- Email: [email protected]
Intracerebroventricular interleukin-1 receptor antagonist blocks the enhancement of fear conditioning and interference with escape produced by inescapable shock
BRAIN RESEARCH ELSEVIER
Brain Research 695 (1995) 279-282
Short communication
Intracerebroventricular interleukin-1 receptor antagonist blocks the enhancement of fear conditioning and interference with escape produced by inescapable shock Steven F. Maier *, Linda R. Watkins Neuroscience & Behavior Program and Department of Psychology, Campus Box 345, University of Colorado, Boulder, CO 80309-0345, USA Accepted 11 July 1995
Abstract
Brain interleukin-1 (IL-1) plays a key role in mediating the neural, endocrine, and behavioral consequences of injury and infection. Recent evidence indicates that brain IL-1 may also be important in producing endocrine and neurochemical responses to stressors. The present experiment sought to determine whether intracerebroventricular (i.c.v.) administration of an interleukin-1 receptor antagonist (IL-lra) would block behavioral effects of a stressor. I.c.v. application of hrlL-lra before inescapable shock blocked the subsequent intereference with escape learning and enhancement of fear conditioning normally produced by this treatment. Keywords: lnterleukin-1; Interleukin-1 receptor antagonist; lntracerebroventricular; Rat; Stress; Escape; Fear; Learned helplessness
Tissue injury and infection activate an integrated set of defensive responses called the acute phase response (APR) [2]. The major components of the APR are fever, hypothalamo-pituitary-adrenal activation, brain monoamine release in specific regions, hepatic production of acute phase proteins, alterations in plasma iron, zinc, and copper, and a set of behavioral changes that include decreased social interaction, reduced exploration of novel objects, anorexia, and adipsia [9,11]. Although the APR to inflammatory stimuli is initiated at the periphery, neural mechanisms mediate many of the actual changes that occur. Interleukin113 (IL-1/3) in brain plays a key role. The peripheral administration of inflammatory stimuli such as lipopolysaccharide (cell walls of Gram-negative bacteria) induces IL-1/3 mRNA [1,13] and IL-1/3 bioactivity [22] in discrete brain regions. Furthermore, intracerebroventricular (i.c.v.) and regional IL-1/3 microinjections produce all aspects of the APR including peripheral changes such as leukocytosis and the production of hepatic acute phase proteins [19,20], and blockade of brain IL-1/3 with i.c.v, antiserum or IL-1 receptor antagonist (IL-lra) reduces or eliminates many
* Fax: (1)(303)492-2967; E-mail: [email protected] 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD1 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 9 3 0 - 2
aspects of the APR to peripheral inflammatory stimuli [10,21,24]. The APR to inflammatory stimuli bears a striking resemblance to the neural, endocrine, and behavioral consequences of exposure to stressors. Interestingly, stressors may also be able to activate brain IL-lfl. Restraint has been shown to increase brain IL-1 mRNA [18] and bioactivity [29]. These effects were quite rapid, and anterior hypothalamic application of IL-lra strongly reduced restraint-induced release of NE, 5-HT, and DA in the anterior hypothalamus. The plasma ACTH response was also substantially reduced. These data suggest that central administration of IL-lra might be able to block behavioral effects of stressors as well. Exposure to inescapable tailshock (IS) produces many of the same neural, endocrine, and behavioral changes observed during the APR, as well as others [16]. Two of the best-studied sequalae of IS are enhancement of fear conditioning as measured by freezing and interference with shuttlebox escape learning [15]. The purpose of the present experiment was to determine whether i.c.v. IL-lra would reduce or eliminate these effects. Viral-free, male Sprague-Dawley rats (n = 4 8 ; obtained from Harlan) were housed individually and maintained on a 12 h / 1 2 h light-dark cycle with experiments
S.F. Maier, L.R. Watkins / Brain Research 695 (1995) 279-282
280
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Fig. 1. Mean number of observation periods for which freezing was observed after 2 shocks, across blocks of 2 min. Subjects had received either inescapable shock (IS) or restraint (R) 24 h earlier. Subjects had received hrlL-lra before IS and before testing (BSBT), before R and before testing (BRBT), before IS only (BS), before testing only (BT), or vehicle at all times (VEH).
