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BIOL I~S~'CHL~'I'RY 1951.~9:1200--1208
Effects of Imipramine on the Nocturnal Behavior of Bilateral Olfactory Bulbectomized Rats William J. Giardina and R. J. Radek
Experiments were performed to characterize the circadian behavior of bilateral olfactory bulbectomized (OB) rats and to investigate the effects of imipramine on that behavior. OB and sham-operated (SO) rats were housed individually for 2 weeks in activity monitors on a 13-hr light/11-hr dark cycle. OB rats were significantly more active than SO rats during the dark phase of the cycle, and both groups of rats were equally inactive during the light phase. Seven daily injections of imipramine llO.O mglkg, intraperitoneally (IP)] significantly reduced the nocturnal activity of OB rats, such that OB rats displayed nocturnal activity equivalent to SO rats. Abnormally high nocturnal activity is another important characteristic of the OB rat. This behavioral characteristic may prove to be valuable for the evaluation of novel antidepressive compounds.
Introduction Bilateral olfactory bulbectomy in rats produces a characteristic group of abnormal behaviors referred to as the bulb~ectomy syndrome. Bulbectomy makes rats hyperactive in novel environments, aggressive, irritable, deficient in passive and active avoidance tasks, and muricidal (Caimcross et al 1978; Cairncross et al 1979). Bulbectomy-induced behavior changes are reversed by antidepressive drugs, such as amiaiptyline, nfianserin, and viloxazine (Jancsar and Leonard 1984a; Cairncross et al 1978; Cairncross et al 1979; Willner 1984). Several days of drug administration are needed to normalize behavior in these animals, and the dlag-induced changes then persist for several days after the last dose administered (Jesberger and Richardson 1986). Similarly, in man 1-3 weeks of u'eatment with a tricyc!ic antidepressant is required to achieve a.,~ adequate therapeutic response. Other classes of psychotherapeutic drugs, such as anxiolytics and antipsychotics, do not reverse bulbectomy-induced behavioral deficits (Van Riezen et ai 1977). Olfactory bulbectomized (OB) rats also have elevated circulating corticosterone levels, as depressed patients frequently have, which is lowered by antidepressants (Cairncross et al 1979; Sachar et al 1980). Becaase the OB rat's response to antidepressive drugs shows parallels with the clinical response to antidepressive therapy, the OB rat preparation has received high marks as a screen for potential antidepressive compounds Oesberger and Richardson 1985; Willner 1984). In a review of the neurochemical sequelae of olfactory bulbectomy, Hirsch (1980) From the Department of Pharmacology, Abbott Laboratories, Abbott Park, IL. Address reprint requests to Dr. William J. Giardina, Department of Pharmacology D-46R AP9, Abbott Laboratories, One Abbott Park Road, Abbott Park, IL 60064-3500. Received August 13, 1990; r~vised December ! !, 1990, © 1991 Society of Biological Psychiatry
0006-3223/91/$03.50
Imipramine and Nocturnal Behavior of OB Rats
BIOl.. PSYCHIATRY 1991;29:1200-1208
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concludes that bulbectomy has wide-ranging effects on brain neurotransmitter system. The catecholamines, indoleamines, acetylcholine, aspartate, and glutamate are generally reduced in brain after bulbectomy. The procedure has been reported to cause marked changes in the metabolism of noradrenaline, serotonin, and GABA in amygdaloid cortex and dopamine in the midbrain (Jancsar and Leonard 1984b). Serotonin synthesi~ in brain decreases ~ e r bulbectomy (Neckers et al 1975). The nature of the neurochemical changes that follow bulbectomy are very likely to affect the circadian behavior of the rats. The objectives of the experiments reported here were to characterize the circadian behavior of OB rats in the home-cage environment and to investigate the effects of the tricyclic antidepressant imipramine on that behavior.
