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Effects ofd -Ala2-Met5-enkephalinamide microinjections placed into the bed nucleus of the stria terminalis upon affective defense behavior in the cat
Brain Research, 473 (1988) 147-152 Elsevier
147
BRE 23183
Effects of D-Ala2-MetS-enkephalinamide microinjections placed into the bed nucleus of the stria terminalis upon affective defense behavior in the cat Martin Brutus, Shawki Zuabi and Allan Siegel Department of Neuroscience, UMDNJ-NewJersey MedicalSchool, Newark, NJ 07103 (U.S.A.) (Accepted 19 July 1988) Key words: Affective defense behavior; Bed nucleus of stria terminalis; D-Ala2-MetS-enkephalinamide; Naloxone; Medial hypothalamus
This study examined the effects of intracerebral injections of D-Ala2-Met5-enkephalinamide (DAME) upon hypothalamicaUy elicited hissing behavior in the cat. The bed nucleus of the stria terminalis (BNST) was selected for investigation because of its anatomical connections with the medial hypothalamus, its relatively high concentrations of enkephalins and opiate receptors and its demonstrated ability to modulate hypothalamically elicited aggressive reactions in the eat. DAME microinjected into the BNST in 1.0 or 10.0 pg/0.5 /~l quantities resulted in significant dose dependent increases in mean latencies for elicitation of the hissing response, Suppression of hissing following the 1.0 pg dose of DAME was selectively diminished by prior administration of naloxone. These findings suggest that the opiate receptors within the BNST play a role in the regulation of the hissing component of hypothalamieally elicited affective defense behavior.
Systemically injected naloxone has been shown to profoundly facilitate affective defense behavior elicited from either the hypothalamus 6 or midbrain central gray 34 of the cat. These effects suggest that endogenous opioids play a role in the modulation of aggressive behavior. Furthermore, it is reasonable to assume that such modulation is achieved by the actions of opioids upon those structures situated within the 'limbic-midbrain' which are known to elicit or regulate affective defense behavior 2'3,5,6A5' 29,32,36-38,42
In our research program, we have been attempting to identify the brain sites at which opioid peptides function to control affective defense behavior. In the present study, we have focussed upon the role of the bed nucleus of the stria terminalis (BNST). This structure was selected as a logical candidate for examination because: (1) it contains opioid receptors l's'19 and enkephalin-positive cells, fibers and terminals17'3°'4°'41'44; (2) it receives a major afferent sup-
ply from the amygdala 2°,43, which is known to modulate affective defenseS'1°-12; (3) BNST neurons project directly to the medial hypothalamus 7,9,39,43, an area classically associated with regulation of affective defense; and (4) electrical stimulation of the BNST modulates affective defense behavior 33. The findings reported below constitute an initial study which identifies the BNST as an important component of the limbic forebrain in opioid peptide neuromodulation of affective defense behavior. Eleven adult cats of either sex (Barton Farms, Oxford, NJ) weighing between 2.5 and 4.0 kg were employed in this study. They were maintained on an ad libitum feeding and drinking schedule throughout the duration of the experiments. The cats were anesthesized with sodium pentobarbital (45 mg/kg of b.wt.) prior to aseptic surgical procedures. Seventeen gauge stainless-steel guide tubes were stereotaxically mounted on both sides of the skull directly over the medial hypothalamus, BNST, and caudate nucleus.
