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Chronic mild stress induces widespread decreases in thyroid hormone α1 receptor mRNA levels in brain—Reversal by imipramine
Psychoneuroendocrinology (2009) 34, 281—286
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p s y n e u e n
Chronic mild stress induces widespread decreases in thyroid hormone a1 receptor mRNA levels in brain–—Reversal by imipramine Edward J. Stein a,b, Nylson G. da Silveira Filho c, Danilo C. Machado c, ´bora C. Hipo ´lide c, Karen Barlow a, Jose ´ N. Nobrega a,b,* De a
Neuroimaging Research Section, Centre for Addiction and Mental Health, 250 College Street, Toronto, Ont., M5T 1R8 Canada Pharmacology Department, University of Toronto, Toronto, Ont., Canada c ˜o Paulo (UNIFESP), Sa ˜o Paulo, SP, Brazil Biopsychology Department, Federal University of Sa b
Received 10 June 2008; received in revised form 8 September 2008; accepted 9 September 2008
KEYWORDS Anhedonia; Antidepressant; Depression; Sucrose preference; In situ hybridization
Abstract While considerable clinical evidence implicates thyroid hormones (THs) in depressive illness, the specific nature of this involvement remains unclear. The a1 subtype (TR-a1) is the most abundant TH receptor in brain. Here we investigated changes in TR-a1 mRNA in the chronic mild stress (CMS) model of depression. Rats were exposed to a CMS schedule for 3 weeks, which resulted in a progressive decreases in sucrose preference (an index of anhedonia). They were then treated daily with either imipramine (IMI, 10 mg/kg) or vehicle (VEH) for 2 weeks before being sacrificed for quantitative in situ hybridization analyses of TR-a1 mRNA throughout the brain. Results indicated that CMS followed by VEH induced widespread decreases in TR-a1 mRNA in brain. In contrast, CMS-exposed rats receiving IMI for the last 2 weeks prior to sacrifice showed full recovery of sucrose preference. Furthermore, brain TR-a1 mRNA levels in these animals were similar to those of non-stressed controls receiving either SAL or IMI. These results reveal that TRa1 mRNA brain levels are very sensitive to CMS effects. The reversal of both anhedonic and TR-a1 effects of CMS by IMI suggests that TR-a1 may play a role both in stress-induced depressive symptoms and in their reversal by antidepressant interventions. # 2008 Elsevier Ltd. All rights reserved.
1. Introduction Several lines of evidence have implicated thyroid hormones (THs) in the pathophysiology of depressive illness and in * Corresponding author at: Neuroimaging Research Section, Centre for Addiction and Mental Health, 250 College Street, Toronto, Ont., M5T 1R8 Canada. Tel.: +1 416 979 6917; fax: +1 416 979 4739. E-mail address: [email protected] (J.N. Nobrega).
mechanisms of antidepressant effects. Thus clinical thyroid disorders are often associated with depressive symptomatology and various antidepressant interventions are associated with significant decreases in circulating levels of L-thyroxine (T4) (Bauer et al., 2003; Brouwer et al., 2005; Joffe, 2006). Therapeutically, triiodothyronine (T3) supplementation augments the effects of antidepressant treatments (Debattista, 2006; Lifschytz et al., 2006). While different hypotheses have been advanced concerning the involvement of THs in
0306-4530/$ — see front matter # 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.psyneuen.2008.09.005
282 affective illness (Joffe and Sokolov, 1994), the specific nature of this involvement remains unclear. Part of the difficulty may relate to the fact that TH regulation in brain may be different from TH regulation in peripheral organs (Leonard and Safran, 1994; Sarkar and Ray, 1994). TH receptors (TRs) are transcription factors that regulate gene expression in concert with co-activators and co-repressors (Flamant et al., 2007). While a and b subtypes of TRs are present in glia and neurons (Bradley et al., 1989), more than 70% of TRs in brain are of the a1 subtype (Schwartz et al., 1992). Mice lacking the TR-a1 isoform display hippocampal changes and behavioural abnormalities (Guadano-Ferraz et al., 2003; Venero et al., 2005). Anhedonia, defined as a diminished ability to derive pleasure from normally rewarding stimuli, is a core symptom of depression in humans. In experimental animals, chronic exposure to unpredictable stressors (commonly referred to as chronic mild stress, CMS) results in a reduction in preference for sweet solutions, which is thought to reflect a state of stress-induced anhedonia (Willner, 1997, 2005; Willner et al., 1987). In addition, CMS-exposed animals show increased thresholds for intracranial self-stimulation (Moreau et al., 1995) and decreased preference for places associated with amphetamine or food reward (Papp et al., 1991). They also show impaired immune function (Kubera et al., 1995) and sleep abnormalities similar to those seen in human depression, i.e. decreased latency to first REM episode and increased time spent in REM (Cheeta et al., 1997). Changes in mesoaccumbens dopamine release (Stamford et al., 1991), b-adrenoceptor mediated cAMP responses (Papp et al., 1994a,b) and changes in enkephalin responses to social stress (Bertrand et al., 1997) have also been described in the CMS model. The anhedonic responses as well as a number of behavioural, biochemical and physiological changes induced by CMS are reversed by antidepressant treatments (Cheeta et al., 1997; Papp et al., 1994c; Willner, 1997, 2005). We investigated whether CMS-induced anhedonia is accompanied by changes in brain TR-a1 mRNA levels and whether TR-a1 mRNA is affected by antidepressant drug treatment.
2. Materials and methods 2.1. Subjects Thirty-two male Wistar rats (Biopsychology Dept, UNIFESP, Brazil) were used. Except as described below, they were individually housed and allowed free access to food and water in a light-controlled (8:00—20:00 h) and temperature-controlled room (20 1 8C). All animals weighed approximately 300 g at the start of the experiment.
E.J. Stein et al. 4-h periods of continuous noise (85 dB), three 1-h periods of exposure to a cloth with cat smell, three 13-h periods of continuous illumination, two 16-h periods of partial food deprivation (30 g of food), two 1-h periods with an empty water bottle and two 2-h periods with stroboscopic illumination. The stressors were randomly applied and administered continuously for a period of 4 weeks. A sucrose preference index (SPI = sucrose/sucrose + water consumed during a 1-h test period) was the dependent variable. At the end of 4 weeks, control and CMS groups were each divided into two groups matched for SPI in week 4. Eight control rats and 8 CMS rats received imipramine (IMI) for 2 weeks (10 mg/kg i.p. twice a day), whereas the remainder (n = 8 per group) received 0.9% saline (VEH).
