338
Brain Research, 511 (1990) 338-340 Elsevier
BRES 23999
Granule cells in the ventral dentate gyrus are essential for kainic acid-induced wet dog shakes but not those induced by precipitated abstinence in morphine-dependent rats Clifford L. Mitchell 1, Laura M. Grimes 2 and Jau-Shyong Hong 1 1Laboratory of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (U.S.A.) and 2Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC 27514 (U.S.A.) (Accepted 28 November 1989) Key words: Colchicine; Dentate granule cell; Ventral hippocampus; Wet dog shake; Kainic acid; Morphine; Naltrexone
Wet dog shaking elicited by systemic administration of kainic acid is eliminated by bilateral destruction of ventral dentate granule cells. In contrast, wet dog shaking induced by naltrexone precipitated abstinence in morphine-dependent rats is unaffected by destruction of ventral dentate granule cells. It is concluded that at least two anatomically distinct brain regions modulate wet dog shaking behavior. Paroxysmal shaking of the head, neck and trunk in rats can be induced by a variety of chemical and physical stimuli. This behavior has been referred to as 'wet dog shakes' (WDS) because it resembles the shaking behavior observed in wet dogs 15. Although WDS can be induced in morphine-dependent rats after the administration of an opioid antagonist 14, WDS induced by activation of the limbic system can be attenuated by opioid antagonists 3' 4,6,11
In morphine-dependent rats, WDS are elicited by naloxone, an opioid antagonist, when administered directly to the medial thalamus, or mesodiencephalic junction but not when administered to neocortical, hippocampal, or tegmental areas 19. They are eliminated by transection of the brain at the mid-collicular level but not by lesions anterior to the fasciculus retroflexus is. On the other hand, WDS induced by activation of the limbic system (by electrical stimulation, opioid peptides or kainic acid) are abolished by destruction of dentate granule cells (DGC) of the ventral hippocampal formation 1'2'8'13. There has not, however, been a direct comparison of the effect of granule cell destruction on WDS induced by morphine withdrawal vs. activation of the limbic system with a compound such as kainic acid (KA), a cyclic analog of glutamate. We report here that destruction of ventral DGC has no effect on WDS induced by naltrexone administration to morphine-dependent animals but abolishes KA-induced WDS in these same animals.
Male, Fischer-344 rats weighing 237-280 g were obtained from Charles River Breeding Company. They were housed (4/cage) in plastic cages with cedar chip bedding and maintained on a 12 h light-dark cycle in a temperature and humidity controlled room with access to NIH Diet 31 and water ad lib. For surgery, each animal received pentobarbital, 50 mg/kg, i.p. Injections were made bilaterally into the ventral hippocampus. The coordinates, taken from Paxinos and Watson 16 were 5.8 mm posterior, 4.5 mm lateral and 7.0 mm deep. Anterior-posterior measurements were made with reference to bregma, lateral measurements were made with reference to the sagittal suture, and depth was measured from the skull at the point of placement. All measurements were made with the skull flat. Each animal was injected bilaterally with 0.5/~1 of physiological saline or 2.5/~g colchicine (Sigma, St. Louis, MO) in 0.5 ktl physiological saline per site. Injections were made with a rate of 0.1/~l/min using a 1.0 /~1 Hamilton syringe and a manually operated microinjector (David Kopf Instruments). The microinjector was left in place for 2.0 min to allow diffusion of fluid away from the site. One week later the animals, under ether anesthesia, were implanted between the shoulder blades with a 75 mg morphine pellet. Forty-eight hours later they were implanted with two 75 mg morphine pellets. After an additional 48 h period they were injected with naltrexone, 1 mg/kg, s.c. The number of WDS occurring
Correspondence: C.L. Mitchell, Lab. of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709, U.S.A.