Correspondence: A. Siegel, Department of Neuroscience, New Jersey Medical School, Medical Science Building, Room H512, 185 South Orange Avenue, Newark, NJ 07103, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
148 Coordinates were derived from the atlas of Jasper and A j m o n e - M a r s a n is. In addition, 3 bolts were anchored to the surface of the skull with dental acrylic to provide a means by which the electrode-cannula array could later be attached to a headholder. Details concerning the types of electrodes, cannulae and surgical p r o c e d u r e s utilized are described elsewhere 4'29. All experiments were conducted in a test chamber constructed of wood (61 x 61 x 61 cm) with a clear plexiglas door. Electrical stimuli were generated by a Grass S-88 stimulator and passed through a constant current photoelectric isolation unit (Grass PSIU6) to each animal. M o n o p o l a r stimulation of the hypothalamus consisted of balanced biphasic pulses delivered at 62.5 Hz, with a pulse width of 1 ms per half cycle duration. P e a k - t o - p e a k current was measured on a Tektronix 5113 dual b e a m oscilloscope with differential inputs. A p p r o x i m a t e l y one week following recovery from surgery, an electrode was vertically lowered through a guide tube overlying the hypothalamus and the brain was stimulated at 0.5 mm steps as the electrode was passed through the medial hypothalamus. W h e n affective defense was consistently elicited from a site by electrical stimulation, the electrode was c e m e n t e d in place. Affective defense behavior was elicited during stimulation at a given site located within the rostrocaudal extent of the medial hypothalamus. Responses elicited from any of these sites were of a similar nature, regardless of the anatomical locus of stimulation. Maps of these sites are shown in Fig. 1A. Affective defense behavior is characterized by sympathetic autonomic responses, threat responses such as hissing, growling and unsheathing of the claws and directed attack upon a rat or conspecific 1424'26'36"37. The hissing response was utilized because it is a fundamental component of the affective defense reaction 21 and is always present during the occurrence of this behavior. The latency to hiss was defined as the duration of time required to elicit hissing following the onset of stimulation. After the response was fully characterized as affective defense behavior in the freely moving animal, each cat was restrained in a h e a d h o l d e r similar to one that has been extensively e m p l o y e d in our laboratory 29'35 and elsewhere 2223'38. Painless immobilization was achieved by first placing the animal in a cat bag
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Fig. 1. A: maps of hypothalamic sites from which hissing behavior was elicited by electrical stimulation. B: maps of sites in the bed nucleus of the stria terminalis into which o-Ala2-MetLen kephalinamide (DAME) was microinjected. When DAME was injected into these sites (filled circles), it suppressed the hissing component of hypothalamically elicited affective defense behavior. In contrast, similar injections placed into the caudate nucleus (open circles) had no effects upon this behavior. AC, anterior commissure; AH, anterior hypothalamus; BNST, bed nucleus of the stria terminalis; Cd, caudate nucleus; Fx, Fornix; LH, lateral hypothalamus; MM, mammillary bodies; NA, nucleus accumbens; OC, optic chiasm; OT, optic tract: PH, posterior hypothalamus; PO, preoptic region: RE. nucleus reuniens, VMH, ventromedial hypothalamus.
so that it could lay comfortably, but also move its limbs during the testing periods. Then, the acrylic electrode cap was fastened to a brass h e a d h o l d e r 22'23 so that the position of the head would be fixed in space. This permitted precise intracranial delivery of drugs to telencephalic sites along with controlled stimulation of the hypothalamus. Each cat was exposed to the restrainer at least 10 times prior to experimental testing or until it showed no overt signs of discomfort or stress. Comparisons of the latency to hiss in the restrained and freely moving cat indicated that restraint had no effects upon this response. A f t e r the animals were fully habituated to the headholder, stimulation was applied to the medial
149 hypothalamus at the lowest current level that reliably elicited hissing within 20 s. For each animal, current values delivered to the hypothalamus were held constant during an entire test session. Baseline latencies for hissing ranged from 1.0 to 12.0 s. The current intensity delivered to the hypothalamus ranged from 0.1 to 0.8 mA. Baseline hissing latencies were recorded over a half hour test period prior to the administration of DAME or vehicle into the BNST or caudate nucleus. Following focal intracerebral injections, hissing latencies were again determined over 4 consecutive 30 min periods of time (i.e. 5-30, 30-60, 60-90, 90-120), with 12 stimulation trials delivered in each period. The interstimulus interval was 2 min. The opioid agonist D-Ala2-MetS-enkephalinamide (DAME) was selected for study because of its capacity to act on several opiate receptors 16'2s. DAME
A
(Bachem, Torrence, CA) was dissolved in 0.9% sterile normal saline (0.5MI) and buffered with NaOH to pH 7.4. A 0.5 MI SGE (Scientific Glass Engineering) microsyringe, which had been passed through a cannula implanted into either the BNST or the overlying caudate nucleus, was utilized to inject DAME or an equal volume of vehicle into these structures. Two doses of DAME were tested: 1.0 and 10.0Mg. All animals received both doses of DAME in a randomized order. In 3 cats, BNST sites were also tested following pretreatment with the opiate receptor antagonist naloxone (10 Mg/0.5 MI, pH = 7.4) 15 min prior to delivery of DAME (1/~g) into the same site. In the 15 min period between naloxone pretreatment and delivery of DAME, hissing latencies were recorded in order to determine whether naloxone administration alone could alter latencies for this response. As a control
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Fig. 2. A: microinjections of DAME placed into the BNST resulted in an increase in response latencies for hissing in a dose dependent manner. DAME induced increases in hissing latencies returned to baseline levels in a time dependent manner. Each of the points and error bars on the curves depicted above, represent the mean and S.E.M. for all stimulation trials attempted during each of the 4 halfhour testing periods following the microinjection of DAME or vehicle. Neither vehicle control injections, nor DAME injections placed into the caudate nucleus altered latencies for hissing (n = 6 for BNST; n = 5 for caudate nucleus). B: (I) Effects of DAME: microinjections of DAME placed into BNST significantly suppressed hissing in 3 cases (P < 0.05) in which naloxone pretreatment was later attempted (in II). Vehicle injected alone did not alter latencies for hissing. (II) Pretreatment with naloxone: when naloxone (10 #g) was injected into these 3 BNST sites prior to infusion of DAME (1 Mg), the suppression of hissing seen in 'I' was blocked (P < 0.05). Naloxone, injected into BNST alone, did not alter latencies for hissing (n = 3).
