Hybridization with sense NT-3 and antisense THprobe. Note the lack of silver grains with the use of the sense NT-3 probe. (Bar = 12 j.m.). Neurobiology: Smith et ...
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 8788-8792, September 1995 Neurobiology
Stress and antidepressants differentially regulate neurotrophin 3 mRNA expression in the locus coeruleus MARK A. SMITH*t, SHINYA MAKINOI, MARGARET ALTEMUS§, DAVID MICHELSONt, SUNG-KWAN HONGt, RICHARD KVETNANSKYI, AND ROBERT M. POST* *Biological Psychiatry, tClinical Neuroendocrinology, and §Laboratory of Clinical Science Branches, National Institute of Mental Health, Bethesda, MD 20892; and $Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovakia Communicated by Seymour S. Kety, National Institutes of Health, Bethesda, MD, May 18, 1995
hypothesis is supported by a recent study demonstrating that NT-3 enhances the survival of adult LC noradrenergic neurons in vivo (15). The LC neurons were protected only by NT-3 and not by other neurotrophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin 4 (NT-4), although NT-4 has been shown to affect the survival rate of embryonic LC neurons in vitro (16). Neurotrophic factors are logical candidates for the modulation of LC plasticity as they not only affect neuronal development and survival but also influence phenotypic expression of transmitters and neuropeptides (17) and neurite outgrowth (18). In this regard it is of interest that plasticity of the LC may not be confined to alterations in NE turnover. Electrophysiological evidence suggests that sprouting of LC axons may also occur after chronic stress (19). Classically, target-derived trophic factors are thought to bind to tyrosine receptor kinases and be retrogradely transported to the cell body to modulate neuronal survival. Thus, the LC mnay gain access to NT-3 via innervation of the hippocampal formation and cerebellum, where NT-3 is highly expressed (20-22). On the other hand, the many examples of coexpression of neurotrophic factors and their receptors in the same neuron allows for the possibility that neurotrophic factors may also exert their effects via an autocrine mechanism of action (23-25). Therefore, we investigated whether NT-3 and its receptor Ntrk3 (neurotrophic tyrosine kinase receptor type 3; formerly TrkC) are expressed in the LC. Recent evidence suggests that stress affects the expression of neurotrophic factors in the central nervous system. In the hippocampus, repeated stress increases NT-3 mRNA levels but decreases BDNF mRNA (26). This is opposite to the effects of seizures and ischemia, which decrease NT-3 and increase NGF and BDNF mRNA levels (27-30). In the present study we examined whether stress might also affect neurotrophin mRNA levels in the LC. In addition, we determined whether treatments for depression, such as antidepressant drugs and electroconvulsive seizures (ECS), might affect neurotrophin expression in a manner opposite to that of stress.
ABSTRACT The mechanisms by which stress and antidepressants exert opposite effects on the course of clinical depression are not known. However, potential candidates might include neurotrophic factors that regulate the development, plasticity, and survival of neurons. To explore this hypothesis, we examined the effects of stress and antidepressants on neurotrophin expression in the locus coeruleus (LC), which modulates many of the behavioral and physiological responses to stress and has been implicated in mood disorders. Using in situ hybridization, we demonstrate that neurotrophin 3 (NT-3) is expressed in noradrenergic neurons of the LC. Recurrent, but not acute, immobilization stress increased NT-3 mRNA levels in the LC. In contrast, chronic treatment with antidepressants decreased NT-3 mRNA levels. The effect occurred in response to antidepressants that blocked norepinephrine uptake, whereas serotonin-specific reuptake inhibitors did not