated (20 cm diameter, 7 cm high) clear plastic small rodent metabolism units with wire mesh floors (E=llOO, Econo-metabolism Unit, Maryland Plastics, NY). A short (3. cm ) tunnel provided access to a food hopper in which powder-cd food (Purina mouse chow 5015) was placed. An aluminum ring in front of the hopper restricted entry and prevented the animals from placing their paws in the food. The animals readily consumed the powdered food by licking. Water was provided in a plastic graduated tube placed directly across from the food hopper. After three days of hahituation, the effects of either naloxone (1.0 mg/kg, i.p.> or FMRFamide (0.01, O.lC and 1.0 ug, i.c.v.) pretreatments (10 min) on the food uptake of the smaller mice fcllowing the "resident-intruder" pairing were examined. After the aggressilre encounter, defeated mice (n=lO, in all cases) were returned to the units and their food intakes were recorded over three hours. Saline injected (10 ml/kg, i.p.; 2.0 ~1. i.c.v.) mice (n=lO) serve6 as contrn?s. Determinations were made of food and water consumed at the end of each hour. Fresh food (2.0 gm) was provided each hour. P.ny food that was lost by scatter or spillage was collected and corrected for -in the hourly food intake determinations. In addition, the activities of the mice and time course of food intake were noted. Control determinations were made using young mice (n=5) given saline i.p., i.c.v. or not injected, all of which did not undergo social conflict. Data were analysed using Analysis Keuls multiple range tests.
487
of variance
and Student-Kewman-
RESULTS Social Conflict and Nociceptive Responses. Defeated i.c.v.-saline treated mice were analgesic, displaying significantly (pcO.01) greater latencies to respond to the aversive stimulus than control saline, treated mice not undergoing aggressive encounters (Fig. 1A). With regards to the control animals, there were no significant differences between the latencies of response of i.p.- or i.c.v.-saline injected or uninjected mice. Defeated mice receiving naloxone or FMRFamide displayed significantly (~~0.01 for 1.0 ug FMRFamide) lower dose-dependent latencies of response to the thermal stimulus than did the control_, defeated mice. Naloxone and FMRFamide (1.0 and 10 ug) suppressed defeat-induced analgesia (Fig. lA), the thermal response latencies of the injected animals being identical to those of undefeated animals. FMRFamide (0.01 and 0.10 ug) significantly reduced (160.05), but did not block defeat-induced analgesia. The number of bites required to produce the defeat posture was significantly reduced (pcO.01) in naloxone injected mice, as compared to saline-treated animals (Fig. 1R). FMRFamide caused a significant (pc.01 for 0.10 pg) dose-dependent decrease in the number of bites required to elicit defeat. FMRFamide (1.0 and 10 1-18)had similar effects to naloxone. FMRFamide at the lowest dose had no significant effect on the number of bites required to induce defeat. Social Conflict and Food Intake. Ccntrol, defeated mice were observed not to eat or drink for approximately 20-30 min after the agonistic encounter: during this period the mice were engaged in grooming activities. In contrast, unpaired mice which did not experience any aggressive encounters showed no such similar behaviors. In consequence, during the first and second hour after the conflict situation, the defeated mice consumed significantly (~0.01) more food than did the unpaired control mice (Fig. 2). These differences disappeared during the third hour. Naloxone pre-treatment significantly (p
488
.lO
Sal. - y
1.0
FMRFamide
0
10 ,
Nx.
kg’1
i.c.v
Sal.
,
.Ol
10
1.0
FMRFamide
10
I
Nx.
[pg)
Figure 1 Effects of FMRFamide (.Ol-10 ,_g, i.c.v.1 and naloxone hydrochloride (Nx., 1.0 mg/kg i.p.) on A. thermal response latencies of subordinate mice following aggressive encounters, and B. the number of bites required to obtain defeat in subordinate mice. U represents control saline (2.0~1, i.c.v.) mice which have not experienced any aggressive encounters, D represents uninjected defeated mice and Sal. represents saline (2.0 ~1, i.c.v.) treated defeated mice. Vertical lines represent two standard errors of the mean. N=lO, in all cases.
