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Impaired fear extinction learning in adult heterozygous BDNF knock-out mice
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Impaired fear extinction learning in adult heterozygous BDNF knock-out mice
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Laura Psotta, Volkmar Lessmann 1, Thomas Endres 1,⇑ Institute of Physiology, Medical Faculty, Otto-von-Guericke-University Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany
a r t i c l e
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Article history: Received 29 October 2012 Revised 25 March 2013 Accepted 25 March 2013 Available online xxxx Keywords: Brain-derived neurotrophic factor Fear extinction Aging Fear conditioning
a b s t r a c t Brain-derived neurotrophic factor (BDNF) is a crucial regulator of neuroplasticity, which underlies learning and memory processes in different brain areas. To investigate the role of BDNF in the extinction of amygdala-dependent cued fear memories, we analyzed fear extinction learning in heterozygous BDNF knock-out mice, which possess a reduction of endogenous BDNF protein levels to 50% of wild-type animals. Since BDNF expression has been shown to decline with aging of animals, we tested the performance in extinction learning of these mice at 2 months (young adults) and 7 months (older adults) of age. The present study shows that older adult heterozygous BDNF knock-out mice, which have a chronic 50% lack of BDNF, also possess a deficit in the acquisition of extinction memory, while extinction learning remains unaffected in young adult heterozygous BDNF knock-out mice. This deficit in extinction learning is accompanied by reduction of BDNF protein in the hippocampus, amygdala and the prefrontal cortex. Ó 2013 Elsevier Inc. All rights reserved.
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Studying the processes underlying acquisition and modulation of fear memories is probably one of the most important research areas in the field of behavioral neuroscience. Anxiety disorders such as phobias and posttraumatic stress disorders (PTSDs), are among the most commonly diagnosed mental health problems (Breslau, Peterson, Poisson, Schultz, & Lucia, 2004), and there is overwhelming evidence that patients suffering these diseases are impaired either in the learning and storage of fear memory or in fear extinction learning (Jovanovic & Ressler, 2010; Mahan & Ressler, 2012). The classical Pavlovian fear conditioning paradigm is an adequate model to analyze the mechanisms of fear memory. Pairing an unconditioned stimulus (US), e.g. an aversive electric foot shock, with an initially neutral, ineffectual stimulus, e.g. a tone, results in a learning process that the neutral stimulus is a predictor for the occurrence of the US and thus the previously neutral stimulus is turned into a conditioned stimulus (CS). However, if the CS is presented repeatedly without the US, the conditioned response diminishes again until almost no remaining fear response can be observed. This phenomenon is called extinction of fear memory and is supposed to be a form of new learning that results in an inhibition of the original fear memory trace, since the original fear behavior can return after a delay of time (spontaneous recovery), or if the CS is presented in a different context than the extinction context (i.e., renewal; for reviews see e.g. Maren, 2011; Quirk & Mueller, 2008).
⇑ Corresponding author. Fax: +49 0 391 67 15819. 1
E-mail address: [email protected] (T. Endres). These authors shared senior authorship.
Brain-derived neurotrophic factor (BDNF) is a protein which is known to play a critical role in neuronal differentiation and survival (Davey & Davies, 1998; Lewin & Barde, 1996). Furthermore, BDNF is also involved in the induction and expression of activitydependent synaptic plasticity (like e.g. long-term potentiation) thus regulating processes involved in learning and memory formation (see e.g. Bekinschtein et al., 2008; Cowansage, LeDoux, & Monfils, 2010; Gottmann, Mittmann, & Lessmann, 2009). BDNF also plays a critical role in the acquisition and consolidation of amygdala dependent fear memory (Endres & Lessmann, 2012; Ou & Gean, 2006; Ou, Yeh, & Gean, 2010; Rattiner, Davis, French, & Ressler, 2004; Rattiner, Davis, & Ressler, 2005) and very recently, it has been shown that BDNF is also involved in the extinction of fear memories (for review see: Andero & Ressler, 2012). Fear extinction training induces a strong increase in BDNF mRNA in the infralimbic medial prefrontal cortex (IL; Bredy et al., 2007) and BDNF-infusion in the IL facilitated extinction learning even without extinction training (Peters, Dieppa-Perea, Melendez, & Quirk, 2010). Furthermore, a region-specific genetic knock-down of BDNF in the hippocampus impairs fear extinction (Heldt, Stanek, Chhatwal, & Ressler, 2007). Chhatwal and colleagues showed that extinction training leads to an increase of BDNF mRNA within the basolateral amygdala (BLA), and that interfering with BDNF signaling by overexpressing non-functional TrkB receptors (tTrkB) in the amygdala, prevents the retention of extinction memory, but not the within-session extinction, suggesting a role of BDNF in the consolidation of extinction memory (Chhatwal, Stanek-Rattiner, Davis, & Ressler, 2006). Furthermore, Soliman et al. (2010) showed impaired fear extinction learning in mice that carry a heterozygous or homozygous mutation in the BDNF gene (Val/Met and Met/Met) which affects BDNF secretion (Egan et al.,
