Asp Forum - Derived Neurotrophic Factor Transcripts and Cyclic AMP Response Element

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ï»¿Derived Neurotrophic Factor Transcripts and Cyclic AMP Response ElementStressorSpecific Regulation of Distinct BrainDerived Neurotrophic Factor Transcripts and Cyclic AMP Response ElementBinding Protein Expression in the Postnatal and Adult Rat HippocampusAmrita Nair1,3, Krishna C Vadodaria1,3, Sunayana B Banerjee1, Madhurima Benekareddy1, Brian G Dias1, Ronald S Duman2 and Vidita A Vaidya11Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India2Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USACorrespondence: Dr VA Vaidya, Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.Received 3 August 2006; Revised 27 September 2006; Accepted 12 October 2006; Published online 13 December 2006.Top of pageAbstractStress regulation of brainderived neurotrophic factor (BDNF) is implicated in the hippocampal damage observed in depression. BDNF has a complex gene structure with four 5' untranslated exons (I with unique promoters, and a common 3' coding exon (V). To better understand the stress regulation of BDNF, we addressed whether distinct stressors differentially regulate exonspecific BDNF transcripts in the postnatal and adult hippocampus. The early life stress of maternal separation (MS) resulted in a time pointdependent differential upregulation of BDNF transcripts restricted to early postnatal life (P14BDNF II, P21BDNF IV, V). In adulthood, distinct stressors regulated BDNF transcripts in a signature manner. Immobilization stress, administered once, decreased all BDNF splice variants but had differing effects on BDNF I/II (increase) and III/IV (decrease) when administered chronically. Although immobilization stress reduced BDNF (V) mRNA, chronic unpredictable stress did not influence total BDNF despite altering specific BDNF transcripts. Furthermore, a prior history of MS altered the signature pattern [url=http://www.libreria-apogeo.it/abercrom...]abercrombie milano[/url] in which adultonset stress regulated specific BDNF transcripts. We also examined the expression of cyclic AMP response elementbinding protein (CREB), an upstream transcriptional activator of BDNF, and observed a CREB induction in the postnatal hippocampus <a href="http://www.dtdrivingschool.co.uk/giusep..."&g... zanotti shoes</a> following MS. As a possible consequence of enhanced CREB and BDNF expression following MS, we examined hippocampal progenitor proliferation and observed a significant increase restricted to early life. Patients suffering from major depression or posttraumatic stress disorder have been reported to exhibit hippocampal volumetric loss (Bremner et al, 2000; Smith, 2005). In animal models, sustained exposure to stress is known to induce dendritic atrophy within the hippocampal CA subfields (Magarinos et al, 1996; Vyas et al, 2002) and to decrease neurogenesis in the dentate gyrus (DG) (Mirescu and Gould, 2006; Pham et al, 2003). The form and extent of stressinduced hippocampal damage is thought to depend upon the timing, type, duration, and frequency of the stressor (Pacak and Palkovits, 2001; Radley and Morrison, 2005). In addition, stress responses are governed by an individual's prior history of stress exposure. Indeed, early life adverse experience is known to alter adult responses to stress (Ladd et al, 2000), thus contributing to the generation of individual differences in vulnerability, not only to stress but also to stressrelated psychopathology (Heim and Nemeroff, 2002).Although it is evident that sustained stress, both in early life and adulthood, can adversely affect hippocampal structure and function (Brunson et al, 2003; Buwalda et al, 2005; Magarinos et al 1996; Mirescu and Gould, 2006; Vyas et al, 2002), the molecular underpinnings for this remain unclear. Decreased expression of the neurotrophin brainderived neurotrophic factor (BDNF) has been implicated in stressinduced hippocampal damage and dysfunction (Duman, 2004; GomezPinilla and Vaynman, 2005). BDNF, which is expressed at the highest levels in the hippocampus, plays a vital role in hippocampal development and continues to shape adult hippocampal structure and function (Branchi et al, 2004; Lu and Gottschalk, 2000). The rat BDNF gene through the alternate splicing of four distinct 5' exons (I to a common 3' exon (V), and by the use of two separate polyadenylation sites, can generate eight unique BDNF transcripts (Timmusk et al, 1993). The 5' exons, each with their unique promoter, remain untranslated, with only the common 3' coding exon (V) generating the mature BDNF protein. The complex gene structure of BDNF appears to permit the distinct BDNF promoters to be differentially recruited to generate both a regionspecific basal expression (Timmusk et al, 1993) and a stimulusevoked regulation during development and in adulthood (Lauterborn et al, 1996; Sathanoori et al, 2004). Multiple BDNF transcripts are known to be differentially regulated in response to distinct stimuli such as activity, exercise and antidepressant administration (Dias et al, 2003; RussoNeustadt et al, 2000; Timmusk et al, 1995). Thus