150 for volume effects here, vehicle alone was microinjected into the BNST in amounts equal to the combined volume of the naloxone and D A M E solutions that had been injected in the experimental condition. Response latencies were then again determined. At the completion of the experiments, animals were perfused with 0.9% saline and 10% formalin. Brains were sectioned at 50 ~m and stained with cresyl violet for localization of electrode and cannula tips. D A M E injected into 7 sites located in the BNST (n = 6 cats, see Fig. 1B) suppressed the hissing response as indicated by marked increases in the latency of this response following stimulation of the hypothalamus. D A M E placed into the BNST produced a dose dependent increase in hissing latencies (Fig. 2A). An analysis of variance which compared the drug dose for vehicle, 1.0 and 10.0/~g of D A M E , indicated that the response latencies were significantly increased in the first (F = 6.13, df = 3,19, P < 0.01) and second 30 min periods (F = 4.32, df = 3,18, P < 0.025) after the injection. Maximal effects of D A M E administration were attained in the first half hour after the injections. During this time, a dose of 1/~g of D A M E produced an increase of over 100% in the mean latency to hissing (P < 0.025). The 10 ~g dose of D A M E increased the mean latency by almost 300%. Both increases were calculated as changes relative to baseline control values. In the third half-hour period following injection with either dose of drug, response latencies were no longer found to be significantly different from baseline values. Control injections of the vehicle had no effect on hissing latency at any time point. In 3 of the BNST sites examined, naloxone (10/xg) injected 15 min prior to administration of a minimum effective dose of DAME, blocked the suppressive effects of D A M E (P < 0.05). These results are summarized in Fig. 2B. Naloxone, though blocking the action of D A M E , did not itself alter hissing latencies. In contrast to the effects of D A M E microinjections at BNST sites, injections of D A M E (1 ktg)
This research was supported by NIH Grant NS 07941-19 awarded by the National Institutes of Neurological and Communicative Diseases and Stroke. We would like to thank Drs. Henry Edinger and Hreday Sapru for their valuable suggestions for the improvement of this manuscript.
1 Atweh, S.F. and Kuhar, M., Autoradiographic localization of opiate receptor in rat brain. III. The telencephalon, Brain Research, 134 (1977) 393-405. 2 Bandler, R. and McCullock, T., Afferents to a midbrain periaqueductal region in the defense reaction in the cat as
revealed by horseradish peroxidase. II. The diencephalon, Behav. Brain Res., 13 (1984) 279-285. 3 Bandler, R., Identification of hypothalamic and midbrain neurons mediating aggressive and defensive behavior by intracerebral microinjections of excitatory amino acids. In R.
placed into the caudate nucleus (n = 5 cats) failed to alter the hissing latencies at any of the time periods examined (Fig. 2A). The present study demonstrates that D A M E infused into the BNST suppresses the hissing component of hypothalamically elicited affective defense behavior in a dose dependent manner. This effect lasts for about one hour, probably due to the fact that D A M E is known to resist degradation by brain enzymes 27 for approximately this period of time. Similar injections placed into the caudate nucleus fail to alter response latencies. Since prior administration of naloxone into the BNST effectively eliminates the suppressive effects of D A M E upon this type of aggressive behavior, it may be concluded that D A M E interacts with opioid receptors on BNST neurons to inhibit hissing behavior elicited by medial hypothalamic stimulation. Our findings suggest that the BNST may form a link in an endogenous opioid system which descends from the amygdala 9'13'2°,3°'36,38,40,43 to inhibit hypothalamically elicited affective defense. A possible mechanism underlying opioid modulation of this response within the BNST has been suggested from studies in which iontophoretic application of D A M E has been shown to alter neuronal firing patterns within this structure 25'31. These electrophysiological findings imply that the suppression of affective defense behavior we have observed may have resulted from the capacity of D A M E to suppress neuronal activity within the BNST. Inhibitory enkephalinergic modulation of BNST path neurons which project to the medial hypothalamus 7,39,43 may serve to modify the activity of hypothalamic neurons associated with the expression of affective defense behavior.