489
0.6 I ti
5 Q) 0.4 % z B 9 0.2
72
i.p ia
0.0 (r
I c
U
i.p i.cr _
D
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D
$1
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Figure 2 Effects of FMRFamide (.Ol-1.0 bg, 1.c.v.) and naloxone hydrochloride (10 mglkg, i.p.) on the three hour food intake of defeated (D) mice. Saline injected (10 ml/kg, i.p.; 2.0 ~1, i.c.v.) and non-injected (C) defeated mice were used as controls. U represents the food intakes of unpaired mice not undergoing any aggressive encounters. N=lO, for Nx and FMRFamide treated mice; N=5 for control individuals. Vertical lines represent two standard errors of the mean.
490
was reduced to approximately IO-20 min. There was no consistent latency to the initiation of feeding following treatment with naloxone or the higher doses of FMRFamide. Post-conflict grooming in the naloxone and FMRFamide treated defeated mice was also reduced. DISCUSSION The present results show clearly that FMRFamide inhibits the analgesic and ingestive responses arising from the stress of social conflict and defeat in mice. As defeat-induced analgesia and feeding are associated with activation of opiate mechanisms (14), the present results provide strong evidence that FMRFamide opposes the central effects of endogenous opiates. The effects of FMRFamide on defeat-induced analgesia and feeding are analogous to the inhibitory actions of the prototypic opiate antagonist, naloxone (14). The results of these studies support then, the suggestion that either FKRFamide, or PMRFamide-like immunoreactive peptides present in the mammalian central nervous system (4,5), may have neuromodulatory or regulatory antagonistic actions on endogenous opioid systems. The central actions of FMRFamide on defeat-induced, opioid analgesia in mice are analogous to those observed in the rat, where injection of either FMRFamide or FMRFamide-like immunoreactive material into the spinal cord antagonizes morphine-induced analgesia (7). In addition, FHRFamide has also been demonstrated to inhibit opioid-mediated increases in nociceptive thresholds and analgesia in invertebrates (16). Together, these findings support the hypothesis that FMRFamide, or FMRFamide-like peptides, antagonize opioid-mediated analgesic mechanisms. Defeat-induced analgesia has been shown to be independent of the number of bites received by the vanquished mouse (13). The reduced number of bites obtained after treatment with either FMRFamide or naloxone does not, then, account for the lowered nociceptive thresholds that are observed in the defeated individuals. In addition, it has also been demonstrated that it is the opioid activation resulting from the defeat experience which determjnes the final nociceptive response (14). Thus, mice which receive a low number of bites can still display a high nociceptive threshold (12). The present observation does, however, indicates that FMRFamide and naloxone have modulatory and/or suppressive effects on opioid influenced agonistic encounters and aggressive interactions. Defeat-induced feeding is also suppressed in a dose-dependent manner by FMRFamide. This effect is similar to the inhibitory actions cf FMRFamide on morphine- and food-deprivation-induced feeding in mice (8). These inhibitory actions of E'MRFamideon opiate-induced feeding are analogous to those of naloxone. As noted above, there is evidence that food-deprivation- and defeat-induced feeding are mediated prjmarily by opiate mechanisms (11). Thus, the results of the present study also support the hypothesis that either FMRFamide, or PMRFamide-like peptides, can antagonize opioid-mediated ingestive responses. It should also be noted that the use of powdered food in the present study
491
obviates against feeding and the suppressive actions of FMRFamide being secondary to non-specific alterations in locomotor and oral behaviors. Furthermore, neither FMRFamide nor naloxone have any evident effects on the basal food-intake of free-feeding mice (8). Extensive FMRFamide-like immunoreactivity has been observed in the hippocampus, midbrain, brainstem and various nuclei of the hypothalamus (4,5). These brain regions have been implicated in a variety of behaviors and physiolgical activities that are mediated by endogenous opioid systems (17-19). Presumably, after i.c.v. injections, FMRFamide reached receptive elements or components of neuronal circuitry able to inhibit opioid systems that are activated by the stress of the agonistic encounter and ensuing defeat. This inhibitory role of FMRFamide may involve direct antagonism of endogenous epioid effects (7), activation of inhibitory neuronal pathways through excitatory neuronal effects (5,6), or actions on other neurotransmitters or neuromodulators involved in the expression of opioid effects. The similarities of response between FMRFamide and naloxone, as well as the absence of any evident effects in control animals, reduces the possibility that FMRFamide is causing non-specific disruptions of the analgesia measures and feeding. Although the mechanisms of action remains to be clarified, the present observations strongly suggest that FMRFamide, or FMRFamide-like peptides, may interact with endogenous opioid systems and modify their behavioral and physiological consequences. In conclusion, these observations provide further support for social conflict being a biologically relevant model for the investigations of endogenous opioid system activation, control and effects. ACKNOWLEDGEMENTS We thank Andrew Mathers for technical assistance. This research was supported by a National Science and Engineering Research Council of Canada grant (S222Al) to M.K. REFERENCES 1.
