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Responses to cold, heat, and pain increase locomotion in rats and are attenuated by pinealectomy
Physiology& Behavior,Vol. 55, No. 3, pp. 583-586, 1994 Copyright© 1994ElsevierScienceLtd Printedin the USA.All fightsreserved 0031-9384/94$6.00 + .00
Pergamon
Responses to Cold, Heat, and Pain Increase Locomotion in Rats and Are Attenuated by Pinealectomy J. I. C H U A N G A N D M. T. LIN 1
Department of Physiology, National Cheng Kung University Medical College, Tainan city, Taiwan, Republic of China Received 22 F e b r u a r y 1993 CHUANG, J. I. AND M. T. LIN. Responses to cold, heat, and pain increaselocomotion in rats and are attenuatedby pinealectomy. PHYS1OL BEHAV 55(3) 583-586, 1994.--The effects of pinealectomy on locomotor behavior responses to cold, heat or pain were assessed in freely moving rats. External cold (4°C) or heat (36°C) stress produced increases of locomotion (including horizontal and vertical movement, and total distance traveled), increases of number of turnings (including both clockwise and counterclockwise), and decreases of postural freezing in rats. In addition, pain (produced by intradermal injection of normal saline) was also shown to produce increases of locomotion (including horizontal and vertical movement, and total distance traveled) and decreases of postural freezing in rats. The increases of locomotion (including horizontal and vertical movement, and total distance traveled), as well as the decreases of postural freezing induced by either cold or pain, were attenuated by pretreatment of animals with pinealectomy. The heat-induced increasesof vertical movement as well as the decreases of postural freezing were also attenuated by pinealectomy. The results indicate that these nonphotic, stress-provokingstimuli act through the pineal gland to induce escape behaviors to try to get out of the stressed conditions in rats. Pineal gland
Cold
Heat
Pain
Locomotion
Behavior
THE activity of the mammalian pineal gland is primarily under the control of the environmental photoperiodl. Several nonphotic, stress-provoking stimuli, such as ambient temperature, forced immobilization, forced swimming and insulin-induced hypoglycemia, have been shown to influence the pineal activity in a variety of small rodent species (17,20,22-24,27,30-35). In addition, a bulk of evidence indicates that a number of behavioral responses exerted by some stress-provoking stimuli (such as cold, heat and pain) are mediated by the pineal gland (1,9,10,14,15,18,20). Recently, we have provided a new modularized infrared light matrix systen with high resolution for measuring animal behaviors (36). It can record the sequences of animal's activity in a computer-aided system with a resolution of 0.2 s in time and 1.6 cm in space, and permanently stores all the resulting data in file. In the current study, we have attempted to apply this system to assess the behavioral responses to cold, heat, or pain in shamoperated rats and in pinealectomized rats to assess any possible involvement of the pineal gland in behavioral responses induced by cold, heat, or pain.
1To whom requests for reprints should be addressed.
583
METHOD Male Sprague-Dawley rats were used in the present experiments. Upon receipt from the supplier (Animal Resource Center, National Cheng Kung University Medical College, Tainan city, Taiwan, ROC), the animals were housed in pairs in a temperature-regulated (25 + I°C) room at a 12:12 h light:dark cycle with water and food ad lib. The light was turned on at 0600 h and turned offat 1800 h. Pinealectomy was performed according to the method of Hoffman and Reiter (16) and Kuszak and Rodin (19) at 1 month after birth (body weight about 120 g). The animal under pentobarbital sodium (50 mg/kg, IP) anesthesia was mounted on a stereotaxic instrument. A midline longituidinal incision was made exposing the occipital and parietal bones of the skull. Then a bone disc (4 m m in diameter) was removed just above the confluence of the superior sagittal and transverse sinuses. The bone piece was then lifted off the underlying dura from its rostral edge, exposing the superior saglttal vein (SSV), the transverse sinus (TS), and the confluens sinus (CS). The pineal gland was removed by inserting an open pair of forceps into the junction between the two sinuses, grasping the stalk, and removing gland and stalk in one motion. Excessive
584
CHUANG AND LIN
hemorrhage was prevented by rapidly returning the bone disc to its roiginal position and by the application of collagen hemostatic felt. Finally, the incision was sutured and iodine was applied over the wound. Sham-operated animals had identical suregry, but no damage was applied to the gland. A comparison of weight gain between sham-operated and pinealectomized animals showed no significant difference. One month (b.wt. 250300 g) after pinealectomy or sham operation the animals were subjected to experimentation. After killing the animals, it was found that surgical extirpation had destroyed the gland in all cases. The behavioral apparatus used were four activity chambers equipped with an infrared light matrix system as detailed elsewhere (36). In brief, the first part of the system was an IBM PCAT compatible microcomputer which was used as the controller for data acquisition and as the analyzer for data location. The second part of the system was the infrared light-emitting and receiving circuits. The two kinds of devices adopted were the infrared light-emitting diode (IRLED) and phototransistor. Twenty-four IRLED or phototransistors were fixed on an acrylic bar with 1.6 cm interval between two neighboring devices. For detecting the horizontal and vertical movements simultaneously, we installed