Psychopharmacology(1992) 108:23 26
Psychopharmacology © Springer-Verlag 1992
Chronic imipramine treatment normalizes levels of tyrosine hydroxylase in the locus coeruleus of chronically stressed rats Kathleen R. MeUa, Eric J. Nestler, and Ronald S. Duman Laboratory of Molecular Psychiatry,Department of Psychiatry,Yale University School of Medicine and the RibicoffResearch Facilities of the Connecticut Mental Health Center, 34 Park Street, New Haven, CT 06508, USA Received June 24, 1991 / Final version October 14, 1991
Abstract. Previous studies have demonstrated that chronic stress increases and antidepressant treatments decrease levels of tyrosine hydroxylase (TH) in locus coeruleus (LC). In the present study, the influence of chronic antidepressant treatment on the induction of TH immunoreactivity in response to cold stress is examined. It was found that chronic imipramine pretreatment (18 days) attenuated the induction of TH in response to cold stress, resulting in levels of TH immunoreactivity not different from control. In contrast, imipramine pretreatment for 1 or 7 days was not sufficient to normalize the stress-induced elevation of TH immunoreactivity. These findings raise the possibility that the therapeutic action of antidepressants may be derived, in part, from the ability of these treatments to normalize levels of TH and thereby the function of the NE neurotransmitter system under conditions of stress. Key words: Tyrosine hydroxylase - Locus coeruleus Stress - Antidepressants - Imipramine
Stress is known to precipitate and/or exacerbate clinical depression in some patients (Winokur 1979; Dohrenwend and Dohrenwend 1980; Loyd 1980; Rabkin 1982; Breslau and Davis 1986). Moreover, exposure of rats to chronic stress produces many of the motivational, neuroendocrine, and behavioral concomitants seen in human depression (Katz 1981, 1982; Katz and Sibel 1982). For these reasons, chronic stress has been considered a useful animal model for human affective disorders (Willner 1984). In this regard, it is noteworthy that chronic treatment with clinically effective antidepressants such as tricyclics (Katz and Hersh 1981), monoamine oxidase inhibitors (Katz et al. 1981), atypical antidepressants (iprindole, buproprion, mianserin) (Katz Abbreviations: locus coeruleus, LC; norepinephrine, NE; tyrosine
hydroxylase,TH Offprint requests to: R.S. Duman
and Sibel 1982a), or electroconvulsive seizures (Katz 1981), block the behavioral effects of chronic intermittent unpredictable stress. In agreement with the time course for the therapeutic actions of clinical treatment, chronic but not acute antidepressant treatments have been found effective in this regard (Katz and Baldrighi 1982; Katz and Sibel 1982b). One of the most consistently and widely studied neurochemical targets of stress and antidepressant treatments is the norepinephrine (NE) neurotransmitter system. Prolonged exposure to stress can result in increased levels of norepinephrine (NE) in the locus coeruleus (LC) and its terminal regions (Stone and McCarty 1983; Adell et al. 1988, 1989; cf Stone 1975). Chronic stress also regulates tyrosine hydroxylase (TH), the rate limiting enzyme for the synthesis of NE, by increasing levels of mRNA, protein, and enzyme activity (Thoenen 1970; Zigmond et al. 1974; Melia et al. 1992; Richard et al. 1988). In contrast, chronic antidepressant treatments have been reported to decrease levels of NE and its metabolites, enzyme activity, and levels of TH mRNA and protein in LC (Roffler-Tarlov et al. 1973; Segal et al. 1974; Adell et al. 1988, 1989; Nestler et al. 1990). These findings are of particular interest since numerous clinical studies have demonstrated dysfunction of the NE system in depression (for review see Gold et al. 1988). In the present study, the influence of chronic antidepressant treatment on the ability of stress to alter the NE system was examined. A previous study has demonstrated that chronic antidepressant treatment prevented the stressed induced increase in levels of NE in midbrain and striatum but the mechanisms involved were not determined (Adell et al. 1989). In the present study, the influence of antidepressant treatments and stress on levels of TH protein in the LC were determined by immunoblot analysis. The results of the present study support the conclusion that antidepressant treatment is capable of normalizing increases of the NE system in response to chronic stress and indicate that this effect occurs at least in part through regulation of TH expression in LC.
