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Effects of bright light on responsiveness to a muscarinic agonist in rats selectively bred for endogenously increased cholinergic function
139
Psychiatry Research, 33:139-150
Elsevier
Effects of Bright Light on Responsiveness to a Muscarinic Agonist in Rats Selectively Bred for Endogenously Increased Cholinergic Function David H. Overstreet, Amir H. Rezvani Received
November
Steven
C. Dilsaver,
David S. Janowsky,
and
20, 1989; revised version received May 29, 1990; accepted June 2, 1990.
Abstract. The Flinders Sensitive Line (FSL) was derived from the Sprague-Dawley rat by selectively breeding those animals exhibiting a high level of sensitivity to an anticholinesterase. The Flinders Resistant Line (FRL) was simultaneously developed as a control line. These lines exhibit nonoverlapping distributions of their thermic responsiveness to oxotremorine. Bright light prevents the development of supersensitivity to oxotremorine occurring as a result of forced stress or treatment with a muscarinic receptor antagonist in the rat. The authors now report that treatment with bright light during the regular photoperiod (i.e., a time that does not produce a phase-shift or free-running) differentially affects the hypothermic response and activity-suppressing effect of oxotremorine in both the FSL and FRL. Both lines exhibit decreased hypothermia without reduction in motor activity in response to oxotremorine following 6 days of treatment with bright light. The magnitude of blunting of the hypothermic response was greater in the FSL than the FRL. These findings suggest that (1) studies of the effects of bright light are contingent on the end point one measures and (2) the capacity of this treatment to blunt the hypothermic response to a muscarinic agonist is greater in an animal model with endogenously hyperactive muscarinic cholinergic systems. Key Words. Affective disorders, bright light, cholinergic sensitivity, forced swim test, Flinders Sensitive Line, Flinders Resistant Line, hypothermia, muscarinic.
Exposure to full-spectrum bright artificial light potently reduces the hypothermic response to the muscarinic agonist oxotremorine in normal (Dilsaver and Majchrzak, 1987), stressed (Flemmer et al., in press), and amitriptyline-treated (Dilsaver et al., 1989) Sprague-Dawley rats. The capacity of bright light to produce subsensitivity of a muscarinic cholinergic mechanism could contribute to its effectiveness in the treatment of winter depression (for review, see Dilsaver, 1989; Wehr and Rosenthal, 1989). Recent studies also indicate that treatment with bright light alters adrenergic and nicotinic mechanisms (see Dilsaver, 1989). Treatment with bright light blunts the agonist-induced responsiveness of muscarinic and nicotinic cholinergic mechanisms
David H. Overstreet, Ph.D., is Associate Professor, School of Biological Sciences, Flinders University of South Australia. Steven C. Dilsaver, M.D., was Professor of Psychiatry and Neuroscience, and Director of the Psychopharmacology Program, Department of Psychiatry, The Ohio State University, Columbus, OH. David S. Janowsky, M.D., is Professor and Chairman, Department of Psychiatry, and Director of the Center for Alcohol Studies, University of North Carolina, Chapel Hill, NC. Amir H. Rezvani, Ph.D., is Director of the Center for Alcohol Studies, University of North Carolina. (Reprint requests to Dr. S.C. Dilsaver, Dept. of Psychiatry, University of Texas School of Medicine, P.O. Box 20708, Houston, TX 77225, USA.) 0165-17Xl,‘YO/$O3.50
Q IYYO Elsevier Scientific Publishers Ireland
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140 when the animals are treated during either a fraction or the entirety of the regular photoperiod (Dilsaver, 1988, 1989). This study assesses the effect of treatment with bright light during the entirety of the regular photoperiod in the Flinder’s Sensitive Line (FSL) relative to the Flinders Resistant Line (FRL). The FSL was bred to be endogenously supersensitive to muscarinic agonists (Overstreet et al., 1986, 1988; Pepeet al., 1988; Walliset al., 1988). The FRL is relatively insensitive to the effects of muscarinic agonists and anticholinesterases, and was simultaneously bred to be a control line for all studies involving the FSL. The FSL was recently set forth as an animal model of depression (Overstreet, 1986, 1989; Shiromani et al., 1988; Overstreet et al., 1988). It is particularly useful in testing hypotheses stemming from a muscarinic-cholinergic or muscarinic-cholinergicaminergic interaction theory of the etiology of affective illness (for review, see Dilsaver, 1986a, 19866; Janowsky and Risch, 1987). The hypothermic effect of oxotremorine is mediated by an effect on the hypothalamus (Lomax and Jenden, 1966). It is not known whether the effects of bright light are limited to functions mediated by the hypothalamus. The hypothalamus is directly affected by light via input from the retinohypothalamic pathway (Moore and Lenn, 1972; Johnson et al., 1988). The FSL provides an opportunity to assess the effect of treatment with bright light on the oxotremorine-induced hypothermia and motoric inhibition in an open field, and immobility in the forced swim test (Overstreet, 1986; Overstreet et al., 1986). The FSL exhibits a profound reduction in movement in an open field in response to oxotremorine and greater immobility in the forced swim test than the FRL. Methods Animals. The FSL and FRL rats used in this study came from a breeding colony maintained at the Center for Alcohol Studies at the University of North Carolina in Chapel Hill. This colony was derived from colonies at the Flinders University of South Australia. Male rats, about 80 days of age and weighing 350-450 g at the beginning of study, were used in all experiments. These animals were housed individually and given free access to food and water. The animals were maintained on a 12:12 hour reverse light-dark cycle. The intensity of ambient lighting during the light phase (10 p.m. to 10 a.m) was 400 lux. The vivarium is illuminated with red light during subjective night. The rat cannot perceive the color red, yet the intensity of illumination is sufficient to allow all required experimental manipulations. Conditions Prevailing During Measurement of Dependent Variables. Oxotremorine was injected during the light phase in all previous studies focusing on the effect of bright light on thermic responsiveness to this agonist (Dilsaver, 1989). Similarly, thermic responsiveness was measured during the light phase in this study. The effect of this muscarinic agonist on motor behavior in an open field (crossings in I min) was measured in a room illuminated with red light. The mean rate of crossings greatly increases and the SD falls substantially when open field behavior is measured during the dark phase (Dilsaver et al., submitted). Immobility in the forced swim test was measured under the lighting conditions prevailing during the last 2 hours of the photoperiod (between 8 a.m. and 10 a.m.) in this study. Pharmaceutical Sigma Chemical syringe.