2.3. Tissue preparation and in situ hybridization (ISH) At the end of the sixth week, animals were sacrificed by decapitation. Brains were rapidly removed and frozen over dry ice. Twenty-micron cryostat sections were cut throughout the brain, thaw-mounted on Fisher Superfrost slides and stored at 80 8C. 35S-UTP-labeled riboprobes were prepared from primers complementary to bases 1591—1609 and 2030—2011 GenBank Accession # M18028.1. Probes were diluted to a concentration of 200,000 cpm/ml in hybridization solution containing: 50% formamide, 35% Denhardt’s solution, 10% dextran sulfate, 0.1 SSC, salmon sperm DNA (300 mg/ml), yeast tRNA (100 mg/ml), and DTT (40 mM). Slides were incubated in plastic mailers overnight at 60 8C. After hybridization, the sections were rinsed in 4 SSC at 60 8C, treated in RNase A (20 mg/ml) solution at 45 8C for 40 min, washed with agitation in decreasing concentrations of SSC containing 25 g/ml sodium thiosulfate, dipped in water, dehydrated in 70% ethanol, and air-dried. The slides were exposed to Hyperfilm b-Max film (Amersham, Quebec) for 4 weeks at 4 8C along with calibrated radioactivity standards. Probe specificity was confirmed by testing labeled sense and scrambled probes, both of which produced no measurable signal on film. Likewise, slides processed without radiolabeled probe resulted in no quantifiable image on film. All densitometric analyses were performed on coded films using MCID software (Imaging Research, St. Catharines, Ontario). Illumination was adjusted to ensure equal film background in all cases and a standard curve was generated to translate optical density to mCi/gT. For each brain region, multiple optical density readings were obtained and averaged for each section, then averaged for each subject and then for each experimental group.
2.4. Data analysis 2.2. Chronic mild stress (CMS) procedure Animals were initially trained to drink a 1% sucrose solution using a two-bottle choice procedure. The CMS regimen consisted of a regular weekly schedule of 11 different stressors including the following: two periods of total food and water deprivation (31 and 15 h), two 18-h periods in a soiled cage (wet bedding), two periods of cage tilt (458— 608), one period of paired housing (18-h), two 5-h and one
Total fluid intake and SPI data for the 4 weeks prior to drug administration were analyzed with two-way repeated measures ANOVAs. Data for the drug injection period were analyzed as 2 2 2 designs with weeks as a within-subject factor and Drug and Stress as between-subject factors. Densitometric data from ISH films were analyzed as 2 2 ANOVA designs (with Stress and Imipramine as factors) followed, where warranted, by independent t-tests.
Changes in brain a1 thyroid hormone receptor mRNA in a model of depression
283
3. Results 3.1. Behavioural data As expected, CMS induced a significant decline in SPI over 4 weeks (Fig. 1) (CMS main effect F 1,30 = 11.5, p < 0.002; weeks: F 3,90 = 2.79, p < 0.04; CMS weeks: F 3,90 = 12.5, p < 0.001). This was reversed in weeks 5—6 by imipramine (IMI main effect and IMI CMS interaction, both p < 0.001). No main effect of weeks ( p > 0.29) was seen, but significant CMS weeks ( p < 0.01), IMI weeks ( p < 0.01), and CMS IMI weeks ( p < 0.004) interactions were detected. These significant interactions simply reflect the fact that preference declined in the CMS-vehicle group but remained constant in the other three groups. During this phase the previous difference between Control and CMS groups was accentuated, as mean SPI in CMS + VEH rats was significantly lower than those in the other groups (all p < 0.004). In contrast, CMS + IMI rats reverted to sucrose preference levels identical to those of the Control + IMI and Control + VEH groups (Fig. 1). In this experiment IMI effects were seen as early as 1 week after initiation of treatment, in contrast to the more commonly observed gradual effects of the antidepressant in this model. This was an uncharacteristic occurrence, since in other experiments with this protocol we do not see effects of IMI until the second or third week of treatment. Total fluid consumption did not vary significantly among groups during the first phase (data not shown); in particular, no significant main effect of CMS was detected ( p = 0.364). During the drug treatment phase, the only significant ANOVA component was a main effect of drug ( p = 0.037).
Fig. 1 Effects of chronic unpredictable stress and imipramine on sucrose preference. For weeks 1—4, N = 16 per group. By week 2 sucrose preference was significantly lower in CMS animals compared to controls (***p < 0.002) and this effect persisted on weeks 3 and 4 (***p < 0.001). For weeks 5—6, N = 8 per group. ***p < 0.004 CMS + VEH vs. the other three groups.
3.2. Changes in TR-a1 mRNA The distribution of TR-a1 mRNA in brain agreed well with previous descriptions (Bradley et al., 1989). Hybridization data and ANOVA results are shown in Table 1. In all brain areas where a significant stress drug interaction was detected (23 of 30 areas, 77%), values for the CMS + VEH group were significantly lower than those for both the Control + VEH group and the CMS + IMI groups ( p < 0.05). The CMS + IMI group did not differ significantly from non-stressed groups in any area. Fig. 2 illustrates these effects at two separate brain levels. Comparisons between Control + VEH and Control + IMI groups yielded no significant differences, although the latter
Fig. 2 Representative coronal brain sections illustrating of the effects of chronic mild stress accompanied by imipramine treatment or vehicle on TR-a1 mRNA at two different brain levels (upper row: approximately 1.00 mm from bregma; lower row: approximately 3.5 mm from bregma). A general decrease in TR-a1 mRNA levels is apparent in the CMS + VEH group, whereas levels in the CMS + IMI group are similar to those in the no-stress control group. Calibration bar = 2.5 mm.
284 Table 1
E.J. Stein et al. Changes in TR-a1 mRNA after chronic mild stress (CMS) and imipraminea. Control
CMS
Vehicle
Imipramine b
Vehicle
Imipramine
Caudate-putamen N. Accumbens
137.0 3.1 150.6 5.4
129.7 5.0 140.4 4.5
123.1 4.9*,# 132.0 4.4*,#
138.2 1.4 152.4 3.1
Amygdala Anterior cortical n. Basolateral n. Posterior cortical n.
152.3 8.1 148.2 6.0 177.1 12.0
137.1 7.2 136.7 8.6 140.0 7.4
131.6 5.7# 130.7 3.9*,# 165.3 16.9
153.8 4.2 149.5 2.0 175.4 5.7
Cortex Infralimbic Cingulate Piriform Frontal II/III Frontal IV Frontal V/VI Parietal II/III Parietal IV Parietal V/VI Occipital Temporal Entorhinal
198.6 7.5 170.2 6.6 184.1 9.8 176.0 10.2 137.4 4.7 158.8 6.6 175.5 9.6 135.4 3.9 160.9 6.5 163.3 8.5 158.1 8.2 156.3 8.8
181.0 12.8 155.5 6.7 162.7 9.7 163.1 9.7 130.3 5.7 149.0 7.3 158.0 9.1 126.5 5.4 151.2 7.7 152.9 9.4 147.8 5.6 140.7 3.9
150.2 5.5*,# 141.2 4.7*,# 152.0 6.7 * 142.0 5.8*,# 119.1 4.0*,# 135.6 6.1 133.4 3.7*,# 115.6 3.2*,# 133.0 3.6*,# 133.5 3.3*,# 131.9 3.8# 142.9 5.4
175.2 3.8 160.7 3.4 164.9 4.2 159.3 3.3 133.3 1.0 150.8 1.6 154.7 2.8 132.5 1.2 155.2 2.1 158.9 4.2 154.5 2.1 154.0 3.6
Hypothalamus Paraventricular n. Ventromedial n.