339
Fig. 1. The intactness of the granule cells was determined with Cresyl violet stain. In rats injected with saline in ventral hippocampus, no damage was observed (arrow B). However, rats injected with colchicine exhibit loss of granule cells in ventral (D) but not dorsal (C) dentate. Dorsal and ventral sections were taken from the same animals (A-D). over the next hour was recorded. Nine days later, all animals received K A , 8 mg/kg, s.c. and observed for W D S over a 2 h period. They were then anesthetized
The m e a n values + S.E.M. for the n u m b e r of WDS are shown in Table I. Ventral colchicine lesions had no effect on W D S induced by naltrexone in morphine
with pentobarbital sodium, 50 mg/kg, i.p. Three days later they were again anesthetized with pentobarbital sodium and perfused intracardially with 0.85% saline followed by 10% neutral buffered formalin. The brains were removed, blocked, and m o u n t e d in paraffin. They were subsequently sliced at 10/~m. O n e of every 10 slices was m o u n t e d and one in every 20 slices was stained with Cresyl violet for analysis of lesion damage. Animals were discarded from further analysis if their lesions were unilateral or involved another structure. Thus, upon completion of the histological analysis, a total of 12 animals r e m a i n e d for statistical analysis: 7 ventral saline and 5 ventral colchicine. Fig. 1 illustrates typical histological effects seen in saline and colchicine treated animals. Data were analyzed using M a n n - W h i t n e y U-tests 17. Comparisons made were no. of WDS elicited by (a) naltrexone and (b) K A in the ventral saline vs. ventral colchicine groups, with P ~< 0.05 as the level of significance.
d e p e n d e n t rats. In contrast, these rats exhibited no W D S when injected with KA. It is becoming increasingly apparent that at least two anatomically distinct brain regions modulate W D S be-
TABLE I N u m b e r o f wet dog shakes
Numbers represent mean + S.E.M. of WDS occurring over a 1 h period for naltrexone, 1 mg/kg, s.c., and a 2 h period for kainic acid, 8 mg/kg, s.c.; all animals received three 75 mg/kg morphine pellets over the course of 6 days prior to naltrexone; the number of animals per group (n) is shown in parentheses.
Naltrexone Kainic acid
Ventral saline (n = 7)
Ventral colchicine (n = 5)
23 + 2.2 90 + 18
22 + 2.7 0 + 0*
* Statistically different from the corresponding ventral saline control group by Mann-Whitney U-test, P < 0.003, 2-tailed.
340 havior. O n e site lies in the mesodiencephalic region of the brain (rostral to the inferior colliculus and caudal to the fasciculus retroflexus). It is this region which is responsible for the WDS elicited by naloxone administration to morphine d e p e n d e n t rats t8-21. The other area lies in the ventral dentate gyrus. In this area, opioid peptides elicit WDS 12'13. Moreover, lesions of dentate granule cells block W D S induced by intrahippocampal administration of opioid peptides 13, stimulation of the perforant path t and systemically administered K A 8. That endogenous opioids contribute to KA-induced W D S is supported by the fact that they are attenuated by the opioid antagonist naloxone 6"7. It is apparent, however, that non-opioid mechanisms may also contribute to 1 Barnes, M.I. and Mitchell, C.L., Colchicine lesions of ventral, but not dorsal, dentate granule cells attenuate wet dog shakes elicited by perforant path stimulation, Brain Research, submitted. 2 Barnes, M.I. and Mitchell, C.L., Differential effects of colchicine lesions of dentate granule cells on wet dog shakes and seizures elicited by direct hippocampal stimulation, Brain Research, submitted. 