Agents. Oxotremorine (base) and methyl-atropine were purchased from Company (St. Louis, MO). Both drugs were injected S.C. using a l-ml insulin
141 Treatment With Bright Artificial Light. Full-spectrum bright artificial light was emitted from a bank (Duro Test Co., North Bergen, NJ) of eight 122-cm long VitaLite tubes suspended 50 cm above the animals. Temperature under the light unit at the level of the animals was 24 “C, 1 o C higher than in the room. This particular unit has been used in several previously published reports on the neurobiological effects of bright light (Dilsaver, 1989). The intensity of lighting in the plane of the rats’ eyes was 7,400 lux in this and all other published reports. While 7,400 lux is a high intensity of illumination, actual exposure of the rat to bright light is limited consequent to the period of exposure being its sleep phase. The rat sleeps during most of the light phase. Sprague-Dawley rats treated with this particular light unit at an intensity of 7,400 lux exhibit a profound reduction in thermic responsiveness to oxotremorine, which is reversible within 10 days of discontinuing treatment (Dilsaver, 1989). Measurement of Temperature. Core body temperature was measured by the insertion of a thermistor probe 6 cm into the rectum. Output was presented on a Bailey’s digital telethermometer (Senortrek Instruments). Measurement of Open Field Behavior. Open field behavior (i.e, number of crossings) was measured in a wooden chamber which was 60 cm x 60 cm with a wall of 12 cm on all sides. The chamber was divided into 36 squares of 100 cm2 and was covered with Plexiglas. Oxotremorine Challenge. The hypothermic response to oxotremorine is centrally mediated. This is evident from the fact that it is blocked by a muscarinic receptor antagonist which crosses the blood-brain barrier but not by charged molecules with effects limited to the periphery (Dilsaver et al., 1987). Lomax and Jenden (1966) have presented evidence that oxotremorineinduced hypothermia is mediated by an effect on the hypothalamus. Light directly activates the retinohypothalamic tract-a neuroanatomic pathway extending into the hypothalamus. Methyl-atropine (2 mgi kg) was injected S.C. before the administration of oxotremorine to block the peripheral effects of the muscarinic agonist. The addition of another methyl group to the nitrogen atom in atropine produces a positively charged molecule with limited lipid solubility. This results in its being an antagonist at peripheral but not central muscarinic receptor sites. Oxotremorine, 0.2 mg/ kg, s.c., was injected 15 min after the injection of methyl-atropine. Core temperature was measured 30 min after the injection of oxotremorine. This measurement was recorded (1) before performing any experimental manipulation (i.e., at baseline) and (2) after 3 and/or 6 days of treatment with bright light as opposed to standard lighting. Each of the two groups of FSL (FSL, and FSL2) and FRL (FRL, and FRL,) rats were challenged with oxotremorine, and core body temperature was recorded after a 5-day hiatus during which they were housed under standard lighting conditions. The data obtained at this point indicated that there were no meaningful differences in thermic responsiveness within each line. A crossover was therefore performed. In the period following the crossover, Phase 2, the groups that were initially exposed to bright light (FSL, and FRL,) were subjected to standard lighting conditions, and those originally housed under standard conditions of lighting (FSL, and FRL,) were subjected to bright light during the entire photoperiod for 6 days. We were interested in the time course of the development of subsensitivity to the hypothermic response to oxotremorine. The rats were therefore challenged 3 and 6 days following the start of exposure to bright light in Phase 2. Majchrzak and Dilsaver (1990) recently reported that thermic responsiveness to oxotremorine is unaffected by repeated injections of this agonist at 48-hour intervals at a dose slightly-higher than that used here (0.25 vs. 0.20 mg/kg, i.p). There was no evidence that treatment with bright light attenuated the oxotremorine-induced reduction in crossings in an open field or immobility in the forced swim test during Phase 1 of the study. Consequently, these parameters were not measured during Phase 2. Forced Swim Test. All of the animals were maintained under identical lighting conditions for 1 more day after the measurement of thermic responsiveness to oxotremorine so that immobil-
142 ity in the forced swim test could be measured 24 hours later. The rats were individually placed in the swim tank, and immobility was then immediately recorded over a IO-min period using a stopwatch. The rats were partially dried with a towel and then placed under a heat lamp for 10 min after completion of this test. Table I outlines the course of events in this study. Analysis of Data. Each pharmacological challenge produced data for four independent groups-the FSL-light, FSL-control, FRL-light, and FRL-control groups. These data were subjected to a two-way analysis of variance (ANOVA). Thermic responsiveness was measured both in the course of treatment with bright light and under the standard lighting conditions in the vivarium. We were interested in determining whether the main effects for treatment with bright light and sequence of treatment were significant (i.e., treatment with bright light first vs. treatment with bright light second). The repeated measures design inherent in the crossover phase permitted us to assess this question. The difference in the responsiveness of each animal under these two conditions was entered into a two-way ANOVA for repeated measures. All measures of variance in the text refer to the standard deviation (SD). The critical value of (Ywas set at p < 0.05, two-tailed.
Table 1. Sequence
of experimental
events
Days of studv 1
2-8
Event Baseline
measurement
of hypothermic
response
to oxotremorine
FSLt and FRLl groups are treated with bright light during entire regular photoperiod FSLz and FRLz are maintained under standard vivarium
4
Baseline recording of open field behavior bright light for 3 days
7
All groups are rechallenged with oxotremorine. and hypothermic response and open field behavior are measured
8
Forced swim test (FSLt light for 7 days.1
9-13
13
and FRLt treated
(FSLt
conditions
and FRLl treated
with
with bright
All groups are subjected to 5 days of washout groups subjected to control conditions)
iall
Hypothermic response to oxotremorine measured in all groups to determine whether they are all at their original baseline (measured on day 1 J
14-19
FSLt and FRLl are now subjected to control and FSLz and FRLz are treated with bright
16, 19
Hypothermic
Note. FSL = Flinders Sensitwe
response
to oxotremorine
Line. FRL = Flinders
Resistant
conditions, light
measured
in all groups
Line.
Results Effect of Bright Light on Thermic
Responsiveness
to a Muscarinic
The baseline temperatures of all the rats (38 “C-39 “C) were within the range when temperature is measured using a rectal probe. Baseline temperature differ significantly between groups (range = 38.6 “C-38.8 “C).
Agonist. expected did not
143 The oxotremorine-induced hypothermic response was quantitatively similar for both FSL and FRL groups at the start of the study, 5 days after the discontinuation of treatment with bright light, and after 3 days of treatment with bright light during Phase 2. FSL groups exhibited significantly greater hypothermic responses than the FRL groups regardless of whether they were treated with bright light during Phase 1 or 2 of the study (mean hypothermic response f SD = 2.5 k 0.4 “C vs. 2.4 k 0.3 “C). The hypothermic response of the FRL groups did not differ by order of treatment (mean hypothermic responses + SD = 1.1 f 0.3 ‘C vs. 1.0 ?r 0.3 “C). There was no overlap between the FSL and FRL groups (i.e., the FSL animal which exhibited the least response to oxotremorine exhibited a response of greater magnitude than any of the FRL animals). Table 2 summarizes the hypothermic response data. Table 2. Change in mean hypothermic oxotremorine relative to baseline
responsiveness
(“C + SD) to
Baseline response
Response Phase 1
Response during washout
Response Phase 2 day 3
FSLl
-
Decreased
Baseline
Baseline
Baseline
FSL2
-
Baseline
Baseline
Baseline
Decreased
FRLl
Decreased
Baseline
Baseline
Baseline
FRL7
Baseline
Baseline
Baseline
Baseline
Group
Response Phase 2 day 6
Note. FSL = Flinders Sensitive Lme. FRL = Flinders Resistant Line. The FSLf and FRLI groups were treated with bright light first and exposed to the control condition second. The reverse applies to the FSLz and FRLz groups. There was no effect of order of treatment for either line. The FSL rat exhibiting the lowest responsiveness had a larger response to oxotremorine both at baseline and after treatment with bright light for 6 days than the most responsive of the FRL animals. “Decreased” Indicates that the hypothermic response of a group was significantly less than at its baseline. “Baseline” indicates that the hypothermic response of a given group is statistically similar to its response to oxotremorine before the start of the study. The only condition associated with bluntmg of the hypothermic response was that of treatment with bright light for 6 days, Three days was not sufficient (Phase 2, day 4. groups FSLz and FRLzJ. Treatment with bright light produced significant blunting of the hypothermic response in both the FSL and the FRLs groups. The magnitude of the effect of bright light was significantly greater in the FSL groups.
Fig. 1 illustrates the results of the various oxotremorine challenges after 6 days of treatment with bright light. The data are presented as the difference between the hypothermic response between the control and light conditions. Positive scores indicate that the treatment with bright light was associated with blunting of the hypothermic response within a group. Three of the four groups exhibited clear-cut blunting. The exception was the FRL group which was first treated with bright light. Analysis of the data using a two-way ANOVA indicated that there was a significant effect of line. The magnitude of blunting of the hypothermic response to oxotremorine was greater in the FSL than FRL rats (F= 6.13; u”= I, 29;~ < 0.05). There was also an order effect. The groups treated with bright light first (i.e., in Phase 1) exhibited less of a hypothermic response to oxotremorine in Phase 2 (F= 10.58; df = 1,29;p
144 carry-over or order effect could manifest itself by a greater hypothermic response to oxotremorine of the FSL, and FRL2 (the control groups in Phase 1) relative to the FSLr and FRL, groups (the control groups in Phase 2) in Phase 2. That is, the light-treated FSL, and FRL, groups might exhibit attenuation of the hypothermic response to oxotremorine in Phase 2 relative to the FSL, and FRL, groups in Phase 1 due to previous treatment with bright light. Fig. 1 illustrates the mean difference f SD between the hypothermic responsiveness of each group of rats to oxotremorine under the control (exposure to ambient lighting in the vivarium during the regular photoperiod) and experimental (following 6 days of treatment with bright light) conditions. A positive difference indicates that treatment with bright light attenuated the hypcthermic response to oxotremorine. Fig. 1. Results of experiments assessing effects of treatment on hypothermic response in FSL and FRL rats
with bright light
1.60, 1.40 G 0-
1.20
5 z b
0.60
1.00
:
0.60
.r p
0.40
g
0.20
66
0.00 -0.20 -0.40
r-
J FSL2
FSLl
FRL2
FRLl
Values were derived by subtracting the hypothermrc response under control and experimental conditions. A positive value indicates that the mean change in hypothermic response I+ SC to oxotremorine diminished following 6 days of treatment with bright light. CL indicates that a particular group of rats was maintained under control conditions during Phase 1 and bright light after the crossover. LC indicates the reverse. FSL = Flinders Sensitive Line. FRL = Flinders Resistant Line.
