Repeated stress increases catalytic TrkB mRNA in rat hippocampus

Neuroscience Letters 267 (1999) 81±84

Repeated stress increases catalytic TrkB mRNA in rat hippocampus Masashi Nibuya a, Michihiro Takahashi a, David S. Russell b, Ronald S. Duman a, c,* a

Department of Psychiatry, Laboratory of Molecular Psychiatry, Yale University School of Medicine, Connecticut Mental Health Center, New Haven, CT 06508, USA b Department of Neurology, Laboratory of Molecular Psychiatry, Yale University School of Medicine, Connecticut Mental Health Center, New Haven, CT 06508, USA c Department of Pharmacology, Laboratory of Molecular Psychiatry, Yale University School of Medicine, Connecticut Mental Health Center, New Haven, CT 06508, USA Received 2 January 1999; received in revised form 18 March 1999; accepted 22 March 1999

Abstract Northern blot analysis was utilized to distinguish between catalytic and truncated TrkB mRNA on the basis of transcript size. Repeated (10 days), but not acute, immobilization stress signi®cantly increased levels of catalytic TrkB mRNA, but did not in¯uence expression of truncated TrkB transcripts in rat hippocampus. Exposure to another paradigm, a combination of different, unpredictable stressors, also increased levels of catalytic, but not truncated, TrkB mRNA. In situ hybridization analysis demonstrated that chronic stress up-regulated TrkB mRNA in CA1 and CA3 pyramidal and dentate gyrus granule cells layers of hippocampus. As previously reported, both acute and chronic immobilization stress decreased expression of BDNF mRNA, suggesting that up-regulation of catalytic TrkB mRNA may be a compensatory adaptation to repeated stress. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Brain-derived neurotrophic factor; TrkB; Tyrosine kinase; Stress; Depression; Antidepressant

The expression of brain-derived neurotrophic factor (BDNF) in hippocampus is dramatically down-regulated in response to acute, as well as repeated immobilization stress [16]. Decreased expression of BDNF could contribute to the atrophy and, in extreme cases, death of stress-sensitive CA3 pyramidal neurons in hippocampus, and could play a role in stress related psychiatric illnesses [4]. The latter possibility is supported by recent brain imaging studies which demonstrate that the volume of hippocampus is decreased in patients with depression or posttraumatic stress disorder [3,15]. The action of BDNF is mediated by a membrane bound receptor containing intrinsic tyrosine kinase activity, referred to as TrkB [6,8]. Alterations in the expression of TrkB could thereby in¯uence neuronal responses to stress. In addition to catalytic TrkB, there is a truncated form of the receptor which lacks the tyrosine kinase domain. In the brain, catalytic TrkB appears to be expressed primarily in neurons, and truncated TrkB is expressed in both neurons * Corresponding author. Tel.: 11-203-974-7726; fax: 11-203974-7724. E-mail address: [email protected] (R.S. Duman)

and glia [6]. Although the exact function of truncated TrkB is unknown, it could serve to: inactivate BDNF that is released into the synapse; act as a reserve of BDNF for later use [6]; or serve as a negative regulator of the full length receptor [5]. Expression of TrkB is also activity dependent and regulated by excitatory and inhibitory neurotransmitter systems [6,17]. In the present study, we extend the previous work on regulation of BDNF by examining the in¯uence of repeated stress on expression of catalytic and truncated TrkB mRNA in hippocampus. Male Sprague±Dawley rats (150±200 g) (CAMM, Wayne, NJ) were group housed and maintained on a 12:12 h light/dark cycle with food and water freely available. Rats were administered immobilization stress (45 min) once or repeatedly (10 days) by placing them in plastic immobilization bags as previously described [11±13]. For the unpredictable stress paradigm, rats were subjected to a series of stressors which differed each day for a 10-day period as previously described [12,18]. The stressors included cage rotation (50 min), swim stress (4 min), cold isolation (60 min), lights on overnight, isolation housing (overnight), lights off for 2 h during normal daytime, and

0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 33 5- 3

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Fig. 1. Repeated stress increases catalytic TrkB mRNA in rat hippocampus. Rats were administered immobilization stress for 10 days. Levels of BDNF and TrkB mRNA in hippocampus were determined by Northern blot analysis. Representative autoradiograms are shown in the upper panel. The levels of BDNF (4.4 and 1.8 kb) and catalytic (9.0 kb) and truncated (7.5 kb) TrkB mRNA were quanti®ed by densitometry. Cyclophilin was used to correct for differences in total RNA per lane. The results are expressed as percent of control and are the mean ^ SEM of eight separate animals in the control and stress groups. *P , 0:05 compared with control (Student's t-test).

