- Email: [email protected]
Quinpirole attenuates the striatal Fos expression induced by escape behavior
Brain Research 785 Ž1998. 347–350
Short communication
Quinpirole attenuates the striatal Fos expression induced by escape behavior W.M. Struthers, David Wirtshafter
)
Department of Psychology (M r C 285), The UniÕersity of Illinois at Chicago, 1007 West Harrison Street, Chicago, IL 60612, USA Accepted 4 November 1997
Abstract Several studies have shown that the D 2-like dopamine receptor agonist quinpirole is able to markedly potentiate the striatal Fos expression induced by D1 agonists. The present study examined the effects of quinpirole on the striatal Fos-like immunoreactivity ŽFLI. induced by escape behavior. Male rats were pretreated with either saline or quinpirole Ž0.156, 0.625, 1.25 or 2.5 mgrkg. and 30 min later, placed in a shuttle box and required to crossover every 30 s in order to escape mild footshock. Animals were sacrificed 30 min following the completion of a 1-h block of escape trials and sections through the striatum were processed for FLI. Pretreatment with quinpirole produced a marked, dose-dependent, attenuation of escape-induced FLI in the striatum. These findings demonstrate that quinpirole affects the striatal Fos expression induced by shuttling in a very different fashion than it does that induced by D1 agonists, and further support the view that dopaminergic mechanisms play an important role in behaviorally induced striatal Fos expression. q 1998 Elsevier Science B.V. Keywords: Quinpirole; c-fos; Shuttle box; Striatum; Dopamine; D 2 receptor; Caudate-putamen; Immediate-early genes
Many recent studies have used the expression of the proto-oncoprotein Fos as a marker for neurons affected by various treatments, and this technique has proved to be an especially powerful tool for examining the functional role of dopamine in the striatum. Striatal Fos expression can be enhanced by stimulation of D1-like dopamine receptors w9,13–15,19,20x, but the role of D 2-like receptors appears to be more complex. Administration of selective D 2 antagonists results in robust striatal Fos expression w6,14,15x, suggesting that basal stimulation of D 2-receptors acts to inhibit production of the Fos protein. In contrast, the D 2-like agonist quinpirole markedly potentiates the Fos expression induced by the partial D 1 agonist SKF-38393 w10,13x, demonstrating that stimulation of D 2-receptors can also facilitate Fos synthesis under some conditions. These findings are in agreement with a large body of data demonstrating the presence of interactive effects of stimulation of D 1 and D 2 dopamine receptors w3x. When combined with the full D 1 agonist A-77636, which induces Fos expression by itself, quinpirole produces a regionally and compartmentally specific mixture of excitatory and inhibitory effects w19,20x, which results in the production of
) Corresponding [email protected]
author.
Fax:
q 1-312-413-2631;
E-mail:
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 3 7 8 - 4
a ‘patchy’ pattern of striatal labeling similar to that seen after administration of the indirect dopamine agonist amphetamine w1,8,20x. Although most studies of immediate early genes in the basal ganglia have focused on the effects of pharmacological agents, several workers have observed that striatal Fos expression can also be induced by behavioral manipulations w5,7,11,12,17,18x. These results suggest that examination of Fos expression may provide a technique for studying the physiological functioning of the striatum, and for determining the extent to which the principles derived from pharmacological induction studies are applicable to the normal operation of this structure. Recent work has suggested that stimulation of D 1-receptors plays a critical role in behaviorally induced Fos expression w11x. Since the striatal response to D 1 agonists can be greatly modified by coadministration of the D 2 -like agonist quinpirole w10,13,19,20x, we decided, in the current experiment, to examine the effects of quinpirole pretreatment on behaviorally induced Fos expression. For this purpose, we examined rats who were required to shuttle to escape from a mild footshock. Twenty-seven adult Sprague–Dawley derived male rats Ž375–500 g. obtained from the University of Illinois at Chicago Psychology Department’s breeding colony were used. Animals were housed individually in hanging wire