Price, D.A. and Greenberg, M.J. (1977). The structure of a molluscan cardioexcitatory neuropeptide. Science 197: 670-671.
2.
Cottrell, A. (1982). Fl$RFamideneuropeptides simultaneously increase and decrease K currents in an identified neurone. Nature 296: 87-89.
3.
Nagle, G.T. (1981). The molluscan cardioactive neuropeptide FMRFamide: subcellular localization in bivalve ganglia. Journal of Neurobiology 12: 599-611.
4.
Chronwall, B,M,, Olschowaka, J.A. and O'Donohue, L. (1984). Histochemical localization of FMRFamide-like immunoreactivity in the rat brain. Peptides 5: 569-584.
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5.
G'Donohue, T.L., Bishop, J.F., Chronwall, B.M., Groome, J. and Watson, W.H. (1984). Characterization and distribution of FMRFamide immunoreactivity in the rat central nervous system. Peptides 5: 563-568.
6.
Gayton, R.J. (1982). Mammalian neuronal actions 06 FMRE;amideand the structurally related opioid Met-endephalin-Arg -Phe . Nature 298: 276-276.
7.
Tang, J., Yang, Y.T. and Costa, E. (1984). Inhibition of spontaneous and opiate-modified nociception by an endogenous neuropeptide with Phe-Met-Arg-Phe-NH*-like immunoreactivity. Proceedings of the National Academy of Sciences (U.S.A.) 81: 5002-5005.
8.
Kavaliers, M., Hirst, M. and Mathers, A. (1985). Inhibitory influences of FMRFamide on morphine and deprivati_on-inducedfeeding. Neuroendocrinology (in press).
9.
Amir, S., Brown, Z.W. and Amit. Z. (1980). The role of endorphins in stress: evidence and speculations. Neuroscience and Riobehaviora! Reviews 4: 77-86.
10. Bodnar, R.J., Kelly, D.D., Brutus, M., and Glusman, M. (1980). Stress-induced analgesia: neural and hormonal determinants. Neuroscience and Biobehaviornl Reviews 4: 87-100. 11. Terman, G.W., Shavit, Y., Lewis, ,T.W.,Cannon. J.T. and Liebeskind, J.C. (1984). Intrinsic mechanisms of pain inhibition: activation by stress. Science 226: 1270-1277. 12. Miczek, K.A. and Thompson, M.L. (1984). Analgesia resulting from defeat in a social confrontation: The role of endogenous opioids in the brain. In: Bandler, R. (ed.) Neurology and neurobiology, Vol. 12, Modulation of sensorimotor activity during alterations in behavioral states, Alan R. Liss Press, New York, p. 431-456. 13. Miczek, K.A., Thompson, M.L. and Shuster, I,. (1982). Opioid-like analgesia in defeated mice. Science 215: 1520-1522. 14. Teskey, G.C., Lavaliers, M. and Hirst, M. (1984). Social conflict activates analgesic and ingestive behaviors in male mice. Life Sciences 35: 303-315. 1.5.Haley, T.J. and McCormick, W.G. (1957). Pharmacological effects produced by intracerebral injections of drugs in the conscious mouse. British Journal of Pharmacology 12: 12-15. 16. Kavaliers, M., Hirst, M. and Teskey, G.C. (1485). The effects of opioid and FMRFamide peptides on snail thermal behavior. Neuropharmacology (in press).