24
Materials and methods Animals. Male Sprague Dawley rats (initial weight at onset of chronic antidepressant treatments was 150-200 g) were housed three per cage in wire mesh cages (35 × 20.5 x 17 cm) in a colony room with a 12 h light-dark cycle (lights on 7:00 a.m., off7 : 00 p.m.) maintained at 21 ° C. All animals were allowed ad libitum access to food and water throughout each experiment. Procedure. During the 5-day cold stress, animals were housed singly in plastic cages (28 x 15 x 15 cm) with wood chips for bedding, in a large cold room (2.5 x 2.5 x 2.5 m) maintained at 4 ° C. This stress paradigm has been reported to result in increased levels of TH immunoreactivity in LC (Richard et al. 1988; Melia et al. 1992). To determine whether chronic antidepressant treatment alters the induction of LC TH by chronic stress, colony housed animals were injected with imipramine (IP, 10 mg/kg) for 1, 7, or 18 days and then exposed to cold stress as described above. Daily injections of imipramine were continued throughout the 5 days of stress so that animals received a total of 6, 12, or 23 daily injections. Naive animals housed three per cage (without wood chips) at 21 ° C were used for the control group in this experiment. Animals were decapitated after the treatments and TH levels were assayed in the LC, substantia nigra (SN) and ventral tegmentum (VTA). Immunoblottino of TH. Animals were decapitated, and the LC, SN, and VTA were excised from 1 ram-thick coronal sections of brain by obtaining punches with a 14- (LC) or 15- (VTA and SN) gauge, blunt syringe needle. The tissue was sonicated in 100 lal 2% SDS. Samples were normalized for protein content using the method of Lowry et al. (1951) and adjusted to contain 50 mM TRIS (pH 6.7), 2% SDS, 4% glycerol, and 2% 2-mercaptoethanol, with bromophenol blue as a marker. The samples were then subjected to onedimensional SDS-polyacrylamide gel electrophoresis (with 7.5% acrylamide/0.3 % bisacrylamide in the resolving gels). Proteins were electrophoretically transferred to nitrocellulose paper and then subjected to immunoblot analysis using a polyclonal antibody to TH (1:2000) (generously supplied by Dr. J. Haycock, Louisiana State University), as previously described (Guitart et al. 1990). Resulting filters were washed for 2 h in immunoblot buffer and then dried and autoradiographed with the use of intensifying screens (Dupont). These conditions result in specific immunolabeling of TH protein on both one- and two-dimensional gels (Guitart et al. 1990). Levels of immunolabeling were quantified by densitometry or by counting excised bands in a gamma counter. These two methods yielded similar results. Under the immunolabeling conditions used, levels of TH-immunoreactivity were linear over a three-fold range of tissue concentration. Data analysis and presentation. Data were analyzed using Analysis of variance (ANOVA) for primary analyses and the Scheffe F-test for post hoc comparisons. Where appropriate, between subject t-tests were used to compare results of two groups. Data are presented as the mean percent change from control + / - SEM, as indicated.
Results T h e influence o f c h r o n i c c o l d stress a n d i m i p r a m i n e t r e a t m e n t s o n levels o f T H i m m u n o r e a c t i v i t y in L C were e x a m i n e d . C o l d stress was c h o s e n b e c a u s e it p r o d u c e s s o m e n e u r o e n d o c r i n e c h a n g e s similar to t h o s e seen in d e p r e s s e d p a t i e n t s ( V e r n i k o s et al. 1982) a n d b e c a u s e it results in a reliable increase in levels o f T H m R N A a n d p r o t e i n in L C ( R i c h a r d et al. 1988). C h r o n i c e x p o s u r e to c o l d (5 d a y s at 4 ° C) p r o d u c e d a statistically significant increase [t(10) = - 2.37, P < 0.05] in T H - i m m u n o r e a c t i v i t y
A
TH'~
'~ ~
Treatment
+
B 5o •~ ~ . .~, o
-,
g
-T-
25
-2s-
~ g -50. -75 ' -lOO -4-
Stress
IMI
Stress
+ IMI
Fig. 2. The effect of imipramine pretreatment for l, 7 or 18 days on ments on TH-immunoreactivity in the LC. Rats were either exposed to cold stress (5 days), treated with imipramine for 18 days (10 mg/kg, IP) or pretreated with imipramine and then cold stressed in the continued presence of antidepressant treatment. Levels of TH immunoreactivity were determined in LC by immunoblot analysis as described in Materials and methods. Autoradiograms representative of the effect of each treatment condition are shown (A). Levels of TH immunoreactivity were quantitated by densitometry of the resulting autoradiograms and the results are presented as percent change from control (B). Each point is the mean+SEM of 6-12 separate determinations. * P < 0.05, significantly different from control (Student's t-test)