1074-7427/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nlm.2013.03.003
Please cite this article in press as: Psotta, L., et al. Impaired fear extinction learning in adult heterozygous BDNF knock-out mice. Neurobiology of Learning and Memory (2013), http://dx.doi.org/10.1016/j.nlm.2013.03.003
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2003). In addition, systemic application of the TrkB-receptor agonist 7,8-Dihydroxyflavone was able to foster fear extinction learning (Andero et al., 2010). In conclusion, the above mentioned studies suggest that BDNF is an important regulator of fear extinction learning of amygdala-dependent cued fear memories. Given the known physiological decline of endogenous BDNF protein expression with aging (Boger et al., 2011; Carreton et al., 2012; Silhol, Bonnichon, Rage, & Tapia-Arancibia, 2005) we here focused on the role that diminished endogenous BDNF levels exert on fear extinction during early aging. In a recent study we could show, that heterozygous BDNF knock-out mice (BDNF+/ mice), which retain roughly 50% of BDNF protein levels compared to their wild-type littermates, display an age-dependent deficit in fear learning (Endres & Lessmann, 2012): if the mice were 3 months of age or beyond, the ability to consolidate fear memories was impaired. Based on these results we hypothesized that these mice might exhibit also an age-dependent deficit in fear extinction learning. Several studies have shown that the protein levels of BDNF in the brain decreases with increasing age (see e.g. Boger et al., 2011; von Bohlen & Halbach, 2010). Thus, in young BDNF+/ mice the level of BDNF might still be adequate to form new extinction memory traces, whereas during aging the quantity of BDNF might fall below a critical threshold, so that no extinction memory can be formed. In order to test this, we analyzed fear extinction learning in young adult (2 months) and older adult (7 months) BDNF+/ mice and their wild-type littermates. In addition, we quantified BDNF protein levels in brain areas which are well known to be essential for fear extinction learning, i.e. basolateral amygdala, hippocampus and the mPFC, using an enzyme-linked immunosorbent assay (ELISA). To test our hypothesis, we used 2 or 7 months old male BDNF+/ mice (Korte et al., 1995), which were backcrossed to a C57BL/6J genetic background (Charles River, Sulzfeld, Germany) for more than 10 generations. Wild-type littermates (WT) served as controls. The experimental group sizes were between eight and twelve mice per genotype. Mice were group-housed (2–4 per cage) in a temperature controlled room (22 °C) with a 12 h light–dark cycle (lights on at 7 a.m.) with food and water access ad libitum. All experimental procedures were performed in accordance with the ethical guidelines for the use of animals in experiments and were approved by the local animal care committee (Landesverwaltungsamt SachsenAnhalt, IPHY/G/01-872/08). For the fear conditioning experiments we used an automated setup that assessed the activity of the animals via an array of infra-red light beams (Fear conditioning system, TSE systems GmbH, Bad Homburg, Germany) which was located in a separate testing room. For the 2 months old mice we used an 8 kHz sine tone (30 s, 75 dB, SPL) as CS, that was paired three times with a co-terminating scrambled foot shock (1 s, 0.7 mA). Since we knew from our previous experiments that this protocol was not able to induce fear in BDNF+/ mice, which are older than 3 months of age (Endres & Lessmann, 2012), we first established a fear conditioning protocol that could compensate this learning deficit in older BDNF+/ mice. It turned out that a protocol applying five CS–US pairings with a shock intensity of 0.4 mA was able to induce a comparable amount of conditioned fear in older BDNF+/ mice and their wildtype littermates, as observed with three pairings in the younger animals. In addition, we verified that the two conditioning protocols induced a comparable amount of conditioned fear and a similar extinction of fear in young WT mice (F6,133 = 1.6, p > 0.05, data not shown). On the first day of the experiment, animals were fear conditioned according to the above mentioned protocols and 24 h later the animals underwent extinction training. The extinction training was identical for both age groups and was performed in a different context than the fear conditioning. It consisted of 28 CS presentations with a consistent inter-stimulus interval of 5 s. On day three we tested the animals for extinction memory by exposing them to