Methods Male, alamo Sprague-Dawley rats weighing 150 to 200 g were anesthetized with sodium pentobarbital (50 mg/kg, IP) and placed in a stereotaxic apparatus. The main olfactory bulbs were removed by aspiration through two burr holes (2 mm diameter), which were placed 7 mm anterior to bregma and 2 mm lateral to the saggital suture. Sterile gelfoam was placed into the open skull and the skin flaps were sutured with autoclips. The surgical procedure was identical for the sham-operated (SO) rats except for the actual removal of the olfactory bulbs. The extent of the olfactory bulb ablation was verified by gross examination of the brain at the end of an experiment. OB ~ d SO rats were housed individually in cages (26 cm x 18 cm × 18 cm) in the vivarium for at least 7 days following surgery. A 13-hr light (5:30 AM to 6:30 PM)/I lhr dark (6:30 PM to 5:30 AM) cycle was maintained throughout the recovery period. After recovery, animals were transferred to 16 Omnitech Animal Activity Monitor cages (40.5 c m x 40.5 cm x 30.0 cm), which were in a quiet, isolated room in the pharmacology laboratory. One animal was placed in each cage. Sawdust bedding covered the floor of each plastic monitor cage. Food and water were pr,Jvided ad libitum throughout an experiment. The animals were handled only when the sawdus~ bedding was replaced on days 3, 6, 9, and 12. In the first experiment, eight OB and eight SO rats were placed in the cages. The 13hr light/1 l-hr dark cycle was continued without haterruption from the vivarium. In the second experiment, 12 OB and 4 SO rats were placed in the monitors on a 24-hr light/0hr dark cycle for 7 days before reinstating t_he ! 3-hx !ight/i 1-hr dark cycle begun in the vivarium. In both experiments the rats were placed in the monitors at 2 PM on day I. Locomotive activity (total inches traveled in the cage) was recorded every hour, 23 hr per day (1 hr, 10 AM tO l I AM, during the light phase was used for resupplying water and food and for periodic changing of the sawdust bedding), for 14 days. The effects of imipramine at 3.0, 10.0, and 15.0 mg/kg IP on home-cage activity of OB rats were investigated in the third, fourth, and fifth experiments. In each experiment, 16 OB rats were placed in the activity cages for 16 days on the 13-hr light/11-hr dark cycle. Injections of either vehicle (n = 8) or imipramine (n = 8) began on day four, and were made between 10 AM and 11 AM. In a sixth experiment, the effects of imiprarrfine (10.0 mg/kg IP) (n = 8) and vehicle (n = 8) on the locomotive behavior of SO rats were evaluated. Locomotive activity was measured every hour, 23 hr per day, throughout the experiments. A one- or two-factor, repeated measures, analysis of variance (ANOVA) was used for statistical evaluation of the data. The one-factor test was used for the analysis of changes
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BIOL PSYCHIATRY 1~t;29:120~-1208
W.J. Giardina and R.J. Radek
in locomotive behavior over time within a group. The two-factor test was used for the analysis of differences in locomotive behavior between OB and SO lesioned animals and for the analysis of differences between vehicle and imipramine treatments in either OB or SO rats.
Results OB and SO rats were very active during the first hour in the monitors as shown at 3 PM for day 1 in Figure 1. Locomotive activity decreased greatly for both groups during the remainder of the light cycle. During the dark phase of the cycle on day 1, both groups were very active, and with the onset of the light phase at 5:30 AM, both groups were again inactive. On days 2 through 14 the OB rats showed greater levels of locomotive activity than SO rats during the dark phase of the activity cycle and both groups of rats were equally inactive during the light phase as shown in Figure 2. Both groups increased locomotive activity immediately before the onset of the dark phase. However, the OB rats were more active than the SO rats during the early and late hours of the dark phase as shown, for example, by the activity on days 10 and 14 in Figure 1. The effects of 24-hr light followed by a change to the fight-dark cycle on locomotive activity are shown in Figure 3. In this experiment, the lights were on 24 hr a day for the first 7 days after the animals were taken from the vivarium. During the first 7 days, OB and SO rats had equally low activity scores from 5:30 AM to 6:30 PM, the normally righted phase of their cycle, and equally high activity scores ~ m 6:30 PM to 5:30 AM, the normally darkened phase of their cycle. The 24-hr activity patterns were nearly identical for both groups on these days as shown for day 2 in Figure 4. With the reinstatement of the light-dark cycle on day 8, the SO rats did not change their activity levels in the dark from 6:30 PM to 5:30 AM, whereas the OB rats became clearly more active than the SO rats in the dark phase as shown in Figure 3. Both groups of animals continued to remain relatively inactive during the light phase from 5:30 AM to 6:30 PM. As shown in Figure 4 for day 12, the pattern of activity from 6:30 PM to 5:30 Art clearly changed for both OB and SO rats when switched to the light-dark cycle, and, as in the ~rst experiment: the OB rats were more active than the SO rats during the early and late hours ¢,f the dark phase. Seven daily injections of imipramine at i0.0 mg/kg IP were required to markedly decrease the dark phase hyperactivity of the OB rats as shown in Figure 5. The final injection of imipramine was given on day 12. The OB rats became hyperactive again on day 15. Figure 5 also shows that the daily administration of imipramine (10.0 mg/kg iP) did not affect the locomotive activity of SO rats during the dark phase. A lower dose of irnipramine (3.0 mg/kg IP) administered daily for 16 days was ineffective. A larger dose nf 15 mg/kg IP also reversed dark phase hyperactivity z~,i 9 daily injections.