Behavioral Data. Table 3 summarizes the effects of treatment with bright light on mobility (number of crossings) in an open field following the injection of oxotremorine (0.2 mg/kg KC.) and immobility in the forced swim test. Treatment with bright light was associated with a significant increase in crossings in the FSL group and a decrease in the FRL group at baseline (i.e., when not administered oxotremorine and before treatment with bright light) (see column 1 of Table 3, F = 4.65; df= 1, 29; p < 0.05). However, treatment with bright light did not decrease the motoric inhibiting effects of oxotremorine (column 2 in Table 3). The two-way ANOVA revealed a highly significant difference between the lines (F= 25.28; df = 1,29; p < 0.001). The FSL animals exhibited greater reduction in movement when challenged with oxotremorine. However, there was no effect of treatment with bright light (F = 0.03; df = 1, 29; p > 0.8). Thus, treatment with bright light did not alter the oxotremorine-induced inhibition of motor behavior, though it produced a dramatic blunting of the hypothermic response to this muscarinic agonist.
145 Table 3. Effects of treatment with bright light on activity and immobility FSL and FRL rats Activity at baseline1 (lines/min)
Treatment status FSL/control FSL/bright
light
FRL/control FRL/briaht
liaht
Note. Results are presented
Activity after oxotremorine2 (lines/min)
in the
Immobility in forced swim3 (set)
23.3 f 4.4
6.6 k 3.6
428.5 f 34.9
41.1 + 4.1
7.0 k 2.4
367.7 2 27.0
31.4 + 2.9
19.7 xk 1.5
150.0 2 30.1
35.1 k 4.6
18.3 i 4.4
223.4 i: 27.7
as mean + SD. FSL = Flinders
Sensitive
Lrne. FRL = Flanders Resistant
Line.
1. Recorded after 3 days of exposure to bright light of the FSL2 and FRL2 groups in Phase 2 of the study. 2. Recorded after 6 days of treatment of the FSLz and FRL2 groups with bright light in Phase 2. Methyl-atroprne 12 mg/kg I was given 15 min before the injection of oxotremonne IO.2 mg/kgl. Number of crossings (I” 1 min in a 60 X 60 cm open field) was recorded 15 mm after the injection of oxotremorme. 3. Recorded after 7 days of exposure to bright light 17400 1~x1.
The results of the forced swim test were similar to the open field component of the study. Both of the FSL groups were significantly more immobile than the FRL groups. Treatment with bright light did not alter immobility (column 3 of Table 3). Statistical analysis once again confirmed the highly significant difference between lines (F= 46.07; df = 1, 25; p < 0.00 1) and the absence of an effect of exposure to bright light (F= 0.02; df = 1, 25;~ > 0.8). The increased and decreased immobility of the FRL and FSL rats, respectively, in the forced swim test led to a significant interaction between line and treatment condition (F = 4.35; df = 1, 25; p < 0.05). Discussion The reduction of the oxotremorine-induced hypothermic response in FSL rats treated with full-spectrum bright light replicates previous findings (Dilsaver and Majchrzak, 1987; Dilsaver, 1989; Dilsaver et al., 1989). This report extends the data base to include the effect of light treatment on a line of rats with a genetically transmitted supersensitivity to muscarinic agonists. The capacity of treatment with bright light to subsensitize the rat to muscarinic agonist-induced hypothermia was more robust in the FSL than in the FRL rats (see Fig. 1). Dilsaver et al. have observed a similar phenomenon. The capacity of bright light to produce subsensitivity to oxotremorine is more impressive in rats subjected to a manipulation enhancing thermic responsiveness to oxotremorine such as treatment with amitriptyline (Dilsaver et al., 1989) or forced stress (Flemmeret al., in press) than in naive animals. Sprague-Dawley rats treated with amitriptyline (15 mg/ kg, i.p., twice daily for 7 days) exhibit an increase in their mean hypothermic response to oxotremorine (1.0 mg/kg, i.p.) from 1.3 f 0.3 “C (n 10) to 2.6 * 0.6 “C. Treatment with bright light for 6 hours daily during the phase-delay portion of the phaseresponse curve (5-l 1 p.m.) for 7 days at the identical intensity used in the study reported here produced complete normalization of thermic responsiveness to oxotremorine (mean hypothermic response f SD = 1.3 f 0.3 “C). Prolongation of the photoperiod from 5 p.m. to 11 p.m. did not account for this finding. In fact, simply prolonging the photoperiod with dull light was associated with an additional increq
146 ment in the hypothermic response to oxotremorine. Forced swim stress at 12 “C once daily for 10 min resulted in an increase in the mean hypothermic response to oxotremorine (0.25 mg/ kg) from 0.2 f 0.4 “C to 0.9 f 0.2 “C. Treatment with bright light (at the intensity used in the study reported here for 8 hours daily during the regular photoperiod) lowered the hypothermic response to 0.6 f 0.2 “C despite continuation of daily forced swim stress. The crossover design allowed us to assess the possibility that there are order effects of long-term treatment with bright light. The observation that the groups treated with bright light in Phase 1 (FSL, and FRL,) exhibited less of a hypothermic response to oxotremorine in Phase 2 than did the control groups in Phase 1 (FSL, and FRL,) suggests that bright light may have long-term effects. Exposure to bright light dramatically alters retinal photoceptors (Katz and Eldred, 1989). The data reported here suggest that the effects of treatment with bright light may linger for lo-14 days (the time difference between the two oxotremorine challenges). In contrast, Dilsaver and associates at Ohio State University (see Dilsaver, 1989, for a review) have always found that the muscarinic cholinergic mechanism(s) affecting oxotremorine-induced hypothermia return to their baseline level of sensitivity to this agonist within a week. The Ohio State group has used the Sprague-Dawley rat exclusively. There may be line differences. Additional experiments are required to determine the duration of the muscarinic agonist-induced subsensitization mediated by treatment with bright light in the FSL, FRL, and Sprague-Dawley rats, The tendency of bright light to normalize sensitivity to a muscarinic agonist in the FSL rats is consistent with the possibility that it has “antidepressant” properties. State-dependent or trait-related supersensitivity of brain muscarinic mechanisms may be involved in the pathophysiology of depression (Janowsky and Risch, 1987). However, antidepressants characteristically counter immobility in the forced swim test (Borsini and Meli, 1988; Duncan et al., 1986). One would a priori expect treatment with bright light to do so if it indeed had antidepressant properties. FSL rats exhibit much greater immobility in the forced swim test at baseline than do FRL rats (Overstreet, 1989; Overstreet et al., 1986). This study does not provide evidence that exposure to bright light decreases immobility of the FSL rats in the forced swim test (see Table 3). It merely suggests that treatment with bright light during the regular photoperiod has selective effects on muscarinic mechanisms. The results of this study must be conservatively interpreted for the reasons discussed below. Some observers have questioned the value of using the rat in studies of the biological properties of bright light. There is a valid concern that results generated using this nocturnal animal may not be useful in addressing concerns about the effects of bright light on a diurnal animal such as man. We would agree that the rat is not the best animal model to use in studying the neurobiology of bright light. It is, however, the most conveniently used animal in the initial phase of a long-term project due to cost and ease of handling. An example relevant to the interpretation of the results presented here is illustrative. Light has phasic properties that were not assessed in this study. Peck and Dilsaver (submitted; see also review by Dilsaver, 1990) demonstrated that it is essential to treat the rat with bright light during a particular portion of the 24-hour day if one is to produce a classic effect of the somatic treatments for depression. These investigators demonstrated that treatment with bright light either