the acrylic bar pair (both emitting and receiving bars were precisely aligned at a distance of 45 cm) along the xaxis, y-axis and z-axis (as shown in Fig. 1). The following locomotor behaviors were measured using this system: I. horizontal fine movement time (HFMT in s; time elapsed for a horizontal displacement of less than 1.6 cm); 2. horizontal gross movement time (HGMT in s; time elapsed for a horizontal displacement of greater than 1.6 cm); 3. vertical movement time (VMT in s; time elapsed for vertical displacement in which one or more infrared light beams on the z-axis were blocked; 4. freezing time (FT in s; time elapsed when the animal did not have HFMT, HGMT, or VMT); 5. total distance traveled (TDT in cm; the total sum of all HGMT); 6. clockwise turning (CT in counts); and 7. anticlockwise turning (ACT in counts). Each rat was tested only once. While the rat was being injected, the open field was washed. The rat was then returned to the open field for observation right after injection. All behavioral testings were prepared in the light phase of the diurnal cycle during the period of 1000-1400 h. In the first series of experiments, animals were exposed to a selected ambient temperature (Ta) for 90 min. In another series of experiments, animals were acclimatized to the open field for the first 30 rain and tested nociceptively for the last 60 min. RESULTS
Three groups of either the sham-operated rats (n = 12 per group) or the pinealectomized rats (n = 12 per group) were exposed to a selected Ta of either 22°C, 36°C, or 4°C for 90 rain, and the values of their behavioral parameters in the last 60 rain are summarized in Table 1 As compared to those of Ta = 22°C group, the animals of either the Ta = 36°C group, or the T~ = 4°C group had a higher value ofHFMT, HGMT, VMT, TDT, CT, or ACT, but a lower value of FT. Table 1 also shows that all the values of behavioral parameters obtained from the pinealectomized rats were not significantly different from those of the sham-operated rats at Ta = 22°C. However, as compared to those of the sham-operated rats, the pinealectomized rats had a
IR t r o n s m i t t e r (o)
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,, ............
F--*(X,Y,Z)
Y1. .....
k
1 ....
........
.ITX4 x,l x2
IR receiver
X5
IF (X4-X3)<(X2-X I)
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(b) Z AXIS-> L. °.-1
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1
......
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X AXIS-, 45cm
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FIG. I. The block diagrams showing the animal cage and emitting and receiving acrylic bar pairs installed along the x, y, and z axis. They also illustrate the start and end locations in areas where the corresponding infrared beams are consistently blocked. The locations can be used to calculate the equivalent center coordinates of x and y axis.
lower value of VMT, but a higher value of FT in the heat (Ta -- 36°C). In the cold (Ta = 4°C) the pinealectomized rats had a lower value of either HFMT, HGMT, VMT, or TDT, but a higher value of FF, as compared to those of the sham-operated rats. In another series of experiments, 30 rain after the animals had been acclimatized to the open field at Ta = 22°C, pain was induced by intradermal injection of normal saline in two groups of the sham-operated and the pinealectomized rats. Table 2 presents summaries of the mean and the standard error values for each of the measured parameters collected from 12 sham-operated rats and 12 pinealectomized rats in response to pain. As compared to those (without intradermal injection) at Ta = 22°C of the sham-operated rats, pain produced a higher value of either HFMT, HGMT, VMT, or TDT, but a lower value of FT. These figures also show that the pain-induced increases of locomotion as well as decreases of postural freezing are significantly attenuated by pinealectomy. Both the CT and the ACT were not affected by painful stimulation. DISCUSSION
In the current results, either cold (4°C), heat (36°C), or pain produce increases of locomotion (including horizontal and vertical movement, and total distance traveled) and/or number of turnings (including clockwise and counterclockwise) in freely moving rats. On the other hand, the postural freezing (which represents the total time elapsed when the animal did not have horizontal and vertical movements during the observation period) was decreased following cold, heat, or pain stimulation. The results indicate that these non-
PINEALS A N D L O C O M O T I O N
585
TABLE 1 EFFECTS OF PINEALECTOMY ON VARIOUS ACTIVITY RESPONSES TO DIFFERENT AMBIENT TEMPERATURES(Ta: 22°C, 4°C, AND 36°C) Treatment Sham operation Ta: 22°C Ta: 36°C Ta: 4°C Pinealectomy Ta:22°C Ta: 36°C Ta: 4°C
FT (0.2 s)
HFMT (0.2 s)
HGMT (0.2 s)
VMT (0.2 s)
CT (Counts)
ACT (Counts)
TDT (cm)
17908 _+ 14 16751 + 86* 17160 -+ 87*
9 -+ 3 397 + 20* 139 _+ 16"
5+ 2 331 _+ 38* 85 + 13"
19 _+ 10 477 _+ 77* 579 + 64*
2 -+ 1 13 + 2* 15 _+ 2*
2 _+ 1 14 _+ 2* 15 + 2*
36 _ 15 2104 + 190" 615 + 81"
17930_+ 4 17046 _+ 90*t 17625 _+ 44"~"
5--- 2 342 _+ 32* 68 _+ 10"I"
1+ 1 308 _+ 44* 37 _+ 6"~-
3+ 2 254 _+ 43"1" 223 _+ 33"t
1_+1 11 _+ 2 17 _+ 2*
2_+1 9 _+ 2* 16 _+ 2*
15_+ 5 1890 _+ 236* 297 _+ 46*5"
The values are expressed as means + SEM of 12 rats for each group. FT: postyral freezing time; HFMT: horizontal fine movement time; HGMT: horizontal gross movement time; VMT: vertical movement time; CT: clockwise turning time; ACT: anticlockwise turning time; TDT: total distance traveled. * Significantly different from corresponding control values (Ta: 22°C of each group), p < 0.05 (one-way ANOVA). ~"Significantly different from the corresponding control values (sham-operated group), p < 0.05 (one-way ANOVA).