Discussion The circadian behavior of the OB rat has not been extensively investigated. Sieck (1972) studied the behavior of OB and SO rats in small home cages (8.3 cm square) under continual darkness for 8 days, and observed inconsistent activity patterns among the OB and SO rats. Araki et al (1980) measured the locomotiv~ activity of OB rats that were
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Figure 1. Locomotive activity of OB and SO rats for each hour in the home cage on days 1, 10, and 14. Ordinate: mean (n = 8) distance traveled in inches. Abscissa: Each hour of the 13-hr light (5:30 ~ to 6:30 PM)/I l-hr dark (6:30 PM tO 5:30 AM) cycle. Two factor repeated measures A N O V A indicated no difference between the OB and SO groups on day 1 and a significant difference (p < 0.05) between the groups on days 10 and 14.
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BIOL PSYCHIATRY 1991;29:1200-1208
W J . Giardina and R.]. Radek
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housed on a 12-hr light/12-hr dark cycle. They removed the rats from their home cages and placed them in circular activity monitors for 24 hr on the 3rd, 7th, and 15th day after bulbectomy. On the 7th day, OB rats were more active than the SO rats in the dark, and on the 15th day, the OB rats were more active than the SO animals in both the dark and light phases of the cycle. While both of these studies involved 24-hr behavioral observations, neither study truly examined the natural circadian behavior of OB rats in the home-c,:ge environment. The res~:~ts of the experiments reported here show that the nocturnal behavior of OB rats in the home cage is abnormal. OB rats are hyperemotional and hyperreactive animals as indicated by high defecation rates, exaggerated responses to novel stimuli, and elevated corticosterone levels (Sieck and Gordon 1972; Sieck et al 1973; Cairncross et al 1979; Cairncross et al 1977a). Nevertheless, the OB rats showed the same behavior as SO rats in lighted conditions after they habituated to the cages. In contrast tG the SO rats, the OB rats remained exceptionally active in the dark. They showed an exaggeration of the locomotive activity increase in the early and late hours of the dark phase of the cycle. While the OB rat shows a maladaptive response when placed in novel environments or exposed to novel stimuli, the results of the experiments reported here indicate that the OB rat continues to show abnormal behavioral patterns even after prolonged exposure to the same environment. When changed from a 24-hr light environment to the light-dark
Imipramine and Nocturnal Behavior of OB Rats
BIOL PSYCHIATRY 1991 ;29:1200-1208
1205
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Figure 3. Cumulative locomotive activity of the OB and SO rats during the 24-hr light/0-hr dark cycle on days I through 7 and during the 13-hr light/l l-hr dark cycle on days 8 through 14. Legend: Dark Phase = 6:30 PM to 5:30 AM, the normally darkened phase of the cycle; Light Phase = 5:30 AM to 6:30 PM, the normally lighted phase of the cycle. Ordinate: mean (OB: n = 12; SO: n = 4) of the total distance traveled by each animal in inches. Abscissa: Days I through 14. Two factor repeated measures ANOVA indicated a significant difference 07 < 0.05) between the OB and SO rats during the dark phase and no difference between the groups during the light phase. One-factor repeated measures ANOVA indicated a significant change with days in the OB group and no change with days in the SO group during the dark phase. Neither group showed a change with days in the light phase.