conducted during the light part of the cycle. All rats were anaesthetized (55 m g / k g sodium pentobarbital, supplemented with Metofane) and stereotaxically implanted (AP =-1.5 from bregma, M L = 0 . D V = - 5 . 5 ) with a chronic stainless steel guide cannula (26-gauge) that terminated in the third ventricle. Ventricle placement was verified at the time of surgery by the presence of free gravity flow of saline through PE tubing attached to the cannula. Sterile stainless steel stylets (33-gauge) were inserted into cannulae after surgery. Details of surgery and verification of cannulae placement can be found in Maier et al. [17]. Two weeks after surgery, the rats were divided into 6 groups of 8 rats each. Four groups received IS while restrained in Plexiglas tubes 17.5 cm in length and 7.0 cm in diameter. Fixed electrodes were applied to the tail which extended from the rear of the tubes. IS consisted of 100 5-s 1.0 mA tailshocks, occuring on the average of once every 60-s. The remaining 2 groups were restrained in the apparatus for an equal period of time but did not receive shock. Twenty-four hours later all subjects received fear conditioning in a shuttlebox (for description see [15,17]) followed by escape training. The subjects were first observed for 10 min and freezing scored. Each rat was observed every 8-s and scored as freezing or not freezing. To be scored as freezing the rat had to have all 4 paws on the grid with no movement of the body or vibrissae except that required for respiration. This is a highly unambiguous and stereotyped response in the rat (inter-rater r = 0.94) and has been argued to reflect conditioned fear [3]. The observer was blind to group membership. The 10 min observation period was followed by 2 gridshocks (0.8 mA scrambled gridshock) separated by 60-s. Each gridshock could be terminated by a single crossing of the shuttlebox (FR-1). Freezing was then assessed for 20 min. This observation period was followed by 3 further shocks that could be terminated by a single crossing of the shuttlebox, followed by 25 escape trials during which 2 crossings (FR-2) were required to terminate each shock. If an escape response had not occurred by 30-s on a given trial the trial was automatically terminated and a 30-s latency recorded.
These procedures have been in standard use and detailed rationale and description can be found in [15,17]. The groups differed in drug treatment given before IS or restraint (R), and before subsequent shuttlebox testing. One of the IS and one of the R groups received human recombinant IL-lra (donated by Amgen) 10 min before IS or R, immediately after IS or R, and 10 min before shuttlebox testing. IL-lra was administered i.c.v, at a dose of 100/zg in 1 /zl. I.c.v. microinjection was made over a 1 min period using a 33-gauge microinjector inserted through the indwelling guide cannula. The injector was left in place for 2 min after an injection. A second IS group received IL-lra before and after the IS, but only vehicle (Synergen CSE buffer) before the shuttlebox testing. A third IS group received vehicle before and after IS, but IL-lra before the shuttlebox testing. The remaining IS and R groups received vehicle at each point. There was no freezing before the 2 shocks in the shuttlebox. Fig. 1 depicts the amount of freezing in the 20 min period after the 2 shocks. All groups showed substantial freezing, indicating fear conditioning to the contextual cues of the apparatus. The conditioned fear extinguished across the 20 min observation period. As is typical, prior IS potentiated the amount of fear conditioning. IL-lra given before IS and before testing blocked this potentiation, as did IL-lra given before IS only. IL-lra administered before the testing but not before the IS had no effect. Importantly, IL-lra by itself had no impact on fear conditioning in R controls. Repeated measures ANOVA yielded reliable effects of groups (F5,41 = 5.84, P < 0.0005) and the interaction of groups with trials (F45,369 = 1.53, P < 0.02). Newman-Keuls post-hoe pairwise comparisons ( P < 0.05) indicate that the IS groups given vehicle and IL-lra before testing differed from all of the other groups which did not differ among themselves. Shuttlebox escape latencies are shown in Fig. 2. As has repeatedly been observed [15,17], there were no group differences in FR-1 escape latencies either before or after the observation period. This means that all groups received equal shock durations before the fear measurement. Large
>_ u
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BP
AP
1 2 3 4 5 B L O C K S OF 5 TRIALS
Fig. 2. Mean shuttlebox escape latencies for the 2 FR-I trials preceding the observation period (BP), the 3 FR-1 trials after the observatuion period (AP), and blocks of 5 FR-2 trials. Subjects had received either inescapable shock (IS) or restraint (R) 24 h earlier. Subjects had received hrlL-lra before IS and before testing (BSBT), before R and before testing (BRBT), before IS only (BS), before testing only (BT), or vehicle at all times (VEH).