147 during the regular photoperiod or 2 hours during the phase-advance portion (l-3 a.m.) of the phase-response curve has an effect opposite that of treatment with bright light for 2 hours during the phase-delay portion (9-l 1 p.m.) of the phase-response curve. The development of subsensitivity to the cw,-agonist clonidine (a classic effect of the somatic treatments for depression) requires treatment during the phase-delay portion of the phase-response curve in the Sprague-Dawley rat (see review by Dilsaver, 1989). Treatment with bright light during the entire regular photoperiod (6 a.m. to 6 p.m.) for 7 days produced enhancement of the thermic response to clonidine. Treatment for 2 hours nightly (l-3 a.m.) during the phase-advance portion of the phase-response curve for 7 nights did not alter thermic responsiveness to clonidine. However, treatment with a pulse of bright light from 1 a.m. to 3 a.m. for 12 consecutive nights was associated with an increase in the hypothermic response to clonidine. Only one condition produced decreased sensitivity to the a,-agonist, the classic effect we anticipated. Treatment with bright light for 2 hours nightly for 7 consecutive nights during the phase-delay portion of the phase-response curve (9-l 1 p.m.) produced decreased sensitivity to clonidine. Obtaining this effect required that the rat both be exposed to normal ambient lighting during the regular photoperiod and receive a 2-hour pulse during the phase-delay portion of the phase-response curve. The nightly administration of a 2-hour pulse of bright light between 9 p.m. and 11 p.m. while maintaining the animals in a darkened environment for 22 hours daily increased responsiveness to clonidine (Dilsaver, 1989). The observation that the administration of bright light decreased responsiveness to clonidine, albeit only under specific conditions, indicates that this treatment shares a property characteristic of antidepressants. However, the preclinical data have greater value than simply demonstrating that bright light has this property. Our preclinical observations can be used to design a clinical study testing the hypothesis that treatment with bright light produces decreased thermic responsiveness to clonidine in patients with winter depression. Our protocol assessing the effect of bright light on change in responsiveness of patients with winter depression to an cY,-agonist is a direct extension of the preclinical work on this topic. Clonidine safely produces hypothermia in human subjects (Glue et al., 1988). The effect of time of treatment with bright light on the thermic response to clonidine can be assessed in control subjects and patients with winter depression. However, we expect that treatment with bright light in the early morning (during the phase-advance portion as opposed to the phase-delay portion of the phase-response curve) will produce the classic effect of a somatic treatment for depression. This hypothesis is based on the results of the meta-analysis by Terman et al. (1989) of the outcome data from studies in which bright light was used to treat winter depression. They concluded that treatment with bright light in the early morning (the phaseadvance portion of the phase-response curve) was most effective in the treatment of winter depression. Thus, we do not expect results of treatment with bright light to be identical in the rat and human subjects with winter depression. However, the rat is a useful model to (1) the extent that it allowed us to demonstrate that a classic effect of a somatic treatment for depression can be produced by treatment with bright light and (2) simultaneously
148
suggested a means of identifying whether this effect occurs in patients with winter depression. The protocol used for the administration of bright light in this study did not produce a phase delay or advance. There is an important implication of the finding that the effect of treatment with bright light on the thermic response of the rat to clonidine is contingent on the timing of treatment with bright light. It suggests that if bright light at an adequate intensity, daily duration, and number of days were to be used to phaseshift the rat, the FSL animals might exhibit an increase in crossings in an open field following the injection of oxotremorine and increase mobility in the forced swim stress test. The data obtained in this study suggest that treatment with bright light during the entire regular photoperiod has a selective effect in the rat. The treatment protocol produced subsensitivity to the hypothermic effect of oxotremorine but did not alter motor behavior (crossings in an open field following the injection of oxotremorine or immobility in the swim test) of FSL rats. This circumscribed result may simply reflect the treatment protocol. Phase-shift studies are now indicated. Phase shifts could affect interacting muscarinic and nicotinic, cholinergic and aminergic systems (Janowsky et al., 1972; Dilsaver, 1986a, 19866; Janowsky and Risch, 1987; Wallis et al., 1988). Investigators interested in the role of muscarinic mechanisms in the neurobiology of depression assume that these systems interact and that depression is not merely due to aberrant muscarinic mechanisms (Dilsaver, 1986a, 1986b, 1989; Janowsky and Risch, 1987). The effect of treatment with bright light on hypothalamically mediated behaviors can be assessed in rats with behavioral deficits that are at least analogous to abnormalities in patients with severe depressive illness. Diminished hedonic capacity is a cardinal characteristic of endogenous depression (Spitzer et al., 1977) or major depression with melancholia (American Psychiatric Association, 1987). The FSL exhibits deficits in self-stimulation which, at least anthropomorphically if not in actuality, are related to the neurobiology of depression. The demonstration that treatment with bright light diminishes the magnitude of this deficit is a logical extension of work presented in this report. Acknowledgment. This Abnormalities in Affective
work was supported Illness).
by MH-005503-05
(Muscarinic
Receptor
149
References American Psychiatric Association. DSM-III-R: Diagnostic and Statistical Manual of Mental Disorders. 3rd ed., revised. Washington, DC: APA, 1987. Borsini, F., and Meli, A. Is the forced swimming test a suitable model for revealing antidepressant activity? Psychopharmacology, 94:147-160, 1988. Dilsaver, S.C. Cholinergic mechanisms in depression. Brain Research Reviews, 11:285-316, 1986a. Dilsaver, S.C. Cholinergic-monoaminergic interaction in the pathophysiology of depression. International Clinical Psychopharmacology, 1: 181-198, 19866. Dilsaver, S.C. Artificial light and nicotine subsensitivity. Biological Psychiatry, 24:437-440, 1988. Dilsaver, S.C. Neurobiologic effects of bright light. Brain Research Reviews, 14:311-334, 1989. Dilsaver, S.C.; Alessi, N.E.; and Snider, R.M. Amitriptyline supersensitizes a central muscarinic mechanism. Biological Psychiatry, 22~495 507, 1987. Dilsaver, S.C., and Majchrzak, M.J. Bright artificial light subsensitizes a central muscarinic mechanism. Life Sciences, 41:2607-2614, 1987. Dilsaver, S.C.; Majchrzak, M.J.; and Flemmer, D.D. Bright light blocks amitriptyline induced cholinoceptor supersensitivity. Biological Psychiatry, 261416-423, 1989. Dilsaver, SC.; Peck, J.A.; Miller, S.H.; Hoh, J.; Jaeckle, R.S.; and Traumata, D. Chronic inescapable swim stress enhances the motoric inhibiting effects of a muscarinic agonist. Pharmacology, Biochemistry and Behavior, submitted. Duncan, G.E.; Breese, G.R.; Criswell, H.; Stumpf, W.E.; Mueller, R.A.; and Covery, J.B. Effects of antidepressant drugs injected into the amygdala on behavioral responses of rats in the forced swim test. Journal of Pharmacology and Experimental Therapeurics, 238:758-762, 1986. Flemmer, D.D.; Dilsaver, S.C.; and Peck, J.A. Bright light blocks forced stress induced supersensitivity of a central muscarinic mechanism. Pharmacology, Biochemistry and Behavior, in press. Glue, P.; Sellman, J.D.; Joyce, R.P.; Nicolls, G.; and Nutt, D.J. The hypothermic response to clonidine is absent in alcohol withdrawal but returns in abstinence. Biological Psychiatry, 24:102-104, 1988. Janowsky, D.S.; El-Yousef, M.K.; Davis, J.D.; and Sekerke, H.D. A cholinergic adrenergic hypothesis of depression and mania. Lancer, 11:632-635, 1972. Janowsky, D.S., and Risch, SC. Cholinergic mechanisms in depression. In: Meltzer, H.Y., ed. Psychopharmacology: A Generation of Progress. New York: Raven Press, 1987. pp. 527-534. Johnson, R.F.; Morin, L.P.; and Moore, R.Y. Retinohypothalamic projections in the hamster and rat demonstrated using cholera toxin. Brain Research, 462:1301-1312, 1988. Katz, M.L., and Eldred, G.E. Failure of vitamin E to protect the retina against damage from bright cyclic light exposure. Investigative Ophthalmology and Visual Science, 30:29-36, 1989.