3 Bloom, E, Segal, D., Ling, N. and Guillemin, R., Endorphins: profound behavioral effects in rats suggest new etiological factors in mental illness, Science, 194 (1976) 630-632. 4 Drust, E.G. and Connor, J.D., Pharmacological analysis of shaking behavior induced by enkephalins, thyrotropin-releasing hormone or serotonin in rats: evidence for different mechanisms, J. Pharmacol. Exp. Ther., 224 (1983) 148-154. 5 Frush, D.P. and McNamara, J.O., Evidence implicating dentate granule cells in wet dog shakes produced by kindling stimulations of entorhinal cortex, Exp. Neurol., 92 (1986) 102-113. 6 Fuller, T.A. and Olney, J.W., Effects of morphine or naloxone on kainic acid neurotoxicity, Life Sci., 24 (1979) 1793-1798. 7 Grimes, L., Hong, J., McGinty, J., Mitchell, C., Obie, J. and Tilson, H., Enkephalin contained in dentate granule ceils is important for kainic acid-induced wet dog shakes. In J.W. Holaday, P.-Y. Law and A. Herz (Eds.), Progress in Opioid Research: Proceedings of the 1986 International Narcotics Research Committee, NIDA Research Monograph No. 75, Rockville, 1986, pp. 481-484. 8 Grimes, L.M., Earnhardt, T.S., Mitchell, C.L., Tilson, H.A. and Hong, J.S., Granule cells in the ventral, but not dorsal, dentate gyrus are essential for kainic acid-induced wet dog shakes, Neurosci. Lett., submitted. 9 Lal, H., Gianutsos, G. and Puri, S., A comparison of narcotic analgesics with neuroleptics on behavioral measures of dopa-
KA-induced WDS. Numerous agents (e.g. G A B A m i m e tic, norepinephrine, atropine and antiserotonergic) reduce these WDS 9'1°. W h e t h e r these agents are acting on the D G C of the ventral dentate gyrus or at some other site to reduce these WDS has not been studied. In view of the fact that granule cell discharge appears essential for W D S elicited by limbic seizures 5, we suggest that any agent which acts to reduce this discharge would be effective in reducing K A - i n d u c e d WDS. Obviously, further research is needed to clarify the contributions of opioid and non-opioid mechanism to K A - i n d u c e d WDS. This research was supported in part by training Grant 5T32-ES-07126 (L.M.G.). We thank Mrs. Loretta Moore for manuscript preparation. minergic activity, Life Sci., 17 (1975) 29-34. 10 Lal, H. and Numan, R., Blockade of morphine-withdrawal body shakes by haloperidol, Life Sci., 18 (1975) 163-168. 11 Lanthorn, T. and Isaacson, R.L., Studies of kainate induced wet dog shakes in the rat, Life Sci., 22 (1978) 171-178. 12 Lee, P.H.K., Obie, J. and Hong, J.S., Opioids induce convulsions and wet dog shakes in rats: mediation by hippocampal mu, but not delta or kappa opioid receptors, J. Neurosci., 9 (1989) 692-697. 13 Lee, P.H.K. and Hong, J.S., Ventral hippocampal dentate granule cell lesions enhance motor seizures but reduce wet dog shakes induced by mu opioid receptor agonist, Neuroscience, in press. 14 Martin, W.R., Opioid antagonists, Pharmacol. Rev., 19 (1967) 464-521. 15 Martin, W.R., Wikler, A., Eades, C.G. and Pescor, ET., Tolerance to and physical dependence on morphine in rats, Psychopharmacologia, 4 (1963) 247-260. 16 Paxinos, G. and Watson, C., The Brain in Stereotaxic Coordinates, 2nd ed., Academic Press, New York, 1986, Plates 33 and 49. 17 Siegel, S., Nonparametric Statistics, McGraw-Hill, New York, 1956, pp. 312. 18 Wei, E., Brain lesions attenuating 'wet shakes' behavior in morphine-abstinent rats, Life Sci., 12 (1973) 385-392. 19 Wei, E., Loh, H. and Way, E.L., Neuroanatomical correlates of morphine dependence, Science, 177 (1972) 616-617. 20 Wei, E., Loh, H. and Way, E., Neuroanatomical correlates of wet shake behavior in the rat, Life Sci., 12 (1973) 489-496. 21 Wei, E., Sigel, S. and Way, E., Regional sensitivity of the rat brain to the inhibitory effects of morphine on wet shake behavior, J. Pharmacol. Exp. Ther., 193 (1975) 56-63.