photic, stress-provoking stimuli may result in escape behaviors to try to get out of the stressed condition. It was thought at one time that the rat became more active as a means of thermoregulating during cold exposure (5,6,29). Because body temperature of the rat may decrease during cold exposure, it was reasoned that the observed increase in motor activity during cold exposure was a thermoregulatory response to increase thermogenesis from skeletal muscle (5,6). However, it now seems clear that increased motor activity is not a thermoregulatory response to produce heat under conditions of heat loss. For example, Campbell and Lynch (5) found that the fooddeprived rats maintained at a warm Ta of 31 °C to prevent development of hypothermia were more active than ad lib-fed rats. Furthermore, rats normally run more during the dark phase of the circadian cycle, a period when their body temperature is normally elevated (3,4). The effect of raising Ta on motor activity in the rat is occasionally unpredictable; escape behavior may manifest an elevation in running activity in the heat (8). These observations, together with the present results, tend to support the concept that escape behavior manifests an elevation in locomotion as well as a depression in postural freezing in either the cold or heat. Evidence has accumulated to indicate that acute cold exposure may modulate pineal activity, which is controlled primarily by the L ' D cycle. Acute cold exposure has been shown to elevate plasma catecholamine (2) and to induce ultrastructural changes, indicative of increased synthetic and secretory activity in mouse pinealocytes (18). In addition, midlight levels of serum and pineal melatonin rose in golden hamsters acutely exposed to cold (27), similar to rats (23,33). However, during the dark phase, pineal
N-acetyltransferase activity (NAT) (but not melatonin content) in rats was depressed following 2 h of cold exposure (30). Pineal N A T activity in long-day white-footed mice exposed to cold soon after onset of darkness was depressed, whereas mice exposed to cold later at night had slightly elevated enzyme activity (32). The thermogenic adaptations initiated by Siberian hamsters in preparation for winter include an increased capacity for nonshivering thermogenesis, growth of winter pelage, and spontaneous daily torpor (12,26). These effects are mediated by the pineal gland (11,28). Heat exposure depressed the pineal N A T activity in rats (22,35). Even though the role of stress in affecting pineal activity remains equivocal, it is possible that cold or heat load as a natural stress factor influences pineal N A T activity via circulating norepinephrine. The present results showed that the increases of locomotion (including both horizontal and vertical movement), as well as decreases of postural freezing induced by cold or pain, were attenuated following pinealectomy. In addition, the heat-induced increases of vertical m o v e m e n t as well as decreases of postural freezing were attenuated by pinealectomy. Our recent results provided new evidence to suggest that pineal activation enhances sympathetic efferent activity and results in hypertension and tachycardia in rats (7). Thus, it appears that these stress-provoking stimuli activate both the pineal gland and the sympathetic-adrenal system and lead to escape behaviors. The pineal can be thought to mediate several behavioral adjustments elicited by these stress-provoking stimuli in the environment. In fact, rats displayed remarkable shivering over the trunk and the limbs in the the cold. On the other hand, when exposed
TABLE 2 EFFECT OF PINEALECTOMY ON VARIOUS ACTlVITY RESPONSES PRODUCED BY INTRADERMAL INJECTION OF NORMAL SALINE IN RATS AT ROOM TEMPERATURE (22°C) Treatment
FF (0.2 s)
HFMT (0.2 s)
HGMT (0.2 s)
VMT (0.2 s)
CT (Counts)
ACT (Counts)
TDT (cm)
Sham operation Pinealectomy
17483 + 44 17880 + 49*
90 _+ 9 43 + 14"
46 -+_6 18 + 8*
332 + 35 105 _+ 31"
5+ 2 2+ 1
4 ___2 2 _+ 1
355 + 40 165 _ 66*
The values are expressed as mean _ SEM of 12 rats for each group. * Significantly different from corresponding control values (sham operation), p < 0.05 (one-way ANOVA).