the extinction context where they received three CS presentations after a habituation phase of 180 s. To rule out that older BDNF+/ animals show a reduced exploratory activity which might interfere with the analysis of freezing behavior as an indicator of fear behavior, we performed open field experiments with both genotypes. The animals were exposed to an open field arena (30 30 45 cm) under dim light conditions for 10 min. The distance traveled by the animals was recorded and analyzed by a video tracking system (Any-maze, Stoelting Co). In order to compare the baseline BDNF protein levels in the extinction learning relevant brain areas between the two genotypes as well as between the different age groups, we used a second set of animals (n = 4–8 per age/genotype), which were sacrificed and the brains were removed and sliced using a brain matrix (AgnTho’s AB, Lidingö, Sweden). Probes of the amygdala, the hippocampus and the mPFC were taken with a soft tissue biopsy puncher (Zivic instruments, Pittsburgh PA, USA) and stored at 80 °C. BDNF protein levels were measured with the BDNF Quantikine ELISA kit (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s instruction. During the first three presentations of the CS in the extinction session, young BDNF+/ mice as well as their wild-type littermates showed a robust and comparable amount of conditioned fear behavior in response to the CS. With ongoing CS presentations the freezing behavior declined (Fig. 1A, phase: F5,84 = 5.21, p = 0.0003) and there were no differences between the two genotypes (genotype: F1,84 = 0.07, p = 0.79; genotype phase: F5,84 = 0.62, p = 0.68), indicating successful fear learning as well as successful within-session extinction regardless of the genotype (Fig. 1A). In the 7 months old animals we observed also a comparable extent of conditioned freezing in both genotypes during the first three CS presentations, indicating successful fear learning even in the adult BDNF+/ mice. However, with ongoing CS presentations the BDNF+/ mice did not show a reduction in freezing behavior contrary to their wild-type littermates, indicating no within-session extinction in these animals (Fig. 1C). An ANOVA revealed no significant general effect of the number of CS presentation (phase: F5,126 = 1.59, p = 0.16), indicating such an unsuccessful extinction learning. But we observed a significant effect of the factor genotype (F1,126 = 18.64, p < 0.0001), indicating a different extinction performance between WT and BDNF+/ mice. However, we did not see a significant interaction between these two factors (genotype x phase: F5,126 = 1.26, p = 0.28). Twenty-four hours after the extinction training, we re-exposed the animals to the CS in the extinction context in order to test their extinction memory. Consistent with our results for the within-session extinction, we observed successful extinction memory in young animals for both genotypes, since BDNF+/ as well as the wild-type mice showed significantly less freezing in response to the CS compared to the beginning of the extinction training (paired t-test comparison, p’s < 0.05) (Fig. 1B). In contrast to the results for young animals but in accordance with our observation regarding the within-session extinction, in the 7 months old animals we observed a successful extinction memory only in wild-type mice (p < 0.05) whereas the BDNF+/ mice still showed a high amount of freezing behavior in response to the CS, which was not different from the freezing behavior at the beginning of the extinction training (p > 0.05). This result further demonstrates the impairment in fear extinction learning in 7 months old BDNF+/ mice. Importantly, the increased freezing in response to the CS in the 7 months old BDNF+/ mice is not due to any generalization of fear in these animals since we observed a similar low amount of freezing during the habituation period of the extinction memory test as in their wild-type littermates (Fig. 1D, p > 0.05). Although we verified before that our stronger conditioning protocol used for the older animals induced similar fear learning and
Please cite this article in press as: Psotta, L., et al. Impaired fear extinction learning in adult heterozygous BDNF knock-out mice. Neurobiology of Learning and Memory (2013), http://dx.doi.org/10.1016/j.nlm.2013.03.003
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Fig. 1. Fear extinction learning in 2 (A and B) and 7 (C and D) months old BDNF+/ and wild-type mice. (A) 2 months old animals showed no difference in extinction learning between the two genotypes, during the extinction training. Both genotypes showed a reduction of freezing behavior within the training session. (B) In the extinction memory test that was performed 24 h after the extinction training the 2 months old animals showed intact extinction memory and no elevated freezing behavior during the habituation phase of the memory test. (C) In contrast to the young animals, 7 months old BDNF+/ mice were not able to extinguish the cued fear memory, since they showed no reduction in freezing behavior during the extinction training. (D) In the extinction memory test, the 7 months old BDNF+/ mice did not show reduced cued fear behavior. In addition, they showed no elevated freezing during the habituation of the memory test, thus excluding a generalized fear behavior. (Indicates significant difference between BDNF+/ and wild- type mice, # indicates significant difference in comparison to the freezing behavior expressed at the beginning of the extinction training (CS1-3)).