in the L C o f a p p r o x i m a t e l y 45 % (Fig. 1, A a n d B), b u t no significant c h a n g e in T H - i m m u n o r e a c t i v i t y in either the S N o r V T A ( d a t a n o t shown). I n c o n t r a s t to the effects o f c o l d stress, r e p e a t e d a d m i n i s t r a t i o n (18 days) o f the tricyclic a n t i d e p r e s s a n t , i m i p r a m i n e (10 m g / k g , IP), p r o d u c e d a significant decrease [ t ( 1 2 ) = 2 . 2 8 , P < 0 . 0 5 ] in T H - i m m u n o r e a c t i v i t y in the L C (Fig. 1, A a n d B), b u t no significant c h a n g e in T H - i m m u n o r e a c t i v ity in S N o r V T A ( d a t a n o t shown). These results c o n f i r m a n d e x t e n d the results o f p r e v i o u s w o r k f r o m o u r labo r a t o r y (Nestler et al. 1990). T h e influence o f a n t i d e p r e s s a n t t r e a t m e n t o n induction o f T H in L C b y c h r o n i c c o l d stress was e x a m i n e d next. P r e t r e a t m e n t w i t h i m i p r a m i n e for 18 d a y s f o l l o w e d by 5 days of cold exposure and continued imipramine t r e a t m e n t c o m p l e t e l y n o r m a l i z e d the i n d u c t i o n o f T H b y c h r o n i c stress (Fig. 1, A a n d B). I n c o n t r a s t , p r e t r e a t m e n t with i m i p r a m i n e for 1 o r 7 d a y s d i d n o t alter stressedi n d u c e d increases in levels o f T H ; in fact, the levels o f T H in L C o f stressed rats p r e t r e a t e d for 1 o r 7 d a y s were significantly i n c r e a s e d (Fig. 2) relative to c o n t r o l [ A N O V A a n d p o s t h o c analysis using the Scheffe F-test: F(1,32)=9.02, P
Psychopharmacology © Springer-Verlag 1992
Chronic imipramine treatment normalizes levels of tyrosine hydroxylase in the locus coeruleus of chronically stressed rats Kathleen R. MeUa, Eric J. Nestler, and Ronald S. Duman Laboratory of Molecular Psychiatry,Department of Psychiatry,Yale University School of Medicine and the RibicoffResearch Facilities of the Connecticut Mental Health Center, 34 Park Street, New Haven, CT 06508, USA Received June 24, 1991 / Final version October 14, 1991
Abstract. Previous studies have demonstrated that chronic stress increases and antidepressant treatments decrease levels of tyrosine hydroxylase (TH) in locus coeruleus (LC). In the present study, the influence of chronic antidepressant treatment on the induction of TH immunoreactivity in response to cold stress is examined. It was found that chronic imipramine pretreatment (18 days) attenuated the induction of TH in response to cold stress, resulting in levels of TH immunoreactivity not different from control. In contrast, imipramine pretreatment for 1 or 7 days was not sufficient to normalize the stress-induced elevation of TH immunoreactivity. These findings raise the possibility that the therapeutic action of antidepressants may be derived, in part, from the ability of these treatments to normalize levels of TH and thereby the function of the NE neurotransmitter system under conditions of stress. Key words: Tyrosine hydroxylase - Locus coeruleus Stress - Antidepressants - Imipramine
Stress is known to precipitate and/or exacerbate clinical depression in some patients (Winokur 1979; Dohrenwend and Dohrenwend 1980; Loyd 1980; Rabkin 1982; Breslau and Davis 1986). Moreover, exposure of rats to chronic stress produces many of the motivational, neuroendocrine, and behavioral concomitants seen in human depression (Katz 1981, 1982; Katz and Sibel 1982). For these reasons, chronic stress has been considered a useful animal model for human affective disorders (Willner 1984). In this regard, it is noteworthy that chronic treatment with clinically effective antidepressants such as tricyclics (Katz and Hersh 1981), monoamine oxidase inhibitors (Katz et al. 1981), atypical antidepressants (iprindole, buproprion, mianserin) (Katz Abbreviations: locus coeruleus, LC; norepinephrine, NE; tyrosine
hydroxylase,TH Offprint requests to: R.S. Duman
