Reduction of learned helplessness by central administration of quaternary naltrexone

Physiology&Behavior,Vol. 51, pp. 1075-1078, 1992

0031-9384/92 $5.00 + .00 Copyright© 1992 PergamonPressLtd.

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BRIEF COMMUNICATION

Reduction of Learned Helplessness by Central Administration of Quaternary Naltrexone J O S H U A E. B L U S T E I N , *1 W A Y N E G. W H I T E H O U S E , ~ " D A N I E L J. C A L C A G N E T T I , : ~ J O S E P H R. T R O I S I II,§ D A V I D L. M A R G U L E S t A N D P H I L I P J. BERSHi-

*Beaver College, Glenside, PA, ?Temple University, Philadelphia, PA, ¢Northeastern Ohio University College of Medicine, Rootstown, OH, and §Johns Hopkins University, Baltimore, MD Received 16 August 1991 BLUSTEIN, J. E., W. G. WHITEHOUSE, D. J. CALCAGNETTI, J. R. TROISI II, D. L. MARGULES AND P. J. BERSH. Reduction of learned helplessness by central administration of quaternary naltrexone. PHYSIOL BEHAV 51(5) 1075-1078, 1992.--Prior research has established that escape impairment resulting from prior inescapable shock (IS) could be reversed by the peripheral administration of the opiate antagonist naltrexone (NTX), but not the quaternary form of naltrexone (QNTX), which when systemically administered, does not readily pass the blood-brain barrier. As it was unclear whether the failure of systemically administered QNTX to reduce shuttle escape deficits following exposure to IS could be attributed to reasons other than the restricted access of QNTX to receptor sites in the brain, rats were affixed with chronic indwelling ventricular cannulae to allow direct brain administration of QNTX. The present experiment found a significant attenuation of the escape deficit produced by prior inescapable shock following the intracerebroventricular (ICV) administration of QNTX ( 10 ug/rat). These data provide further evidence of a mediational role for central opiate receptors in the expression of escape interference following inescapable shock. Quaternary naltrexone

Intracerebroventricular

Learned helplessness

WHETHER or not organisms can control aversive events has widespread behavioral and physiological consequences. Exposure to uncontrollable (inescapable) shock may impair subsequent learning of responses to escape shock (10) and Pavlovian conditioned responses (1), in addition to activating various neurochemical alterations that result in decreased activity (2), diminished responsiveness to painful stimuli (7), and increased stress symptomatology (14). Because these aftereffects are not observed following exposure to equivalent amounts of controllable shock, the uncontrollability of the shock, and not the shock per se, is thought to be responsible for these consequences. Recent studies have suggested a potential opioid basis for the finding that animals exposed to uncontrollable (inescapable) shock exhibit subsequent difficulty in acquiring a novel escape response, an effect commonly referred to as the learned helplessness phenomenon (10). It has been reported that the opiate antagonist naltrexone can block escape deficits in a dose-dependent manner, when administered prior to inescapable shock (5), or 24 h later, before testing animals for acquisition of a shockescape response (16). In contrast to its effect on the escape performance of rats receiving inescapable shock, naltrexone has been found to impair otherwise competent shuttlebox escape acquisition in animals trained with escapable shock, as well as

in shock-naive rats (16). More recently we (15) found that subcutaneously (SC) delivered QNTX (a methyl derivative of naltrexone which does not readily cross the blood brain barrier) (3) in doses as high as 50 mg/kg failed to block escape deficits in inescapably shocked rats. However, even 1 mg/kg of naltrexone attenuated interference with escape acquisition produced by prior inescapable shock. At the same time, both compounds induced escape deficits in animals previously trained with escapable shock and in no-shock control animals. Collectively, these findings suggest that the mechanism for learned helplessness is centrally located, whereas the escape impairment induced by naltrexone in animals previously trained with escapable shock appears to be peripherally mediated. Recently, several studies have applied the methodology of administering QNTX ICV and comparing its effectiveness to peripheral administration (4,6). The present study applied this same method to investigate two hypotheses. First, if the failure of subcutaneously administered QNTX to ameliorate the learned helplessness effect (in doses as high as 50 mg/kg) is due to its inability to gain access to central opioid receptors, then ICV administration of QNTX should reliably reduce the learned helplessness effect compared to animals administered subcutaneous QNTX. Second, if the induction of escape deficits by

1Requests for reprints should be addressed to Joshua E. Blustein, Department of Psychology, Beaver College, Glenside, PA, 19038.