far, the stressdependent regulation of distinct [url=http://www.libreria-apogeo.it/b...]gucci borse outlet[/url] BDNF transcripts is poorly understood. Given the complexity of the BDNF gene, little is known about the transcription factors that regulate exonspecific BDNF promoters to influence gene expression. However, several studies both in vivo and in vitro implicate the transcription factor cyclic AMP response elementbinding protein (CREB) in contributing to the regulation of BDNF expression (Barco et al, 2005; Conti et al, 2002). In addition, specific stressors in adult life have been reported to regulate hippocampal CREB expression (Alfonso et al, 2006; Song et al, 2006) and a decline in CREB expression has been observed in depressed patients (Blendy, 2006; Lai et al, 2003; Yamada et al, 2003). Besides its role in the regulation of BDNF, CREB has been shown to influence both structural and synaptic plasticity in the hippocampus (Josselyn and Nguyen, 2005; Nakagawa et al, 2002). Given the crucial role that BDNF and its <a href="http://www.libreria-apogeo.it/rep..."&... borse hermes</a> upstream transcriptional activator CREB have been suggested to play in stressrelated hippocampal damage (Blendy, 2006; Duman, 2004), understanding their regulation by distinct stressors in postnatal and adult life is critical.We hypothesized that the stress regulation of BDNF splice variants, as well as CREB expression, may be dependent upon the timing of stress exposure during the lifespan of an animal, the nature of the stressor, its duration, and frequency. In the present study, we have analyzed the influence of early life stress and of distinct adultonset stressors on the expression of multiple BDNF transcripts and CREB mRNA in the hippocampus. In addition, we have also addressed whether a history of early life maternal separation (MS) substantially alters the pattern of regulation of specific BDNF splice variants and CREB mRNA following adultonset stress. Our results raise the intriguing possibility that stressorspecific differences in the regulation of BDNF and CREB, as well as influences on these modulators by early life experience, may contribute to the generation of individual differences in hippocampal vulnerability to stress.Top of pageMATERIALS AND METHODSAnimalsSprague rats bred in our animal facility were used for all experiments. The animals were grouphoused and maintained on a 12 h light/dark cycle (lights on at [url=http://www.libreria-apogeo.it/burbe...]burberry bologna[/url] 0700 hours) with ad libitum access to food and water. All experimental procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the TIFR Institutional Animal Ethics Committee.Adult Stress ParadigmsThe stress paradigms, both acute and chronic, were performed on adult male animals of postnatal day 60 (P60). As previously described (Nibuya et al, 1999), acute immobilization stress (n=10/group) involved placing the animals in plastic restraint stress cone bags (Harvard Apparatus) for 2 h on a single day, and killing them at the end of this 2h stress period. Animals subjected to chronic immobilization stress paradigm (n=5/group) received the immobilization stress 2 h daily for a 10 day period, and were killed at the end of the stress period on the final day. In the chronic unpredictable stress (CUS) experiment (n=7 adult males were subjected to two stressors daily for a period of 10 days (Ortiz et al, 1996), and killed at the end of the final stressor. The type, order, and time of administration of the stressors were randomly generated, justifying the unpredictable nature of this stress paradigm. The stressors were cage rocking, swim stress, lights on overnight, cold isolation, lights off for 3 h during the light period of the light/dark cycle, overnight social isolation, food and water deprivation overnight, and immobilization for 1 h. Control animals were handled and left in the home cage.Maternal SeparationFor the MS experiments, pregnant dams were individually housed and randomly allocated to experimental or control groups. Litters in the MS groups were removed from the home cage for a 3h period (0900 hours hours), and placed in a beaker lined with soft bedding. The beakers were placed on a euthermic pad maintained at 37 and were kept in an isolated room. The dam was placed in a fresh cage for the duration of the separation. After termination of the 3 h separation, the pups [url=http://www.dtdrivingschool.co.uk/giusep...]giuseppe zanotti shoes[/url] were <a href="http://www.libreria-apogeo.it/b...">gucci borse outlet</a> returned to the home cage, followed by the dam. Ac MS involved separating the litters on P7 for 3 or 6 h, and killing the pups at the end of the separation period (n=4/group). Each MS group had its own control litter. To examine the influence of chronic MS (Chr MS), litters were separated for 3 h daily from P2 to P14, and killed at different time points following the end of the MS on P14. The time points for killing of the litters were: P14 (n=4 (immediately following the end of the 3 h separation period on P14) and P21 (n=3 [url=http://www.libreria-apogeo.it/rep...]replica borse hermes[/url] for ctrl and 5 