alter NT-3 levels. Electroconvulsive seizures also decreased NT-3 expression in the LC as well as the hippocampus. Ntrk3 (neurotrophic tyrosine kinase receptor type 3; formerly TrkC), the receptor for NT-3, is expressed in the LC, but its mRNA levels did not change with stress or antidepressant treatments. Because NT-3 is known to be trophic for LC neurons, our results raise the possibility that some of the effects of stress and antidepressants on LC function and plasticity could be mediated through NT-3. Moreover, the coexpression of NT-3 and its receptor in the LC suggests the potential for autocrine mechanisms of action. The behavioral and physiological responses to stress closely resemble the symptoms of clinical depression (1). Stress can precipitate episodes of depression (2), and chronic, uncontrollable stress has been proposed as a model of depression (3). How stress might exacerbate depression in vulnerable patients is not known. However, the locus coeruleus (LC), which plays a major role in behavioral arousal in response to novel or stressful stimuli (4), has long been a focus of investigation. Stress increases the firing rate of LC noradrenergic neurons as well as levels of norepinephrine (NE) and tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines (5-9). As a consequence, chronic stress increases the magnitude of NE release in response to a novel stressor (10). In contrast, tricyclic antidepressants (TCAs) decrease LC firing (11) and TH mRNA and protein in the LC (12, 13). Pretreatment with imipramine for 18 days, but not 1 or 7 days, prevents the induction of TH in the LC by cold stress (14). Generally, both the therapeutic and neurochemical effects of antidepressants require several weeks to develop. The mechanisms underlying LC adaptation in response to stress and antidepressants are not presently known. However, a possible candidate for the regulation of LC plasticity might be a neurotrophic factor such as neurotrophin 3 (NT-3). This
MATERIALS AND METHODS Animals and Experimental Groups. Male Sprague-Dawley rats (280-320 g) (Taconic Farms) were immobilized by taping their limbs to a board as described (26, 31). Some animals were adrenalectomized (ADX) and implanted with a 42.5-mg 21-dayrelease corticosterone pellet (Innovative Research of America). Sham ADX rats were implanted with a placebo pellet. Then each Abbreviations: NT-3 and NT-4, neurotrophins 3 and 4; BDNF, brainderived neurotrophic factor; LC, locus coeruleus; ECS, electroconvulsive seizures; NGF, nerve growth factor; TH, tyrosine hydroxylase; TCA, tricyclic antidepressant; ADX, adrenalectomized; NE, norepi-
nephrine. tTo whom reprint requests should be made at: Biological Psychiatry Branch, National Institute of Mental Health, Building 10, Room 3N212, Bethesda, MD 20892.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8788
group of rats was subjected to immobilization stress (2 hr/day) for 1 or 7 consecutive days and immediately sacrificed after the last immobilization. Corticosterone was measured by radioimmunoassay with a kit from ICN. Female Sprague-Dawley rats (175-200 g) were given daily i.p. injections of imipramine hydrochloride (5 mg/kg in saline) for 8 wk. Controls were injected with saline. Rats were sacrificed 24 hr after the last injection. In another experiment, male rats (200-225 g) were given i.p. injections of 5 mg of desipramine, fluoxetine, or trazodone per kg of body weight for 8 wk. Rats were sacrificed 1 hr after their last injection and compared with saline-injected controls. In a third experiment, male rats (175-200 g) were implanted s.c. with an Alzet pump delivering either saline or desipramine (10 mg/kg per day) for 3 wk. Some animals were stressed by immobilization for 2 hr on days 18-21 and sacrificed immediately after the last immobilization. All drugs were from Sigma except fluoxetine, which was generously donated by Eli Lilly. ECS was induced