151 Bandler (Ed.), Modulation of Sensorimotor Activity During Alteration in Behavioral States, Liss, New York, 1984, pp. 369-392. 4 Block, C.H., Siegel, A. and Edinger, H.M., Effects of amygdaloid stimulation upon trigeminal sensory fields of the lip that are established during hypothalamically elicited quiet biting attack in the cat, Brain Research, 197 (1980) 39-55. 5 Brutus, M., Shaikh, M.B., Edinger, H. and Siegel, A., Effects of experimental temporal lobe seizures upon hypothalamically elicited aggressive behavior in the cat, Brain Research, 366 (1986) 53-63. 6 Brutus, M. and Siegel, A., Effects of the opiate antagonist naloxone upon hypothalamically elicited affective defense behavior in the cat, submitted. 7 Dalsass, M. and Siegel, A., The bed nucleus of the stria terminalis: electrophysiological properties and responses to amygdaioid and hypothalamic stimulation, Brain Research, 425 (1987) 346-350. 8 Dalsass, M. and Siegel, A., Opioid peptide regulation of neurons in the bed nucleus of the stria terminalis, Soc. Neurosci. Abstr., 14 (1988) 402. 9 DeOlmos, J.S. and Ingram, W.R., The projection field of the stria terminalis in the rat brain: an experimental study, J. Comp. Neurol., 146 (1972) 303-334. 10 Egger, M.D. and Flynn, J.P., Effects of electrical stimulation of the amygdala on hypothalamically elicited attack behavior in cat, J. Neurophysiol., 26 (1963) 705-720. 11 Egger, M.D. and Flynn, J.P., Amygdaloid suppression of hypothalamically elicited attack behavior, Science, 136 (1962) 43-44. 12 Egger, M.D. and Flynn, J.P., Further studies on the effects of amygdaloid stimulation and ablation on hypothalamically elicited attack behavior in cats. In W.R. Adey and T. Tokizane (Eds.), Structure and Function of the Limbic System, Progress in Brain Research, Vol. 27, Elsevier, Amsterdam, 1967, pp. 165-182. 13 Fernandez de Molina, A. and Hunsperger, R.W., Central representation of affective reactions in forebrain and brain stem: electrical stimulation of amygdala, stria terminalis and adjacent structures, J. Physiol. (Lond.), 145 (1959) 251-265. 14 Flynn, J.P., Neural basis of threat and attack. In R.G. Grennell and S. Gabay (Eds.), Biological Foundation of Psychiatry, Raven, New York, 1976, pp. 273-295. 15 Fuchs, S.A.G., Edinger, H.M. and Siegel, A., The organization of hypothalamic pathways mediating affective defense behavior in the cat, Brain Research, 330 (1985) 77-92. 16 Goeders, N.E., Lane, J.D. and Smith, J.E., Self-administration of methionine-enkephalin into the nucleus accumbens, Pharmacol. Biochem. Behav., 20 (1984) 451-455. 17 Gros, C., Pradelles, P., Humbert, J., Dray, F., LeGal LaSalle, G. and Ben Ari, Y., Regional distribution of met-enkephalin within the amygdaloid complex and bed nucleus of the stria terminalis, Neurosci. Lett., 10 (1978) 193-196. 18 Jasper, H.H. and Ajmone-Marsan, C.A., Stereotaxic Atlas of the Cat Diencephalon, National Research Council of Canada, Ottawa, 1954. 19 Kuhar, M., Pert, C.B. and Snyder, S.H., Regional distribution of opiate receptor binding in monkey and human brain, Nature (Lond.), 245 (1973) 447-450. 20 Krettek, J.E. and Price, J.L., Amygdaloid projections to subcortical structures within the basal forebrain and brain-