348
W.M. Struthers, D. Wirtshafterr Brain Research 785 (1998) 347–350
mesh cages with food and water freely available. Animals were maintained at 208C and tested during the light phase of the cycle Žlights onroff: 0700 hr1900 h.. The shuttle box measured 25.5 cm H = 20.5 cm W = 61 cm L and was divided into two compartments of equal size by a partition containing a 10 = 10-cm doorway. The floor consisted of stainless steel bars 1.7 mm in diameter spaced 1.3 cm apart. The position of the subjects was tracked by means of photocells positioned 8 cm from the divider in each of the two compartments. Escape latencies were recorded to the nearest 0.1 s. A constant power shock scrambler ŽBRSrForinger, SGS-001. was used to provide current to the floor bars. Animals were given subcutaneous injections of either saline Ž n s 6. or of quinpirole ŽEli Lilly, Indianapolis, IN. at doses of 0.156 mgrkg Ž n s 5., 0.625 mgrkg Ž n s 4., 1.25 mgrkg Ž n s 5. or 2.5 mgrkg Ž n s 5.. Twenty minutes later, the animals were placed into the shuttle box. After a 5-min adaptation period, rats received a mild footshock Ž0.05 W. which persisted until they escaped to the opposite side of the shuttle box. Escape trials were separated by 30 s and escape latencies from the onset of shock were recorded to the nearest 0.1 s. Escape training continued for 1 h, after which the animals were returned to their home cages. Additional control animals were injected with saline Ž n s 2. and simply returned to their home cages and sacrificed 95 min later. Animals were perfused 30 min after being replaced in their home cages. Subjects were anesthetized with sodium pentobarbital Ž100 mgrkg. and perfused transcardially with 100 ml of saline followed by 750 ml of 10% formalin in 0.1 N phosphate-buffered saline ŽPBS, pH 7.2.. The brains were removed and placed in fixative for 2 h at 48C. Brains were then transferred PBS containing 20% sucrose for at least 12 h. Brains were removed from the sucrose solution and rapidly frozen with Histofreeze spray ŽFischer Scientific, Pittsburgh, PA., after which 35-m m cryostat sections through the striatum were collected. The tissue was rinsed thoroughly in PBS and processed for FLI using methods similar to those we have previously described in detail w19x. Briefly, the tissue was incubated on a rotator for 72 h at 48C in a polyclonal rabbit anti-Fos antibody ŽAb5, Oncogene Research Products, Cambridge, MA. at a dilution of 50,000 = prepared in Tris-buffered saline containing 2% goat blocking serum, 0.2%, Triton X-100 and 0.05% sodium azide. The tissue was then rinsed and incubated in a biotinylated goat anti-rabbit secondary Ž200 = ; Vector Laboratories, Burlingame, CA. in the same buffer for 2 h at room temperature. After further rinsing, the tissue was incubated in the Vectastain Elite ABC reagent ŽVector Laboratories. in PBS for 1.5 h at room temperature. After three rinses in PBS, the tissue was developed using the nickel intensified diaminobenzadine technique. Sections were then rinsed again, air-dried and coverslipped with Eukit ŽCalibrated Instruments,
Hawthorne, NY.. Control sections were prepared by omitting the primary antibody, in which case no labeling could be observed. Sections through the striatum at a level slightly anterior to the disappearance of the longitudinal fissure in the septum w19x were examined. Fields in the dorsal striatum neighboring the lateral ventricle measuring 1.0 = 1.0 mm were captured and digitized using a Leica Q500 imaging system. Using standard image analysis techniques, the numbers of labeled cells were automatically measured. The effect of quinpirole pretreatment on mean escape latencies is shown in Fig. 1A. Quinpirole tended to produce a slight increase in escape latencies, but this effect did not approach statistical significance Ž F s 1.028, df s 4,20, p ) 0.4.. Almost no striatal labeling could be observed in subjects returned to their home cages following saline injections. In contrast, robust Fos-like-immunoreactivity ŽFLI. was present in the striatum of saline-injected animals who
Fig. 1. Effects of saline and quinpirole on escape latency and FLI in the striatum. ŽA. Effects of saline and quinpirole treatment on latency to escape in a shuttle box escape paradigm. ŽB. Imaging analysis of the number of cells displaying FLI in the striatum per mm2 after 1 h of forced escape behavior with saline or quinpirole treatment.