in male Sprague-Dawley rats (225-250 g) by electrical stimulation (80 mA for 0.5 sec) once per day for 5 consecutive days via earclips. Rats were sacrificed 4 hr after the last seizure. Ear clips were attached to control animals, which did not receive electrical stimulation. In Situ Hybridization. A riboprobe for rat NT-3 was generated by polymerase chain reaction from 1 ,ug of reversetranscribed rat hippocampal mRNA. The sense primer for NT-3 was a 28-mer containing nucleotides 158-177 of the rat NT-3 cDNA (32), anXba I restriction site along with a GG cap. The antisense primer was a 28-mer complementary to nucleotides 615-634 of the rat NT-3 cDNA and included a Sal I restriction site along with a GG cap. After 35 cycles at 94°C for 1 min, 51°C for 2 min, and 72°C for 3 min, the fragment of -500 bp was subcloned into pGEM-4Z (Promega). Northern blot analysis confirmed that the NT-3 probe hybridized to a single RNA band at about 1.3 kb as expected (33). Frozen brain sections (15 jim) at the level of the LC were fixed in paraformaldehyde, and delipidated as described (34). Antisense NT-3 riboprobe was labeled with 35S-labeled UTP by using T7 RNA polymerase according to the manufacturer (Ambion). Brain sections were hybridized overnight at 56°C with 1 X 106 cpm of riboprobe in 50 Al of hybridization buffer containing 20 mM Tris HCl (pH 7.4), 50% formamide, 300 mM NaCl, 1 mM EDTA (pH 8), 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 250 ,ug of yeast tRNA and 250 ,ug of total RNA per ml, 10 mg of salmon sperm DNA per ml, 10% dextran sulfate, 100 mM dithiothreitol, 0.1% SDS, and 0.1% sodium thiosulfate. Sections were then rinsed four times for 5 min each in 4x SSC (1x SSC = 0.15 M NaCl/0.015 M sodium citrate, pH 7) and treated with 20 ,ug of RNase A (Boehringer Mannheim) per ml of 0.5 M NaCl/1 mM EDTA/10 mM Tris, pH 8, for 30 min at 24°C. After rinsing for 5 min each in lx, 0.5X, and 0.1 x SSC at 24°C, slides were washed in 0.1 x SSC twice for 30 min each at 65°C. The slides were rinsed in increasing concentrations of ethanol containing 300 mM ammonium acetate and apposed to Amersham Hyperfilm-f3max for 4-10 days. Autoradiograms were analyzed by image analysis software as described (7, 26). Optical density was corrected for film nonlinearity by using 14C standards matched to 35S brain paste standards and converted to dpm/mg of wet tissue. In situ hybridization with adenosine 5'-[a-(35S)thio]triphosphate-labeled 48-mer oligonucleotide probes directed against rat TH and Ntrk3/TrkC was performed as described (7, 26). Double Labeling in Situ Hybridization. A 280-bp cDNA specific for rat TH (35) (provided by E. J. Lewis, University of Oregon) was transcribed into cRNA labeled with digoxigeninUTP according to the manufacturer's instructions (Genius Kit, Boehringer Mannheim). Sense and antisense NT-3 riboprobes were labeled with [33P]UTP. Frozen sections through the LC were fixed and defatted as above, and digoxigenin-labeled TH
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transcribed from about 50 ng of plasmid and 2 x 106 cpm of 33P-labeled NT-3 were added in 50 ,ul of hybridization buffer (without dithiothreitol). Slides were hybridized overnight and washed exactly as above. Digoxigenin-labeled probe was visualized by preincubating slides in buffer (100 mM Tris/150 mM NaCl, pH 7.5) containing 3% normal goat serum and 0.3% Triton X-100 for 30 min and then adding alkaline phosphataseconjugated rabbit anti-digoxigenin antibody (Boerhinger Mannheim) (1:500). After incubation with antibody for 3 h at 24°C, the slides were washed and then incubated overnight in buffer (100 mM Tris/100 mM NaCl/50 mM MgCl2, pH 9.5) containing nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate, and levamisole (0.24 mg each per ml) per manufacturer's instructions. Slides were dipped in Ilford K.SD emulsion, developed after 3 weeks, and covered with a glycerol-applied coverslip.