S.F. Maier, L.R. Watkins / Brain Research 695 (1995) 279-282
group differences emerged on the FR-2 escape trials. Prior IS strongly interfered with FR-2 escape. The mean 20-s latencies indicate that most of the subjects completely failed to escape on most trials. The IS groups given IL-lra both before IS and testing and before IS only failed to show this escape deficit. IL-lra administered before shuttlebox testing had no effect. Again, IL-lra by itself had no effect on the escape measure. In summary, i.c.v. IL-lra blocked the potentiation of fear conditioning and the interference with escape learning produced by IS when it was given immediately before and after the IS. IL-lra preceding the testing had no effect. It is very likely that this effect was mediated in the brain rather than by leakage to the periphery. IL-lra is a large peptide [8] and so not able to penetrate the blood-brain barrier [6]. In addition, the dose of IL-lra used is far below that found to have effects after peripheral injection [6]. Indeed we have attempted to block the IS-induced fear and shuttle escape deficits with peripheral injections of IL-lra and have used doses up to 100 mg/kg. All have been without effect. These data suggest that brain IL-1 is an important link between at least some stressors and their behavioral consequences, just as it is a key link between infection and the APR. They further suggest that there may be considerable overlap between the circuitries responsible for illness and stress, as originally proposed by Selye [27]. Although the present experiment does not implicate any particular mechanism, it is noteworthy that many APRs to both peripheral inflammatory stimuli and central administration of IL-1 depend on the induction of corticotrophic releasing hormone (CRH). Both peripheral inflammatory stimuli and central IL-1 injection induce hypothalamic mRNA for CRH [10,30] and CRH release [25]. Furthermore, central application of antiserum to CRH and CRH receptor antagonists blocks many aspects of the APR to both peripheral and central stimulation [7,10,11,23,31]. An extensive literature documents the critical role of central CRH in mediating the behavioral effects of stressors [12]. Thus IL-1 may be a link between stressors and the activation of CRH systems, possibly by acting to release NE and 5-HT onto CRH-containing neurons in the hypothalamus [28] and elsewhere [14]. Finally, it should be noted that these effects of IL-1 may occur because IL-1 leads to the production of generalized excitatory mediators such as prostaglandins [5] and nitric oxide [4] from glial cells and astrocytes. Indeed, IL-1 receptors and IL-1 mRNA are contained both in neurons and glia (for review see [26]), and either cell type could play the critical role in IL-1 stress mechanisms.
Acknowledgements This research was supported by NIMH Grants MH50479 and MH45045, and RSDA MH 00314.
281
References [1] Ban, E., Haour, F. and Lenstra, R., Brain interleukin-1 gene expression induced by peripheral lipopolysaccharide administration, Cytokine, 4 (1992) 48-54. [2] Baumann, J.H, and Gauldie, J., The acute phase response, lmmunol. Today, 15 (1994) 74-81. [3] Blanchard, D.C. and Blanchard, R.J., lnnnate and conditioned reactions to threat in rats with amygdala lesions, J. Comp. Physiol. Psychol., 81 (1972) 281-290. [4] Colton, C.A., Snell, J., Chernysko, O.and Gilbert, D.L., Induction of superoxide anion and nitric oxide production in microglia, Ann. NY Acad. Sci, 738 (1994) 54-56. [5] Dinarello, C.A., Modalities for reducing interleukin-I activity in fisease, lmmunol. Today, 14 (1993) 260-264. [6] Dinarello, C.A. and Thompson, R.C., Interleukin-1 and interleukin-1 antagonism, Blood, 77 (1991) 1627-1652. [7] Dunn, A.J., Antoon, M. and Chapma, Y., Reductions of exploratory behavior by intraperitoneal injection of interleukin-1 involves brain corticotropin releasing hormone, Brain Res. Bull., 26 (1991) 539542. [8] Eisenberg, S.P., Evans, R.J., Arend, N.P. et al., Primary structure and functional expression from eDNA of a known interleukin-1 receptor antagonist, Nature, 343 (1990) 341-346. [9] Hart, B.L., Biological basis of the behavior of sick animals, Neurosci. Biobehat,. Ret,., 12, 123-137 (1988). [10] Kakucska, I., Qi, Y., Clark, B.D. and