W.M. Struthers, D. Wirtshafterr Brain Research 785 (1998) 347–350
Fig. 2. Photomicrographs of the medial striatum after saline or quinpirole treatment followed by shock-induced escape behavior Ž63=, bar s 0.1 mm.. ŽA. Escape-induced FLI after saline, ŽB. Effects of quinpirole Ž2.5 mgrkg. pretreatment on escape-induced FLI.
shuttled prior to sacrifice ŽFig. 2A.. Darkly stained cells were observed primarily in the medial and dorsal regions of the striatum and no obvious ‘patchiness’ was apparent. Intense staining was also evident in a number of other telencephalic regions including the lateral septum, the claustrum and the medial frontal cortex. Administration of the D 2 agonist quinpirole produced a marked, dose-dependent, attenuation of FLI in the striatum of rats engaged in escape behavior ŽFig. 1B and Fig. 2B.. A one-way analysis of variance ŽANOVA. indicated that there was a significant effect of quinpirole dose on the number of cells displaying FLI in the striatum Ž F s 27.506, df s 4,20, p - 0.001.. Post-hoc analysis using the Tukey technique revealed that all doses of quinpirole attenuated FLI relative to saline injections Ž p - 0.05.. Additionally, significantly more cells were present in rats given the lowest dose of quinpirole Ž0.156 mgrkg. than in those given the two highest doses Ž1.25 mgrkg and 2.5 mgrkg; p - 0.05.. The results of this study demonstrate that escape behavior in a shuttle box elicits robust FLI in the striatum, perhaps reflecting the locomotor behavior generated by the
349
animals during the escape trials. It is possible that the stress of being shocked or of being placed in a novel environment may have also contributed to the observed responses w5,7,12x. Other workers have observed striatal Fos expression in animals subjected to immobilization stress and in subjects forced to swim w5x, or to run in a treadmill or running wheel w11,18x. We have also observed striatal Fos expression in male rats following sexual behavior w17x, suggesting that aversiveness is not essential for the induction of FLI. Further studies will be necessary to determine precisely which aspects of the present test situation were responsible for the elicitation of striatal FLI, but our results suggest that forced shuttling may provide a useful model for the physiological induction of immediate early gene expression in the striatum. This procedure for inducing Fos expression has the advantage that the behavior itself is relatively resistant to disruption by pharmacological treatments; in the current study, for example, quinpirole, even at a very high dose, did not significantly effect escape latencies. The most important result of the current study is the finding that the D 2-like agonist quinpirole produces a very pronounced, dose-dependent, attenuation of escape-induced striatal FLI. Since quinpirole did not markedly alter escape latencies, this result suggests that striatal Fos expression does not play an essential role in the performance of the shuttling response. In contrast to the suppression observed here, quinpirole has been shown to potentiate striatal Fos expression in animals injected with SKF-38393 w10,13,15x. Quinpirole also potentiates the response to the full D 1 agonist A-77636 in the lateral striatum and within patches in the medial and rostral striatum w19,20x. The current results, therefore, indicate that