RESULTS Colocalization of NT-3 and TH mRNAs in the LC. NT-3 mRNA was found to be colocalized with TH mRNA in noradrenergic neurons of the LC by using combined radioactive and nonradioactive in situ hybridization (Fig. 1A). Hybridization with a NT-3 sense probe produced no specific signal in LC neurons (Fig. 1B). NT-3 was present in the majority of the neurons in the dorsal portion of the adult LC, whereas labeling was less intense in the ventral portion. Very few TH-positive neurons in the subcoerulear portion of the LC expressed NT-3. NT-3 mRNA did not appear to be present in glia. BDNF mRNA was also present in LC neurons at levels just above the limit of detection (results not shown). NGF and NT-4 mRNAs were not detectable in the LC. Using oligonucleotide probes we were able to detect Ntrk3/TrkC but not Ntrkl/TrkA or Ntrk2/TrkB mRNAs as in previous observations (36). A
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FIG. 1. Combined radioactive (NT-3) and nonradioactive (TH) in situ hybridization in the LC. (A) NT-3 coexpression in TH neurons.
The dark-reaction product throughout the cells indicates digoxigeninlabeled antisense TH probe, while the small black silver grains reflect 33P-labeled antisense NT-3 hybridization. Note the prominent coexpression of the two mRNA species in neurons indicated by long arrows. The small arrow indicates a neuron expressing TH but not NT-3. (B) Hybridization with sense NT-3 and antisense TH probe. Note the lack of silver grains with the use of the sense NT-3 probe. (Bar = 12 j.m.)
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Effect of Stress and Glucocorticoids on NT-3 mRNA. To determine if stress affected NT-3 expression in the LC, rats were stressed by immobilization for 2 hr once or on 7 consecutive days. An increase in NT-3 mRNA in the LC in response to repeated immobilizations is shown in Figs. 2 and 3. In rats that were repeatedly stressed, NT-3 mRNA levels increased 40 + 7% over those in unstressed controls (P < 0.001) (see Fig. 4). [In comparison, LC TH mRNA levels increase about 35% in response to repeated immobilization (9).] Acute stress up to 3 hr in duration was not sufficient to induce NT-3 in the hippocampus or LC. We also examined rats 4 hr after the end of a single 2-hr immobilization period and again found no change in NT-3 mRNA levels. The increase in NT-3 in response to repeated stress was relatively transient as levels returned to baseline within 24 hr after the last immobilization. NT-3 mRNA levels did not significantly increase in the cerebellum in response to immobilization stress. We next examined whether administration of 10 mg of corticosterone would mimic the effects of stress on NT-3 expression. Despite the fact that corticosterone levels produced 2 hr after injection of 10 mg of corticosterone were greater than those measured in stressed animals (350-675 ng/ml), corticosterone administration did not cause a significant increase in NT-3 mRNA levels in the LC. Likewise in ADX rats, NT-3 mRNA levels were not altered in the LC (see Fig. 4). To examine further whether the effects of repeated stress on NT-3 expression were dependent on glucocorticoids, we stressed ADX rats whose endogenous glucocorticoids had been removed and replaced with a 42.5-mg corticosterone pellet. This pellet maintained plasma corticosterone levels near those measured in unstressed sham-operated rats (26). Again NT-3 mRNA expression was significantly increased only by repeated stress and only in animals that were adrenally intact (Fig. 5). Effects of Antidepressants and ECS on Neurotrophins. Administration of imipramine for 8 weeks significantly re-
FIG. 3. Dark-field photomicrographs of NT-3 in situ hybridization in the LC from an unstressed rat (A) and a rat stressed seven times (B). White silver grains indicate the hybridization of radiolabeled NT-3 probe to BDNF mRNA. (Bar = 70 tim.)