493
17. Morley, J.E., Levine, A.S., Yim, G.K.W. and Lowy, M.T. (1983).
Opiate modulation of appetite. Neuroscience and Biobehavioral Revl.ews7: 281-305. 18. Basbaum, A.I. and Fields, R.L. (1984). Endogenous pain control systems: Brainstem spinal pathways and endorphin circuitry. Annual Review of Neuroscience 7: 309-338. 19. Cuello, A.C. (1983). Central distribution of opioid peptides. British Medical Bulletin 39: 11-16.
Accepted 25/6/85
494
485-494,
1985
FMRFAMIDE, A PUTATIVE ENDOGFNOUS OPIATE ANTAGONIST: EVIDENCE FROM SUPPRESSION OF DEFEAT-INDUCED ANALGESIA AND FEEDING IN MICE Martin Kavaliers and Maurice Hirst Department of Zoology and Department of Pharmacology and Toxicology, University of Western Ontario, London, Ontario, Canada N6A 5B7 [reprints to MK] ABSTRACT Social conflict and defeat in mice leads to an activation of endogenous opiate systems. The effects of intracerebroventricular administration of the peptide FMRFamide (Phe-Met-Arg-Phe-NH*) and the opiate antagonist naloxone, on agrressive encounters, defeat-induced analgesia and defeat-induced feeding were examined in male mice. Both substances reduced the number of bites required to cause defeat in subordinate mice during aggressive encounters, as well as suppressing the subsequent defeat-induced analgesia. Administration of FMRFamide or naloxone also reduced defeat-induced feeding. These results indicate that FMRFamide (or FMRFamide-like neuropeptides) may function as endogenous opioid antagonists. INTRODUCTION The peptide Phe-Met-Arg-Phe-NH2 (FMRFamide) has been isolated from the ganglia of the Venus clam, Macrocallista nimbosa (1) and found to evoke a variety of cardiac and neuronal actions in invertebrates (l-3). FMRFamide-like immunoreactivity was subsequently demonstrated to have wide distribution in the central nervous system of mammals, with the highest concentrations occurring in the spinal cord and hypothalamus (4,5). This broad distribution led to the suggestion that FMRFamide, or FMRFamide-like peptides, may have regulatory roles in the mammalian central nervous system, which involve autonomic and endocrine regulation, as well as pain modulation (5). This speculation received support with the findings that iontophoretically applied FMRFamide has excitatory effects on mammalian neurons (6). it was demonstrated recently that both FMRFamide and an immunoreactive FMRFamide-like peptide purified from bovine brain could antagonize morphine-induced analgesia in rats (7). In addition, Tang et al. (7) showed that intrathecal or intracerebroventricular (i.c.v.)injections of antibody directed against FMRFamide induced long lasting analgesia and decreased tolerance to morphine-induced analgesia. These investigations suggested that FMRFamide, or related peptides in the 485
spinal cord, may affect pain threshold by functioning as endogenous opiate antagonists. Subsequently, Kavaliers -et al. (8) showed that i.c.v. injections of FMRFamide suppressed both morphine- and food deprivation-induced feeding in the mouse, in a manner analogous to that obtained after treatment with the opiate antagonist, naloxone. These data provide further support for the suggestion that either FMRFamide, or FMRFamide-like peptides, may function as physiological opiate antagonists. There is substantial evidence that after exposure to either physically or psychologically stressful situations endogenous opioid activity is increased in mammals (9-12). In both wild and laboratorybred mice, intraspecific aggressive interactions can result in the display of a specific defeat posture in the vanquished individual (12-14). This behavior has been considered to represent a generalized natural biological response to the stress of social confrontation (14). Defeat has been shown to result in activation of endogenous opiate systems (12), and the display of naloxone senstive analgesic and ingestive responses (14). In the present study we describe the effects of FMRFamide on the analgesic, behavioral and ingestive responses of mice subjected to the stress of social conflict and defeat. We show that FMRFamide can suppress these endogenous opiate activated responses, in a manner analogous to that observed with naloxone, and consistent with the proposal that FMRFamide, or related peptides, function as endogenous opiate antagonists. MATERIALS AND METHODS Animals Small (2-3 months