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QNTX in escapably shocked rats is due to its peripheral activity, then ICV administration of QNTX should fail to induce escape deficits in these animals. If QNTX acts to reduce the learned helplessness efl~ct following its ICV administration, then its failure to act as an antagonist when peripherally administered is likely to be the result of its inability to readily cross the blood brain barrier. A single dose of 10 #g/rat was selected based upon pilot work.

0.5 cm in diameter and measuring 0.95 cm from center to center, through which shock was delivered. Scrambled constant-currem AC shock (1.0 mA) was delivered simultaneously to both sides of the shuttle box by a Coulbourn Instruments E l 3- l 6 shocker. All programming equipment was located in a room adiacenl to the experimental chambers.

METHOD

At the start of pretraining, rats were randomly assigned to a triad, consisting of an escapable shock (ES), a yoked inescapable shock (IS), and a no-shock (NS) rat. Animals in a given triad were placed in their respective chambers, in which ES and IS rats received 80 shocks delivered on a variable time 60-s schedule. Following 1 s of exposure to inescapable shock ES animals could terminate the shock with a single depression of the response lever. Failure to emit a single lever press response resulted in shock lasting for 30 s. Inescapable shock rats were yoked to their ES partners such that they received the identical intensity and duration of shock; however, their behavior had no effect on shock termination. No-shock animals remained in the chamber for the duration of the session, but did not receive shock. Twenty'four hours following shock pretraining, all subjects were given shock-escape testing in a shuttle box. Fifteen minutes prior to the test. one-half of the animals in each controllability condition received QNTX (either 10 mg/kg SC, or 10 ug/rat ICV), whereas the remaining animals received the saline vehicle either SC or ICV. The first 5 trials required a single crossing response (FR 1) to terminate the shock, whereas the subsequent 30 trials required a double crossing response (FR 2) to terminate the shock. Failure to meet the response requirement within 60 s resulted in automatic shock termination. A variable time shock schedule with a mean of 60 s and a range of 10-110 s was used. Shock intensity for both pretraining and test phases was t.0 mA.

Subjects Eighty-seven female rats of Long-Evans descent (200-280 g) served as subjects. Subjects were individually housed in hanging stainless steel wire mesh cages and provided ad lib food and water. All manipulations were conducted during the light phase of a 10:14 h light:dark cycle.

Surgery, Drugs, and Injections Rats were anesthetized with subcutaneous injections of ketamine hydrochloride ( 1 mg/ml per kg body weight). A 22-gauge stainless steel outer cannula (Plastics One, Roanoke, VA) was stereotaxically implanted with the tip targeted within the right lateral ventricle (coordinates used were 0.4 mm posterior to bregma, 1.4 mm lateral to midline, and 3.1 mm ventral to the surface of the cortex), with the skull leveled between lambda and bregma. Following surgery, the rats were permitted a 7-12 day recovery interval prior to shock training. The QNTX was dissolved in 0.9% saline (2.5 #g/ul). The ICV injections were delivered by backloading the drug up a 27-gauge internal cannula (Plastic One, Roanoke, VA) into PE-50 tubing (Intramedic No. 7406) and inserting the internal cannula into the restrained rat's external cannula. An injection volume of 4 tzl was delivered by a 5 ul syringe (Hamilton No. 7105) mounted in a repeating pushbutton device (Hamilton No. PB 00-1) at a rate of 4 ul/ 40s. The total dose of QNTX given was 10 ug/rat. Saline (0.9%) served as the vehicle as well as the control injection. One-half of the animals received subcutaneous injections (SC) of either QNTX (10 mg/kg) or the vehicle saline.

Apparatus Pretraining was carried out in two identical operant chambers, with the dimensions 30.2 x 24.0 × 36.8 cm (L X W X H). The front and back wall of each chamber was constructed of stainless steel, whereas the ceiling and two side walls were made of clear Plexiglas. A stainless steel lever protruded 2.5 cm into each chamber from the left corner of the front wall, and required a force of 10 g (0.1 N) to depress. The chamber was also equipped with a 7.5-W houselight, located in the center of the ceiling. The floor consisted of a grid of stainless steel bars affixed perpendicularly to the side walls, 0.5 cm in diameter and spaced 1.8 crn apart (center to center). Scrambled constant-current AC shocks, 1.0 mA in intensity, were delivered through the stainless steel bars comprising the grid floor of each chamber by a Coulbourn Instruments Model E 13-16 solid state shocker]distributor. No shocks were delivered in the third chamber. All chambers were enclosed within sound attenuating cubicles. Escape testing was conducted in a two-way shuttle box, 46.0 × 19.0 × 22.5 (L × W × H), enclosed in a sound-attenuating chamber. The inside of the shuttle box consisted of stainless steel end walls and Plexiglas sidewalls and ceiling. A stainless steel partition, with rounded archway, 6.0 × 7.0 cm, bisected the chamber. Each half had a house light mounted on its end wall to provide diffuse illumination and 10 stainless steel grids,