quinpirole interacts with the striatal Fos expression induced by shuttling in a very different fashion than it does that induced by D 1 agonists. Inhibitory effects of D 2 stimulation on Fos expression have, however, also been observed previously. Quinpirole pretreatment has been shown to greatly attenuate the striatal response to a number of nondopaminergic agents including fenfluramine, morphine and pilocarpine w4x, and has also been shown to suppress the staining induced by A-77636 in the matrix compartment of the rostral medial striatum w19,20x. Further work will be required to determine whether or not these various inhibitory effects of quinpirole on Fos expression are mediated through a single mechanism. The simplest explanation of the current results is that shuttle escape behavior leads to an increase in striatal dopamine release w16x, which consequently induces Fos expression as a result of stimulation of D 1 dopamine receptors. Quinpirole might then inhibit the Fos response by an action on inhibitory D 2 autoreceptors, which would be expected to reduce stimulated dopamine release. In this model, quinpirole would be functioning as an indirect D 1 antagonist. Since D 1-mediated Fos expression is more prominent in striatonigral than in striatopallidal neurons
350
W.M. Struthers, D. Wirtshafterr Brain Research 785 (1998) 347–350
w14x, this model makes specific predictions about which population of striatal cells express FLI in response to shuttling. Another possibility is that the effects of quinpirole might be due to direct activation of post-synaptic D 2-receptors. For example, D 2-receptors appear to inhibit Fos expression in striatopallidal cells w14x, and Fos expression in these neurons can be induced by stimulation of the frontal cortex w2x, a region that appears to be intensely activated during shuttle escape behavior. Further studies are clearly necessary to clarify the mechanism through which quinpirole exerts its effects on striatal FLI, but the current results demonstrate both that it is possible to conduct pharmacological studies on behaviorally induced Fos expression, and that dopamine plays an important role in these effects.
w7x
w8x
w9x
w10x
w11x
w12x
Acknowledgements w13x
The authors thank Eli Lily for their generous gift of quinpirole. This work was supported by NIH NS33992. w14x
References w1x K.E. Asin, D. Wirtshafter, Amphetamine induces Fos-like immunoreactivity in the striatum of primates, Brain Res. 719 Ž1996. 138–142. w2x S. Berretta, H.B. Parthasarathy, A.M. Graybiel, Local release of GABAergic inhibition in the motor cortex induces immediate-early gene expression in indirect pathway neurons of the striatum, J. Neurosci. 17 Ž1997. 4752–4763. w3x D. Clark, F.J. White, Review: D1 dopamine receptor: the search for function: a critical evaluation of the D1 rD 2 dopamine receptor classification and its functional implications, Synapse 1 Ž1987. 347–388. w4x D.F. Cook, D. Wirtshafter, The D 2 agonist quinpirole attenuates striatal c-fos expression induced by serotonergic, opiate and muscarinic agonists, Soc. Neurosci. Abstr. 22 Ž1996. 1089. w5x W.E. Cullinan, J.P. Herman, D.F. Battaglia, H. Akil, S.J. Watson, Pattern and time course of immediate early gene expression in rat brain following acute stress, Neuroscience 64 Ž1994. 477–505. w6x M. Dragunow, G.S. Robertson, R.L.M. Faull, H.A. Robertson, K.