586
C H U A N G A N D LIN
to heat, rats displayed increased grooming and salivary spreading, increased locomotion, and relaxed postural extension (t3,25). It was also showen that pinealectomized rats were found to tolerate heat stress less than pineal-intact controls (15). Evaporative cooling was also reduced in pinealectomized hamsters (15), suggesting that the pineal gland is involved in the control of this pathway for heat loss. In the heat, the grooming typically begin with paw licking and face washing with the wet paws, followed during longer bouts of licking of the abdomen, sides, and tail.
It is unknown whether the increases of locomotion as well as the inhibition of postural freezing in the heat, as shown in the current results, is related to grooming and salivary spreading in rats. ACKNOWLEDGEMENTS The work reported here was supported by research grants from the National Science Council of Republic of China. The authors wish to thank Miss Y. Y. Chan for her excellent technical assistance.
REFERENCES 1. Axelrod J. The pineal gland: A neurochemical transducer. Science 184:1341-1348; 1974. 2. Benedict, C. R.; Filenz, M.; Stanford, S. C. Plasma noradrenaline levels during exposure to cold. J. Physiol. (Lond.) 269:47P-48P; 1977. 3. Bolles, R. C.; Duncan, P. M.; Grossen, N. E.; Matter, C. F. Relationship between activity level and body temperature in the rat. Psychol. Rep. 23:991-994; 1968. 4. Bolles, R. C.; Duncan, P. M. Daily course of activity and subcutaneous body temperature in hungry and thirsty rats. Physiol. Behav. 4:87-89; 1967. 5. Campbell, B. A.; Lynch, G. S. Activity and thermoregulation during food deprivation in the rat. Physiol. Behav. 2:311-313; 1967. 6. Campbell, B. A.; Lynch, G. S. Influence of hunger and thirst on the relationship between spontaneous activity and body temperature. J. Comp. Physiol. Psychol. 65:492-498; 1968. 7. Chuang, J. 14 Lin, M. T. Pineal stimulation produces both hypertension and tachycardia in rats. Brain Res. Bull. (in press). 8. Finger, F. W. Relation of general activity in rats to environmental temperature. Percept. Mot. Skills 43:875-890; 1976. 9. Gassori, O.; Van Ree, J. M. Beta-endorphin-(10-16) antagonizes behavioral responses elicited by melatonin following injection into the nucleus accubens of rats. Life Sci. 37:357-364; 1985. 10. Glass, J. D.; Lynch, G. R. The effect of superficial pinealectomy on reproduction and brown fat in the adult white-footed mouse, Peromyscus leucopus. J. Comp. Physiol. B 144:145-152; 1981. I I. Goldman, B. D.; Darrow, J. M. The pineal gland and mammalian photoperiodism. Neuroendocrinology 37:386-396; 1983. 12. Goldman, B. D.; Darrow, J. M.; Duncan, M. J.; Yogev, L. Photoperiod, reproductive hormones, and winter torpor in three hamster soecies. In: Heller, H. C.; Wang, L. C. H.; Musacchia, X. J., eds. Living in the cold: Physiological and biochemical adaprarions. New York: Elsevier; 1986:341-350. 13. Gordon, C. J. Thermal biology of the laboratory rat. Physiol. Behav. 47:963-991; 1990. 14. Harrow, H. J.; Darnell, D. K.; Phillips, J. A. Pinealectomy in ground squirrels: Effect on behavioral and physiological responses to heat stress. Physiol. Behav. 28:501-504; 1982. 15. Heldmaier, G.; Lynch, G. R. Pineal involvement in thermoregulation and acclimatization. Pineal Res. Rev. 4:97-139; 1986. 16. Hoffman, R. A.; Reiter, R. J. Rapid pinealectomy in hamsters and other small rodents. Anat. Rec. 153:19-22; 1965. 17. Joshi, B.; Troianim M.E.; Milin, J.; Nurnburger, F.; Reiter, R. J. Adrenal-mediated depression of N-acetyl-transferase activity and melatonin levels in the rat pineal gland. Life Sci. 38:1573-1580; 1986. 18. Kachi, T. Pineal actions on the autonomic system. Pineal Res. Rev. 5:217-263; 1987. 19. Kuszak, J.; Rodin, M. A new technique of pinealectony for adult rats. Experientia 33:283-284; 1977. 20. Lynch, H. J.; Eng, J. P.; Wurtman, R. S. Control of pineal indole biosynthesis by changes in sympathetic tone caused by factor other than environmental lighting. Proc. Natl. Acad. Sci. USA 70:17041707; 1973.