150 Lomax, P., and Jenden, D. J. Hypothermia following systemic and intracerebral injections of oxotremorine in the rat. Neuropharmacology. 5:353-359, 1966. Majchrzak, M.J., and Dilsaver, SC. Chronic treatment with ethanol produces supersensitivity to a muscarinic agonist. Progress in Nemo-Psychopharmacology and Biological Psychiatry,
14:379-386,
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Moore, R.Y., and Lenn, N.J. A retinohypothalamic projection in the rat. Journal of Comparative Neurology, 146: I-14, 1972. Overstreet, D. H. Selective breeding for increased cholinergic function: Development of a new animal model of depression. Biological Psychiatry, 21:49-58, 1986. Overstreet, D.H. Genetic animal model of depression with cholinergic supersensitivity. In: Lerer, B., and Gershon, E., eds. New Directions in Affective Disorders. New York: SpringerVerlag, 1989. pp. 67-70. Overstreet, D.H.; Janowsky, D.S.; Gillin, J.C.; Shiromani, P.J.; and Sutin, E.L. Stressinduced immobility in rats with cholinergic supersensitivity. Biological Psychiatry, 21:657-664, 1986.
Overstreet. D.H.; Russell, R.W.; Cracker, A.D.; Janowsky, D.S.; and Gillin, J.C. Genetic and pharmacological models of cholinergic supersensitivity. Experientia, 44:465-472, 1988. Peck, P.A., and Dilsaver, SC. Treatment with bright light during the phase-delay portion of the PRC produces subsensitivity to clonidine. Pharmacology, Biochemistry and Behavior, submitted. Pepe, S.; Overstreet, D.H.; and Cracker, A.D. Enhanced benzodiazepine responsiveness in rats with increased cholinergic function. Pharmacology, Biochemistry and Behavior, 31:15-20, 1988.
Shiromoni, J.P.; Overstreet, D.J.; Levy, D.; Goodrich, CA.; Campbell, S.S.; and Gillin, J.C. Increased REM sleep in rats selectively bred for cholinergic hyperactivity. Neuropsychophurmucology, 1:127-133, 1988. Spitzer, R.L.; Endicott, J.; and Robins, E. Research Diagnostic Criteria. New York: New York State Psychiatric Institute, 1977. Terman, M.; Terman, J.S.; Quitkin, F.M.; McGrath, P.J.; Stewart, J.W.; and Rafferty, B. Light therapy for seasonal affective disorder: A review of efficacy. Neuropsychopharmucology, 2: l-22,
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Wallis, E.; Overstreet, D.H.; and Cracker, A.D. Selective breeding for increased cholinergic function: Increased serotonergic sensitivity. Pharmacology, Biochemistry and Behavior, 3 1:345-350,
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N.E. Seasonality
and affective
illness. American
Journal
of
Psychiatry Research, 33:139-150
Elsevier
Effects of Bright Light on Responsiveness to a Muscarinic Agonist in Rats Selectively Bred for Endogenously Increased Cholinergic Function David H. Overstreet, Amir H. Rezvani Received
November
Steven
C. Dilsaver,
David S. Janowsky,
and
20, 1989; revised version received May 29, 1990; accepted June 2, 1990.
Abstract. The Flinders Sensitive Line (FSL) was derived from the Sprague-Dawley rat by selectively breeding those animals exhibiting a high level of sensitivity to an anticholinesterase. The Flinders Resistant Line (FRL) was simultaneously developed as a control line. These lines exhibit nonoverlapping distributions of their thermic responsiveness to oxotremorine. Bright light prevents the development of supersensitivity to oxotremorine occurring as a result of forced stress or treatment with a muscarinic receptor antagonist in the rat. The authors now report that treatment with bright light during the regular photoperiod (i.e., a time that does not produce a phase-shift or free-running) differentially affects the hypothermic response and activity-suppressing effect of oxotremorine in both the FSL and FRL. Both lines exhibit decreased hypothermia without reduction in motor activity in response to oxotremorine following 6 days of treatment with bright light. The magnitude of blunting of the hypothermic response was greater in the FSL than the FRL. These findings suggest that (1) studies of the effects of bright light are contingent on the end point one measures and (2) the capacity of this treatment to blunt the hypothermic response to a muscarinic agonist is greater in an animal model with endogenously hyperactive muscarinic cholinergic systems. Key Words. Affective disorders, bright light, cholinergic sensitivity, forced swim test, Flinders Sensitive Line, Flinders Resistant Line, hypothermia, muscarinic.
Exposure to full-spectrum bright artificial light potently reduces the hypothermic response to the muscarinic agonist oxotremorine in normal (Dilsaver and Majchrzak, 1987), stressed (Flemmer et al., in press), and amitriptyline-treated (Dilsaver et al., 1989) Sprague-Dawley rats. The capacity of bright light to produce subsensitivity of a muscarinic cholinergic mechanism could contribute to its effectiveness in the treatment of winter depression (for review, see Dilsaver, 1989; Wehr and Rosenthal, 1989). Recent studies also indicate that treatment with bright light alters adrenergic and nicotinic mechanisms (see Dilsaver, 1989). Treatment with bright light blunts the agonist-induced responsiveness of muscarinic and nicotinic cholinergic mechanisms
David H. Overstreet, Ph.D., is Associate Professor, School of Biological Sciences, Flinders University of South Australia. Steven C. Dilsaver, M.D., was Professor of Psychiatry and Neuroscience, and Director of the Psychopharmacology Program, Department of Psychiatry, The Ohio State University, Columbus, OH. David S. Janowsky, M.D., is Professor and Chairman, Department of Psychiatry, and Director of the Center for Alcohol Studies, University of North Carolina, Chapel Hill, NC. Amir H. Rezvani, Ph.D., is Director of the Center for Alcohol Studies, University of North Carolina. (Reprint requests to Dr. S.C. Dilsaver, Dept. of Psychiatry, University of Texas School of Medicine, P.O. Box 20708, Houston, TX 77225, USA.) 0165-17Xl,‘YO/$O3.50
Q IYYO Elsevier Scientific Publishers Ireland
I td
140 when the animals are treated during either a fraction or the entirety of the regular photoperiod (Dilsaver, 1988, 1989). This study assesses the effect of treatment with bright light during the entirety of the regular photoperiod in the Flinder’s Sensitive Line (FSL) relative to the Flinders Resistant Line (FRL). The FSL was bred to be endogenously supersensitive to muscarinic agonists (Overstreet et al., 1986, 1988; Pepeet al., 1988; Walliset al., 1988). The FRL is relatively insensitive to the effects of muscarinic agonists and anticholinesterases, and was simultaneously bred to be a control line for all studies involving the FSL. The FSL was recently set forth as an animal model of depression (Overstreet, 1986, 1989; Shiromani et al., 1988; Overstreet et al., 1988). It is particularly useful in testing hypotheses stemming from a muscarinic-cholinergic or muscarinic-cholinergicaminergic interaction theory of the etiology of affective illness (for review, see Dilsaver, 1986a, 19866; Janowsky and Risch, 1987). The hypothermic effect of oxotremorine is mediated by an effect on the hypothalamus (Lomax and Jenden, 1966). It is not known whether the effects of bright light are limited to functions mediated by the hypothalamus. The hypothalamus is directly affected by light via input from the retinohypothalamic pathway (Moore and Lenn, 1972; Johnson et al., 1988). The FSL provides an opportunity to assess the effect of treatment with bright light on the oxotremorine-induced hypothermia and motoric inhibition in an open field, and immobility in the forced swim test (Overstreet, 1986; Overstreet et al., 1986). The FSL exhibits a profound reduction in movement in an open field in response to oxotremorine and greater immobility in the forced swim test than the FRL. Methods Animals. The FSL and FRL rats used in this study came from a breeding colony maintained at the Center for Alcohol Studies at the University of North Carolina in Chapel Hill. This colony was derived from colonies at the Flinders University of South Australia. Male rats, about 80 days of age and weighing 350-450 g at the beginning of study, were used in all experiments. These animals were housed individually and given free access to food and water. The animals were maintained on a 12:12 hour reverse light-dark cycle. The intensity of ambient lighting during the light phase (10 p.m. to 10 a.m) was 400 lux. The vivarium is illuminated with red light during subjective night. The rat cannot perceive the color red, yet the intensity of illumination is sufficient to allow all required experimental manipulations. Conditions Prevailing During Measurement of Dependent Variables. Oxotremorine was injected during the light phase in all previous studies focusing on the effect of bright light on thermic responsiveness to this agonist (Dilsaver, 1989). Similarly, thermic responsiveness was measured during the light phase in this study. The effect of this muscarinic agonist on motor behavior in an open field (crossings in I min) was measured in a room illuminated with red light. The mean rate of crossings greatly increases and the SD falls substantially when open field behavior is measured during the dark phase (Dilsaver et al., submitted). Immobility in the forced swim test was measured under the lighting conditions prevailing during the last 2 hours of the photoperiod (between 8 a.m. and 10 a.m.) in this study. Pharmaceutical Sigma Chemical syringe.