and Sibel 1982a), or electroconvulsive seizures (Katz 1981), block the behavioral effects of chronic intermittent unpredictable stress. In agreement with the time course for the therapeutic actions of clinical treatment, chronic but not acute antidepressant treatments have been found effective in this regard (Katz and Baldrighi 1982; Katz and Sibel 1982b). One of the most consistently and widely studied neurochemical targets of stress and antidepressant treatments is the norepinephrine (NE) neurotransmitter system. Prolonged exposure to stress can result in increased levels of norepinephrine (NE) in the locus coeruleus (LC) and its terminal regions (Stone and McCarty 1983; Adell et al. 1988, 1989; cf Stone 1975). Chronic stress also regulates tyrosine hydroxylase (TH), the rate limiting enzyme for the synthesis of NE, by increasing levels of mRNA, protein, and enzyme activity (Thoenen 1970; Zigmond et al. 1974; Melia et al. 1992; Richard et al. 1988). In contrast, chronic antidepressant treatments have been reported to decrease levels of NE and its metabolites, enzyme activity, and levels of TH mRNA and protein in LC (Roffler-Tarlov et al. 1973; Segal et al. 1974; Adell et al. 1988, 1989; Nestler et al. 1990). These findings are of particular interest since numerous clinical studies have demonstrated dysfunction of the NE system in depression (for review see Gold et al. 1988). In the present study, the influence of chronic antidepressant treatment on the ability of stress to alter the NE system was examined. A previous study has demonstrated that chronic antidepressant treatment prevented the stressed induced increase in levels of NE in midbrain and striatum but the mechanisms involved were not determined (Adell et al. 1989). In the present study, the influence of antidepressant treatments and stress on levels of TH protein in the LC were determined by immunoblot analysis. The results of the present study support the conclusion that antidepressant treatment is capable of normalizing increases of the NE system in response to chronic stress and indicate that this effect occurs at least in part through regulation of TH expression in LC.
24
Materials and methods Animals. Male Sprague Dawley rats (initial weight at onset of chronic antidepressant treatments was 150-200 g) were housed three per cage in wire mesh cages (35 × 20.5 x 17 cm) in a colony room with a 12 h light-dark cycle (lights on 7:00 a.m., off7 : 00 p.m.) maintained at 21 ° C. All animals were allowed ad libitum access to food and water throughout each experiment. Procedure. During the 5-day cold stress, animals were housed singly in plastic cages (28 x 15 x 15 cm) with wood chips for bedding, in a large cold room (2.5 x 2.5 x 2.5 m) maintained at 4 ° C. This stress paradigm has been reported to result in increased levels of TH immunoreactivity in LC (Richard et al. 1988; Melia et al. 1992). To determine whether chronic antidepressant treatment alters the induction of LC TH by chronic stress, colony housed animals were injected with imipramine (IP, 10 mg/kg) for 1, 7, or 18 days and then exposed to cold stress as described above. Daily injections of imipramine were continued throughout the 5 days of stress so that animals received a total of 6, 12, or 23 daily injections. Naive animals housed three per cage (without wood chips) at 21 ° C were used for the control group in this experiment. Animals were decapitated after the treatments and TH levels were assayed in the LC, substantia nigra (SN) and ventral tegmentum (VTA). Immunoblottino of TH. Animals were decapitated, and the LC, SN, and VTA were excised from 1 ram-thick coronal sections of brain by obtaining punches with a 14- (LC) or 15- (VTA and SN) gauge, blunt syringe needle. The tissue was sonicated in 100 lal 2% SDS. Samples were normalized for protein content using the method of Lowry et al. (1951) and adjusted to contain 50 mM TRIS (pH 6.7), 2% SDS, 4% glycerol, and 2% 2-mercaptoethanol, with bromophenol blue as a marker. The samples were then subjected to onedimensional SDS-polyacrylamide gel electrophoresis (with 7.5% acrylamide/0.3 % bisacrylamide in the resolving gels). Proteins were electrophoretically transferred to nitrocellulose paper and then subjected to immunoblot analysis using a polyclonal antibody to TH (1:2000) (generously supplied by Dr. J. Haycock, Louisiana State University), as previously described (Guitart et al. 1990). Resulting filters were washed for 2 h in immunoblot buffer and then dried and autoradiographed with the use of intensifying screens (Dupont). These conditions result in specific immunolabeling of TH protein on both one- and two-dimensional gels (Guitart et al. 1990). Levels of immunolabeling were quantified by densitometry or by counting excised bands in a gamma counter. These two methods yielded similar results. Under the immunolabeling conditions used, levels of TH-immunoreactivity were linear over a three-fold range of tissue concentration. Data analysis and presentation. Data were analyzed using Analysis of variance (ANOVA) for primary analyses and the Scheffe F-test for post hoc comparisons. Where appropriate, between subject t-tests were used to compare results of two groups. Data are presented as the mean percent change from control + / - SEM, as indicated.