21. Matsushima, S.; Morisawa, Y. Effects of acute cold exposure on the ultrastructure of the mouse pinealocyte. Cell Tissue Res. 195:461469; 1978. 22. Nir, I.; Hirschmann, N. Pineal N-acetyltransferase depression in rats exposed to heat. Experientia 34:1645-1646; 1978. 23. Oxenkrug, G. F.; Mclntyre, I. M. Stress-induced synthesis of melatonin: Possible involvement of the endogenous monoamine oxidase inhibitor (tribulin). Life Sci. 37:1743-1746; 1985. 24. Reiter, R. J. Pineal response to stress: Implications for reproductive physiology. In: Pancheri, P.; Zidhella, L., eds. Biorhythms and stress in the physiopathology of reproduction. New York: Hemisphere; 1988:215-226. 25. Roberts, W. W. Differential thermosensor control of thermoregulatory grooming, locomotion and relaxed postural extension. Ann NY Acad. Sci. 525:363-374; 1988. 26. Ruby, N. F.; Ibuka, N.; Barnes, B. M.; Zucker, I. Suprachiasmatic nuclei influence torpor and circadian temperature rhythms in hamsters. Am. J. Physiol. 257:R210-R215; 1989. 27. Shiu, S. Y. W.; Pang, S. F. Cold stress induced rise in mid-light levels of serum and pineal melatonin in male hamsters. J. Steroid Biochem. 20B:1467; 1984. 28. Steinlechner, S.; Heldmaier, G.; Weber, C.; Role, T. R. Role of photoperiod: Pineal gland interaction in torpor control. In: Heller, H. C.; Wang, L. C. H.; Musacchia, X. J. Living in the cold: Physiological and biochemical adaprarions. New York: Elsevier; 1986:301307. 29. Stevensom, J. A.; Rixon, R. H. Environmental temperature and deprivation of food and water on the spontaneous activity of rats. Yale J. Biol. Med. 29:575-584; 1957. 30. Tannenbaum, M. G.; Reiter, R. J.; Vaughan, M. K.; Troiani, M. E.; Gonzalez-Brito, A. Effects of short-term cold exposure on pineal biosynthetic function in rats. Cryobiology 25:227-232; 1988. 31. Tannenbaum, M. G.; Reiter, R. J.; Hurlbut, E. C.; Vaughan, M. K.; Gonzalez-Brito, A.; Troiani, M. E. Pineal sensitivity to nighttime swimming stress changes during the active season in Richardson's ground squirrels (Sperrnophilus richardsonii). J. Exp. Zool. 250:298-303; 1989. 32. Tannenbaum, M. G.; Haigh, G. R.; Vaughan, M. K.; Reiter, R. J. Effects of acute cold exposure at night on pineal N-aeetyltransferase activity and melatonin content in white-footed mice, Peromyscus leucopus. Comp. Biochem. Physiol. 95A:363-366; 1990. 33. Torres, G.; Haak, H. A.; Lytle, L. D. Catecholaminergic mechanisms mediate hypothermia-induced elevations in pineal gland N-acetyltransferase in neonatal rats. J. Pineal Res. 6:43-53; 1989. 34. Troiani, M. E.; Reiter, R. J.; Vayghan, M. K.; Oaknin, S.; Vayghan, G. M. Forced swimming depresses night-time melatonin content without changing N-acetyltransferase activity in the rat pineal gland. Neuroendocrinology 47:55-60; 1988. 35. Welker, H. A.; Vollrath, L. The effects of a number of short-term exogenous stimuli on pineal serotonin-N-acetyl-transferase activity in rats. J. Neural Transm. 59:69-80; 1984. 36. Young, M. S.; Li, Y. C.; Lin, M. T. A modularized infrared light matrix system with high resolution for measuring animal behaviors. Physiol. Behav. 53:545-551; 1993.