Procedur e

ttLstology At the conclusion of the experiment, all subjects were sacrificed by pentobarbital overdose and cannula placements were verified. The cannulated subjects were injected ICV with 2 ~1 of ink (Hunt/Speedball India ink No. 3338) and the rats were perfused transcardially with 10% buffered formalin. The brains were removed, and coronal sections were made along the cannula tract. Cannulae placements were visually inspected for the presence of ink lining the ventricular spaces. Three animals were excluded from the statistical analysis due to incorrect cannula placements. RESULTS A one-way analysis of variance carried out on the performance of ES rats during training was not statistically reliable (F < 1.0). Accordingly, all treatment groups received comparable amounts of preshock. An analysis of variance conducted on the mean latency to complete the FR 1 response revealed a significant main effect for controllability, F(2, 72) = 4.34, p < 0.02. Neither the drug factor nor the drug × controllability interaction approached significance. Newman-Keuls post hoc tests (p < 0.05) revealed that NS animals were faster to escape than either ES or IS animals. No reliable differences were found between the latter groups. Figure 1 illustrates the mean latency to complete the FR 2 response as a function of controllability, injection route, and drug type. These data are of primary interest, because FR 2 escape has been found to be selectively impaired by prior inescapable shock (9). Three planned non-orthogonal contrasts,

CENTRAL OPIATES AND HELPLESSNESS

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CONTROLLABILllY

FIG. I. Mean escape latencies and standard errors during FR 2 escape trials at three levels of controllability as a function of route of administration and drug (10 mg/kg SC and 10 #g/rat ICV) or vehicle (0.9% saline).

using the Dunn-Bonferroni correction procedure, revealed that, when data were pooled across injection route, FR 2 latency was reliably longer for saline IS animals than for ES animals, t(72) = 6.02, p < 0.001, and NS animals, t(72) = 8.39, p < 0.001, whereas ES and NS animals did not reliably differ (t > 1.0). Thus, the conditions of the present experiment produced the typical learned helplessness effect for saline-treated animals. The central issue of the present experiment was addressed by three planned orthogonal contrasts. First, IS animals given QNTX showed a reliable reduction in escape interference when the drug was administered ICV, F(1, 72) = 24.1, p < 0.001, compared to SC. In addition, QNTX impaired escape performance in ES and NS animals when subcutaneously administered, F( l, 72) = 13.96, p < 0.001, and F(1, 72) = 8.75, p < 0.01 respectively, relative to ICV. DISCUSSION In an earlier set of experiments (15), systemic injection of NTX blocked escape impairment produced by prior exposure to inescapable shock, but its quaternary derivative was without effect. This implies that these effects of inescapable shock are principally mediated by opiate receptors in the brain, because the actions of moderate doses of quaternary opiate antagonists

are normally restricted to the periphery (13) and, therefore, ought to be inetficacious in antagonizing centrally mediated opioid actions. However, this conclusion is based on the absence of an effect, which could be attributed to factors other than the restricted access of a quaternary opioid compound to receptor sites in the brain. The present study demonstrated that ICV administered QNTX produced a reliable reduction of escape latencies in those animals previously exposed to inescapable shock. Our claim that shuttle escape deficits following inescapable shock are centrally mediated is strengthened by the fact that a central dose nearly 300 times less than the peripheral dose reliably reduced escape deficits. In contrast, the same compound peripherally injected failed to affect escape latencies in animals otherwise identically treated. Clearly, these data strengthen the interpretation that the failure of peripherally administered QNTX to improve FR 2 shuttle escape performance in rats previously exposed to inescapable shock is the consequence of its poor access to central opioid receptors (15). Furthermore, the disruption of escape acquisition by opiate antagonists in rats exposed to escapable shock or to no shock (15,16) was confirmed here to be the result of an action of these antagonists at receptor sites in the peripheral nervous system. Thus, the ICV administration of QNTX did not impair escape acquisition among ES and NS rats, whereas SC administration did. A major question these data raise concerns the brain mechanism(s) and receptor site(s) that play a role in impairing escape performance following exposure to inescapable shock. Earlier work (8) demonstrated that lesions of the ventromedial septum either attenuated or eliminated several inescapable shock-induced phenomena, including escape deficits. Autoradiographic identification of receptor sites has revealed dense populations o f m u opioid receptors in the medial nucleus of the septum (l 1). More recently, it has been demonstrated that animals exposed to inescapable shock, but not escapable shock, show decreased opioid binding at the mu receptor in the brain (12). Taken together, these data suggest a role for mu receptors of the medial septum in learned helplessness. Although it is still not entirely clear which brain opioid pathways and receptor types (or subtypes) mediate escape deficits following exposure to inescapable shock, data from the present study and others (15) provide strong evidence that escape deficits following exposure to inescapable shock are centrally mediated through an endogenous opioid system. ACKNOWLEDGEMENT We would like to thank Dr. H. Merz of Boehringer-lngelheim for his generous donation of naltrexone methobromide.