Results T h e influence o f c h r o n i c c o l d stress a n d i m i p r a m i n e t r e a t m e n t s o n levels o f T H i m m u n o r e a c t i v i t y in L C were e x a m i n e d . C o l d stress was c h o s e n b e c a u s e it p r o d u c e s s o m e n e u r o e n d o c r i n e c h a n g e s similar to t h o s e seen in d e p r e s s e d p a t i e n t s ( V e r n i k o s et al. 1982) a n d b e c a u s e it results in a reliable increase in levels o f T H m R N A a n d p r o t e i n in L C ( R i c h a r d et al. 1988). C h r o n i c e x p o s u r e to c o l d (5 d a y s at 4 ° C) p r o d u c e d a statistically significant increase [t(10) = - 2.37, P < 0.05] in T H - i m m u n o r e a c t i v i t y
A
TH'~
'~ ~
Treatment
+
B 5o •~ ~ . .~, o
-,
g
-T-
25
-2s-
~ g -50. -75 ' -lOO -4-
Stress
IMI
Stress
+ IMI
Fig. 2. The effect of imipramine pretreatment for l, 7 or 18 days on ments on TH-immunoreactivity in the LC. Rats were either exposed to cold stress (5 days), treated with imipramine for 18 days (10 mg/kg, IP) or pretreated with imipramine and then cold stressed in the continued presence of antidepressant treatment. Levels of TH immunoreactivity were determined in LC by immunoblot analysis as described in Materials and methods. Autoradiograms representative of the effect of each treatment condition are shown (A). Levels of TH immunoreactivity were quantitated by densitometry of the resulting autoradiograms and the results are presented as percent change from control (B). Each point is the mean+SEM of 6-12 separate determinations. * P < 0.05, significantly different from control (Student's t-test)
in the L C o f a p p r o x i m a t e l y 45 % (Fig. 1, A a n d B), b u t no significant c h a n g e in T H - i m m u n o r e a c t i v i t y in either the S N o r V T A ( d a t a n o t shown). I n c o n t r a s t to the effects o f c o l d stress, r e p e a t e d a d m i n i s t r a t i o n (18 days) o f the tricyclic a n t i d e p r e s s a n t , i m i p r a m i n e (10 m g / k g , IP), p r o d u c e d a significant decrease [ t ( 1 2 ) = 2 . 2 8 , P < 0 . 0 5 ] in T H - i m m u n o r e a c t i v i t y in the L C (Fig. 1, A a n d B), b u t no significant c h a n g e in T H - i m m u n o r e a c t i v ity in S N o r V T A ( d a t a n o t shown). These results c o n f i r m a n d e x t e n d the results o f p r e v i o u s w o r k f r o m o u r labo r a t o r y (Nestler et al. 1990). T h e influence o f a n t i d e p r e s s a n t t r e a t m e n t o n induction o f T H in L C b y c h r o n i c c o l d stress was e x a m i n e d next. P r e t r e a t m e n t w i t h i m i p r a m i n e for 18 d a y s f o l l o w e d by 5 days of cold exposure and continued imipramine t r e a t m e n t c o m p l e t e l y n o r m a l i z e d the i n d u c t i o n o f T H b y c h r o n i c stress (Fig. 1, A a n d B). I n c o n t r a s t , p r e t r e a t m e n t with i m i p r a m i n e for 1 o r 7 d a y s d i d n o t alter stressedi n d u c e d increases in levels o f T H ; in fact, the levels o f T H in L C o f stressed rats p r e t r e a t e d for 1 o r 7 d a y s were significantly i n c r e a s e d (Fig. 2) relative to c o n t r o l [ A N O V A a n d p o s t h o c analysis using the Scheffe F-test: F(1,32)=9.02, P