Pergamon
Responses to Cold, Heat, and Pain Increase Locomotion in Rats and Are Attenuated by Pinealectomy J. I. C H U A N G A N D M. T. LIN 1
Department of Physiology, National Cheng Kung University Medical College, Tainan city, Taiwan, Republic of China Received 22 F e b r u a r y 1993 CHUANG, J. I. AND M. T. LIN. Responses to cold, heat, and pain increaselocomotion in rats and are attenuatedby pinealectomy. PHYS1OL BEHAV 55(3) 583-586, 1994.--The effects of pinealectomy on locomotor behavior responses to cold, heat or pain were assessed in freely moving rats. External cold (4°C) or heat (36°C) stress produced increases of locomotion (including horizontal and vertical movement, and total distance traveled), increases of number of turnings (including both clockwise and counterclockwise), and decreases of postural freezing in rats. In addition, pain (produced by intradermal injection of normal saline) was also shown to produce increases of locomotion (including horizontal and vertical movement, and total distance traveled) and decreases of postural freezing in rats. The increases of locomotion (including horizontal and vertical movement, and total distance traveled), as well as the decreases of postural freezing induced by either cold or pain, were attenuated by pretreatment of animals with pinealectomy. The heat-induced increasesof vertical movement as well as the decreases of postural freezing were also attenuated by pinealectomy. The results indicate that these nonphotic, stress-provokingstimuli act through the pineal gland to induce escape behaviors to try to get out of the stressed conditions in rats. Pineal gland
Cold
Heat
Pain
Locomotion
Behavior
THE activity of the mammalian pineal gland is primarily under the control of the environmental photoperiodl. Several nonphotic, stress-provoking stimuli, such as ambient temperature, forced immobilization, forced swimming and insulin-induced hypoglycemia, have been shown to influence the pineal activity in a variety of small rodent species (17,20,22-24,27,30-35). In addition, a bulk of evidence indicates that a number of behavioral responses exerted by some stress-provoking stimuli (such as cold, heat and pain) are mediated by the pineal gland (1,9,10,14,15,18,20). Recently, we have provided a new modularized infrared light matrix systen with high resolution for measuring animal behaviors (36). It can record the sequences of animal's activity in a computer-aided system with a resolution of 0.2 s in time and 1.6 cm in space, and permanently stores all the resulting data in file. In the current study, we have attempted to apply this system to assess the behavioral responses to cold, heat, or pain in shamoperated rats and in pinealectomized rats to assess any possible involvement of the pineal gland in behavioral responses induced by cold, heat, or pain.
1To whom requests for reprints should be addressed.
583
METHOD Male Sprague-Dawley rats were used in the present experiments. Upon receipt from the supplier (Animal Resource Center, National Cheng Kung University Medical College, Tainan city, Taiwan, ROC), the animals were housed in pairs in a temperature-regulated (25 + I°C) room at a 12:12 h light:dark cycle with water and food ad lib. The light was turned on at 0600 h and turned offat 1800 h. Pinealectomy was performed according to the method of Hoffman and Reiter (16) and Kuszak and Rodin (19) at 1 month after birth (body weight about 120 g). The animal under pentobarbital sodium (50 mg/kg, IP) anesthesia was mounted on a stereotaxic instrument. A midline longituidinal incision was made exposing the occipital and parietal bones of the skull. Then a bone disc (4 m m in diameter) was removed just above the confluence of the superior sagittal and transverse sinuses. The bone piece was then lifted off the underlying dura from its rostral edge, exposing the superior saglttal vein (SSV), the transverse sinus (TS), and the confluens sinus (CS). The pineal gland was removed by inserting an open pair of forceps into the junction between the two sinuses, grasping the stalk, and removing gland and stalk in one motion. Excessive
584
CHUANG AND LIN
hemorrhage was prevented by rapidly returning the bone disc to its roiginal position and by the application of collagen hemostatic felt. Finally, the incision was sutured and iodine was applied over the wound. Sham-operated animals had identical suregry, but no damage was applied to the gland. A comparison of weight gain between sham-operated and pinealectomized animals showed no significant difference. One month (b.wt. 250300 g) after pinealectomy or sham operation the animals were subjected to experimentation. After killing the animals, it was found that surgical extirpation had destroyed the gland in all cases. The behavioral apparatus used were four activity chambers equipped with an infrared light matrix system as detailed elsewhere (36). In brief, the first part of the system was an IBM PCAT compatible microcomputer which was used as the controller for data acquisition and as the analyzer for data location. The second part of the system was the infrared light-emitting and receiving circuits. The two kinds of devices adopted were the infrared light-emitting diode (IRLED) and phototransistor. Twenty-four IRLED or phototransistors were fixed on an acrylic bar with 1.6 cm interval between two neighboring devices. For detecting the horizontal and vertical movements simultaneously, we installed the acrylic bar pair (both emitting and receiving bars were precisely aligned at a distance of 45 cm) along the xaxis, y-axis and z-axis (as shown in Fig. 1). The following locomotor behaviors were measured using this system: I. horizontal fine movement time (HFMT in s; time elapsed for a horizontal displacement of less than 1.6 cm); 2. horizontal gross movement time (HGMT in s; time elapsed for a horizontal displacement of greater than 1.6 cm); 3. vertical movement time (VMT in s; time elapsed for vertical displacement in which one or more infrared light beams on the z-axis were blocked; 4. freezing time (FT in s; time elapsed when the animal did not have HFMT, HGMT, or VMT); 5. total distance traveled (TDT in cm; the total sum of all HGMT); 6. clockwise turning (CT in counts); and 7. anticlockwise turning (ACT in counts). Each rat was tested only once. While the rat was being injected, the open field was washed. The rat was then returned to the open field for observation right after injection. All behavioral testings were prepared in the light phase of the diurnal cycle during the period of 1000-1400 h. In the first series of experiments, animals were exposed to a selected ambient temperature (Ta) for 90 min. In another series of experiments, animals were acclimatized to the open field for the first 30 rain and tested nociceptively for the last 60 min. RESULTS
Three groups of either the sham-operated rats (n = 12 per group) or the pinealectomized rats (n = 12 per group) were exposed to a selected Ta of either 22°C, 36°C, or 4°C for 90 rain, and the values of their behavioral parameters in the last 60 rain are summarized in Table 1 As compared to those of Ta = 22°C group, the animals of either the Ta = 36°C group, or the T~ = 4°C group had a higher value ofHFMT, HGMT, VMT, TDT, CT, or ACT, but a lower value of FT. Table 1 also shows that all the values of behavioral parameters obtained from the pinealectomized rats were not significantly different from those of the sham-operated rats at Ta = 22°C. However, as compared to those of the sham-operated rats, the pinealectomized rats had a
IR t r o n s m i t t e r (o)
.....