stem in the cat, J. Comp. Neurol., 178 (1978) 225-254. 21 Leyhausen, P., Cat Behavior. The Predatory and Social Behavior of Domestic and Wild Cats, Garland, STPM, New York, 1979. 22 MacDonald, M.F., Fessock, L. and Brown, S.H., Ethanol and the neural substrates for affective defense in the cat, Q. J. Stud. Alcohol, 32 (1971) 406-419. 23 MacDonnell, M.F. and Flynn, J.P., Control of sensory fields by stimulation of hypothalamus, Science, 152 (1966) 1406-1408. 24 Maeda, H., Behavioral construction of hypothalamic rage in cats, Fukuoka Acta. Med., 67 (1976) 364-373. 25 Matsui, H. and Yamamoto, C., Neuronal sensitivity to opiate and opioid peptides in the bed nucleus of the stria terminalis: effects of chronic treatment with morphine, Neuropharmacology, 23 (1984) 755-762. 26 Nakao, H., Brain Stimulation and Learning, Switch-off Behavior, Fisher, Jena, 1971. 27 Pert, C.B., Pert, A., Chang, J.-K. and Fong, B.T.W., (DAlaZ)-Met-Enkephalinamide: a potent long lasting synthetic pentapeptide analgesic, Science, 194 (1976) 330-332. 28 Phillips, A.G. and Lafiane, F., Reward produced by microinjection of (D-AIa2), MetS-enkephalinamide into the ventral tegmental area, Behav. Brain Res., 5 (1982) 225-229. 29 Pott, C.B., Kramer, S.Z. and Siegel, A., Central gray modulation of affective defense is differentially sensitive to naloxone, Physiol. Behav., 40 (1987) 207-213. 30 Rao, R., Yamano, M., Shinsaka, S., Shinohara, A. and Tohya, M., Origin of leucine-enkephalin fibers and their two main afferent pathways in the bed nucleus of the stria terminalis in the rat, Exp. Brain Res., 65 (1987) 411-420. 31 Sawada, S. and Yamamoto, C., Postsynaptic inhibitory actions of catecholamines and opioid peptides in the bed nucleus of the stria terminalis, Exp. Brain Res., 41 (1981) 264-270. 32 Shaikh, M.B., Barrett, J.A. and Siegel, A., The pathways mediating affective defense and quiet biting attack behavior from the midbrain central gray of the cat: an autoradiographic study, Brain Research, 437 (1987) 9-25. 33 Shaikh, M.B., Brutus, M., Siegel, H.E. and Siegel, A., Regulation of feline aggression by the bed nucleus of the stria terminalis, Brain Res. Bull., 16 (1986) 179-182. 34 Shaikh, M.B. and Siegel, A., Naloxone induced modulation of feline aggression elicited from the midbrain, Pharmacol. Biochem. Behav., in press. 35 Shaikh, M.B., Shaikh, A.B. and Siegel, A., Opioid peptides within the midbrain periaqueductal gray suppress affective defense behavior in the cat, Peptides, in press. 36 Siegel, A. and Edinger, H.M., Role of the limbic system in hypothalamically elicited attack behavior, Neurosci. Biobehay. Rev., 7 (1983) 395-407. 37 Siegel, A. and Edinger, H.M., Neural control of aggression and rage behavior. In P. Morgane and J. Panksepp (Eds.), Handbook of the Hypothalamus, Vol. 3, Dekker, New York, 1981, pp. 203-240. 38 Stoddard-Apter, S.L. and MacDonald, M.P., Septal and amygdalar efferents to the hypothalamus which facilitate hypothalamically elicited intraspecific aggression and associated hissing in the cat. An autoradiographic study, Brain Research, 193 (1980) 19-32. 39 Swanson, L.W., and Cowan, W.M., The connections of the septal region in the rat, J. Comp. Neurol., 186 (1979) 621-656.
152 40 Uhl, G., Kuhar, M. and Snyder, S., Enkephalin-containing pathway: amygdaloid efferents in the stria terminalis, Brain Research, 149 (1978) 223-228. 41 Wamsley, J K . , Young, W.S., III and Kuhar, M.J., Immunohistochemical localization of enkephalin in rat forebrain, Brain Research, 190 (1980) 153-174. 42 Wasman, M. and Flynn, J.P., Directed attack elicited from hypothalamus, Arch. Neurol., 61 (1962) 220-227.
43 Watson, K.E., Jr., Troiano, K., Poulakos, J., Weiner, S., Block, C.H. and Siegel, A., A [t4C]2-deoxyglucose analysis of the functional neural pathways of the limbic forebrain in the rat. I. The amygdala, Brain Res. Rev., 5 (1983) 1-44. 44 Zamir, N., Palkovitz, M. and Brownstein, M., Distribution of immunoreactive Met-enkephalin-Arg6-Gly7-Leus and Leu-Enkephalin in discrete regions of rat brain, Brain Research, 326 (1985) 1-8.