duced NT-3 mRNA in the LC (Fig. 4). Treatment with desipramine, which primarily blocks NE reuptake, also reduced NT-3 mRNA levels in the LC (Figs. 4 and 6). In contrast, the serotonin-specific reuptake blockers (fluoxetine and trazodone) did not affect NT-3 mRNA in the LC (Fig. 4). NT-3 mRNA levels in the dentate gyrus were not affected by any of the antidepressants (Fig. 4). Treatment for 2 weeks with the benzodiazepine alprazolam (10 mg/kg i.p. twice a day) also did not affect NT-3 mRNA levels (results not shown). None of the drugs affected the levels of Ntrk3/TrkC mRNA in the LC or hippocampus. Another treatment for depression, electroconvulsive seizures (ECS), decreased NT-3 mRNA levels in both the LC and the dentate gyrus (Fig. 4). There was no effect of ECS on Ntrk3/TrkC mRNA in the LC, although ECS did increase Ntrk3/TrkC mRNA in the hippocampal formation from 853 ± 24 to 1482 ± 108 dpm/mg (P < 0.001). ECS also increased BDNF mRNA in the LC from 47 ± 12 to 150 ± 16 dpm/mg (P < 0.001) and in the dentate gyrus from 445 ± 40 to 5175 ± 1060 dpm/mg (P < 0.01). To examine whether antidepressants would block the stressinduced increase in NT-3 mRNA in the LC, rats were pretreated with desipramine for 3 weeks and then stressed by immobilization on 4 consecutive days. Desipramine signifi*
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FIG. 2. Autoradiographs of NT-3 mRNA in the LC. Note the increase in NT-3 hybridization in the LC from the rat stressed on 7 consecutive days (B) compared with the unstressed control rat (A). (x8.)
FIG. 4. Effects of stress, glucocorticoids, antidepressants, and ECS on NT-3 mRNA levels in the LC and dentate gyrus. Results are expressed as the mean % of the control group ± SEM and are derived from 6-20 separate determinations. *, Results are significantly different from those of the control group (P < 0.05). Treatments: Stress, 2 hr of immobilization on 7 consecutive days; ADX, adrenalectomy;
Cort, corticosterone; Fluox, fluoxetine; Trazo, trazodone; Desip, desipramine; Imip, imipramine; ECS, five consecutive electroconvulsive seizures.
Neurobiology: Smith et aL 300 ]
Proc. Natl. Acad. Sci. USA 92 (1995)
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FIG. 5. Effects of single or repeated (seven times) stress on NT-3 mRNA in the LC in intact rats (Sham) or ADX rats with corticosterone replacement (ADX/Cort). *, Results (n = 6-10 rats per group) were significantly different from those of the control group (P < 0.01).
cantly blocked the stress-induced increase in TH mRNA but only partially attenuated the increase in LC NT-3 mRNA. Although there was a significant drug effect (P < 0.05) and a significant stress effect (P < 0.0001) on NT-3 in the LC, the drug-stress interaction by two-way ANOVA was not significant. However, there was a trend (P = 0.12) for NT-3 mRNA levels in the LC to be less in the desipramine/stress group compared with the saline/stress group. Desipramine had no effect on NT-3 in the dentate gyrus. See Table 1.
DISCUSSION The principal finding of this study is that NT-3 mRNA expression occurs in noradrenergic neurons of the LC and is regulated in an opposite manner by stress and antidepressants. We have also confirmed that BDNF mRNA is present in the LC as recently described (37, 38). NT-3 and BDNF are also present in dopaminergic neurons in the substantia nigra and ventral tegmental area (39), and BDNF, NT-3, and NT-4, but not NGF, enhance the survival of fetal dopaminergic neurons (40). In a similar fashion, the survival of cultured LC neurons is enhanced by NT-3 and NT-4 but not by NGF (16). Moreover, it has recently been shown that NT-3 was the only member of the NGF family capable of rescuing adult LC neurons from 6-hydroxydopamine toxicity in vivo (15). The reason NGF, BDNF, and NT-4 may not have been effective may be because in the adult LC the only tyrosine receptor kinase present is Ntrk3/TrkC, the receptor for NT-3 (36). However, it should be pointed out that other growth factors such as ciliary neurotrophic factor may also influence LC survival and differentiation (41). Growth factors such as NT-3 may be relevant to the profound loss of LC neurons, which occurs in Alzheimer disease (42). Because some of the targets of LC noradrenergic neurons express NT-3, it was assumed that the source of the NT-3 that
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