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w20x
Jansen, D 2 dopamine receptor antagonists induce Fos and related proteins in rat striatal neurons, Neuroscience 37 Ž1990. 287–294. G.E. Duncan, D.J. Knapp, G.R. Breese, Neuroanatomical characterization of Fos induction in rat behavioral models of anxiety, Brain Res. 713 Ž1996. 79–91. A.M. Graybiel, R. Moratalla, H.A. Robertson, Amphetamine and cocaine induced drug-specific activation of the c-fos gene in striosome-matrix compartments and limbic subdivisions of the striatum, Proc. Natl. Acad. Sci. USA 87 Ž1990. 6912–6916. G.J. LaHoste, J.F. Marshall, Rapid development of D1 and D 2 dopamine receptor supersensitivity as indicated by striatal and pallidal Fos expression, Neurosci. Lett. 179 Ž1994. 153–156. G.J. LaHoste, J. Yu, J.F. Marshall, Striatal Fos expression is indicative of dopamine D1 rD 2 synergism and receptor supersensitivity, Proc. Natl. Acad. Sci. USA 90 Ž1993. 7451–7455. I. Liste, M.J. Guerra, H.J. Caruncho, J.L. Labandeira-Garcia, Treadmill running induces striatal Fos expression via NMDA glutamate and dopamine receptors, Exp. Brain Res. 115 Ž1997. 458–468. M. Papa, M.P. Pellicano, H. Welzl, A.G. Sadile, Distributed changes in c-fos and c-jun immunoreactivity in the rat brain associated with arousal and habituation to novelty, Brain Res. Bull. 32 Ž1993. 509–515. M.L. Paul, A.M. Graybiel, J.C. David, H.A. Robertson, D1-like and D 2 -like dopamine receptors synergistically activate rotation and c-fos expression in the dopamine-depleted striatum in a rat model of Parkinson’s disease, J. Neurosci. 12 Ž1992. 3729–3742. G.S. Robertson, S.R. Vincent, H.C. Fibiger, D1 and D 2 dopamine receptors differentially regulate c-fos expression in striatonigral and striatopallidal neurons, Neuroscience 49 Ž1992. 285–296. H.A. Robertson, M.L. Paul, R. Moratalla, A.M. Graybiel, Expression of the immediate early gene c-fos in basal ganglia: induction by dopaminergic drugs, Can. J. Neurol. Sci. 19 Ž1991. 380–383. S.G. Speciale, J.D. Miller, B.A. McMillen, D.C. German, Activation of specific central dopamine pathways: locomotion and footshock, Brain Res. Bull. 16 Ž1986. 33–38. W.M. Struthers, D. Wirtshafter, Copulation induces Fos-like-immunoreactivity in the striatum of male rats, Soc. Neurosci. Abstr. 21 Ž1995. 282.2. W.M. Struthers, D. Wirtshafter, Forced or voluntary locomotor activity elicits Fos-like immunoreactivity in the rat striatum, Soc. Neurosci. Abstr. 22 Ž1996. 1086. D. Wirtshafter, K.E. Asin, Interactive effects of stimulation of D1 and D 2 receptors on Fos-like immunoreactivity in the normosensitive rat striatum, Brain Res. Bull. 35 Ž1994. 85–91. D. Wirtshafter, G. Schardt, K.E. Asin, Compartmentally specific effects of quinpirole on the striatal Fos expression induced by stimulation of D1-dopamine receptors in intact rats, Brain Res. 771 Ž1997. 271–277.