Brain Research, 511 (1990) 338-340 Elsevier
BRES 23999
Granule cells in the ventral dentate gyrus are essential for kainic acid-induced wet dog shakes but not those induced by precipitated abstinence in morphine-dependent rats Clifford L. Mitchell 1, Laura M. Grimes 2 and Jau-Shyong Hong 1 1Laboratory of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (U.S.A.) and 2Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC 27514 (U.S.A.) (Accepted 28 November 1989) Key words: Colchicine; Dentate granule cell; Ventral hippocampus; Wet dog shake; Kainic acid; Morphine; Naltrexone
Wet dog shaking elicited by systemic administration of kainic acid is eliminated by bilateral destruction of ventral dentate granule cells. In contrast, wet dog shaking induced by naltrexone precipitated abstinence in morphine-dependent rats is unaffected by destruction of ventral dentate granule cells. It is concluded that at least two anatomically distinct brain regions modulate wet dog shaking behavior. Paroxysmal shaking of the head, neck and trunk in rats can be induced by a variety of chemical and physical stimuli. This behavior has been referred to as 'wet dog shakes' (WDS) because it resembles the shaking behavior observed in wet dogs 15. Although WDS can be induced in morphine-dependent rats after the administration of an opioid antagonist 14, WDS induced by activation of the limbic system can be attenuated by opioid antagonists 3' 4,6,11
In morphine-dependent rats, WDS are elicited by naloxone, an opioid antagonist, when administered directly to the medial thalamus, or mesodiencephalic junction but not when administered to neocortical, hippocampal, or tegmental areas 19. They are eliminated by transection of the brain at the mid-collicular level but not by lesions anterior to the fasciculus retroflexus is. On the other hand, WDS induced by activation of the limbic system (by electrical stimulation, opioid peptides or kainic acid) are abolished by destruction of dentate granule cells (DGC) of the ventral hippocampal formation 1'2'8'13. There has not, however, been a direct comparison of the effect of granule cell destruction on WDS induced by morphine withdrawal vs. activation of the limbic system with a compound such as kainic acid (KA), a cyclic analog of glutamate. We report here that destruction of ventral DGC has no effect on WDS induced by naltrexone administration to morphine-dependent animals but abolishes KA-induced WDS in these same animals.
Male, Fischer-344 rats weighing 237-280 g were obtained from Charles River Breeding Company. They were housed (4/cage) in plastic cages with cedar chip bedding and maintained on a 12 h light-dark cycle in a temperature and humidity controlled room with access to NIH Diet 31 and water ad lib. For surgery, each animal received pentobarbital, 50 mg/kg, i.p. Injections were made bilaterally into the ventral hippocampus. The coordinates, taken from Paxinos and Watson 16 were 5.8 mm posterior, 4.5 mm lateral and 7.0 mm deep. Anterior-posterior measurements were made with reference to bregma, lateral measurements were made with reference to the sagittal suture, and depth was measured from the skull at the point of placement. All measurements were made with the skull flat. Each animal was injected bilaterally with 0.5/~1 of physiological saline or 2.5/~g colchicine (Sigma, St. Louis, MO) in 0.5 ktl physiological saline per site. Injections were made with a rate of 0.1/~l/min using a 1.0 /~1 Hamilton syringe and a manually operated microinjector (David Kopf Instruments). The microinjector was left in place for 2.0 min to allow diffusion of fluid away from the site. One week later the animals, under ether anesthesia, were implanted between the shoulder blades with a 75 mg morphine pellet. Forty-eight hours later they were implanted with two 75 mg morphine pellets. After an additional 48 h period they were injected with naltrexone, 1 mg/kg, s.c. The number of WDS occurring
Correspondence: C.L. Mitchell, Lab. of Molecular and Integrative Neuroscience, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709, U.S.A.