,m,odules.71"
,, ............
F--*(X,Y,Z)
Y1. .....
k
1 ....
........
.ITX4 x,l x2
IR receiver
X5
IF (X4-X3)<(X2-X I)
modules.
(b) Z AXIS-> L. °.-1
b.' tt 2
then X=(X2+Xl)/2 Y=(Y2+Y1)/2
1
......
2 om
X AXIS-, 45cm
I~
FIG. I. The block diagrams showing the animal cage and emitting and receiving acrylic bar pairs installed along the x, y, and z axis. They also illustrate the start and end locations in areas where the corresponding infrared beams are consistently blocked. The locations can be used to calculate the equivalent center coordinates of x and y axis.
lower value of VMT, but a higher value of FT in the heat (Ta -- 36°C). In the cold (Ta = 4°C) the pinealectomized rats had a lower value of either HFMT, HGMT, VMT, or TDT, but a higher value of FF, as compared to those of the sham-operated rats. In another series of experiments, 30 rain after the animals had been acclimatized to the open field at Ta = 22°C, pain was induced by intradermal injection of normal saline in two groups of the sham-operated and the pinealectomized rats. Table 2 presents summaries of the mean and the standard error values for each of the measured parameters collected from 12 sham-operated rats and 12 pinealectomized rats in response to pain. As compared to those (without intradermal injection) at Ta = 22°C of the sham-operated rats, pain produced a higher value of either HFMT, HGMT, VMT, or TDT, but a lower value of FT. These figures also show that the pain-induced increases of locomotion as well as decreases of postural freezing are significantly attenuated by pinealectomy. Both the CT and the ACT were not affected by painful stimulation. DISCUSSION
In the current results, either cold (4°C), heat (36°C), or pain produce increases of locomotion (including horizontal and vertical movement, and total distance traveled) and/or number of turnings (including clockwise and counterclockwise) in freely moving rats. On the other hand, the postural freezing (which represents the total time elapsed when the animal did not have horizontal and vertical movements during the observation period) was decreased following cold, heat, or pain stimulation. The results indicate that these non-
PINEALS A N D L O C O M O T I O N
585
TABLE 1 EFFECTS OF PINEALECTOMY ON VARIOUS ACTIVITY RESPONSES TO DIFFERENT AMBIENT TEMPERATURES(Ta: 22°C, 4°C, AND 36°C) Treatment Sham operation Ta: 22°C Ta: 36°C Ta: 4°C Pinealectomy Ta:22°C Ta: 36°C Ta: 4°C
FT (0.2 s)
HFMT (0.2 s)
HGMT (0.2 s)
VMT (0.2 s)
CT (Counts)
ACT (Counts)
TDT (cm)
17908 _+ 14 16751 + 86* 17160 -+ 87*
9 -+ 3 397 + 20* 139 _+ 16"
5+ 2 331 _+ 38* 85 + 13"
19 _+ 10 477 _+ 77* 579 + 64*
2 -+ 1 13 + 2* 15 _+ 2*
2 _+ 1 14 _+ 2* 15 + 2*
36 _ 15 2104 + 190" 615 + 81"
17930_+ 4 17046 _+ 90*t 17625 _+ 44"~"
5--- 2 342 _+ 32* 68 _+ 10"I"
1+ 1 308 _+ 44* 37 _+ 6"~-
3+ 2 254 _+ 43"1" 223 _+ 33"t
1_+1 11 _+ 2 17 _+ 2*
2_+1 9 _+ 2* 16 _+ 2*
15_+ 5 1890 _+ 236* 297 _+ 46*5"
The values are expressed as means + SEM of 12 rats for each group. FT: postyral freezing time; HFMT: horizontal fine movement time; HGMT: horizontal gross movement time; VMT: vertical movement time; CT: clockwise turning time; ACT: anticlockwise turning time; TDT: total distance traveled. * Significantly different from corresponding control values (Ta: 22°C of each group), p < 0.05 (one-way ANOVA). ~"Significantly different from the corresponding control values (sham-operated group), p < 0.05 (one-way ANOVA).