Lechan, R.M., Endotoxin-induced corticotropin releasing hormone gene expression in the hypothalamic paraventricular nucleus is mediated centrally by interleukin- 1, Endocrinology, 133 (1993) 815-821. [11] Kent, S., Bluthe, R.M., Kelley, K.W. and Dantzer, R., Sickness behavior as a new target for drug development, Trends Pharmacol. Sci., 13 (1992) 24-28. [12] Koob, G.F., The behavioral neuroendocrinology of corticotropin-releasing factor. In C.B. Nemeroff (Eds.), Neuroendocrinology, CRC Press, Boca Raton, 1992, pp. 353-365. [13] Laye, S., Parnet, P., Goujon, E. and Dantzer, R., Peripheral administration of lipopolysaccharide induces the expression of cytokine transcripts in the brain and pituitary of micc, Mol. Brain Res., 27 (1994) 157-162. [14] Linthorst, A.C., Flachskamm, C., Holsboer, F. and Reul, J.M.. Local administration of recombinant human interleukin-1 beta in the rat hippocampus increases serotonergic neurotransmission, hypothalarno-pituitary-adrenocortical activity, and body temperature, Endocrinology, 135 (1994) 520-532. [15] Maier, S.F., The role of fear in mediating the shuttle escape learning deficit produced by inescapapble shock, J. Exp. P~vchol.: Anim. Behat,. Proc., 16 (1990) 137-150. [16] Maier, S.F., Learned helplessness, fear, and anxiety. In: C. Stanfordand P. Salmon (Eds.), Stress: from Synapse to Syndrome, Academic Press, London, 1993, pp. 207-248. [17] Maier, S.F., Chlordiazepoxide microinjected into the region of the dorsal raphe nucleus eliminates the interference with escape responding produced by inescapable shock whether administered before inescapable shock or escape testing, Beha~,. Neurosci., 108 (1994) 121-130. [18] Minami, M., Kuraishi, Y., Yamaguchi, T., Nakai, S., Hirai, Y. and Satoh, M., Immobilization stress induces interlekin-1/3 mRNA in the rat hypothalamus, Neurosci. Lett., 123 (1991) 254-256. [19] Morimoto, A., Murakami, N., Nakamori, T., Sakata, Y. and Watanabe, T., Brain regions involved in the development of acute phase responses accompanying fever in rabbits, J. Physiol., 416 (1989) 645-657. [20] Morimoto, A., Watanabe, T., Sakata, Y. and Murakami, N., Leukocytosis induced by microinjection of endogenous pyrogen or interleukin-I into the preoptic and anterior hypothalamus, Brain Res., 475 (1988) 345-348.
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[21] Plata-Salaman, C.R. and French-Mullen, S.M., lntracerebroventricular administration of a specific IL-1 receptor antagonist blocks food and water intake suppression induced by lipopolysaccharide, Physiol. Behav., 51 (1992) 1277-1279. [22] Quan, N., Sundar, S.K. and Weiss, J.M., Induction of interleukin-1 in various brain regions after peripheral and central administration of lipopolysaccharide, J. Neuroimmunol., 49 (1994) 125-134. [23] Rothwell, N.J., CRF is involved in the pyrogenic and thermogenic effects of interleukin-1 beta in the rat, Am. J. Physiol., 256 (1989) Elll-Ell5. [24] Rothwell, N.J., Functions and mechanisms of interleukin-1 in the brain, Trends Pharmacol. Sci., 12 (1991) 430-436. [25] Sapolsky, E., Rivier, C., Yamamoto, G., Plotsky, P. and Vale, W., Interlekin-1 srtimulates the secretion of corticotrophin releasing factor, Science, 238 (1987) 422-524. [26] Schobitz, B., De Kloet, E.R. and Holsboer, F., Gene expression and
[27] [28]
[29]
[30]
[31]
function of interleukin-1, interleukin-6, and tumor necrosis factor in the brain, Prog. Neurobiol., 44 (1994) 397-432. Selye, H., Stress, Acta, Montreal, 1950. Shintani, F., Kanaba, S., Nakai, T. et al., Interelukin-1/3 augments release of norepinephrine, dopamine, and serotonin in the rat hypothalamus, J. Neurosci., 13 (1993) 3574-3581. Shintani, F., Nakai, T., Kanba, S., et al., Involvement of interleukin-I in immobilization stress-induced increases in plasma ACTH and in release of hypothalamic monoamines in the rat, J. Neurosci., 15 (1995) 1961-1970. Suda, T., Tozawa, F., Mouri, T., Demura, H. and Shizume, K., Interleukin-1 stimulates corticotropin-releasing factor gene expression in the hypothalamus, Endocrinology, 126 (1990) 1223-1228. Uehara, A., Sekiya, C., Takasugi, Y., Namiki, M. and Arimura, A., Anorexia induced by interlekin-l: involvement of corticotropin-rcleasing factor, Am. J. Physiol., 257 (1989) R613-R617.