147
BRE 23183
Effects of D-Ala2-MetS-enkephalinamide microinjections placed into the bed nucleus of the stria terminalis upon affective defense behavior in the cat Martin Brutus, Shawki Zuabi and Allan Siegel Department of Neuroscience, UMDNJ-NewJersey MedicalSchool, Newark, NJ 07103 (U.S.A.) (Accepted 19 July 1988) Key words: Affective defense behavior; Bed nucleus of stria terminalis; D-Ala2-MetS-enkephalinamide; Naloxone; Medial hypothalamus
This study examined the effects of intracerebral injections of D-Ala2-Met5-enkephalinamide (DAME) upon hypothalamicaUy elicited hissing behavior in the cat. The bed nucleus of the stria terminalis (BNST) was selected for investigation because of its anatomical connections with the medial hypothalamus, its relatively high concentrations of enkephalins and opiate receptors and its demonstrated ability to modulate hypothalamically elicited aggressive reactions in the eat. DAME microinjected into the BNST in 1.0 or 10.0 pg/0.5 /~l quantities resulted in significant dose dependent increases in mean latencies for elicitation of the hissing response, Suppression of hissing following the 1.0 pg dose of DAME was selectively diminished by prior administration of naloxone. These findings suggest that the opiate receptors within the BNST play a role in the regulation of the hissing component of hypothalamieally elicited affective defense behavior.
Systemically injected naloxone has been shown to profoundly facilitate affective defense behavior elicited from either the hypothalamus 6 or midbrain central gray 34 of the cat. These effects suggest that endogenous opioids play a role in the modulation of aggressive behavior. Furthermore, it is reasonable to assume that such modulation is achieved by the actions of opioids upon those structures situated within the 'limbic-midbrain' which are known to elicit or regulate affective defense behavior 2'3,5,6A5' 29,32,36-38,42
In our research program, we have been attempting to identify the brain sites at which opioid peptides function to control affective defense behavior. In the present study, we have focussed upon the role of the bed nucleus of the stria terminalis (BNST). This structure was selected as a logical candidate for examination because: (1) it contains opioid receptors l's'19 and enkephalin-positive cells, fibers and terminals17'3°'4°'41'44; (2) it receives a major afferent sup-
ply from the amygdala 2°,43, which is known to modulate affective defenseS'1°-12; (3) BNST neurons project directly to the medial hypothalamus 7,9,39,43, an area classically associated with regulation of affective defense; and (4) electrical stimulation of the BNST modulates affective defense behavior 33. The findings reported below constitute an initial study which identifies the BNST as an important component of the limbic forebrain in opioid peptide neuromodulation of affective defense behavior. Eleven adult cats of either sex (Barton Farms, Oxford, NJ) weighing between 2.5 and 4.0 kg were employed in this study. They were maintained on an ad libitum feeding and drinking schedule throughout the duration of the experiments. The cats were anesthesized with sodium pentobarbital (45 mg/kg of b.wt.) prior to aseptic surgical procedures. Seventeen gauge stainless-steel guide tubes were stereotaxically mounted on both sides of the skull directly over the medial hypothalamus, BNST, and caudate nucleus.
Correspondence: A. Siegel, Department of Neuroscience, New Jersey Medical School, Medical Science Building, Room H512, 185 South Orange Avenue, Newark, NJ 07103, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
148 Coordinates were derived from the atlas of Jasper and A j m o n e - M a r s a n is. In addition, 3 bolts were anchored to the surface of the skull with dental acrylic to provide a means by which the electrode-cannula array could later be attached to a headholder. Details concerning the types of electrodes, cannulae and surgical p r o c e d u r e s utilized are described elsewhere 4'29. All experiments were conducted in a test chamber constructed of wood (61 x 61 x 61 cm) with a clear plexiglas door. Electrical stimuli were generated by a Grass S-88 stimulator and passed through a constant current photoelectric isolation unit (Grass PSIU6) to each animal. M o n o p o l a r stimulation of the hypothalamus consisted of balanced biphasic pulses delivered at 62.5 Hz, with a pulse width of 1 ms per half cycle duration. P e a k - t o - p e a k current was measured on a Tektronix 5113 dual b e a m oscilloscope with differential inputs. A p p r o x i m a t e l y one week following recovery from surgery, an electrode was vertically lowered through a guide tube overlying the hypothalamus and the brain was stimulated at 0.5 mm steps as the electrode was passed through the medial hypothalamus. W h e n affective defense was consistently elicited from a site by electrical stimulation, the electrode was c e m e n t e d in place. Affective defense behavior was elicited during stimulation at a given site located within the rostrocaudal extent of the medial hypothalamus. Responses elicited from any of these sites were of a similar nature, regardless of the anatomical locus of stimulation. Maps of these sites are shown in Fig. 1A. Affective defense behavior is characterized by sympathetic autonomic responses, threat responses such as hissing, growling and unsheathing of the claws and directed attack upon a rat or conspecific 1424'26'36"37. The hissing response was utilized because it is a fundamental component of the affective defense reaction 21 and is always present during the occurrence of this behavior. The latency to hiss was defined as the duration of time required to elicit hissing following the onset of stimulation. After the response was fully characterized as affective defense behavior in the freely moving animal, each cat was restrained in a h e a d h o l d e r similar to one that has been extensively e m p l o y e d in our laboratory 29'35 and elsewhere 2223'38. Painless immobilization was achieved by first placing the animal in a cat bag