134.3 2.9 130.5 4.3
124.8 6.1 124.2 8.1
122.2 2.9# 121.1 4.1*,#
135.7 1.3 138.5 2.1
Hippocampus CA1 CA2/CA3 Dentate gyrus Subiculum
192.4 9.0 183.8 7.7 276.2 14.2 154.5 8.8
167.7 17.4 171.6 15.0 244.1 30.5 153.2 13.4
154.0 5.0 147.1 6.6 217.1 8.8*,# 136.3 3.4
168.6 7.4 170.1 7.4 255.8 14.3 155.5 3.6
Subthalamic n. Subst. nigra—P. compacta Superior colliculus Periaqueductal gray Pontine n. n. Solitary tract Cerebellum: granular I
146.7 6.5 131.6 8.6 128.8 4.7 126.8 5.2 155.5 9.9 148.0 11.8 209.3 11.3
137.9 6.5 121.9 9.6 124.9 6.8 117.5 5.1 170.0 15.2 120.7 7.8 202.6 13.4
129.2 3.1*,# 109.7 4.1 111.4 3.1*,# 108.7 3.2*,# 160.9 19.5 113.4 2.3*,# 190.0 6.3#
149.2 1.6 124.8 2.4 131.0 1.9 140.4 11.9 157.0 6.9 121.0 2.0 226.9 7.1
a
Values are means S.E.M. in nCi/g. N = 8 per group. A significant stress main effect was seen only in cortical areas (cingulate, frontal layers 2/3, infralimbic, parietal layers 2/3 and parietal layer 5, all p’s < 0.05) and in the nucleus of the solitary tract ( p < 0.04). A main effect of Drug Treatment was not seen in any region. A significant Stress Drug Treatment interaction was seen in 23 of the 30 regions, exceptions being layers 5/6 of the frontal cortex; hippocampal CA1, CA2, CA3 and subicular regions, substantia nigra and the pontine nucleus (all p’s > 0.05). b None of the Control + IMI means are different from Control + VEH means. * Significantly different from Control + VEH group ( p < 0.05). # Significantly different from CMS + IMI group ( p < 0.05).
group showed a clear trend towards decreased TR-a1 mRNA levels (Table 1). In some brain regions (e.g. entorhinal cortex, paraventricular and ventromedial hypothalamus, posterior cortical amygdaloid nucleus), the decrease in TR-a1 mRNA after IMI was in fact comparable in magnitude to the decrease induced by the stress treatment.
4. Discussion We found that decreases in sucrose preference induced by CMS, as well as the reversal of this effect by imipramine, were paralleled by changes in TR-a1 mRNA levels in brain.
In rats exposed to CMS and then given vehicle injections, TR-a1 mRNA levels were significantly decreased throughout the brain, whereas in CMS-exposed rats given imipramine TR-a1 mRNA levels were similar to those in normal nonstressed controls. This suggests that IMI reversed both the anhedonic behaviour and the TR-a1 decreases induced by CMS. The possibility that food restriction and/or body weight losses (typically 10% of controls) could have contributed to the ISH results appears unlikely, since find that other animals maintained at 85% of their body weight show no changes in TR-a1 mRNA (data not shown). Furthermore, we found no
Changes in brain a1 thyroid hormone receptor mRNA in a model of depression changes in TR-b1 mRNA in these animals (data not shown). If the observed a1 ISH changes were somehow related to body weight changes, we might expect them to affect other subtypes of TH receptors as well. Could the altered expression of TR-a1 in brain be secondary to altered levels of T3 in brain? Expression of TR-a1 has been found to decrease with increased levels of T3 in cultured GH3 cells (Hahn et al., 1999b), but in adult rat brain no changes in TR-a1 expression were seen in response to variations in thyroid axis activity (Hodin et al., 1990). Nonetheless, it is conceivable that CMS may have increased T3 concentrations within various brain regions which, in turn, could have produced a decrease in TR-a1 expression in those areas. Consistent with this possibility, increased 50 DII activity as well as elevated T3 concentrations were found in selective brain regions in response to various forms of acute stress (Baumgartner et al., 1998). In plasma, CMS significantly increased total T3 and total T4 without affecting concentrations of free T4 or TSH (Kioukia et al., 2000). In future studies it would be of interest to quantitate regional brain T3 and T4 levels following CMS and antidepressant treatments. It is also possible that the observed mRNA changes occurred independently of alterations in brain levels of T3 or T4. TH receptor binding to DNA is not ligand-dependent and TRs, including TR-a1, are thought to have constitutive suppressive effects on gene expression in the unliganded state (Flamant et al., 2007). Irrespective of mechanism, an intriguing possibility suggested by the current data is that the effects of IMI on TR-a1 may be different in CMS-exposed rats vs. non-stressed animals. In normal non-stressed rats IMI tended to reduce brain TR-a1 mRNA, whereas in CMS-exposed rats not only did IMI cause no further decreases in TR-a1 mRNA but instead seems to have brought TR-a1 back to baseline control levels. A previous report found that chronic lithium in normal euthyroid rat brain alters TR-a1 mRNA levels in a regionally specific manner (Hahn et al., 1999a). Our current findings confirm that TR-a1 levels are sensitive to repeated antidepressant drug interventions but show that in the case of IMI these effects are much more pronounced in stressed than in normal control rats. Recently, the first evidence has been provided of altered emotional reactivity associated with structural brain changes in TR-a1-deficient mice (Guadano-Ferraz et al., 2003; Venero et al., 2005), clearly signaling an important role for this receptor in behaviour. Our results further suggest that TR-a1 is strongly sensitive to chronic stress and may potentially play a role in the pathophysiology on depressive sates. Preliminary observations indicate that TR-a1 is also downregulated in the Flinders Sensitive Line of rats, although not in learned helplessness, another model of depression (unpublished observations). Therefore, regardless of mechanisms involved, it is likely that the involvement of TR-a1 in stress effects may be associated with specific behavioural symptoms, such as anhedonia, rather than being a general marker for stressrelated abnormalities.
Conflict of interest None.
285
Role of funding source Supported in part by funds from the Ontario Mental Health Foundation (OMHF, Canada). The OMHF had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
Acknowledgement We thank Roger Raymond for help in designing probes and developing the in situ hybridization protocols.