Brain Research 695 (1995) 279-282
Short communication
Intracerebroventricular interleukin-1 receptor antagonist blocks the enhancement of fear conditioning and interference with escape produced by inescapable shock Steven F. Maier *, Linda R. Watkins Neuroscience & Behavior Program and Department of Psychology, Campus Box 345, University of Colorado, Boulder, CO 80309-0345, USA Accepted 11 July 1995
Abstract
Brain interleukin-1 (IL-1) plays a key role in mediating the neural, endocrine, and behavioral consequences of injury and infection. Recent evidence indicates that brain IL-1 may also be important in producing endocrine and neurochemical responses to stressors. The present experiment sought to determine whether intracerebroventricular (i.c.v.) administration of an interleukin-1 receptor antagonist (IL-lra) would block behavioral effects of a stressor. I.c.v. application of hrlL-lra before inescapable shock blocked the subsequent intereference with escape learning and enhancement of fear conditioning normally produced by this treatment. Keywords: lnterleukin-1; Interleukin-1 receptor antagonist; lntracerebroventricular; Rat; Stress; Escape; Fear; Learned helplessness
Tissue injury and infection activate an integrated set of defensive responses called the acute phase response (APR) [2]. The major components of the APR are fever, hypothalamo-pituitary-adrenal activation, brain monoamine release in specific regions, hepatic production of acute phase proteins, alterations in plasma iron, zinc, and copper, and a set of behavioral changes that include decreased social interaction, reduced exploration of novel objects, anorexia, and adipsia [9,11]. Although the APR to inflammatory stimuli is initiated at the periphery, neural mechanisms mediate many of the actual changes that occur. Interleukin113 (IL-1/3) in brain plays a key role. The peripheral administration of inflammatory stimuli such as lipopolysaccharide (cell walls of Gram-negative bacteria) induces IL-1/3 mRNA [1,13] and IL-1/3 bioactivity [22] in discrete brain regions. Furthermore, intracerebroventricular (i.c.v.) and regional IL-1/3 microinjections produce all aspects of the APR including peripheral changes such as leukocytosis and the production of hepatic acute phase proteins [19,20], and blockade of brain IL-1/3 with i.c.v, antiserum or IL-1 receptor antagonist (IL-lra) reduces or eliminates many
* Fax: (1)(303)492-2967; E-mail: [email protected] 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD1 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 9 3 0 - 2
aspects of the APR to peripheral inflammatory stimuli [10,21,24]. The APR to inflammatory stimuli bears a striking resemblance to the neural, endocrine, and behavioral consequences of exposure to stressors. Interestingly, stressors may also be able to activate brain IL-lfl. Restraint has been shown to increase brain IL-1 mRNA [18] and bioactivity [29]. These effects were quite rapid, and anterior hypothalamic application of IL-lra strongly reduced restraint-induced release of NE, 5-HT, and DA in the anterior hypothalamus. The plasma ACTH response was also substantially reduced. These data suggest that central administration of IL-lra might be able to block behavioral effects of stressors as well. Exposure to inescapable tailshock (IS) produces many of the same neural, endocrine, and behavioral changes observed during the APR, as well as others [16]. Two of the best-studied sequalae of IS are enhancement of fear conditioning as measured by freezing and interference with shuttlebox escape learning [15]. The purpose of the present experiment was to determine whether i.c.v. IL-lra would reduce or eliminate these effects. Viral-free, male Sprague-Dawley rats (n = 4 8 ; obtained from Harlan) were housed individually and maintained on a 12 h / 1 2 h light-dark cycle with experiments
S.F. Maier, L.R. Watkins / Brain Research 695 (1995) 279-282
280
I
~= m~
.= 0
•
•-----C>-,--
IS-VEH
~
,S-BS IS-BT R-VEH R-BRBT
, 2
•
, 4
-
, 6
,
, 8
-
, 10
2 MIN B L O C K S
Fig. 1. Mean number of observation periods for which freezing was observed after 2 shocks, across blocks of 2 min. Subjects had received either inescapable shock (IS) or restraint (R) 24 h earlier. Subjects had received hrlL-lra before IS and before testing (BSBT), before R and before testing (BRBT), before IS only (BS), before testing only (BT), or vehicle at all times (VEH).