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Fig. 1. A: maps of hypothalamic sites from which hissing behavior was elicited by electrical stimulation. B: maps of sites in the bed nucleus of the stria terminalis into which o-Ala2-MetLen kephalinamide (DAME) was microinjected. When DAME was injected into these sites (filled circles), it suppressed the hissing component of hypothalamically elicited affective defense behavior. In contrast, similar injections placed into the caudate nucleus (open circles) had no effects upon this behavior. AC, anterior commissure; AH, anterior hypothalamus; BNST, bed nucleus of the stria terminalis; Cd, caudate nucleus; Fx, Fornix; LH, lateral hypothalamus; MM, mammillary bodies; NA, nucleus accumbens; OC, optic chiasm; OT, optic tract: PH, posterior hypothalamus; PO, preoptic region: RE. nucleus reuniens, VMH, ventromedial hypothalamus.
so that it could lay comfortably, but also move its limbs during the testing periods. Then, the acrylic electrode cap was fastened to a brass h e a d h o l d e r 22'23 so that the position of the head would be fixed in space. This permitted precise intracranial delivery of drugs to telencephalic sites along with controlled stimulation of the hypothalamus. Each cat was exposed to the restrainer at least 10 times prior to experimental testing or until it showed no overt signs of discomfort or stress. Comparisons of the latency to hiss in the restrained and freely moving cat indicated that restraint had no effects upon this response. A f t e r the animals were fully habituated to the headholder, stimulation was applied to the medial
149 hypothalamus at the lowest current level that reliably elicited hissing within 20 s. For each animal, current values delivered to the hypothalamus were held constant during an entire test session. Baseline latencies for hissing ranged from 1.0 to 12.0 s. The current intensity delivered to the hypothalamus ranged from 0.1 to 0.8 mA. Baseline hissing latencies were recorded over a half hour test period prior to the administration of DAME or vehicle into the BNST or caudate nucleus. Following focal intracerebral injections, hissing latencies were again determined over 4 consecutive 30 min periods of time (i.e. 5-30, 30-60, 60-90, 90-120), with 12 stimulation trials delivered in each period. The interstimulus interval was 2 min. The opioid agonist D-Ala2-MetS-enkephalinamide (DAME) was selected for study because of its capacity to act on several opiate receptors 16'2s. DAME
A
(Bachem, Torrence, CA) was dissolved in 0.9% sterile normal saline (0.5MI) and buffered with NaOH to pH 7.4. A 0.5 MI SGE (Scientific Glass Engineering) microsyringe, which had been passed through a cannula implanted into either the BNST or the overlying caudate nucleus, was utilized to inject DAME or an equal volume of vehicle into these structures. Two doses of DAME were tested: 1.0 and 10.0Mg. All animals received both doses of DAME in a randomized order. In 3 cats, BNST sites were also tested following pretreatment with the opiate receptor antagonist naloxone (10 Mg/0.5 MI, pH = 7.4) 15 min prior to delivery of DAME (1/~g) into the same site. In the 15 min period between naloxone pretreatment and delivery of DAME, hissing latencies were recorded in order to determine whether naloxone administration alone could alter latencies for this response. As a control
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Fig. 2. A: microinjections of DAME placed into the BNST resulted in an increase in response latencies for hissing in a dose dependent manner. DAME induced increases in hissing latencies returned to baseline levels in a time dependent manner. Each of the points and error bars on the curves depicted above, represent the mean and S.E.M. for all stimulation trials attempted during each of the 4 halfhour testing periods following the microinjection of DAME or vehicle. Neither vehicle control injections, nor DAME injections placed into the caudate nucleus altered latencies for hissing (n = 6 for BNST; n = 5 for caudate nucleus). B: (I) Effects of DAME: microinjections of DAME placed into BNST significantly suppressed hissing in 3 cases (P < 0.05) in which naloxone pretreatment was later attempted (in II). Vehicle injected alone did not alter latencies for hissing. (II) Pretreatment with naloxone: when naloxone (10 #g) was injected into these 3 BNST sites prior to infusion of DAME (1 Mg), the suppression of hissing seen in 'I' was blocked (P < 0.05). Naloxone, injected into BNST alone, did not alter latencies for hissing (n = 3).