Agents. Oxotremorine (base) and methyl-atropine were purchased from Company (St. Louis, MO). Both drugs were injected S.C. using a l-ml insulin
141 Treatment With Bright Artificial Light. Full-spectrum bright artificial light was emitted from a bank (Duro Test Co., North Bergen, NJ) of eight 122-cm long VitaLite tubes suspended 50 cm above the animals. Temperature under the light unit at the level of the animals was 24 “C, 1 o C higher than in the room. This particular unit has been used in several previously published reports on the neurobiological effects of bright light (Dilsaver, 1989). The intensity of lighting in the plane of the rats’ eyes was 7,400 lux in this and all other published reports. While 7,400 lux is a high intensity of illumination, actual exposure of the rat to bright light is limited consequent to the period of exposure being its sleep phase. The rat sleeps during most of the light phase. Sprague-Dawley rats treated with this particular light unit at an intensity of 7,400 lux exhibit a profound reduction in thermic responsiveness to oxotremorine, which is reversible within 10 days of discontinuing treatment (Dilsaver, 1989). Measurement of Temperature. Core body temperature was measured by the insertion of a thermistor probe 6 cm into the rectum. Output was presented on a Bailey’s digital telethermometer (Senortrek Instruments). Measurement of Open Field Behavior. Open field behavior (i.e, number of crossings) was measured in a wooden chamber which was 60 cm x 60 cm with a wall of 12 cm on all sides. The chamber was divided into 36 squares of 100 cm2 and was covered with Plexiglas. Oxotremorine Challenge. The hypothermic response to oxotremorine is centrally mediated. This is evident from the fact that it is blocked by a muscarinic receptor antagonist which crosses the blood-brain barrier but not by charged molecules with effects limited to the periphery (Dilsaver et al., 1987). Lomax and Jenden (1966) have presented evidence that oxotremorineinduced hypothermia is mediated by an effect on the hypothalamus. Light directly activates the retinohypothalamic tract-a neuroanatomic pathway extending into the hypothalamus. Methyl-atropine (2 mgi kg) was injected S.C. before the administration of oxotremorine to block the peripheral effects of the muscarinic agonist. The addition of another methyl group to the nitrogen atom in atropine produces a positively charged molecule with limited lipid solubility. This results in its being an antagonist at peripheral but not central muscarinic receptor sites. Oxotremorine, 0.2 mg/ kg, s.c., was injected 15 min after the injection of methyl-atropine. Core temperature was measured 30 min after the injection of oxotremorine. This measurement was recorded (1) before performing any experimental manipulation (i.e., at baseline) and (2) after 3 and/or 6 days of treatment with bright light as opposed to standard lighting. Each of the two groups of FSL (FSL, and FSL2) and FRL (FRL, and FRL,) rats were challenged with oxotremorine, and core body temperature was recorded after a 5-day hiatus during which they were housed under standard lighting conditions. The data obtained at this point indicated that there were no meaningful differences in thermic responsiveness within each line. A crossover was therefore performed. In the period following the crossover, Phase 2, the groups that were initially exposed to bright light (FSL, and FRL,) were subjected to standard lighting conditions, and those originally housed under standard conditions of lighting (FSL, and FRL,) were subjected to bright light during the entire photoperiod for 6 days. We were interested in the time course of the development of subsensitivity to the hypothermic response to oxotremorine. The rats were therefore challenged 3 and 6 days following the start of exposure to bright light in Phase 2. Majchrzak and Dilsaver (1990) recently reported that thermic responsiveness to oxotremorine is unaffected by repeated injections of this agonist at 48-hour intervals at a dose slightly-higher than that used here (0.25 vs. 0.20 mg/kg, i.p). There was no evidence that treatment with bright light attenuated the oxotremorine-induced reduction in crossings in an open field or immobility in the forced swim test during Phase 1 of the study. Consequently, these parameters were not measured during Phase 2. Forced Swim Test. All of the animals were maintained under identical lighting conditions for 1 more day after the measurement of thermic responsiveness to oxotremorine so that immobil-
142 ity in the forced swim test could be measured 24 hours later. The rats were individually placed in the swim tank, and immobility was then immediately recorded over a IO-min period using a stopwatch. The rats were partially dried with a towel and then placed under a heat lamp for 10 min after completion of this test. Table I outlines the course of events in this study. Analysis of Data. Each pharmacological challenge produced data for four independent groups-the FSL-light, FSL-control, FRL-light, and FRL-control groups. These data were subjected to a two-way analysis of variance (ANOVA). Thermic responsiveness was measured both in the course of treatment with bright light and under the standard lighting conditions in the vivarium. We were interested in determining whether the main effects for treatment with bright light and sequence of treatment were significant (i.e., treatment with bright light first vs. treatment with bright light second). The repeated measures design inherent in the crossover phase permitted us to assess this question. The difference in the responsiveness of each animal under these two conditions was entered into a two-way ANOVA for repeated measures. All measures of variance in the text refer to the standard deviation (SD). The critical value of (Ywas set at p < 0.05, two-tailed.
Table 1. Sequence
of experimental
events
Days of studv 1
2-8
Event Baseline
measurement
of hypothermic
response
to oxotremorine
FSLt and FRLl groups are treated with bright light during entire regular photoperiod FSLz and FRLz are maintained under standard vivarium
4
Baseline recording of open field behavior bright light for 3 days
7
All groups are rechallenged with oxotremorine. and hypothermic response and open field behavior are measured
8
Forced swim test (FSLt light for 7 days.1
9-13
13
and FRLt treated
(FSLt
conditions
and FRLl treated
with
with bright
All groups are subjected to 5 days of washout groups subjected to control conditions)
iall
Hypothermic response to oxotremorine measured in all groups to determine whether they are all at their original baseline (measured on day 1 J
14-19
FSLt and FRLl are now subjected to control and FSLz and FRLz are treated with bright
16, 19
Hypothermic
Note. FSL = Flinders Sensitwe
response
to oxotremorine
Line. FRL = Flinders
Resistant
conditions, light
measured
in all groups
Line.
Results Effect of Bright Light on Thermic
Responsiveness
to a Muscarinic
The baseline temperatures of all the rats (38 “C-39 “C) were within the range when temperature is measured using a rectal probe. Baseline temperature differ significantly between groups (range = 38.6 “C-38.8 “C).