for MS) (7 days following the termination of the Chr MS period). Separate agematched litters served as controls for the two MS groups in the time point experiment. To study the interaction of Chr MS during early life and adult exposure to acute or chronic immobilization stress, two experiments were performed in which litters were first randomly allocated to one of four cohorts: control, acute/chronic immobilization stress, Chr MS, and Chr MS+acute/chronic immobilization stress (n=4 Although the first two litters were left undisturbed in early life, the Chr MS and Chr MS+acute/chronic immobilization stress litters were subjected to MS from P2 to P14 as described earlier. All animals were weaned at P21, and males from a single litter were grouphoused. The animals were left undisturbed until P60, when males of the acute/chronic immobilization stress and Chr MS+acute/chronic immobilization stress groups were subjected to a 2h immobilization stress for a single day (acute immobilization stress) or daily for 10 days (chronic immobilization stress). Animals from the control and Chr MS groups were left undisturbed until the time of killing. All groups were killed at the end of the final 2h stress period.Behavioral TestsMS animals were put through the novelty suppressed feeding (NSF) test in adulthood to examine anxiety behavior (Santarelli et al, 2003). The NSF test was performed in a black plexiglass chamber (60 40 30 cm) for a period of 5 min per animal. All animals, both control and MS were deprived of food, but not water, for 36 h before testing. Animals were placed into the corner of the NSF chamber, which had two food pellets placed on a white platform at the center of the chamber. The platform was well lit, whereas the rest of the test chamber was maintained in the dark. The latency to approach was measured as the time taken from placement in the chamber till the animal chewed on the pellet. Latency scores of 300 s were assigned to animals that had not eaten by the end of 5 min when the test was considered terminated. Immediately after the test the animals were returned to their cages and the amount of food eaten over 2 h was measured (home cage consumption). Home cage consumption was the same in control as well as MS animals (Control=140.58 g, MS=14.50.96 g).In Situ HybridizationAll animals were killed by rapid decapitation, the brains removed and frozen on dry ice, before storage at 70 Sections (14mthick) were cut on a cryostat and thaw mounted onto ribonuclease free Probeon plus slides (Electron Microscopy Services, USA). The slides were fixed, acetylated and dehydrated, and stored at 70 In situ hybridization was carried out as described previously (Dias et al, 2003). In brief, rat exonspecific BDNF riboprobes were transcribed using 35Slabeled UTP (Amersham, Buckinghamshire, UK) from transcription competent plasmids provided by Dr Lauterborn (University of California, Irvine, exons I and Dr Rattray (Kings College, London, exon V). The CREB antisense riboprobes were generated from a transcription competent plasmid as described previously (Nibuya et al, 1996). Slides were air dried and exposed to Hyperfilm max (Amersham, UK) for 2 weeks. RNase A pretreatment (40 mg/ml at 37 for 30 min) and competition with excess cold antisense riboprobes for the different BDNF exons or CREB did not yield significant hybridization (data not shown), confirming their specificity.BrdU ImmunohistochemistryNeurogenesis in maternally separated animals was studied using bromodeoxyuridine (BrdU; SigmaAldrich, USA) as a mitotic marker to label proliferating hippocampal progenitors. Litters were randomly allocated to control and Chr MS groups, the latter being subjected to MS from P2 to P14 as described previously. The animals were left undisturbed until P15, P21, or P60, when both control and MS animals were injected with BrdU (100 mg/kg) and killed 2 h later (n=4 Animals were perfused with saline followed by 4% paraformaldehyde, and their brains were removed and allowed to sink in 30% sucrose. The brains were cut on a sliding microtome (Leica) and 50 m thick sections were collected.To detect BrdUpositive hippocampal progenitors, every fifth section of the hippocampus was selected and BrdU immunohistochemistry was performed (Nakagawa et al, 2002). In brief, following DNA denaturation and acid hydrolysis, sections were incubated overnight with mouse antiBrdU antibody (1:500, Boehringer Mannheim, USA) and then exposed to secondary antibody (biotinylated antimouse IgG, 1:500, Vector Laboratories, USA). Signal amplification was carried out with an Avidin complex (Vector) and was detected with diaminobenzidine (Sigma).Quantitation and Data AnalysisLevels of BDNF exonspecific transcripts (I and CREB mRNA were determined using the Macintoshbased Scion Image Software (Scion, USA). To correct for nonlinearity, 14C standards were used for calibration. Equivalent areas of the hippocampal subfieldsDG, CA1, CA3, and CA4were outlined and optical density measurements taken. Hippocampal subfields from both sides of 3 sections from each animal were analyzed to obtain a mean value.Quantitation of the BrdUpositive cells in tissue sections was