150 for volume effects here, vehicle alone was microinjected into the BNST in amounts equal to the combined volume of the naloxone and D A M E solutions that had been injected in the experimental condition. Response latencies were then again determined. At the completion of the experiments, animals were perfused with 0.9% saline and 10% formalin. Brains were sectioned at 50 ~m and stained with cresyl violet for localization of electrode and cannula tips. D A M E injected into 7 sites located in the BNST (n = 6 cats, see Fig. 1B) suppressed the hissing response as indicated by marked increases in the latency of this response following stimulation of the hypothalamus. D A M E placed into the BNST produced a dose dependent increase in hissing latencies (Fig. 2A). An analysis of variance which compared the drug dose for vehicle, 1.0 and 10.0/~g of D A M E , indicated that the response latencies were significantly increased in the first (F = 6.13, df = 3,19, P < 0.01) and second 30 min periods (F = 4.32, df = 3,18, P < 0.025) after the injection. Maximal effects of D A M E administration were attained in the first half hour after the injections. During this time, a dose of 1/~g of D A M E produced an increase of over 100% in the mean latency to hissing (P < 0.025). The 10 ~g dose of D A M E increased the mean latency by almost 300%. Both increases were calculated as changes relative to baseline control values. In the third half-hour period following injection with either dose of drug, response latencies were no longer found to be significantly different from baseline values. Control injections of the vehicle had no effect on hissing latency at any time point. In 3 of the BNST sites examined, naloxone (10/xg) injected 15 min prior to administration of a minimum effective dose of DAME, blocked the suppressive effects of D A M E (P < 0.05). These results are summarized in Fig. 2B. Naloxone, though blocking the action of D A M E , did not itself alter hissing latencies. In contrast to the effects of D A M E microinjections at BNST sites, injections of D A M E (1 ktg)
This research was supported by NIH Grant NS 07941-19 awarded by the National Institutes of Neurological and Communicative Diseases and Stroke. We would like to thank Drs. Henry Edinger and Hreday Sapru for their valuable suggestions for the improvement of this manuscript.
1 Atweh, S.F. and Kuhar, M., Autoradiographic localization of opiate receptor in rat brain. III. The telencephalon, Brain Research, 134 (1977) 393-405. 2 Bandler, R. and McCullock, T., Afferents to a midbrain periaqueductal region in the defense reaction in the cat as
revealed by horseradish peroxidase. II. The diencephalon, Behav. Brain Res., 13 (1984) 279-285. 3 Bandler, R., Identification of hypothalamic and midbrain neurons mediating aggressive and defensive behavior by intracerebral microinjections of excitatory amino acids. In R.
placed into the caudate nucleus (n = 5 cats) failed to alter the hissing latencies at any of the time periods examined (Fig. 2A). The present study demonstrates that D A M E infused into the BNST suppresses the hissing component of hypothalamically elicited affective defense behavior in a dose dependent manner. This effect lasts for about one hour, probably due to the fact that D A M E is known to resist degradation by brain enzymes 27 for approximately this period of time. Similar injections placed into the caudate nucleus fail to alter response latencies. Since prior administration of naloxone into the BNST effectively eliminates the suppressive effects of D A M E upon this type of aggressive behavior, it may be concluded that D A M E interacts with opioid receptors on BNST neurons to inhibit hissing behavior elicited by medial hypothalamic stimulation. Our findings suggest that the BNST may form a link in an endogenous opioid system which descends from the amygdala 9'13'2°,3°'36,38,40,43 to inhibit hypothalamically elicited affective defense. A possible mechanism underlying opioid modulation of this response within the BNST has been suggested from studies in which iontophoretic application of D A M E has been shown to alter neuronal firing patterns within this structure 25'31. These electrophysiological findings imply that the suppression of affective defense behavior we have observed may have resulted from the capacity of D A M E to suppress neuronal activity within the BNST. Inhibitory enkephalinergic modulation of BNST path neurons which project to the medial hypothalamus 7,39,43 may serve to modify the activity of hypothalamic neurons associated with the expression of affective defense behavior.
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