of age, 25-30 gm) and large (6-8 months of age, 40-45 gm) male CF-1 mice (Charles River, Quebec) were used in these studies. The small mice were housed in groups of five, while the larger animals were held individually. All animals were kept under a 12 hour light: 12 hour dark cycle (LD 12:12, L 0700-1900 h, 20 VW/cm') at 22 f 1". Food (Purina mouse chow 5015) and water were available -ad libitum. Experimental Procedures Social Conflict and Nociceptive Responses. At mid-photophase groups of ten different small mice received either intraperitoneal (i.p.) injections of naloxone hydrochloride (1.0 mg/kg, Endo Laboratories, NJ) prepared in saline (10 ml/kg), saline vehicle (10 ml/kg), or i.c.v. injections of 0.01, 0.10, 1.0 and 10 ug (0.17, 1.7, 17 and 170 nmoles, respectively) of FMRFamide (Peninsula Laboratories, CA) dissolved in saline. Using the procedure of Haley and McCormick (15), mice received i.c.v. injections of FMRFamide in 2.0 ~1 (1.0 1.11in each side of the brain) or saline alone which served as a control. The i.c.v. injection interrupted the activity of the animals for approximately 2-3 min, after which they resumed apparently normal behaviors and activity. In order to gain an indication of the regions of the ventricular system to which the drugs penetrated, dilute India ink (2.0 ~1, 1.0 ~1 per side) was injected into over-anesthestized mice. The brains were dissected and
486
the ink was observed
to have penetrated
the ventricular
system.
Thirty minutes after i.p. or i.c.v. injection, a small mouse was placed in the home cage of a larger mouse. The number of bites received by the smaller mouse during this "resident-intruder" pairing was Once recorded until the intruder mouse displayed a full defeat posture. defeat was reached the encounter was terminated. Defeat was characterized by an upright position, limp forepaws, upward-angled head and retracted ears. The same large, aggressive resident mice were used in all of the studies. Results of previous investigations showed that the repeated aponistic interactions had no effects on the aggressive behavior of the larger resident mouse directed towards the small intruder (14). Each smell intruder mouse was used only once. Immediately following the aggressive encounter the nociceptive thresholds of the small intruder mice were assessed using a hot-plate Individual mice were placed onto the heated surface (50 + technique. 0.5'C) and the latency to a paw-licking response was recorded; after which the mouse was quickly removed from the surface and returned to the home cage. Response latencies of groups of ten other mice given saline (10 ml/kg i.p., 2.0 ~1, i.c.v.) but without experience of any social conflict were also determined. In feeding determinations, mice were Social Conflict and Food Intake. housed individually in elevated (20 cm diameter, 7 cm high) clear plastic small rodent metabolism units with wire mesh floors (E=llOO, Econo-metabolism Unit, Maryland Plastics, NY). A short (3. cm ) tunnel provided access to a food hopper in which powder-cd food (Purina mouse chow 5015) was placed. An aluminum ring in front of the hopper restricted entry and prevented the animals from placing their paws in the food. The animals readily consumed the powdered food by licking. Water was provided in a plastic graduated tube placed directly across from the food hopper. After three days of hahituation, the effects of either naloxone (1.0 mg/kg, i.p.> or FMRFamide (0.01, O.lC and 1.0 ug, i.c.v.) pretreatments (10 min) on the food uptake of the smaller mice fcllowing the "resident-intruder" pairing were examined. After the aggressilre encounter, defeated mice (n=lO, in all cases) were returned to the units and their food intakes were recorded over three hours. Saline injected (10 ml/kg, i.p.; 2.0 ~1. i.c.v.) mice (n=lO) serve6 as contrn?s. Determinations were made of food and water consumed at the end of each hour. Fresh food (2.0 gm) was provided each hour. P.ny food that was lost by scatter or spillage was collected and corrected for -in the hourly food intake determinations. In addition, the activities of the mice and time course of food intake were noted. Control determinations were made using young mice (n=5) given saline i.p., i.c.v. or not injected, all of which did not undergo social conflict. Data were analysed using Analysis Keuls multiple range tests.