carried out using a previously described modified unbiased stereology protocol (Nakagawa et al, 2002) on a Zeiss Axioskop microscope. Quantitation was performed on coded sections by an experimenter blind to the study code. Sections spanned the rostrocaudal extent of the hippocampus and every fifth hippocampal section was processed for quantitation (8 sections/animal). BrdUpositive cells within DG were counted as being in the subgranular zone (SGZ)/granule cell layer (GCL) when they were directly touching the SGZ or within it. Cells were counted as hilar <a href="http://www.libreria-apogeo.it/burbe...">burberry bologna</a> when they were further than two cell body widths from the SGZ. The total number of BrdUpositive cells per SGZ/GCL or hilus was estimated by multiplying the total number of BrdU cells counted from every fifth section by the section periodicity (5).Results were subjected to statistical analysis using the Student's ttest for experiments with two groups or twoway analysis of variance (ANOVA) followed by the Bonferroni post hoc test for experiments with four groups. Differences were considered statistically significant at p valuesTop of pageRESULTSThe influence of acute and chronic stressors on the expression of distinct BDNF transcripts and CREB mRNA levels in the postnatal and adult rat brain was examined using in situ hybridization. Densitometric analysis was used to quantify levels of the BDNF splice variants and of CREB mRNA within the DG, CA1, CA3, and CA4 hippocampal subfields.Influence of Ac MS on the Expression of Specific ExonContaining BDNF Transcripts and CREB mRNA in the Postnatal HippocampusThe levels of the distinct exoncontaining BDNF transcripts and CREB mRNA in the hippocampi of animals subjected to an Ac MS for 3 h on P7 are shown in Figure 1. The results show that a single period of separation of pups from the dam for 3 h resulted in a robust and selective upregulation of exon IIIcontaining BDNF transcripts in all the hippocampal subfields (Figure 1c). The regulation of the BDNF splice variants by a single episode of MS depended upon the duration of separation. Ac MS for 6 h on P7 resulted in a small, but significant, downregulation of BDNF exon I mRNA levels in all the hippocampal subfields examined <a href="http://www.libreria-apogeo.it/abercrom...">abercrombie milano</a> (Table 1). The upregulation of BDNF exon III mRNA observed following 3 h MS was lost following a longer MS period of 6 h (Table 1). None of the changes in the specific exoncontaining BDNF transcripts following Ac MS for 3 or 6 h was accompanied with a concomitant change in the total BDNF expression as determined by in situ hybridization for the common BDNF exon V (Figure 1e and Table 1). The expression of CREB was unaltered following an Ac MS for 3 or 6 h (Figure 1f and Table 1). Animals were maternally separated on P7 for 3 h and killed at the end of the 3 h separation period. The levels of the exonspecific BDNF transcripts and CREB mRNA were determined using in situ hybridization and quantitation was performed using densitometric analysis. The levels of the different BDNF transcripts (a) exon I, (b) exon II, (c) exon III, (d) exon IV, (e) exon V, and CREB mRNA (f) within the DG, CA1, CA3, and CA4 hippocampal subfields have been shown along with representative autoradiographic images from control and maternally separated animals. The results are presented as percent of control and are the meanSEM (n=4/group). pttest). The scales of the yaxis differ between the graphs. The different BDNF splice variants and CREB mRNA exhibited a time pointdependent regulation following the Chr MS paradigm (Figures 2 and 3). At P14, when animals were killed immediately following the termination of the final 3h separation, a significant upregulation in BDNF exon II mRNA expression was seen in all the hippocampal subfields (Figure 2b). The expression of BDNF exon IIIcontaining mRNA exhibited a trend towards an increase in the DG (p=0.067), CA3 (p=0.09), and CA4 (p=0.068) in MS animals, but did not achieve significance (Figure 2c). The other BDNF splice variants, as well as the expression of total BDNF mRNA (exon V), remained unchanged in Chr MS animals at P14 (Figure 2). CREB mRNA levels were significantly increased in the DG and CA1, but not the CA3 and CA4, hippocampal subfields in Chr MS animals at P14 (Figure 2f). Animals were maternally separated from P2 to P14 for 3 h daily, and killed on P14 immediately following the termination of the 3 h separation period. Quantitation of the levels of exonspecific BDNF transcripts and CREB mRNA was performed using in situ hybridization and followed by densitometric analysis. The levels of exonspecific BDNF transcripts (a) exon I, (b) exon II, (c) exon III, (d) exon IV, (e) exon V, and CREB mRNA (f) in the hippocampal subfields of the DG, CA1, CA3, and CA4 are graphically represented with autoradiographic images from control and maternally separated animals. The results are presented as percent of con

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Derived Neurotrophic Factor Transcripts and Cyclic AMP Response Element

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