References Bauer, M., London, E.D., Silverman, D.H., Rasgon, N., Kirchheiner, J., Whybrow, P.C., 2003. Thyroid, brain and mood modulation in affective disorder: insights from molecular research and functional brain imaging. Pharmacopsychiatry 36 (Suppl. 3), S215—S221. Baumgartner, A., Hiedra, L., Pinna, G., Eravci, M., Prengel, H., Meinhold, H., 1998. Rat brain type II 50 -iodothyronine deiodinase activity is extremely sensitive to stress. J. Neurochem. 71, 817—826. Bertrand, E., Smadja, C., Mauborgne, A., Roques, B.P., Dauge, V., 1997. Social interaction increases the extracellular levels of [Met]enkephalin in the nucleus accumbens of control but not of chronic mild stressed rats. Neuroscience 80, 17—20. Bradley, D.J., Young 3rd., W.S., Weinberger, C., 1989. Differential expression of alpha and beta thyroid hormone receptor genes in rat brain and pituitary. Proc. Natl. Acad. Sci. U.S.A. 86, 7250—7254. Brouwer, J.P., Appelhof, B.C., Hoogendijk, W.J., Huyser, J., Endert, E., Zuketto, C., Schene, A.H., Tijssen, J.G., Van Dyck, R., Wiersinga, W.M., Fliers, E., 2005. Thyroid and adrenal axis in major depression: a controlled study in outpatients. Eur. J. Endocrinol. 152, 185—191. Cheeta, S., Ruigt, G., van Proosdij, J., Willner, P., 1997. Changes in sleep architecture following chronic mild stress. Biol. Psychiatry 41, 419—427. Debattista, C., 2006. Augmentation and combination strategies for depression. J. Psychopharmacol. 20, 11—18. Flamant, F., Gauthier, K., Samarut, J., 2007. Thyroid hormones signaling is getting more complex: STORMs are coming. Mol. Endocrinol. 21, 321—333. Guadano-Ferraz, A., Benavides-Piccione, R., Venero, C., Lancha, C., Vennstrom, B., Sandi, C., DeFelipe, J., Bernal, J., 2003. Lack of thyroid hormone receptor alpha1 is associated with selective alterations in behavior and hippocampal circuits. Mol. Psychiatry 8, 30—38. Hahn, C.G., Pawlyk, A.C., Whybrow, P.C., Gyulai, L., Tejani-Butt, S.M., 1999a. Lithium administration affects gene expression of thyroid hormone receptors in rat brain. Life Sci. 64, 1793—1802. Hahn, C.G., Pawlyk, A.C., Whybrow, P.C., Tejani-Butt, S.M., 1999b. Differential expression of thyroid hormone receptor isoforms by thyroid hormone and lithium in rat GH3 and B103 cells. Biol. Psychiatry 45, 1004—1012. Hodin, R.A., Lazar, M.A., Chin, W.W., 1990. Differential and tissuespecific regulation of the multiple rat c-erbA messenger RNA species by thyroid hormone. J. Clin. Invest. 85, 101—105. Joffe, R.T., 2006. Is the thyroid still important in major depression? J. Psychiatry Neurosci. 31, 367—368. Joffe, R.T., Sokolov, S.T., 1994. Thyroid hormones, the brain, and affective disorders. Crit. Rev. Neurobiol. 8, 45—63. Kioukia, N., Bekris, S., Antoniou, K., Papadopoulou-Daifoti, Z., Christofidis, I., 2000. Effects of chronic mild stress (CMS) on thyroid hormone function in two rat strains. Psychoneuroendocrinology 25, 247—257.
286 Kubera, M., Basta-Kaim, A., Papp, M., 1995. The effect of chronic treatment with imipramine on the immunoreactivity of animals subjected to a chronic mild stress model of depression. Immunopharmacology 30, 225—230. Leonard, J.L., Safran, M., 1994. Hormonal regulation of type II iodothyronine deiodinase in the brain. In: Wu, V.T.S. (Ed.), Thyroid Hormone Metabolism: Molecular Biology and Alternate Pathways. CRC Press, Boca Raton, FL, pp. 23—44. Lifschytz, T., Segman, R., Shalom, G., Lerer, B., Gur, E., Golzer, T., Newman, M.E., 2006. Basic mechanisms of augmentation of antidepressant effects with thyroid hormone. Curr. Drug. Targets 7, 203—210. Moreau, J.L., Scherschlicht, R., Jenck, F., Martin, J.R., 1995. Chronic mild stress-induced anhedonia model of depression; sleep abnormalities and curative effects of electroshock treatment. Behav. Pharmacol. 6, 682—687. Papp, M., Klimek, V., Willner, P., 1994a. Effects of imipramine on serotonergic and beta-adrenergic receptor binding in a realistic animal model of depression. Psychopharmacology (Berl.) 114, 309—314. Papp, M., Klimek, V., Willner, P., 1994b. Parallel changes in dopamine D2 receptor binding in limbic forebrain associated with chronic mild stress-induced anhedonia and its reversal by imipramine. Psychopharmacology (Berl.) 115, 441—446. Papp, M., Nalepa, I., Vetulani, J., 1994c. Reversal by imipramine of beta-adrenoceptor up-regulation induced in a chronic mild stress model of depression. Eur. J. Pharmacol. 261, 141—147. Papp, M., Willner, P., Muscat, R., 1991. An animal model of anhedonia: attenuation of sucrose consumption and place preference
E.J. Stein et al. conditioning by chronic unpredictable mild stress. Psychopharmacology (Berl.) 104, 255—259. Sarkar, P.K., Ray, A.K., 1994. Synaptosomal T3 content in cerebral cortex of adult rat in different thyroidal states. Neuropsychopharmacology 11, 151—155. Schwartz, H.L., Strait, K.A., Ling, N.C., Oppenheimer, J.H., 1992. Quantitation of rat tissue thyroid hormone binding receptor isoforms by immunoprecipitation of nuclear triiodothyronine binding capacity. J. Biol. Chem. 267, 11794—11799. Stamford, J.A., Muscat, R., O’Connor, J.J., Patel, J., Trout, S.J., Wieczorek, W.J., Kruk, Z.L., Willner, P., 1991. Voltammetric evidence that subsensitivity to reward following chronic mild stress is associated with increased release of mesolimbic dopamine. Psychopharmacology (Berl.) 105, 275—282. Venero, C., Guadano-Ferraz, A., Herrero, A.I., Nordstrom, K., Manzano, J., de Escobar, G.M., Bernal, J., Vennstrom, B., 2005. Anxiety, memory impairment, and locomotor dysfunction caused by a mutant thyroid hormone receptor alpha1 can be ameliorated by T3 treatment. Genes Dev. 19, 2152—2163. Willner, P., 1997. Validity, reliability and utility of the chronic mild stress model of depression: a 10-year review and evaluation. Psychopharmacology (Berl.) 134, 319—329. Willner, P., 2005. Chronic mild stress (CMS) revisited: Consistency and behavioural-neurobiological concordance in the effects of CMS. Neuropsychobiology 52, 90—110. Willner, P., Towell, A., Sampson, D., Sophokleous, S., Muscat, R., 1987. Reduction of sucrose preference by chronic unpredictable mild stress, and its restoration by a tricyclic antidepressant. Psychopharmacology (Berl.) 93, 358—364.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p s y n e u e n