conducted during the light part of the cycle. All rats were anaesthetized (55 m g / k g sodium pentobarbital, supplemented with Metofane) and stereotaxically implanted (AP =-1.5 from bregma, M L = 0 . D V = - 5 . 5 ) with a chronic stainless steel guide cannula (26-gauge) that terminated in the third ventricle. Ventricle placement was verified at the time of surgery by the presence of free gravity flow of saline through PE tubing attached to the cannula. Sterile stainless steel stylets (33-gauge) were inserted into cannulae after surgery. Details of surgery and verification of cannulae placement can be found in Maier et al. [17]. Two weeks after surgery, the rats were divided into 6 groups of 8 rats each. Four groups received IS while restrained in Plexiglas tubes 17.5 cm in length and 7.0 cm in diameter. Fixed electrodes were applied to the tail which extended from the rear of the tubes. IS consisted of 100 5-s 1.0 mA tailshocks, occuring on the average of once every 60-s. The remaining 2 groups were restrained in the apparatus for an equal period of time but did not receive shock. Twenty-four hours later all subjects received fear conditioning in a shuttlebox (for description see [15,17]) followed by escape training. The subjects were first observed for 10 min and freezing scored. Each rat was observed every 8-s and scored as freezing or not freezing. To be scored as freezing the rat had to have all 4 paws on the grid with no movement of the body or vibrissae except that required for respiration. This is a highly unambiguous and stereotyped response in the rat (inter-rater r = 0.94) and has been argued to reflect conditioned fear [3]. The observer was blind to group membership. The 10 min observation period was followed by 2 gridshocks (0.8 mA scrambled gridshock) separated by 60-s. Each gridshock could be terminated by a single crossing of the shuttlebox (FR-1). Freezing was then assessed for 20 min. This observation period was followed by 3 further shocks that could be terminated by a single crossing of the shuttlebox, followed by 25 escape trials during which 2 crossings (FR-2) were required to terminate each shock. If an escape response had not occurred by 30-s on a given trial the trial was automatically terminated and a 30-s latency recorded.