cycle, the OB rats became more active than SO rats m the dark phase of the cycle. These data suggest that following bulbectomy not only the onset but also the daily recurrence of darkness are important variables for the expression of nocturnal hyperactivity in the home-cage environment. The bilateral lesion of olfactory bulb serotonergic nerves by the local intrabulbar injection of a neurotoxin produces a deficit in passive avoidance learning, irritability, and an elevation of corticosterone levels in rats; in contrast, a neurotoxin-induced intrabulbar lesion of adrenergic neurons does not produce such effects (Cairncross et al I977b). These results suggest that altered serotonergic neurotransmission plays a critical role in bulbectomy syndrome. The abnormally high nocturnal locomotive activity of the OB rat may also be due to bulbectomy-induced changes in serotonergic neurotransmission. This hypothesis is supported by the observation that a neurotoxin-induced lesion of fimbrial serotonergic neurons and their terminals in the hippocampus produces nocturnal hyperactivity in rats (Williams and Aznfitia 1981). Agren et al (1986) reported similar circadian rhythmicities for norepinephrine and serotonin in the locus coeruleus and dorsal raphe nucleus of the rat. The acrophases of both amines occurred iroa-nediately before the onset
1206
W.J. Giardina and R.J. Radek
BIOL PSYCHIATRY 1991;29:1200-1208
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Figure 4. Locomotive activity of OB and SO rats for each hour in the home cage on day 2 during the 24-hr light/0-hr dark cycle and on day 12 during the 13-hr lighUl I-hr dark cycle. Ordinate: mean (n = 8 0 B rats and n = 4 SO rats) distance traveled in inches. Abscissa: Each hour of the daily cycle. Two-factor repeated measures ANOVA indicated no difference between the OB and SO groups on day 2 and a significant difference (p < 0.05) between the groups on day 12.
of dark. They postulated an interplay between the two amines in which noradrenergic activity increases serotonin during the light phase and serotonin behaves independently of norepinephrine during the dark phase of the cycle. Because olfactory bulbectomy decreases the synthesis of serotonin in whole-brain as well as affecting the metabolism of the catecho~,amines (Neckers et al 1975; Jancsar and Leonard 1984b), the abnormal noctum~i locomotive behavior of OB rats may reflect a persistent bulbectomy-induced imbalance between the noradrenergic and serotonergic influences in brain during the dark phase of the circadian cycle,
Imipramine and Nocturnal Behavior of OB Rats
BIOL PSYCHIATRY 1991 ;29:12(~- 1208
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Figure 5. Effects of imipramine (10.0 mg/kg !P) on the cumulative locomotive activity of the OB and SO rats dur:mg the I l-hr dark phase of the cycle for each day of the experiment. Ordinate: mean (n = 8) of the total distance traveled by each animal in inches during the dark phase of the cycle. Abscissa: Days 1 through !6. Two-factor repeated measures ANOVA indicated a significant d~fference (p < 0.05) between vehicle and imipramine treatments. There was no significant difference between SO groups receiving vehicle or imipramine.
The noctu:~al activity of the OB rats changed after seven daily morning (11 AM) injections of imipramine to a level equal to that of the SO ~ts. The change persisted for several days after the drug was withdrawn. The reduction in activity was not due to sedation as the daily morning injections of imipramine did not change the nocturnal behavior of SO rats. imipramine may have worked with subchronic administration to restore the balance between the noradrenergic and serotonergic systems in the brain, which was disrupted by the olfactory bulbectomy. It would be very interesting to study the circadian behavior of norepinephrine and serotonin in the OB rat and to determine the influence of antidepressive drugs, such as imipramine, amitryptaline, fluoxetine, and mianserin, on the circadian changes in the biogenic amines of OB rats. The protocol used to evaluate imipramine on the nocturnal behavior of the OB rat may be valuable for evaluation of novel antidepressive compounds as it addresses both efficacy and latency to onset questions in a simple experimental design. The toxicity of a compound can also be assessed in the same experiment by daily observation of OB and SO animals and by the occurrence of unexpected changes in the nocturnal behavior of SO rats. The method has operational advantages over the use of exploratory behavior and passive avoidance learning for such evaluations. Sustained high activity levels during the dark phase of the light-dark cycle appears to be yet another characteristic of the olfactory bulbectomy syndrome in rats. Olfactory bulbectomy-induced physiological, neurochemical, and behavioral changes, with the ex-
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BIOL PSYCHIATRY 1991;29:1200-1208
W.J. Giardina and R.J. Radck
ception of the behavioral work of Araki et al (1980) and Sieck (1972), have been assessed during the light cycle. The abnormally high activity in the dark is very likely a reflection of the bulbectomy-induced changes in neurosubstrates. Behavioral and biochemical measurements made during the dark cycle may reveal additional important and useful characteristics about the OB rat as a screen for potential anfidepressive drags.