food/water deprivation (overnight). For exogenous glucocorticoid treatment, animals were administered a single corticosterone pellet (100 mg, s.c.), and control animals received sham surgery [9]. Animals were decapitated 2 h after the last stress and levels of TrkB and BDNF mRNA were determined by Northern blot or in situ hybridization as previously described [11]. Levels of TrkB and BDNF mRNA were analyzed by outlining the band on Northern blots or the regions of interest on in situ hybridization sections, which were then analyzed on a MacIntosh-based NIH IMAGE analysis program, version 1.52. For each animal, both sides of two individual brain sections were analyzed, for a total of 4 determinations, and the mean ^ SEM was determined. The results were then subjected to Student's t-test, with signi®cance determined at the , 0.05 level. All animal use procedures were in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Yale Animal Care Committee. The in¯uence of stress on TrkB mRNA was determined by Northern blot analysis using a riboprobe that recognizes mRNA transcripts that encode both catalytic and truncated forms of the receptor [8]. Chronic immobilization stress

increased the major catalytic transcript (9.0 kb), but not the major truncated transcript (7.5 kb) (Fig. 1). Minor forms of the catalytic (4.8 kb) and truncated (2.4 and 1.8 kb) transcripts were regulated in a similar manner (data not shown). The in¯uence of repeated unpredictable stress, which consists of exposure to a number of different stressors, on expression of BDNF and TrkB mRNA was also examined. This paradigm is thought to be a better model of chronic stress because the animals are unable to adapt to a single type of stress [18]. Repeated unpredictable stress also increased levels of catalytic, but not truncated, TrkB mRNA in hippocampus (Fig. 2). In contrast to these repeated stress paradigms, a single acute immobilization stress did not signi®cantly in¯uence levels of either catalytic or truncated TrkB mRNA (Fig. 3). Levels of BDNF mRNA were significantly decreased by acute or repeated immobilization stress as previously reported [16], as well as by the repeated unpredictable stress paradigm (Figs. 1±3). To determine which neuronal populations of hippocampus exhibit increased expression of catalytic TrkB mRNA in response to stress, in situ hybridization analysis was conducted using the full length TrkB riboprobe. Although in situ with this probe does not distinguish between catalytic and truncated TrkB mRNA, the Northern blot results

Fig. 2. Repeated unpredictable stress increases catalytic TrkB mRNA. Rats were administered unpredictable stress for 10 days and levels of BDNF and TrkB mRNA were determined by Northern blot analysis 2 h after the initiation of the last stress. The levels of BDNF or TrkB mRNA were quanti®ed by densitometry, and divided by levels of cyclophilin to correct for differences in total RNA per lane. The results are expressed as percent of control and are the mean ^ SEM of six separate animals in the control and stress groups. *P , 0:05 compared with control (Student's t-test).

M. Nibuya et al. / Neuroscience Letters 267 (1999) 81±84

Fig. 3. In¯uence of acute immobilization stress on TrkB and BDNF mRNA. Rats were administered acute immobilization stress (45 min) and levels of BDNF and TrkB mRNA were determined by Northern blot analysis 2 h after the initiation of stress. The level of BDNF or TrkB mRNA was quanti®ed by densitometry, and divided by levels of cyclophilin to correct for differences in total RNA per lane. The results are expressed as percent of control and are the mean ^ SEM of six separate animals in the control and stress groups. *P , 0:05 compared with control (Student's t-test).

demonstrate that any alterations observed should be due to regulation of the catalytic transcripts. In addition, the catalytic TrkB mRNA is expressed in neurons [6], and the major neuronal sub®elds of hippocampus are the cell layers of interest. Repeated immobilization stress signi®cantly increased the expression of TrkB mRNA in the CA3 and CA1 pyramidal and the dentate gyrus granule cell layers of hippocampus (Fig. 4). In contrast, levels of another transcript, PDE4D, were not altered by chronic stress in these regions demonstrating that this effect is relatively speci®c for TrkB (CA1, 94 ^ 3; CA3, 95 ^ 4; dentate gyrus, 92 ^ 3 (percent of sham), mean ^ SEM, n ˆ 6). To determine if the regulation of TrkB mRNA is mediated by elevation of glucocorticoids, rats were administered corticosterone (100 mg pellet, s.c.) for 7 days. This dose results in a four to ®ve-fold increase of glucocorticoid levels, and approximates levels that are observed during stress [7,13]. Corticosterone treatment did not in¯uence levels of TrkB mRNA in the major sub®elds of hippocampus (CA1, 95 ^ 7; CA3, 100 ^ 11; dentate gyrus, 92 ^ 17 (percent of sham), mean ^ SEM, n ˆ 6). The results of this study demonstrate that two different repeated stress paradigms increase the expression of catalytic TrkB mRNA, but do not in¯uence expression of truncated TrkB transcripts in hippocampus. In contrast, acute immobilization stress does not in¯uence expression of either catalytic or truncated TrkB mRNA, indicating that up-regulation of catalytic TrkB mRNA is dependent on repeated stress. Expression of BDNF mRNA is down-regulated by either acute or repeated immobilization as reported by Smith et al. [16]. In addition, we demonstrate that another type of paradigm, unpredictable stress, also decreases BDNF mRNA in hippocampus. In situ hybridization analysis demonstrates that chronic stress decreases levels of TrkB mRNA in CA1 and CA3 pyramidal and dentate gyrus granule cell layers, the same cell layers