487
of variance
and Student-Kewman-
RESULTS Social Conflict and Nociceptive Responses. Defeated i.c.v.-saline treated mice were analgesic, displaying significantly (pcO.01) greater latencies to respond to the aversive stimulus than control saline, treated mice not undergoing aggressive encounters (Fig. 1A). With regards to the control animals, there were no significant differences between the latencies of response of i.p.- or i.c.v.-saline injected or uninjected mice. Defeated mice receiving naloxone or FMRFamide displayed significantly (~~0.01 for 1.0 ug FMRFamide) lower dose-dependent latencies of response to the thermal stimulus than did the control_, defeated mice. Naloxone and FMRFamide (1.0 and 10 ug) suppressed defeat-induced analgesia (Fig. lA), the thermal response latencies of the injected animals being identical to those of undefeated animals. FMRFamide (0.01 and 0.10 ug) significantly reduced (160.05), but did not block defeat-induced analgesia. The number of bites required to produce the defeat posture was significantly reduced (pcO.01) in naloxone injected mice, as compared to saline-treated animals (Fig. 1R). FMRFamide caused a significant (pc.01 for 0.10 pg) dose-dependent decrease in the number of bites required to elicit defeat. FMRFamide (1.0 and 10 1-18)had similar effects to naloxone. FMRFamide at the lowest dose had no significant effect on the number of bites required to induce defeat. Social Conflict and Food Intake. Ccntrol, defeated mice were observed not to eat or drink for approximately 20-30 min after the agonistic encounter: during this period the mice were engaged in grooming activities. In contrast, unpaired mice which did not experience any aggressive encounters showed no such similar behaviors. In consequence, during the first and second hour after the conflict situation, the defeated mice consumed significantly (~0.01) more food than did the unpaired control mice (Fig. 2). These differences disappeared during the third hour. Naloxone pre-treatment significantly (p
488
.lO
Sal. - y
1.0
FMRFamide
0
10 ,
Nx.
kg’1
i.c.v
Sal.
,
.Ol
10
1.0
FMRFamide
10
I
Nx.
[pg)
Figure 1 Effects of FMRFamide (.Ol-10 ,_g, i.c.v.1 and naloxone hydrochloride (Nx., 1.0 mg/kg i.p.) on A. thermal response latencies of subordinate mice following aggressive encounters, and B. the number of bites required to obtain defeat in subordinate mice. U represents control saline (2.0~1, i.c.v.) mice which have not experienced any aggressive encounters, D represents uninjected defeated mice and Sal. represents saline (2.0 ~1, i.c.v.) treated defeated mice. Vertical lines represent two standard errors of the mean. N=lO, in all cases.
489
0.6 I ti
5 Q) 0.4 % z B 9 0.2
72
i.p ia
0.0 (r
I c
U
i.p i.cr _
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D
$1
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Figure 2 Effects of FMRFamide (.Ol-1.0 bg, 1.c.v.) and naloxone hydrochloride (10 mglkg, i.p.) on the three hour food intake of defeated (D) mice. Saline injected (10 ml/kg, i.p.; 2.0 ~1, i.c.v.) and non-injected (C) defeated mice were used as controls. U represents the food intakes of unpaired mice not undergoing any aggressive encounters. N=lO, for Nx and FMRFamide treated mice; N=5 for control individuals. Vertical lines represent two standard errors of the mean.