Chronic mild stress induces widespread decreases in thyroid hormone a1 receptor mRNA levels in brain–—Reversal by imipramine Edward J. Stein a,b, Nylson G. da Silveira Filho c, Danilo C. Machado c, ´bora C. Hipo ´lide c, Karen Barlow a, Jose ´ N. Nobrega a,b,* De a
Neuroimaging Research Section, Centre for Addiction and Mental Health, 250 College Street, Toronto, Ont., M5T 1R8 Canada Pharmacology Department, University of Toronto, Toronto, Ont., Canada c ˜o Paulo (UNIFESP), Sa ˜o Paulo, SP, Brazil Biopsychology Department, Federal University of Sa b
Received 10 June 2008; received in revised form 8 September 2008; accepted 9 September 2008
KEYWORDS Anhedonia; Antidepressant; Depression; Sucrose preference; In situ hybridization
Abstract While considerable clinical evidence implicates thyroid hormones (THs) in depressive illness, the specific nature of this involvement remains unclear. The a1 subtype (TR-a1) is the most abundant TH receptor in brain. Here we investigated changes in TR-a1 mRNA in the chronic mild stress (CMS) model of depression. Rats were exposed to a CMS schedule for 3 weeks, which resulted in a progressive decreases in sucrose preference (an index of anhedonia). They were then treated daily with either imipramine (IMI, 10 mg/kg) or vehicle (VEH) for 2 weeks before being sacrificed for quantitative in situ hybridization analyses of TR-a1 mRNA throughout the brain. Results indicated that CMS followed by VEH induced widespread decreases in TR-a1 mRNA in brain. In contrast, CMS-exposed rats receiving IMI for the last 2 weeks prior to sacrifice showed full recovery of sucrose preference. Furthermore, brain TR-a1 mRNA levels in these animals were similar to those of non-stressed controls receiving either SAL or IMI. These results reveal that TRa1 mRNA brain levels are very sensitive to CMS effects. The reversal of both anhedonic and TR-a1 effects of CMS by IMI suggests that TR-a1 may play a role both in stress-induced depressive symptoms and in their reversal by antidepressant interventions. # 2008 Elsevier Ltd. All rights reserved.
1. Introduction Several lines of evidence have implicated thyroid hormones (THs) in the pathophysiology of depressive illness and in * Corresponding author at: Neuroimaging Research Section, Centre for Addiction and Mental Health, 250 College Street, Toronto, Ont., M5T 1R8 Canada. Tel.: +1 416 979 6917; fax: +1 416 979 4739. E-mail address: [email protected] (J.N. Nobrega).
mechanisms of antidepressant effects. Thus clinical thyroid disorders are often associated with depressive symptomatology and various antidepressant interventions are associated with significant decreases in circulating levels of L-thyroxine (T4) (Bauer et al., 2003; Brouwer et al., 2005; Joffe, 2006). Therapeutically, triiodothyronine (T3) supplementation augments the effects of antidepressant treatments (Debattista, 2006; Lifschytz et al., 2006). While different hypotheses have been advanced concerning the involvement of THs in
0306-4530/$ — see front matter # 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.psyneuen.2008.09.005
282 affective illness (Joffe and Sokolov, 1994), the specific nature of this involvement remains unclear. Part of the difficulty may relate to the fact that TH regulation in brain may be different from TH regulation in peripheral organs (Leonard and Safran, 1994; Sarkar and Ray, 1994). TH receptors (TRs) are transcription factors that regulate gene expression in concert with co-activators and co-repressors (Flamant et al., 2007). While a and b subtypes of TRs are present in glia and neurons (Bradley et al., 1989), more than 70% of TRs in brain are of the a1 subtype (Schwartz et al., 1992). Mice lacking the TR-a1 isoform display hippocampal changes and behavioural abnormalities (Guadano-Ferraz et al., 2003; Venero et al., 2005). Anhedonia, defined as a diminished ability to derive pleasure from normally rewarding stimuli, is a core symptom of depression in humans. In experimental animals, chronic exposure to unpredictable stressors (commonly referred to as chronic mild stress, CMS) results in a reduction in preference for sweet solutions, which is thought to reflect a state of stress-induced anhedonia (Willner, 1997, 2005; Willner et al., 1987). In addition, CMS-exposed animals show increased thresholds for intracranial self-stimulation (Moreau et al., 1995) and decreased preference for places associated with amphetamine or food reward (Papp et al., 1991). They also show impaired immune function (Kubera et al., 1995) and sleep abnormalities similar to those seen in human depression, i.e. decreased latency to first REM episode and increased time spent in REM (Cheeta et al., 1997). Changes in mesoaccumbens dopamine release (Stamford et al., 1991), b-adrenoceptor mediated cAMP responses (Papp et al., 1994a,b) and changes in enkephalin responses to social stress (Bertrand et al., 1997) have also been described in the CMS model. The anhedonic responses as well as a number of behavioural, biochemical and physiological changes induced by CMS are reversed by antidepressant treatments (Cheeta et al., 1997; Papp et al., 1994c; Willner, 1997, 2005). We investigated whether CMS-induced anhedonia is accompanied by changes in brain TR-a1 mRNA levels and whether TR-a1 mRNA is affected by antidepressant drug treatment.
2. Materials and methods 2.1. Subjects Thirty-two male Wistar rats (Biopsychology Dept, UNIFESP, Brazil) were used. Except as described below, they were individually housed and allowed free access to food and water in a light-controlled (8:00—20:00 h) and temperature-controlled room (20 1 8C). All animals weighed approximately 300 g at the start of the experiment.
E.J. Stein et al. 4-h periods of continuous noise (85 dB), three 1-h periods of exposure to a cloth with cat smell, three 13-h periods of continuous illumination, two 16-h periods of partial food deprivation (30 g of food), two 1-h periods with an empty water bottle and two 2-h periods with stroboscopic illumination. The stressors were randomly applied and administered continuously for a period of 4 weeks. A sucrose preference index (SPI = sucrose/sucrose + water consumed during a 1-h test period) was the dependent variable. At the end of 4 weeks, control and CMS groups were each divided into two groups matched for SPI in week 4. Eight control rats and 8 CMS rats received imipramine (IMI) for 2 weeks (10 mg/kg i.p. twice a day), whereas the remainder (n = 8 per group) received 0.9% saline (VEH).