Short communication
Quinpirole attenuates the striatal Fos expression induced by escape behavior W.M. Struthers, David Wirtshafter
)
Department of Psychology (M r C 285), The UniÕersity of Illinois at Chicago, 1007 West Harrison Street, Chicago, IL 60612, USA Accepted 4 November 1997
Abstract Several studies have shown that the D 2-like dopamine receptor agonist quinpirole is able to markedly potentiate the striatal Fos expression induced by D1 agonists. The present study examined the effects of quinpirole on the striatal Fos-like immunoreactivity ŽFLI. induced by escape behavior. Male rats were pretreated with either saline or quinpirole Ž0.156, 0.625, 1.25 or 2.5 mgrkg. and 30 min later, placed in a shuttle box and required to crossover every 30 s in order to escape mild footshock. Animals were sacrificed 30 min following the completion of a 1-h block of escape trials and sections through the striatum were processed for FLI. Pretreatment with quinpirole produced a marked, dose-dependent, attenuation of escape-induced FLI in the striatum. These findings demonstrate that quinpirole affects the striatal Fos expression induced by shuttling in a very different fashion than it does that induced by D1 agonists, and further support the view that dopaminergic mechanisms play an important role in behaviorally induced striatal Fos expression. q 1998 Elsevier Science B.V. Keywords: Quinpirole; c-fos; Shuttle box; Striatum; Dopamine; D 2 receptor; Caudate-putamen; Immediate-early genes
Many recent studies have used the expression of the proto-oncoprotein Fos as a marker for neurons affected by various treatments, and this technique has proved to be an especially powerful tool for examining the functional role of dopamine in the striatum. Striatal Fos expression can be enhanced by stimulation of D1-like dopamine receptors w9,13–15,19,20x, but the role of D 2-like receptors appears to be more complex. Administration of selective D 2 antagonists results in robust striatal Fos expression w6,14,15x, suggesting that basal stimulation of D 2-receptors acts to inhibit production of the Fos protein. In contrast, the D 2-like agonist quinpirole markedly potentiates the Fos expression induced by the partial D 1 agonist SKF-38393 w10,13x, demonstrating that stimulation of D 2-receptors can also facilitate Fos synthesis under some conditions. These findings are in agreement with a large body of data demonstrating the presence of interactive effects of stimulation of D 1 and D 2 dopamine receptors w3x. When combined with the full D 1 agonist A-77636, which induces Fos expression by itself, quinpirole produces a regionally and compartmentally specific mixture of excitatory and inhibitory effects w19,20x, which results in the production of
) Corresponding [email protected]
author.