References Agren H, Koulu M, Saavedra JM, Potter WZ, Linnoila M (1986): Circadian covariation of norepinephrine and serotonin in the locus coeruleus and dorsal raphe nucleus in the rat. Brain Res 397:353-358. Araki H, Yamamoto T, Watanabe S, Ueki S (1980): Changes in sleep-wakefulness pattern following bilateral olfactory bulbectomy in rats. Physiol Behav 24:73-78. Cairncross KD, Wren AF, Cox B, Schnieden H (1977a): Effects of olfactory bulbectomy and domicile on stress-induced corticosterone release in the rat. Physiol 8ehav 19:485-487. Cairncross KD, Cox B, Forster C, Wren A (i977b): The ability of local injection of 6-OHDA, 5.6-DHT and 5,7-DHT into the olfactory bulbs to mimic the effects of bilateral bulbectomy in the rat. Br J Pharmacol 61:145-146. Cairncross KD, Cox B, Forster C, Wren AF (1978): A new model for the detection of antidepressant drugs: Olfactory bulbectomy in the rat compared with existing models. ,/Pharmacol Methods !:131-143. Cairncross KD, Wren AF, Forster C, Cox B, Schnieden H (1979): The effect of psychoactive drugs on plasma corticosterone levels and behaviour in the b~bectom:.~ rat. Pharmacol Biochem Behav 10:355-359. H irsch JD (1980): The neurochemical sequalae of olfactory bulbectomy. Life Sci 26:1551-1559. Jancsar SM, Leonard BE (1984a): The effect of ( __)mianserin and its enantiomets on the behavioural hyperactivity of the olfactory-bulbectomized rat. Neuropharmacology 23:1065-1070. Jancsar SM, Leonard BE (1984b): Changes in neurotransmitter metabolism following olfactory bulbectomy in the rat. Prog Neuro-Psychopharmacol 8iol Psychiatry 8:263-269. Jesberger JA, Richardson JS (1985): Animal models of depression: Farailels and correlates to severe depression in humans. Biol Psychiatry 20:764-784. Jesberger JA, Richardson JS (1986): Effects of antidepressant drugs on the behavior of olfactory bulbectomized and sham-operated rats. Behav Neurosci 100:256-274. Neckers LM, Zarrow MX. Myers MM, Denenberg VH (1975): Influence of olfactory bulbectomy and the serotonergic system upon intermale aggression and maternal behavior in the mouse. Pharmaco! Biochem Behav 3:545-550. Sachar EJ, Asnis G, Nathan S, Halbreich U, Tabrizi MA, Halpren FS (1980): Dextroamphetamine and cortisol in depression. Arch Gen Psychiatry 37:755-757. Sieck MH (1972): The role of the olfactory system in avoidance learning and activity. Physiol Behav 8:705-710. Sieck MH, Gordon BL (1972): Selective olfactory bulb lesions: Reactivity changes and avoidance learning in rats. Physiol Behav 9:545-552. Sieck MH, Turner JF, Gordon BL, Struble RG (1973): Some quantitative measures of activity and reactivity in rats after selective olfactory lesions. Physiol Behav 11:71-79. Van Riezen H, Schnieden H, Wren AF (1977): Olfactory bulb ablation in the rat: Benavioural changes and their reversal by antidepressant drugs. Br J Pharmacol 60:521-528. Williams JH, Azmitia EC (1981): Hippocampal serotonin re-uptake and nocturnal locomotor activity after microinjections of 5,7-DHT in the fomix-fimbria. Brain Res 207:95-107. Willner P (1984): The validity of animal models of depression. Psychopharmacology 83:1-16.