2.3. Tissue preparation and in situ hybridization (ISH) At the end of the sixth week, animals were sacrificed by decapitation. Brains were rapidly removed and frozen over dry ice. Twenty-micron cryostat sections were cut throughout the brain, thaw-mounted on Fisher Superfrost slides and stored at 80 8C. 35S-UTP-labeled riboprobes were prepared from primers complementary to bases 1591—1609 and 2030—2011 GenBank Accession # M18028.1. Probes were diluted to a concentration of 200,000 cpm/ml in hybridization solution containing: 50% formamide, 35% Denhardt’s solution, 10% dextran sulfate, 0.1 SSC, salmon sperm DNA (300 mg/ml), yeast tRNA (100 mg/ml), and DTT (40 mM). Slides were incubated in plastic mailers overnight at 60 8C. After hybridization, the sections were rinsed in 4 SSC at 60 8C, treated in RNase A (20 mg/ml) solution at 45 8C for 40 min, washed with agitation in decreasing concentrations of SSC containing 25 g/ml sodium thiosulfate, dipped in water, dehydrated in 70% ethanol, and air-dried. The slides were exposed to Hyperfilm b-Max film (Amersham, Quebec) for 4 weeks at 4 8C along with calibrated radioactivity standards. Probe specificity was confirmed by testing labeled sense and scrambled probes, both of which produced no measurable signal on film. Likewise, slides processed without radiolabeled probe resulted in no quantifiable image on film. All densitometric analyses were performed on coded films using MCID software (Imaging Research, St. Catharines, Ontario). Illumination was adjusted to ensure equal film background in all cases and a standard curve was generated to translate optical density to mCi/gT. For each brain region, multiple optical density readings were obtained and averaged for each section, then averaged for each subject and then for each experimental group.
2.4. Data analysis 2.2. Chronic mild stress (CMS) procedure Animals were initially trained to drink a 1% sucrose solution using a two-bottle choice procedure. The CMS regimen consisted of a regular weekly schedule of 11 different stressors including the following: two periods of total food and water deprivation (31 and 15 h), two 18-h periods in a soiled cage (wet bedding), two periods of cage tilt (458— 608), one period of paired housing (18-h), two 5-h and one
Total fluid intake and SPI data for the 4 weeks prior to drug administration were analyzed with two-way repeated measures ANOVAs. Data for the drug injection period were analyzed as 2 2 2 designs with weeks as a within-subject factor and Drug and Stress as between-subject factors. Densitometric data from ISH films were analyzed as 2 2 ANOVA designs (with Stress and Imipramine as factors) followed, where warranted, by independent t-tests.
Changes in brain a1 thyroid hormone receptor mRNA in a model of depression
283
3. Results 3.1. Behavioural data As expected, CMS induced a significant decline in SPI over 4 weeks (Fig. 1) (CMS main effect F 1,30 = 11.5, p < 0.002; weeks: F 3,90 = 2.79, p < 0.04; CMS weeks: F 3,90 = 12.5, p < 0.001). This was reversed in weeks 5—6 by imipramine (IMI main effect and IMI CMS interaction, both p < 0.001). No main effect of weeks ( p > 0.29) was seen, but significant CMS weeks ( p < 0.01), IMI weeks ( p < 0.01), and CMS IMI weeks ( p < 0.004) interactions were detected. These significant interactions simply reflect the fact that preference declined in the CMS-vehicle group but remained constant in the other three groups. During this phase the previous difference between Control and CMS groups was accentuated, as mean SPI in CMS + VEH rats was significantly lower than those in the other groups (all p < 0.004). In contrast, CMS + IMI rats reverted to sucrose preference levels identical to those of the Control + IMI and Control + VEH groups (Fig. 1). In this experiment IMI effects were seen as early as 1 week after initiation of treatment, in contrast to the more commonly observed gradual effects of the antidepressant in this model. This was an uncharacteristic occurrence, since in other experiments with this protocol we do not see effects of IMI until the second or third week of treatment. Total fluid consumption did not vary significantly among groups during the first phase (data not shown); in particular, no significant main effect of CMS was detected ( p = 0.364). During the drug treatment phase, the only significant ANOVA component was a main effect of drug ( p = 0.037).
Fig. 1 Effects of chronic unpredictable stress and imipramine on sucrose preference. For weeks 1—4, N = 16 per group. By week 2 sucrose preference was significantly lower in CMS animals compared to controls (***p < 0.002) and this effect persisted on weeks 3 and 4 (***p < 0.001). For weeks 5—6, N = 8 per group. ***p < 0.004 CMS + VEH vs. the other three groups.
3.2. Changes in TR-a1 mRNA The distribution of TR-a1 mRNA in brain agreed well with previous descriptions (Bradley et al., 1989). Hybridization data and ANOVA results are shown in Table 1. In all brain areas where a significant stress drug interaction was detected (23 of 30 areas, 77%), values for the CMS + VEH group were significantly lower than those for both the Control + VEH group and the CMS + IMI groups ( p < 0.05). The CMS + IMI group did not differ significantly from non-stressed groups in any area. Fig. 2 illustrates these effects at two separate brain levels. Comparisons between Control + VEH and Control + IMI groups yielded no significant differences, although the latter
Fig. 2 Representative coronal brain sections illustrating of the effects of chronic mild stress accompanied by imipramine treatment or vehicle on TR-a1 mRNA at two different brain levels (upper row: approximately 1.00 mm from bregma; lower row: approximately 3.5 mm from bregma). A general decrease in TR-a1 mRNA levels is apparent in the CMS + VEH group, whereas levels in the CMS + IMI group are similar to those in the no-stress control group. Calibration bar = 2.5 mm.
284 Table 1
E.J. Stein et al. Changes in TR-a1 mRNA after chronic mild stress (CMS) and imipraminea. Control
CMS
Vehicle
Imipramine b
Vehicle
Imipramine
Caudate-putamen N. Accumbens
137.0 3.1 150.6 5.4
129.7 5.0 140.4 4.5
123.1 4.9*,# 132.0 4.4*,#
138.2 1.4 152.4 3.1
Amygdala Anterior cortical n. Basolateral n. Posterior cortical n.
152.3 8.1 148.2 6.0 177.1 12.0
137.1 7.2 136.7 8.6 140.0 7.4
131.6 5.7# 130.7 3.9*,# 165.3 16.9
153.8 4.2 149.5 2.0 175.4 5.7
Cortex Infralimbic Cingulate Piriform Frontal II/III Frontal IV Frontal V/VI Parietal II/III Parietal IV Parietal V/VI Occipital Temporal Entorhinal
198.6 7.5 170.2 6.6 184.1 9.8 176.0 10.2 137.4 4.7 158.8 6.6 175.5 9.6 135.4 3.9 160.9 6.5 163.3 8.5 158.1 8.2 156.3 8.8
181.0 12.8 155.5 6.7 162.7 9.7 163.1 9.7 130.3 5.7 149.0 7.3 158.0 9.1 126.5 5.4 151.2 7.7 152.9 9.4 147.8 5.6 140.7 3.9
150.2 5.5*,# 141.2 4.7*,# 152.0 6.7 * 142.0 5.8*,# 119.1 4.0*,# 135.6 6.1 133.4 3.7*,# 115.6 3.2*,# 133.0 3.6*,# 133.5 3.3*,# 131.9 3.8# 142.9 5.4
175.2 3.8 160.7 3.4 164.9 4.2 159.3 3.3 133.3 1.0 150.8 1.6 154.7 2.8 132.5 1.2 155.2 2.1 158.9 4.2 154.5 2.1 154.0 3.6
Hypothalamus Paraventricular n. Ventromedial n.