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was reduced to approximately IO-20 min. There was no consistent latency to the initiation of feeding following treatment with naloxone or the higher doses of FMRFamide. Post-conflict grooming in the naloxone and FMRFamide treated defeated mice was also reduced. DISCUSSION The present results show clearly that FMRFamide inhibits the analgesic and ingestive responses arising from the stress of social conflict and defeat in mice. As defeat-induced analgesia and feeding are associated with activation of opiate mechanisms (14), the present results provide strong evidence that FMRFamide opposes the central effects of endogenous opiates. The effects of FMRFamide on defeat-induced analgesia and feeding are analogous to the inhibitory actions of the prototypic opiate antagonist, naloxone (14). The results of these studies support then, the suggestion that either FKRFamide, or PMRFamide-like immunoreactive peptides present in the mammalian central nervous system (4,5), may have neuromodulatory or regulatory antagonistic actions on endogenous opioid systems. The central actions of FMRFamide on defeat-induced, opioid analgesia in mice are analogous to those observed in the rat, where injection of either FMRFamide or FMRFamide-like immunoreactive material into the spinal cord antagonizes morphine-induced analgesia (7). In addition, FHRFamide has also been demonstrated to inhibit opioid-mediated increases in nociceptive thresholds and analgesia in invertebrates (16). Together, these findings support the hypothesis that FMRFamide, or FMRFamide-like peptides, antagonize opioid-mediated analgesic mechanisms. Defeat-induced analgesia has been shown to be independent of the number of bites received by the vanquished mouse (13). The reduced number of bites obtained after treatment with either FMRFamide or naloxone does not, then, account for the lowered nociceptive thresholds that are observed in the defeated individuals. In addition, it has also been demonstrated that it is the opioid activation resulting from the defeat experience which determjnes the final nociceptive response (14). Thus, mice which receive a low number of bites can still display a high nociceptive threshold (12). The present observation does, however, indicates that FMRFamide and naloxone have modulatory and/or suppressive effects on opioid influenced agonistic encounters and aggressive interactions. Defeat-induced feeding is also suppressed in a dose-dependent manner by FMRFamide. This effect is similar to the inhibitory actions cf FMRFamide on morphine- and food-deprivation-induced feeding in mice (8). These inhibitory actions of E'MRFamideon opiate-induced feeding are analogous to those of naloxone. As noted above, there is evidence that food-deprivation- and defeat-induced feeding are mediated prjmarily by opiate mechanisms (11). Thus, the results of the present study also support the hypothesis that either FMRFamide, or PMRFamide-like peptides, can antagonize opioid-mediated ingestive responses. It should also be noted that the use of powdered food in the present study
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obviates against feeding and the suppressive actions of FMRFamide being secondary to non-specific alterations in locomotor and oral behaviors. Furthermore, neither FMRFamide nor naloxone have any evident effects on the basal food-intake of free-feeding mice (8). Extensive FMRFamide-like immunoreactivity has been observed in the hippocampus, midbrain, brainstem and various nuclei of the hypothalamus (4,5). These brain regions have been implicated in a variety of behaviors and physiolgical activities that are mediated by endogenous opioid systems (17-19). Presumably, after i.c.v. injections, FMRFamide reached receptive elements or components of neuronal circuitry able to inhibit opioid systems that are activated by the stress of the agonistic encounter and ensuing defeat. This inhibitory role of FMRFamide may involve direct antagonism of endogenous epioid effects (7), activation of inhibitory neuronal pathways through excitatory neuronal effects (5,6), or actions on other neurotransmitters or neuromodulators involved in the expression of opioid effects. The similarities of response between FMRFamide and naloxone, as well as the absence of any evident effects in control animals, reduces the possibility that FMRFamide is causing non-specific disruptions of the analgesia measures and feeding. Although the mechanisms of action remains to be clarified, the present observations strongly suggest that FMRFamide, or FMRFamide-like peptides, may interact with endogenous opioid systems and modify their behavioral and physiological consequences. In conclusion, these observations provide further support for social conflict being a biologically relevant model for the investigations of endogenous opioid system activation, control and effects. ACKNOWLEDGEMENTS We thank Andrew Mathers for technical assistance. This research was supported by a National Science and Engineering Research Council of Canada grant (S222Al) to M.K. REFERENCES 1.
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Accepted 25/6/85
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