Fax:
q 1-312-413-2631;
E-mail:
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 3 7 8 - 4
a ‘patchy’ pattern of striatal labeling similar to that seen after administration of the indirect dopamine agonist amphetamine w1,8,20x. Although most studies of immediate early genes in the basal ganglia have focused on the effects of pharmacological agents, several workers have observed that striatal Fos expression can also be induced by behavioral manipulations w5,7,11,12,17,18x. These results suggest that examination of Fos expression may provide a technique for studying the physiological functioning of the striatum, and for determining the extent to which the principles derived from pharmacological induction studies are applicable to the normal operation of this structure. Recent work has suggested that stimulation of D 1-receptors plays a critical role in behaviorally induced Fos expression w11x. Since the striatal response to D 1 agonists can be greatly modified by coadministration of the D 2 -like agonist quinpirole w10,13,19,20x, we decided, in the current experiment, to examine the effects of quinpirole pretreatment on behaviorally induced Fos expression. For this purpose, we examined rats who were required to shuttle to escape from a mild footshock. Twenty-seven adult Sprague–Dawley derived male rats Ž375–500 g. obtained from the University of Illinois at Chicago Psychology Department’s breeding colony were used. Animals were housed individually in hanging wire
348
W.M. Struthers, D. Wirtshafterr Brain Research 785 (1998) 347–350
mesh cages with food and water freely available. Animals were maintained at 208C and tested during the light phase of the cycle Žlights onroff: 0700 hr1900 h.. The shuttle box measured 25.5 cm H = 20.5 cm W = 61 cm L and was divided into two compartments of equal size by a partition containing a 10 = 10-cm doorway. The floor consisted of stainless steel bars 1.7 mm in diameter spaced 1.3 cm apart. The position of the subjects was tracked by means of photocells positioned 8 cm from the divider in each of the two compartments. Escape latencies were recorded to the nearest 0.1 s. A constant power shock scrambler ŽBRSrForinger, SGS-001. was used to provide current to the floor bars. Animals were given subcutaneous injections of either saline Ž n s 6. or of quinpirole ŽEli Lilly, Indianapolis, IN. at doses of 0.156 mgrkg Ž n s 5., 0.625 mgrkg Ž n s 4., 1.25 mgrkg Ž n s 5. or 2.5 mgrkg Ž n s 5.. Twenty minutes later, the animals were placed into the shuttle box. After a 5-min adaptation period, rats received a mild footshock Ž0.05 W. which persisted until they escaped to the opposite side of the shuttle box. Escape trials were separated by 30 s and escape latencies from the onset of shock were recorded to the nearest 0.1 s. Escape training continued for 1 h, after which the animals were returned to their home cages. Additional control animals were injected with saline Ž n s 2. and simply returned to their home cages and sacrificed 95 min later. Animals were perfused 30 min after being replaced in their home cages. Subjects were anesthetized with sodium pentobarbital Ž100 mgrkg. and perfused transcardially with 100 ml of saline followed by 750 ml of 10% formalin in 0.1 N phosphate-buffered saline ŽPBS, pH 7.2.. The brains were removed and placed in fixative for 2 h at 48C. Brains were then transferred PBS containing 20% sucrose for at least 12 h. Brains were removed from the sucrose solution and rapidly frozen with Histofreeze spray ŽFischer Scientific, Pittsburgh, PA., after which 35-m m cryostat sections through the striatum were collected. The tissue was rinsed thoroughly in PBS and processed for FLI using methods similar to those we have previously described in detail w19x. Briefly, the tissue was incubated on a rotator for 72 h at 48C in a polyclonal rabbit anti-Fos antibody ŽAb5, Oncogene Research Products, Cambridge, MA. at a dilution of 50,000 = prepared in Tris-buffered saline containing 2% goat blocking serum, 0.2%, Triton X-100 and 0.05% sodium azide. The tissue was then rinsed and incubated in a biotinylated goat anti-rabbit secondary Ž200 = ; Vector Laboratories, Burlingame, CA. in the same buffer for 2 h at room temperature. After further rinsing, the tissue was incubated in the Vectastain Elite ABC reagent ŽVector Laboratories. in PBS for 1.5 h at room temperature. After three rinses in PBS, the tissue was developed using the nickel intensified diaminobenzadine technique. Sections were then rinsed again, air-dried and coverslipped with Eukit ŽCalibrated Instruments,
Hawthorne, NY.. Control sections were prepared by omitting the primary antibody, in which case no labeling could be observed. Sections through the striatum at a level slightly anterior to the disappearance of the longitudinal fissure in the septum w19x were examined. Fields in the dorsal striatum neighboring the lateral ventricle measuring 1.0 = 1.0 mm were captured and digitized using a Leica Q500 imaging system. Using standard image analysis techniques, the numbers of labeled cells were automatically measured. The effect of quinpirole pretreatment on mean escape latencies is shown in Fig. 1A. Quinpirole tended to produce a slight increase in escape latencies, but this effect did not approach statistical significance Ž F s 1.028, df s 4,20, p ) 0.4.. Almost no striatal labeling could be observed in subjects returned to their home cages following saline injections. In contrast, robust Fos-like-immunoreactivity ŽFLI. was present in the striatum of saline-injected animals who
Fig. 1. Effects of saline and quinpirole on escape latency and FLI in the striatum. ŽA. Effects of saline and quinpirole treatment on latency to escape in a shuttle box escape paradigm. ŽB. Imaging analysis of the number of cells displaying FLI in the striatum per mm2 after 1 h of forced escape behavior with saline or quinpirole treatment.
W.M. Struthers, D. Wirtshafterr Brain Research 785 (1998) 347–350
Fig. 2. Photomicrographs of the medial striatum after saline or quinpirole treatment followed by shock-induced escape behavior Ž63=, bar s 0.1 mm.. ŽA. Escape-induced FLI after saline, ŽB. Effects of quinpirole Ž2.5 mgrkg. pretreatment on escape-induced FLI.