REFERENCES 1. Alloy, L. B.; Ehrman, R. N. Instrumental Pavlovian transfer: Learning about response-reinforcer contingencies affects subsequent learning about stimulus-reinforcer contingencies. Learn. Motiv. 12: 109-132; 1981. 2. Anisman, H.; deCatanzaro, D.; Remington, G. Escape performance deficits following exposure to inescapable shock: Deficits in motor response maintenance. J. of Exp. Psychol. Anim. Behav. Proc. 4(3): 197-218; 1978. 3. Brown, D. R.; Goldberg, L. J. The use of quaternary narcotic antagonists in opiate research. Neuropharmacology 24:18 l - 191; 1985. 4. Calcagnetti, D. J.; Helmstetter, F. J.; Fanselow, M. S. Quaternary naltrexone reveals the central mediation of conditional opioid analgeria. Pharmcol. Biochem. Behav. 27:529-531; 1987.

5. Drugan, R. C.; Maier, S. F. Analgesic and opioid involvement in the shock-elicitedactivity and escape deficitsproduced by inescapable shock, Learn. Motiv. 14:30-47; 1983. 6. Fanselow, M. S.; Calcagnetti, D. J.; Helmstetter, F. J. Peripheral versus intracerebroventricular administration of quaternary naltrexone and the enhancement of Pavlovian conditional fear. Brain. Res. 444:147-152; 1988. 7. Grau, J. W.; Hymn, R. L.; Maier, S. F.; Madden, J.; Barchas, J. D. Long-term stress-inducedanalgesiaan activation of the opiate system. Science 213:1409-141 l; 1981. 8. Kelsey, J. E.; Baker, M. D. Ventromedial septal lesions in rats reduce the effectsof inescapable shock on escape performance and analgesia. Behav. Neurosci. 97:945-961; 1983.

1078 9. Maier. S. F.; Albin, R. W.: Testa, T. Failure to learn to escape in rats previously exposed to inescapable shock depends on the nature of escape response. J. of Comp. Physiol. Psychol. 85:581-592: 1973. 10. Maier, S. F.; Seligman, M. E. P. Learned helplessness: Theory and evidence. J. Exp. Psychol. Gen. 105:3-46; 1976. 11. Mansour, A.; Khachaturian, H.; Lewis, M. E.; Akli, H.; Watson, S. J. Autoradiographic differentiation of mu, delta, and kappa opioid receptors in the rat forebrain and midbrain. J. Neurosci. 7:24452464: 1987. 12. Stuckey, J.; Marra, S.; Minor, T.; Insel, R. Changes in mu opiate receptors following inescapable shock. Brain. Res. 476:167-169; 1989.

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13. Valentino, R. J.; Herling, S.; Woods, J. H.; Medzihradsk), [:4 itcrz, H. Quaternary naltrexone: Evidence for the central mediation o1 discriminative stimulus effects of narcotic agonists and antagonisls J, Pharmacol. Exp. Therap. 217:652-659: 1981. 14. Weiss, J. M. Effects of coping responses on stress. J ('ornp. Physical. Psychol. 65:251-260:1968. 15. Whitehouse, W. G.: Blustein, J. E.: Walker, J.; Bersh, P. J.: Margulcs, D. L. Shock controllability and opioid substrates of escape performance and nociception: Differential effects of peripherally and centrally acting naltrexone. Behav. Neurosci. 99:717-733; 1985. 16. Whitehouse, W. G.: Walker, J.; Margules, D. L.: Bersh, P. J. Opiate antagonists overcome the learned helplessness effect but impair competent escape performance. Physiol. Behav. 3(1:73 I--734; 1983.