These procedures have been in standard use and detailed rationale and description can be found in [15,17]. The groups differed in drug treatment given before IS or restraint (R), and before subsequent shuttlebox testing. One of the IS and one of the R groups received human recombinant IL-lra (donated by Amgen) 10 min before IS or R, immediately after IS or R, and 10 min before shuttlebox testing. IL-lra was administered i.c.v, at a dose of 100/zg in 1 /zl. I.c.v. microinjection was made over a 1 min period using a 33-gauge microinjector inserted through the indwelling guide cannula. The injector was left in place for 2 min after an injection. A second IS group received IL-lra before and after the IS, but only vehicle (Synergen CSE buffer) before the shuttlebox testing. A third IS group received vehicle before and after IS, but IL-lra before the shuttlebox testing. The remaining IS and R groups received vehicle at each point. There was no freezing before the 2 shocks in the shuttlebox. Fig. 1 depicts the amount of freezing in the 20 min period after the 2 shocks. All groups showed substantial freezing, indicating fear conditioning to the contextual cues of the apparatus. The conditioned fear extinguished across the 20 min observation period. As is typical, prior IS potentiated the amount of fear conditioning. IL-lra given before IS and before testing blocked this potentiation, as did IL-lra given before IS only. IL-lra administered before the testing but not before the IS had no effect. Importantly, IL-lra by itself had no impact on fear conditioning in R controls. Repeated measures ANOVA yielded reliable effects of groups (F5,41 = 5.84, P < 0.0005) and the interaction of groups with trials (F45,369 = 1.53, P < 0.02). Newman-Keuls post-hoe pairwise comparisons ( P < 0.05) indicate that the IS groups given vehicle and IL-lra before testing differed from all of the other groups which did not differ among themselves. Shuttlebox escape latencies are shown in Fig. 2. As has repeatedly been observed [15,17], there were no group differences in FR-1 escape latencies either before or after the observation period. This means that all groups received equal shock durations before the fear measurement. Large
>_ u
v
20
~
IS-VEH C
IS-BSBT
,z;
~
~S-BS
<~
~ ""Am JL
IS-BT R-VEH R-BRBT
10 z
,
BP
AP
1 2 3 4 5 B L O C K S OF 5 TRIALS
Fig. 2. Mean shuttlebox escape latencies for the 2 FR-I trials preceding the observation period (BP), the 3 FR-1 trials after the observatuion period (AP), and blocks of 5 FR-2 trials. Subjects had received either inescapable shock (IS) or restraint (R) 24 h earlier. Subjects had received hrlL-lra before IS and before testing (BSBT), before R and before testing (BRBT), before IS only (BS), before testing only (BT), or vehicle at all times (VEH).
S.F. Maier, L.R. Watkins / Brain Research 695 (1995) 279-282
group differences emerged on the FR-2 escape trials. Prior IS strongly interfered with FR-2 escape. The mean 20-s latencies indicate that most of the subjects completely failed to escape on most trials. The IS groups given IL-lra both before IS and testing and before IS only failed to show this escape deficit. IL-lra administered before shuttlebox testing had no effect. Again, IL-lra by itself had no effect on the escape measure. In summary, i.c.v. IL-lra blocked the potentiation of fear conditioning and the interference with escape learning produced by IS when it was given immediately before and after the IS. IL-lra preceding the testing had no effect. It is very likely that this effect was mediated in the brain rather than by leakage to the periphery. IL-lra is a large peptide [8] and so not able to penetrate the blood-brain barrier [6]. In addition, the dose of IL-lra used is far below that found to have effects after peripheral injection [6]. Indeed we have attempted to block the IS-induced fear and shuttle escape deficits with peripheral injections of IL-lra and have used doses up to 100 mg/kg. All have been without effect. These data suggest that brain IL-1 is an important link between at least some stressors and their behavioral consequences, just as it is a key link between infection and the APR. They further suggest that there may be considerable overlap between the circuitries responsible for illness and stress, as originally proposed by Selye [27]. Although the present experiment does not implicate any particular mechanism, it is noteworthy that many APRs to both peripheral inflammatory stimuli and central administration of IL-1 depend on the induction of corticotrophic releasing hormone (CRH). Both peripheral inflammatory stimuli and central IL-1 injection induce hypothalamic mRNA for CRH [10,30] and CRH release [25]. Furthermore, central application of antiserum to CRH and CRH receptor antagonists blocks many aspects of the APR to both peripheral and central stimulation [7,10,11,23,31]. An extensive literature documents the critical role of central CRH in mediating the behavioral effects of stressors [12]. Thus IL-1 may be a link between stressors and the activation of CRH systems, possibly by acting to release NE and 5-HT onto CRH-containing neurons in the hypothalamus [28] and elsewhere [14]. Finally, it should be noted that these effects of IL-1 may occur because IL-1 leads to the production of generalized excitatory mediators such as prostaglandins [5] and nitric oxide [4] from glial cells and astrocytes. Indeed, IL-1 receptors and IL-1 mRNA are contained both in neurons and glia (for review see [26]), and either cell type could play the critical role in IL-1 stress mechanisms.
Acknowledgements This research was supported by NIMH Grants MH50479 and MH45045, and RSDA MH 00314.
281
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