134.3 2.9 130.5 4.3
124.8 6.1 124.2 8.1
122.2 2.9# 121.1 4.1*,#
135.7 1.3 138.5 2.1
Hippocampus CA1 CA2/CA3 Dentate gyrus Subiculum
192.4 9.0 183.8 7.7 276.2 14.2 154.5 8.8
167.7 17.4 171.6 15.0 244.1 30.5 153.2 13.4
154.0 5.0 147.1 6.6 217.1 8.8*,# 136.3 3.4
168.6 7.4 170.1 7.4 255.8 14.3 155.5 3.6
Subthalamic n. Subst. nigra—P. compacta Superior colliculus Periaqueductal gray Pontine n. n. Solitary tract Cerebellum: granular I
146.7 6.5 131.6 8.6 128.8 4.7 126.8 5.2 155.5 9.9 148.0 11.8 209.3 11.3
137.9 6.5 121.9 9.6 124.9 6.8 117.5 5.1 170.0 15.2 120.7 7.8 202.6 13.4
129.2 3.1*,# 109.7 4.1 111.4 3.1*,# 108.7 3.2*,# 160.9 19.5 113.4 2.3*,# 190.0 6.3#
149.2 1.6 124.8 2.4 131.0 1.9 140.4 11.9 157.0 6.9 121.0 2.0 226.9 7.1
a
Values are means S.E.M. in nCi/g. N = 8 per group. A significant stress main effect was seen only in cortical areas (cingulate, frontal layers 2/3, infralimbic, parietal layers 2/3 and parietal layer 5, all p’s < 0.05) and in the nucleus of the solitary tract ( p < 0.04). A main effect of Drug Treatment was not seen in any region. A significant Stress Drug Treatment interaction was seen in 23 of the 30 regions, exceptions being layers 5/6 of the frontal cortex; hippocampal CA1, CA2, CA3 and subicular regions, substantia nigra and the pontine nucleus (all p’s > 0.05). b None of the Control + IMI means are different from Control + VEH means. * Significantly different from Control + VEH group ( p < 0.05). # Significantly different from CMS + IMI group ( p < 0.05).
group showed a clear trend towards decreased TR-a1 mRNA levels (Table 1). In some brain regions (e.g. entorhinal cortex, paraventricular and ventromedial hypothalamus, posterior cortical amygdaloid nucleus), the decrease in TR-a1 mRNA after IMI was in fact comparable in magnitude to the decrease induced by the stress treatment.
4. Discussion We found that decreases in sucrose preference induced by CMS, as well as the reversal of this effect by imipramine, were paralleled by changes in TR-a1 mRNA levels in brain.
In rats exposed to CMS and then given vehicle injections, TR-a1 mRNA levels were significantly decreased throughout the brain, whereas in CMS-exposed rats given imipramine TR-a1 mRNA levels were similar to those in normal nonstressed controls. This suggests that IMI reversed both the anhedonic behaviour and the TR-a1 decreases induced by CMS. The possibility that food restriction and/or body weight losses (typically 10% of controls) could have contributed to the ISH results appears unlikely, since find that other animals maintained at 85% of their body weight show no changes in TR-a1 mRNA (data not shown). Furthermore, we found no
Changes in brain a1 thyroid hormone receptor mRNA in a model of depression changes in TR-b1 mRNA in these animals (data not shown). If the observed a1 ISH changes were somehow related to body weight changes, we might expect them to affect other subtypes of TH receptors as well. Could the altered expression of TR-a1 in brain be secondary to altered levels of T3 in brain? Expression of TR-a1 has been found to decrease with increased levels of T3 in cultured GH3 cells (Hahn et al., 1999b), but in adult rat brain no changes in TR-a1 expression were seen in response to variations in thyroid axis activity (Hodin et al., 1990). Nonetheless, it is conceivable that CMS may have increased T3 concentrations within various brain regions which, in turn, could have produced a decrease in TR-a1 expression in those areas. Consistent with this possibility, increased 50 DII activity as well as elevated T3 concentrations were found in selective brain regions in response to various forms of acute stress (Baumgartner et al., 1998). In plasma, CMS significantly increased total T3 and total T4 without affecting concentrations of free T4 or TSH (Kioukia et al., 2000). In future studies it would be of interest to quantitate regional brain T3 and T4 levels following CMS and antidepressant treatments. It is also possible that the observed mRNA changes occurred independently of alterations in brain levels of T3 or T4. TH receptor binding to DNA is not ligand-dependent and TRs, including TR-a1, are thought to have constitutive suppressive effects on gene expression in the unliganded state (Flamant et al., 2007). Irrespective of mechanism, an intriguing possibility suggested by the current data is that the effects of IMI on TR-a1 may be different in CMS-exposed rats vs. non-stressed animals. In normal non-stressed rats IMI tended to reduce brain TR-a1 mRNA, whereas in CMS-exposed rats not only did IMI cause no further decreases in TR-a1 mRNA but instead seems to have brought TR-a1 back to baseline control levels. A previous report found that chronic lithium in normal euthyroid rat brain alters TR-a1 mRNA levels in a regionally specific manner (Hahn et al., 1999a). Our current findings confirm that TR-a1 levels are sensitive to repeated antidepressant drug interventions but show that in the case of IMI these effects are much more pronounced in stressed than in normal control rats. Recently, the first evidence has been provided of altered emotional reactivity associated with structural brain changes in TR-a1-deficient mice (Guadano-Ferraz et al., 2003; Venero et al., 2005), clearly signaling an important role for this receptor in behaviour. Our results further suggest that TR-a1 is strongly sensitive to chronic stress and may potentially play a role in the pathophysiology on depressive sates. Preliminary observations indicate that TR-a1 is also downregulated in the Flinders Sensitive Line of rats, although not in learned helplessness, another model of depression (unpublished observations). Therefore, regardless of mechanisms involved, it is likely that the involvement of TR-a1 in stress effects may be associated with specific behavioural symptoms, such as anhedonia, rather than being a general marker for stressrelated abnormalities.
Conflict of interest None.
285
Role of funding source Supported in part by funds from the Ontario Mental Health Foundation (OMHF, Canada). The OMHF had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
Acknowledgement We thank Roger Raymond for help in designing probes and developing the in situ hybridization protocols.