Agonist. expected did not
143 The oxotremorine-induced hypothermic response was quantitatively similar for both FSL and FRL groups at the start of the study, 5 days after the discontinuation of treatment with bright light, and after 3 days of treatment with bright light during Phase 2. FSL groups exhibited significantly greater hypothermic responses than the FRL groups regardless of whether they were treated with bright light during Phase 1 or 2 of the study (mean hypothermic response f SD = 2.5 k 0.4 “C vs. 2.4 k 0.3 “C). The hypothermic response of the FRL groups did not differ by order of treatment (mean hypothermic responses + SD = 1.1 f 0.3 ‘C vs. 1.0 ?r 0.3 “C). There was no overlap between the FSL and FRL groups (i.e., the FSL animal which exhibited the least response to oxotremorine exhibited a response of greater magnitude than any of the FRL animals). Table 2 summarizes the hypothermic response data. Table 2. Change in mean hypothermic oxotremorine relative to baseline
responsiveness
(“C + SD) to
Baseline response
Response Phase 1
Response during washout
Response Phase 2 day 3
FSLl
-
Decreased
Baseline
Baseline
Baseline
FSL2
-
Baseline
Baseline
Baseline
Decreased
FRLl
Decreased
Baseline
Baseline
Baseline
FRL7
Baseline
Baseline
Baseline
Baseline
Group
Response Phase 2 day 6
Note. FSL = Flinders Sensitive Lme. FRL = Flinders Resistant Line. The FSLf and FRLI groups were treated with bright light first and exposed to the control condition second. The reverse applies to the FSLz and FRLz groups. There was no effect of order of treatment for either line. The FSL rat exhibiting the lowest responsiveness had a larger response to oxotremorine both at baseline and after treatment with bright light for 6 days than the most responsive of the FRL animals. “Decreased” Indicates that the hypothermic response of a group was significantly less than at its baseline. “Baseline” indicates that the hypothermic response of a given group is statistically similar to its response to oxotremorine before the start of the study. The only condition associated with bluntmg of the hypothermic response was that of treatment with bright light for 6 days, Three days was not sufficient (Phase 2, day 4. groups FSLz and FRLzJ. Treatment with bright light produced significant blunting of the hypothermic response in both the FSL and the FRLs groups. The magnitude of the effect of bright light was significantly greater in the FSL groups.
Fig. 1 illustrates the results of the various oxotremorine challenges after 6 days of treatment with bright light. The data are presented as the difference between the hypothermic response between the control and light conditions. Positive scores indicate that the treatment with bright light was associated with blunting of the hypothermic response within a group. Three of the four groups exhibited clear-cut blunting. The exception was the FRL group which was first treated with bright light. Analysis of the data using a two-way ANOVA indicated that there was a significant effect of line. The magnitude of blunting of the hypothermic response to oxotremorine was greater in the FSL than FRL rats (F= 6.13; u”= I, 29;~ < 0.05). There was also an order effect. The groups treated with bright light first (i.e., in Phase 1) exhibited less of a hypothermic response to oxotremorine in Phase 2 (F= 10.58; df = 1,29;p
144 carry-over or order effect could manifest itself by a greater hypothermic response to oxotremorine of the FSL, and FRL2 (the control groups in Phase 1) relative to the FSLr and FRL, groups (the control groups in Phase 2) in Phase 2. That is, the light-treated FSL, and FRL, groups might exhibit attenuation of the hypothermic response to oxotremorine in Phase 2 relative to the FSL, and FRL, groups in Phase 1 due to previous treatment with bright light. Fig. 1 illustrates the mean difference f SD between the hypothermic responsiveness of each group of rats to oxotremorine under the control (exposure to ambient lighting in the vivarium during the regular photoperiod) and experimental (following 6 days of treatment with bright light) conditions. A positive difference indicates that treatment with bright light attenuated the hypcthermic response to oxotremorine. Fig. 1. Results of experiments assessing effects of treatment on hypothermic response in FSL and FRL rats
with bright light
1.60, 1.40 G 0-
1.20
5 z b
0.60
1.00
:
0.60
.r p
0.40
g
0.20
66
0.00 -0.20 -0.40
r-
J FSL2
FSLl
FRL2
FRLl
Values were derived by subtracting the hypothermrc response under control and experimental conditions. A positive value indicates that the mean change in hypothermic response I+ SC to oxotremorine diminished following 6 days of treatment with bright light. CL indicates that a particular group of rats was maintained under control conditions during Phase 1 and bright light after the crossover. LC indicates the reverse. FSL = Flinders Sensitive Line. FRL = Flinders Resistant Line.
Behavioral Data. Table 3 summarizes the effects of treatment with bright light on mobility (number of crossings) in an open field following the injection of oxotremorine (0.2 mg/kg KC.) and immobility in the forced swim test. Treatment with bright light was associated with a significant increase in crossings in the FSL group and a decrease in the FRL group at baseline (i.e., when not administered oxotremorine and before treatment with bright light) (see column 1 of Table 3, F = 4.65; df= 1, 29; p < 0.05). However, treatment with bright light did not decrease the motoric inhibiting effects of oxotremorine (column 2 in Table 3). The two-way ANOVA revealed a highly significant difference between the lines (F= 25.28; df = 1,29; p < 0.001). The FSL animals exhibited greater reduction in movement when challenged with oxotremorine. However, there was no effect of treatment with bright light (F = 0.03; df = 1, 29; p > 0.8). Thus, treatment with bright light did not alter the oxotremorine-induced inhibition of motor behavior, though it produced a dramatic blunting of the hypothermic response to this muscarinic agonist.
145 Table 3. Effects of treatment with bright light on activity and immobility FSL and FRL rats Activity at baseline1 (lines/min)
Treatment status FSL/control FSL/bright
light
FRL/control FRL/briaht
liaht
Note. Results are presented
Activity after oxotremorine2 (lines/min)
in the
Immobility in forced swim3 (set)
23.3 f 4.4
6.6 k 3.6
428.5 f 34.9
41.1 + 4.1
7.0 k 2.4
367.7 2 27.0
31.4 + 2.9
19.7 xk 1.5
150.0 2 30.1
35.1 k 4.6
18.3 i 4.4
223.4 i: 27.7
as mean + SD. FSL = Flinders
Sensitive
Lrne. FRL = Flanders Resistant
Line.
1. Recorded after 3 days of exposure to bright light of the FSL2 and FRL2 groups in Phase 2 of the study. 2. Recorded after 6 days of treatment of the FSLz and FRL2 groups with bright light in Phase 2. Methyl-atroprne 12 mg/kg I was given 15 min before the injection of oxotremonne IO.2 mg/kgl. Number of crossings (I” 1 min in a 60 X 60 cm open field) was recorded 15 mm after the injection of oxotremorme. 3. Recorded after 7 days of exposure to bright light 17400 1~x1.