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where levels of BDNF mRNA are reported to be decreased in response to stress [16]. Levels of both the catalytic and truncated forms of TrkB mRNA are increased by seizures, ischemia, or hypoglycemia [8,11]. These treatments are also reported to increase BDNF mRNA [10], indicating that expression of this neurotrophic factor and its receptor are coordinately regulated under these conditions. Lesions of the major afferent pathways to hippocampus are reported to increase truncated, but not full length, TrkB mRNA in hippocampus, and increase levels of BDNF mRNA [2]. The present ®nding is the ®rst report of selective regulation of full length TrkB mRNA, and the ®rst report that expression of BDNF and TrkB can be regulated in opposite directions. There is little known about the mechanisms underlying the regulation of TrkB mRNA. We found that chronic corticosterone administration did not in¯uence catalytic TrkB mRNA. This is consistent with a previous report demonstrating that adrenalectomy does not in¯uence expression of TrkB [1]. However, it is possible that coriticosterone treatment for longer periods of time could result in regulation of TrkB. Another possibility is that up-regulation of catalytic TrkB mRNA is a compensatory response to prolonged, stress-induced down-regulation of BDNF. This would be similar to the compensatory up-regulation of some G protein coupled receptors that is observed in response to neurotransmitter lesions. Up-regulation of catalytic TrkB could make neurons in the CA1, CA3, and dentate gyrus cell layers more responsive to the lower levels of BDNF that are observed during chronic stress. This could be an important adaptive response, especially in the CA3 pyramidal cell

Fig. 4. In situ hybridization analysis of TrkB mRNA. Rats were administered immobilization stress for 10 days and levels of TrkB mRNA were determined by in situ hybridization analysis. Representative autoradiograms are shown in the upper panel. The level of TrkB mRNA in CA1 and CA3 pyramidal and dentate gyrus (DG) granule cell layers was quanti®ed by densitometry. The results are expressed as percent of control and are the mean ^ SEM of six separate animals in the control and stress groups. *P , 0:05 compared with control (Student's t-test).

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layer where chronic stress is reported to cause atrophy or even death of neurons [7,14]. Alternatively, stress may result in activation of other regulatory pathways that control the expression of catalytic TrkB mRNA. A role for neurotrophic factors and their receptors in stress related psychiatric illnesses is supported by basic and clinical studies [4]. Stress is reported to cause atrophy or even death of CA3 pyramidal neurons in rat hippocampus [7,14], and brain imaging studies demonstrate that the volume of hippocampus is decreased in patients with depression and posttraumatic stress disorder [3,15]. Decreased expression of BDNF in response to stress could contribute to the atrophy and death of hippocampal neurons [16]. The results of the present study raise the possibility that increased expression of the receptor which mediates the action of BDNF, catalytic TrkB, could serve to protect hippocampal neurons from damage. Analysis of TrkB and BDNF expression in post-mortem brain tissue will be necessary to further determine the role of this neurotrophic factor system in the pathophysiology and treatment of affective illness. This work is supported by USPHS grants MH45481, and 2 PO1 MH25642, and by a Veterans Administration National Center Grant for PTSD, VA Hospital in West Haven, Connecticut. [1] Barbany, G. and Persson, H., Adrenalectomy attenuates kainic acid-elicited increases of messenger RNAs for neurotrophins and their receptors in the rat brain. Neuroscience, 54 (4) (1993) 909±922. [2] Beck, K.D., Lamballe, F., Klein, R., Barbacid, M., Schauwecker, P.E., McNeil, T.H., Finch, C.E., Hefti, F. and Day, J.R., Induction of non-catalytic TrkB neurotrophin receptors during axonal sprouting in the adult hippocampus. J. Neurosci., 13 (1993) 4001±4014. [3] Bremner, J.D., Randall, P., Scott, T.M., Bronen, R.A., Seibyl, J.P., Southwick, S.M., Delaney, R.C., McCarthy, G., Charney, D.S. and Innis, R.B., MRI-based measurement of hippocampal volume in patients with combat-related posttraumatic stress disorder. Am. J. Psychiatry, 152 (1995) 973±981. [4] Duman, R.S., Heninger, G.R. and Nestler, E.J., A molecular and cellular theory of depression. Arch. Gen. Psychiatry, 54 (1997) 597±606. [5] Eide, F.F., Vining, E.R., Eide, B.L., Zang, K., Wang, X-Y and

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