References Bauer, M., London, E.D., Silverman, D.H., Rasgon, N., Kirchheiner, J., Whybrow, P.C., 2003. Thyroid, brain and mood modulation in affective disorder: insights from molecular research and functional brain imaging. Pharmacopsychiatry 36 (Suppl. 3), S215—S221. Baumgartner, A., Hiedra, L., Pinna, G., Eravci, M., Prengel, H., Meinhold, H., 1998. Rat brain type II 50 -iodothyronine deiodinase activity is extremely sensitive to stress. J. Neurochem. 71, 817—826. Bertrand, E., Smadja, C., Mauborgne, A., Roques, B.P., Dauge, V., 1997. Social interaction increases the extracellular levels of [Met]enkephalin in the nucleus accumbens of control but not of chronic mild stressed rats. Neuroscience 80, 17—20. Bradley, D.J., Young 3rd., W.S., Weinberger, C., 1989. Differential expression of alpha and beta thyroid hormone receptor genes in rat brain and pituitary. Proc. Natl. Acad. Sci. U.S.A. 86, 7250—7254. Brouwer, J.P., Appelhof, B.C., Hoogendijk, W.J., Huyser, J., Endert, E., Zuketto, C., Schene, A.H., Tijssen, J.G., Van Dyck, R., Wiersinga, W.M., Fliers, E., 2005. Thyroid and adrenal axis in major depression: a controlled study in outpatients. Eur. J. Endocrinol. 152, 185—191. Cheeta, S., Ruigt, G., van Proosdij, J., Willner, P., 1997. Changes in sleep architecture following chronic mild stress. Biol. Psychiatry 41, 419—427. Debattista, C., 2006. Augmentation and combination strategies for depression. J. Psychopharmacol. 20, 11—18. Flamant, F., Gauthier, K., Samarut, J., 2007. Thyroid hormones signaling is getting more complex: STORMs are coming. Mol. Endocrinol. 21, 321—333. Guadano-Ferraz, A., Benavides-Piccione, R., Venero, C., Lancha, C., Vennstrom, B., Sandi, C., DeFelipe, J., Bernal, J., 2003. Lack of thyroid hormone receptor alpha1 is associated with selective alterations in behavior and hippocampal circuits. Mol. Psychiatry 8, 30—38. Hahn, C.G., Pawlyk, A.C., Whybrow, P.C., Gyulai, L., Tejani-Butt, S.M., 1999a. Lithium administration affects gene expression of thyroid hormone receptors in rat brain. Life Sci. 64, 1793—1802. Hahn, C.G., Pawlyk, A.C., Whybrow, P.C., Tejani-Butt, S.M., 1999b. Differential expression of thyroid hormone receptor isoforms by thyroid hormone and lithium in rat GH3 and B103 cells. Biol. Psychiatry 45, 1004—1012. Hodin, R.A., Lazar, M.A., Chin, W.W., 1990. Differential and tissuespecific regulation of the multiple rat c-erbA messenger RNA species by thyroid hormone. J. Clin. Invest. 85, 101—105. Joffe, R.T., 2006. Is the thyroid still important in major depression? J. Psychiatry Neurosci. 31, 367—368. Joffe, R.T., Sokolov, S.T., 1994. Thyroid hormones, the brain, and affective disorders. Crit. Rev. Neurobiol. 8, 45—63. Kioukia, N., Bekris, S., Antoniou, K., Papadopoulou-Daifoti, Z., Christofidis, I., 2000. Effects of chronic mild stress (CMS) on thyroid hormone function in two rat strains. Psychoneuroendocrinology 25, 247—257.
286 Kubera, M., Basta-Kaim, A., Papp, M., 1995. The effect of chronic treatment with imipramine on the immunoreactivity of animals subjected to a chronic mild stress model of depression. Immunopharmacology 30, 225—230. Leonard, J.L., Safran, M., 1994. Hormonal regulation of type II iodothyronine deiodinase in the brain. In: Wu, V.T.S. (Ed.), Thyroid Hormone Metabolism: Molecular Biology and Alternate Pathways. CRC Press, Boca Raton, FL, pp. 23—44. Lifschytz, T., Segman, R., Shalom, G., Lerer, B., Gur, E., Golzer, T., Newman, M.E., 2006. Basic mechanisms of augmentation of antidepressant effects with thyroid hormone. Curr. Drug. Targets 7, 203—210. Moreau, J.L., Scherschlicht, R., Jenck, F., Martin, J.R., 1995. Chronic mild stress-induced anhedonia model of depression; sleep abnormalities and curative effects of electroshock treatment. Behav. Pharmacol. 6, 682—687. Papp, M., Klimek, V., Willner, P., 1994a. Effects of imipramine on serotonergic and beta-adrenergic receptor binding in a realistic animal model of depression. Psychopharmacology (Berl.) 114, 309—314. Papp, M., Klimek, V., Willner, P., 1994b. Parallel changes in dopamine D2 receptor binding in limbic forebrain associated with chronic mild stress-induced anhedonia and its reversal by imipramine. Psychopharmacology (Berl.) 115, 441—446. Papp, M., Nalepa, I., Vetulani, J., 1994c. Reversal by imipramine of beta-adrenoceptor up-regulation induced in a chronic mild stress model of depression. Eur. J. Pharmacol. 261, 141—147. Papp, M., Willner, P., Muscat, R., 1991. An animal model of anhedonia: attenuation of sucrose consumption and place preference
E.J. Stein et al. conditioning by chronic unpredictable mild stress. Psychopharmacology (Berl.) 104, 255—259. Sarkar, P.K., Ray, A.K., 1994. Synaptosomal T3 content in cerebral cortex of adult rat in different thyroidal states. Neuropsychopharmacology 11, 151—155. Schwartz, H.L., Strait, K.A., Ling, N.C., Oppenheimer, J.H., 1992. Quantitation of rat tissue thyroid hormone binding receptor isoforms by immunoprecipitation of nuclear triiodothyronine binding capacity. J. Biol. Chem. 267, 11794—11799. Stamford, J.A., Muscat, R., O’Connor, J.J., Patel, J., Trout, S.J., Wieczorek, W.J., Kruk, Z.L., Willner, P., 1991. Voltammetric evidence that subsensitivity to reward following chronic mild stress is associated with increased release of mesolimbic dopamine. Psychopharmacology (Berl.) 105, 275—282. Venero, C., Guadano-Ferraz, A., Herrero, A.I., Nordstrom, K., Manzano, J., de Escobar, G.M., Bernal, J., Vennstrom, B., 2005. Anxiety, memory impairment, and locomotor dysfunction caused by a mutant thyroid hormone receptor alpha1 can be ameliorated by T3 treatment. Genes Dev. 19, 2152—2163. Willner, P., 1997. Validity, reliability and utility of the chronic mild stress model of depression: a 10-year review and evaluation. Psychopharmacology (Berl.) 134, 319—329. Willner, P., 2005. Chronic mild stress (CMS) revisited: Consistency and behavioural-neurobiological concordance in the effects of CMS. Neuropsychobiology 52, 90—110. Willner, P., Towell, A., Sampson, D., Sophokleous, S., Muscat, R., 1987. Reduction of sucrose preference by chronic unpredictable mild stress, and its restoration by a tricyclic antidepressant. Psychopharmacology (Berl.) 93, 358—364.