339
Fig. 1. The intactness of the granule cells was determined with Cresyl violet stain. In rats injected with saline in ventral hippocampus, no damage was observed (arrow B). However, rats injected with colchicine exhibit loss of granule cells in ventral (D) but not dorsal (C) dentate. Dorsal and ventral sections were taken from the same animals (A-D). over the next hour was recorded. Nine days later, all animals received K A , 8 mg/kg, s.c. and observed for W D S over a 2 h period. They were then anesthetized
The m e a n values + S.E.M. for the n u m b e r of WDS are shown in Table I. Ventral colchicine lesions had no effect on W D S induced by naltrexone in morphine
with pentobarbital sodium, 50 mg/kg, i.p. Three days later they were again anesthetized with pentobarbital sodium and perfused intracardially with 0.85% saline followed by 10% neutral buffered formalin. The brains were removed, blocked, and m o u n t e d in paraffin. They were subsequently sliced at 10/~m. O n e of every 10 slices was m o u n t e d and one in every 20 slices was stained with Cresyl violet for analysis of lesion damage. Animals were discarded from further analysis if their lesions were unilateral or involved another structure. Thus, upon completion of the histological analysis, a total of 12 animals r e m a i n e d for statistical analysis: 7 ventral saline and 5 ventral colchicine. Fig. 1 illustrates typical histological effects seen in saline and colchicine treated animals. Data were analyzed using M a n n - W h i t n e y U-tests 17. Comparisons made were no. of WDS elicited by (a) naltrexone and (b) K A in the ventral saline vs. ventral colchicine groups, with P ~< 0.05 as the level of significance.
d e p e n d e n t rats. In contrast, these rats exhibited no W D S when injected with KA. It is becoming increasingly apparent that at least two anatomically distinct brain regions modulate W D S be-
TABLE I N u m b e r o f wet dog shakes
Numbers represent mean + S.E.M. of WDS occurring over a 1 h period for naltrexone, 1 mg/kg, s.c., and a 2 h period for kainic acid, 8 mg/kg, s.c.; all animals received three 75 mg/kg morphine pellets over the course of 6 days prior to naltrexone; the number of animals per group (n) is shown in parentheses.
Naltrexone Kainic acid
Ventral saline (n = 7)
Ventral colchicine (n = 5)
23 + 2.2 90 + 18
22 + 2.7 0 + 0*
* Statistically different from the corresponding ventral saline control group by Mann-Whitney U-test, P < 0.003, 2-tailed.
340 havior. O n e site lies in the mesodiencephalic region of the brain (rostral to the inferior colliculus and caudal to the fasciculus retroflexus). It is this region which is responsible for the WDS elicited by naloxone administration to morphine d e p e n d e n t rats t8-21. The other area lies in the ventral dentate gyrus. In this area, opioid peptides elicit WDS 12'13. Moreover, lesions of dentate granule cells block W D S induced by intrahippocampal administration of opioid peptides 13, stimulation of the perforant path t and systemically administered K A 8. That endogenous opioids contribute to KA-induced W D S is supported by the fact that they are attenuated by the opioid antagonist naloxone 6"7. It is apparent, however, that non-opioid mechanisms may also contribute to 1 Barnes, M.I. and Mitchell, C.L., Colchicine lesions of ventral, but not dorsal, dentate granule cells attenuate wet dog shakes elicited by perforant path stimulation, Brain Research, submitted. 2 Barnes, M.I. and Mitchell, C.L., Differential effects of colchicine lesions of dentate granule cells on wet dog shakes and seizures elicited by direct hippocampal stimulation, Brain Research, submitted. 