The results of the forced swim test were similar to the open field component of the study. Both of the FSL groups were significantly more immobile than the FRL groups. Treatment with bright light did not alter immobility (column 3 of Table 3). Statistical analysis once again confirmed the highly significant difference between lines (F= 46.07; df = 1, 25; p < 0.00 1) and the absence of an effect of exposure to bright light (F= 0.02; df = 1, 25;~ > 0.8). The increased and decreased immobility of the FRL and FSL rats, respectively, in the forced swim test led to a significant interaction between line and treatment condition (F = 4.35; df = 1, 25; p < 0.05). Discussion The reduction of the oxotremorine-induced hypothermic response in FSL rats treated with full-spectrum bright light replicates previous findings (Dilsaver and Majchrzak, 1987; Dilsaver, 1989; Dilsaver et al., 1989). This report extends the data base to include the effect of light treatment on a line of rats with a genetically transmitted supersensitivity to muscarinic agonists. The capacity of treatment with bright light to subsensitize the rat to muscarinic agonist-induced hypothermia was more robust in the FSL than in the FRL rats (see Fig. 1). Dilsaver et al. have observed a similar phenomenon. The capacity of bright light to produce subsensitivity to oxotremorine is more impressive in rats subjected to a manipulation enhancing thermic responsiveness to oxotremorine such as treatment with amitriptyline (Dilsaver et al., 1989) or forced stress (Flemmeret al., in press) than in naive animals. Sprague-Dawley rats treated with amitriptyline (15 mg/ kg, i.p., twice daily for 7 days) exhibit an increase in their mean hypothermic response to oxotremorine (1.0 mg/kg, i.p.) from 1.3 f 0.3 “C (n 10) to 2.6 * 0.6 “C. Treatment with bright light for 6 hours daily during the phase-delay portion of the phaseresponse curve (5-l 1 p.m.) for 7 days at the identical intensity used in the study reported here produced complete normalization of thermic responsiveness to oxotremorine (mean hypothermic response f SD = 1.3 f 0.3 “C). Prolongation of the photoperiod from 5 p.m. to 11 p.m. did not account for this finding. In fact, simply prolonging the photoperiod with dull light was associated with an additional increq
146 ment in the hypothermic response to oxotremorine. Forced swim stress at 12 “C once daily for 10 min resulted in an increase in the mean hypothermic response to oxotremorine (0.25 mg/ kg) from 0.2 f 0.4 “C to 0.9 f 0.2 “C. Treatment with bright light (at the intensity used in the study reported here for 8 hours daily during the regular photoperiod) lowered the hypothermic response to 0.6 f 0.2 “C despite continuation of daily forced swim stress. The crossover design allowed us to assess the possibility that there are order effects of long-term treatment with bright light. The observation that the groups treated with bright light in Phase 1 (FSL, and FRL,) exhibited less of a hypothermic response to oxotremorine in Phase 2 than did the control groups in Phase 1 (FSL, and FRL,) suggests that bright light may have long-term effects. Exposure to bright light dramatically alters retinal photoceptors (Katz and Eldred, 1989). The data reported here suggest that the effects of treatment with bright light may linger for lo-14 days (the time difference between the two oxotremorine challenges). In contrast, Dilsaver and associates at Ohio State University (see Dilsaver, 1989, for a review) have always found that the muscarinic cholinergic mechanism(s) affecting oxotremorine-induced hypothermia return to their baseline level of sensitivity to this agonist within a week. The Ohio State group has used the Sprague-Dawley rat exclusively. There may be line differences. Additional experiments are required to determine the duration of the muscarinic agonist-induced subsensitization mediated by treatment with bright light in the FSL, FRL, and Sprague-Dawley rats, The tendency of bright light to normalize sensitivity to a muscarinic agonist in the FSL rats is consistent with the possibility that it has “antidepressant” properties. State-dependent or trait-related supersensitivity of brain muscarinic mechanisms may be involved in the pathophysiology of depression (Janowsky and Risch, 1987). However, antidepressants characteristically counter immobility in the forced swim test (Borsini and Meli, 1988; Duncan et al., 1986). One would a priori expect treatment with bright light to do so if it indeed had antidepressant properties. FSL rats exhibit much greater immobility in the forced swim test at baseline than do FRL rats (Overstreet, 1989; Overstreet et al., 1986). This study does not provide evidence that exposure to bright light decreases immobility of the FSL rats in the forced swim test (see Table 3). It merely suggests that treatment with bright light during the regular photoperiod has selective effects on muscarinic mechanisms. The results of this study must be conservatively interpreted for the reasons discussed below. Some observers have questioned the value of using the rat in studies of the biological properties of bright light. There is a valid concern that results generated using this nocturnal animal may not be useful in addressing concerns about the effects of bright light on a diurnal animal such as man. We would agree that the rat is not the best animal model to use in studying the neurobiology of bright light. It is, however, the most conveniently used animal in the initial phase of a long-term project due to cost and ease of handling. An example relevant to the interpretation of the results presented here is illustrative. Light has phasic properties that were not assessed in this study. Peck and Dilsaver (submitted; see also review by Dilsaver, 1990) demonstrated that it is essential to treat the rat with bright light during a particular portion of the 24-hour day if one is to produce a classic effect of the somatic treatments for depression. These investigators demonstrated that treatment with bright light either
147 during the regular photoperiod or 2 hours during the phase-advance portion (l-3 a.m.) of the phase-response curve has an effect opposite that of treatment with bright light for 2 hours during the phase-delay portion (9-l 1 p.m.) of the phase-response curve. The development of subsensitivity to the cw,-agonist clonidine (a classic effect of the somatic treatments for depression) requires treatment during the phase-delay portion of the phase-response curve in the Sprague-Dawley rat (see review by Dilsaver, 1989). Treatment with bright light during the entire regular photoperiod (6 a.m. to 6 p.m.) for 7 days produced enhancement of the thermic response to clonidine. Treatment for 2 hours nightly (l-3 a.m.) during the phase-advance portion of the phase-response curve for 7 nights did not alter thermic responsiveness to clonidine. However, treatment with a pulse of bright light from 1 a.m. to 3 a.m. for 12 consecutive nights was associated with an increase in the hypothermic response to clonidine. Only one condition produced decreased sensitivity to the a,-agonist, the classic effect we anticipated. Treatment with bright light for 2 hours nightly for 7 consecutive nights during the phase-delay portion of the phase-response curve (9-l 1 p.m.) produced decreased sensitivity to clonidine. Obtaining this effect required that the rat both be exposed to normal ambient lighting during the regular photoperiod and receive a 2-hour pulse during the phase-delay portion of the phase-response curve. The nightly administration of a 2-hour pulse of bright light between 9 p.m. and 11 p.m. while maintaining the animals in a darkened environment for 22 hours daily increased responsiveness to clonidine (Dilsaver, 1989). The observation that the administration of bright light decreased responsiveness to clonidine, albeit only under specific conditions, indicates that this treatment shares a property characteristic of antidepressants. However, the preclinical data have greater value than simply demonstrating that bright light has this property. Our preclinical observations can be used to design a clinical study testing the hypothesis that treatment with bright light produces decreased thermic responsiveness to clonidine in patients with winter depression. Our protocol assessing the effect of bright light on change in responsiveness of patients with winter depression to an cY,-agonist is a direct extension of the preclinical work on this topic. Clonidine safely produces hypothermia in human subjects (Glue et al., 1988). The effect of time of treatment with bright light on the thermic response to clonidine can be assessed in control subjects and patients with winter depression. However, we expect that treatment with bright light in the early morning (during the phase-advance portion as opposed to the phase-delay portion of the phase-response curve) will produce the classic effect of a somatic treatment for depression. This hypothesis is based on the results of the meta-analysis by Terman et al. (1989) of the outcome data from studies in which bright light was used to treat winter depression. They concluded that treatment with bright light in the early morning (the phaseadvance portion of the phase-response curve) was most effective in the treatment of winter depression. Thus, we do not expect results of treatment with bright light to be identical in the rat and human subjects with winter depression. However, the rat is a useful model to (1) the extent that it allowed us to demonstrate that a classic effect of a somatic treatment for depression can be produced by treatment with bright light and (2) simultaneously
148
suggested a means of identifying whether this effect occurs in patients with winter depression. The protocol used for the administration of bright light in this study did not produce a phase delay or advance. There is an important implication of the finding that the effect of treatment with bright light on the thermic response of the rat to clonidine is contingent on the timing of treatment with bright light. It suggests that if bright light at an adequate intensity, daily duration, and number of days were to be used to phaseshift the rat, the FSL animals might exhibit an increase in crossings in an open field following the injection of oxotremorine and increase mobility in the forced swim stress test. The data obtained in this study suggest that treatment with bright light during the entire regular photoperiod has a selective effect in the rat. The treatment protocol produced subsensitivity to the hypothermic effect of oxotremorine but did not alter motor behavior (crossings in an open field following the injection of oxotremorine or immobility in the swim test) of FSL rats. This circumscribed result may simply reflect the treatment protocol. Phase-shift studies are now indicated. Phase shifts could affect interacting muscarinic and nicotinic, cholinergic and aminergic systems (Janowsky et al., 1972; Dilsaver, 1986a, 19866; Janowsky and Risch, 1987; Wallis et al., 1988). Investigators interested in the role of muscarinic mechanisms in the neurobiology of depression assume that these systems interact and that depression is not merely due to aberrant muscarinic mechanisms (Dilsaver, 1986a, 1986b, 1989; Janowsky and Risch, 1987). The effect of treatment with bright light on hypothalamically mediated behaviors can be assessed in rats with behavioral deficits that are at least analogous to abnormalities in patients with severe depressive illness. Diminished hedonic capacity is a cardinal characteristic of endogenous depression (Spitzer et al., 1977) or major depression with melancholia (American Psychiatric Association, 1987). The FSL exhibits deficits in self-stimulation which, at least anthropomorphically if not in actuality, are related to the neurobiology of depression. The demonstration that treatment with bright light diminishes the magnitude of this deficit is a logical extension of work presented in this report. Acknowledgment. This Abnormalities in Affective
work was supported Illness).
by MH-005503-05
(Muscarinic
Receptor
149
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