shuttled prior to sacrifice ŽFig. 2A.. Darkly stained cells were observed primarily in the medial and dorsal regions of the striatum and no obvious ‘patchiness’ was apparent. Intense staining was also evident in a number of other telencephalic regions including the lateral septum, the claustrum and the medial frontal cortex. Administration of the D 2 agonist quinpirole produced a marked, dose-dependent, attenuation of FLI in the striatum of rats engaged in escape behavior ŽFig. 1B and Fig. 2B.. A one-way analysis of variance ŽANOVA. indicated that there was a significant effect of quinpirole dose on the number of cells displaying FLI in the striatum Ž F s 27.506, df s 4,20, p - 0.001.. Post-hoc analysis using the Tukey technique revealed that all doses of quinpirole attenuated FLI relative to saline injections Ž p - 0.05.. Additionally, significantly more cells were present in rats given the lowest dose of quinpirole Ž0.156 mgrkg. than in those given the two highest doses Ž1.25 mgrkg and 2.5 mgrkg; p - 0.05.. The results of this study demonstrate that escape behavior in a shuttle box elicits robust FLI in the striatum, perhaps reflecting the locomotor behavior generated by the
349
animals during the escape trials. It is possible that the stress of being shocked or of being placed in a novel environment may have also contributed to the observed responses w5,7,12x. Other workers have observed striatal Fos expression in animals subjected to immobilization stress and in subjects forced to swim w5x, or to run in a treadmill or running wheel w11,18x. We have also observed striatal Fos expression in male rats following sexual behavior w17x, suggesting that aversiveness is not essential for the induction of FLI. Further studies will be necessary to determine precisely which aspects of the present test situation were responsible for the elicitation of striatal FLI, but our results suggest that forced shuttling may provide a useful model for the physiological induction of immediate early gene expression in the striatum. This procedure for inducing Fos expression has the advantage that the behavior itself is relatively resistant to disruption by pharmacological treatments; in the current study, for example, quinpirole, even at a very high dose, did not significantly effect escape latencies. The most important result of the current study is the finding that the D 2-like agonist quinpirole produces a very pronounced, dose-dependent, attenuation of escape-induced striatal FLI. Since quinpirole did not markedly alter escape latencies, this result suggests that striatal Fos expression does not play an essential role in the performance of the shuttling response. In contrast to the suppression observed here, quinpirole has been shown to potentiate striatal Fos expression in animals injected with SKF-38393 w10,13,15x. Quinpirole also potentiates the response to the full D 1 agonist A-77636 in the lateral striatum and within patches in the medial and rostral striatum w19,20x. The current results, therefore, indicate that quinpirole interacts with the striatal Fos expression induced by shuttling in a very different fashion than it does that induced by D 1 agonists. Inhibitory effects of D 2 stimulation on Fos expression have, however, also been observed previously. Quinpirole pretreatment has been shown to greatly attenuate the striatal response to a number of nondopaminergic agents including fenfluramine, morphine and pilocarpine w4x, and has also been shown to suppress the staining induced by A-77636 in the matrix compartment of the rostral medial striatum w19,20x. Further work will be required to determine whether or not these various inhibitory effects of quinpirole on Fos expression are mediated through a single mechanism. The simplest explanation of the current results is that shuttle escape behavior leads to an increase in striatal dopamine release w16x, which consequently induces Fos expression as a result of stimulation of D 1 dopamine receptors. Quinpirole might then inhibit the Fos response by an action on inhibitory D 2 autoreceptors, which would be expected to reduce stimulated dopamine release. In this model, quinpirole would be functioning as an indirect D 1 antagonist. Since D 1-mediated Fos expression is more prominent in striatonigral than in striatopallidal neurons
350
W.M. Struthers, D. Wirtshafterr Brain Research 785 (1998) 347–350
w14x, this model makes specific predictions about which population of striatal cells express FLI in response to shuttling. Another possibility is that the effects of quinpirole might be due to direct activation of post-synaptic D 2-receptors. For example, D 2-receptors appear to inhibit Fos expression in striatopallidal cells w14x, and Fos expression in these neurons can be induced by stimulation of the frontal cortex w2x, a region that appears to be intensely activated during shuttle escape behavior. Further studies are clearly necessary to clarify the mechanism through which quinpirole exerts its effects on striatal FLI, but the current results demonstrate both that it is possible to conduct pharmacological studies on behaviorally induced Fos expression, and that dopamine plays an important role in these effects.
w7x
w8x
w9x
w10x
w11x
w12x
Acknowledgements w13x
The authors thank Eli Lily for their generous gift of quinpirole. This work was supported by NIH NS33992. w14x
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