3 Bloom, E, Segal, D., Ling, N. and Guillemin, R., Endorphins: profound behavioral effects in rats suggest new etiological factors in mental illness, Science, 194 (1976) 630-632. 4 Drust, E.G. and Connor, J.D., Pharmacological analysis of shaking behavior induced by enkephalins, thyrotropin-releasing hormone or serotonin in rats: evidence for different mechanisms, J. Pharmacol. Exp. Ther., 224 (1983) 148-154. 5 Frush, D.P. and McNamara, J.O., Evidence implicating dentate granule cells in wet dog shakes produced by kindling stimulations of entorhinal cortex, Exp. Neurol., 92 (1986) 102-113. 6 Fuller, T.A. and Olney, J.W., Effects of morphine or naloxone on kainic acid neurotoxicity, Life Sci., 24 (1979) 1793-1798. 7 Grimes, L., Hong, J., McGinty, J., Mitchell, C., Obie, J. and Tilson, H., Enkephalin contained in dentate granule ceils is important for kainic acid-induced wet dog shakes. In J.W. Holaday, P.-Y. Law and A. Herz (Eds.), Progress in Opioid Research: Proceedings of the 1986 International Narcotics Research Committee, NIDA Research Monograph No. 75, Rockville, 1986, pp. 481-484. 8 Grimes, L.M., Earnhardt, T.S., Mitchell, C.L., Tilson, H.A. and Hong, J.S., Granule cells in the ventral, but not dorsal, dentate gyrus are essential for kainic acid-induced wet dog shakes, Neurosci. Lett., submitted. 9 Lal, H., Gianutsos, G. and Puri, S., A comparison of narcotic analgesics with neuroleptics on behavioral measures of dopa-
KA-induced WDS. Numerous agents (e.g. G A B A m i m e tic, norepinephrine, atropine and antiserotonergic) reduce these WDS 9'1°. W h e t h e r these agents are acting on the D G C of the ventral dentate gyrus or at some other site to reduce these WDS has not been studied. In view of the fact that granule cell discharge appears essential for W D S elicited by limbic seizures 5, we suggest that any agent which acts to reduce this discharge would be effective in reducing K A - i n d u c e d WDS. Obviously, further research is needed to clarify the contributions of opioid and non-opioid mechanism to K A - i n d u c e d WDS. This research was supported in part by training Grant 5T32-ES-07126 (L.M.G.). We thank Mrs. Loretta Moore for manuscript preparation. minergic activity, Life Sci., 17 (1975) 29-34. 10 Lal, H. and Numan, R., Blockade of morphine-withdrawal body shakes by haloperidol, Life Sci., 18 (1975) 163-168. 11 Lanthorn, T. and Isaacson, R.L., Studies of kainate induced wet dog shakes in the rat, Life Sci., 22 (1978) 171-178. 12 Lee, P.H.K., Obie, J. and Hong, J.S., Opioids induce convulsions and wet dog shakes in rats: mediation by hippocampal mu, but not delta or kappa opioid receptors, J. Neurosci., 9 (1989) 692-697. 13 Lee, P.H.K. and Hong, J.S., Ventral hippocampal dentate granule cell lesions enhance motor seizures but reduce wet dog shakes induced by mu opioid receptor agonist, Neuroscience, in press. 14 Martin, W.R., Opioid antagonists, Pharmacol. Rev., 19 (1967) 464-521. 15 Martin, W.R., Wikler, A., Eades, C.G. and Pescor, ET., Tolerance to and physical dependence on morphine in rats, Psychopharmacologia, 4 (1963) 247-260. 16 Paxinos, G. and Watson, C., The Brain in Stereotaxic Coordinates, 2nd ed., Academic Press, New York, 1986, Plates 33 and 49. 17 Siegel, S., Nonparametric Statistics, McGraw-Hill, New York, 1956, pp. 312. 18 Wei, E., Brain lesions attenuating 'wet shakes' behavior in morphine-abstinent rats, Life Sci., 12 (1973) 385-392. 19 Wei, E., Loh, H. and Way, E.L., Neuroanatomical correlates of morphine dependence, Science, 177 (1972) 616-617. 20 Wei, E., Loh, H. and Way, E., Neuroanatomical correlates of wet shake behavior in the rat, Life Sci., 12 (1973) 489-496. 21 Wei, E., Sigel, S. and Way, E., Regional sensitivity of the rat brain to the inhibitory effects of morphine on wet shake behavior, J. Pharmacol. Exp. Ther., 193 (1975) 56-63.