photic, stress-provoking stimuli may result in escape behaviors to try to get out of the stressed condition. It was thought at one time that the rat became more active as a means of thermoregulating during cold exposure (5,6,29). Because body temperature of the rat may decrease during cold exposure, it was reasoned that the observed increase in motor activity during cold exposure was a thermoregulatory response to increase thermogenesis from skeletal muscle (5,6). However, it now seems clear that increased motor activity is not a thermoregulatory response to produce heat under conditions of heat loss. For example, Campbell and Lynch (5) found that the fooddeprived rats maintained at a warm Ta of 31 °C to prevent development of hypothermia were more active than ad lib-fed rats. Furthermore, rats normally run more during the dark phase of the circadian cycle, a period when their body temperature is normally elevated (3,4). The effect of raising Ta on motor activity in the rat is occasionally unpredictable; escape behavior may manifest an elevation in running activity in the heat (8). These observations, together with the present results, tend to support the concept that escape behavior manifests an elevation in locomotion as well as a depression in postural freezing in either the cold or heat. Evidence has accumulated to indicate that acute cold exposure may modulate pineal activity, which is controlled primarily by the L ' D cycle. Acute cold exposure has been shown to elevate plasma catecholamine (2) and to induce ultrastructural changes, indicative of increased synthetic and secretory activity in mouse pinealocytes (18). In addition, midlight levels of serum and pineal melatonin rose in golden hamsters acutely exposed to cold (27), similar to rats (23,33). However, during the dark phase, pineal
N-acetyltransferase activity (NAT) (but not melatonin content) in rats was depressed following 2 h of cold exposure (30). Pineal N A T activity in long-day white-footed mice exposed to cold soon after onset of darkness was depressed, whereas mice exposed to cold later at night had slightly elevated enzyme activity (32). The thermogenic adaptations initiated by Siberian hamsters in preparation for winter include an increased capacity for nonshivering thermogenesis, growth of winter pelage, and spontaneous daily torpor (12,26). These effects are mediated by the pineal gland (11,28). Heat exposure depressed the pineal N A T activity in rats (22,35). Even though the role of stress in affecting pineal activity remains equivocal, it is possible that cold or heat load as a natural stress factor influences pineal N A T activity via circulating norepinephrine. The present results showed that the increases of locomotion (including both horizontal and vertical movement), as well as decreases of postural freezing induced by cold or pain, were attenuated following pinealectomy. In addition, the heat-induced increases of vertical m o v e m e n t as well as decreases of postural freezing were attenuated by pinealectomy. Our recent results provided new evidence to suggest that pineal activation enhances sympathetic efferent activity and results in hypertension and tachycardia in rats (7). Thus, it appears that these stress-provoking stimuli activate both the pineal gland and the sympathetic-adrenal system and lead to escape behaviors. The pineal can be thought to mediate several behavioral adjustments elicited by these stress-provoking stimuli in the environment. In fact, rats displayed remarkable shivering over the trunk and the limbs in the the cold. On the other hand, when exposed
TABLE 2 EFFECT OF PINEALECTOMY ON VARIOUS ACTlVITY RESPONSES PRODUCED BY INTRADERMAL INJECTION OF NORMAL SALINE IN RATS AT ROOM TEMPERATURE (22°C) Treatment
FF (0.2 s)
HFMT (0.2 s)
HGMT (0.2 s)
VMT (0.2 s)
CT (Counts)
ACT (Counts)
TDT (cm)
Sham operation Pinealectomy
17483 + 44 17880 + 49*
90 _+ 9 43 + 14"
46 -+_6 18 + 8*
332 + 35 105 _+ 31"
5+ 2 2+ 1
4 ___2 2 _+ 1
355 + 40 165 _ 66*
The values are expressed as mean _ SEM of 12 rats for each group. * Significantly different from corresponding control values (sham operation), p < 0.05 (one-way ANOVA).
586
C H U A N G A N D LIN
to heat, rats displayed increased grooming and salivary spreading, increased locomotion, and relaxed postural extension (t3,25). It was also showen that pinealectomized rats were found to tolerate heat stress less than pineal-intact controls (15). Evaporative cooling was also reduced in pinealectomized hamsters (15), suggesting that the pineal gland is involved in the control of this pathway for heat loss. In the heat, the grooming typically begin with paw licking and face washing with the wet paws, followed during longer bouts of licking of the abdomen, sides, and tail.
It is unknown whether the increases of locomotion as well as the inhibition of postural freezing in the heat, as shown in the current results, is related to grooming and salivary spreading in rats. ACKNOWLEDGEMENTS The work reported here was supported by research grants from the National Science Council of Republic of China. The authors wish to thank Miss Y. Y. Chan for her excellent technical assistance.
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