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fear extinction in young WT mice, it might still be possible that the stronger fear conditioning protocol induced a different, more extinction-resistant type of fear learning in BDNF+/ mice. To address this issue, we performed an additional series of experiments, in which we applied the stronger fear conditioning protocol also to 2 months old BDNF+/ mice and observed the same results as seen in response to the weaker fear conditioning protocol: both genotypes showed intact within-session extinction learning (ANOVA: phase: F5,60 = 6.46, p < 0.0001, genotype: F1,60 = 1.16, p = 0.29; genotype phase: F5,60 = 1.01, p = 0.40) as well as a comparable extinction memory (p = 0.23). These results show that 2 months old animals, conditioned with the stronger protocol are able to form reliable fear extinction learning. Another reason that could account for the increased freezing behavior observed in older BDNF+/ mice during the extinction training is a potential generally reduced agility in these animals. To rule this out, we performed an open field test, which clearly showed that there are no differences in spontaneous activity behavior between the two genotypes (p = 0.13, t-test comparison) (Fig. 3). To test whether the age dependent decline in extinction learning is accompanied by reduced expression of BDNF protein, we performed ELISA measurements in different brain areas. The BDNF protein quantification showed a clear overall decline of protein concentration in BDNF+/ mice within the analyzed brain areas (Fig. 2; genotype: F1,58 = 87.7, p < 0.0001). Furthermore the BDNF levels generally decreased with age (age: F1,58 = 26.9, p < 0.0001) and there was a clear difference in BDNF protein levels between the analyzed brain areas (area: F2,58 = 16.6, p < 0.0001). Probably
due to similar changes in all of the different brain areas and genotypes the interaction of these three factors was not significant (genotype age area: F2,58 = 1.4, p = 0.21). In summary these data demonstrate that there is a decline of BDNF protein levels with age in all brain areas investigated in BDNF+/ and wild-type mice, which might underlie the observed learning deficit in adult BDNF+/ mice. In conclusion our results clearly demonstrate that in young adult (2 months) BDNF+/ mice fear extinction learning is still intact, whereas we observed a strong impairment in extinction learning in older adult (7 months) BDNF+/ mice. These older mice neither showed any within-session extinction nor any extinction memory in the memory retrieval test. These results support the notion that BDNF is critically involved in the formation of extinction memories. Furthermore, we could demonstrate that the learning deficit is accompanied by reduction of BDNF protein in three different brain areas which are relevant for extinction learning (Fig. 2), further stressing the impact of reduced levels of endogenous BDNF on fear extinction learning. Our results are in line with a recent study from Soliman and colleagues which demonstrated that mice and humans, carrying the single nucleotide Val66Met polymorphism in the BDNF gene, which leads to a reduced BDNF release, exhibit deficits in fear extinction learning (Soliman et al., 2010). Further evidence for a role of BDNF expression in extinction learning is provided by a study showing that extinction of conditioned fear results in histone acetylation in the BDNF gene promoter region in the prefrontal cortex, resulting in a simultaneous increase in BDNF mRNA expression in the prefrontal cortex (Bredy
Please cite this article in press as: Psotta, L., et al. Impaired fear extinction learning in adult heterozygous BDNF knock-out mice. Neurobiology of Learning and Memory (2013), http://dx.doi.org/10.1016/j.nlm.2013.03.003
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Fig. 2. BDNF protein concentration measured by ELISA in 2 and 7 months old BDNF+/ and wild-type mice in the hippocampus, amygdala and mPFC. BDNF+/ mice showed a significantly lower of BDNF protein level in all brain areas analyzed, and at both ages, compared to wild-type mice. The BDNF protein concentration in the hippocampus, amygdala and mPFC of 2 months old BDNF+/ and wild-type mice was significantly higher than the respective BDNF level in the 7 months mice. Seven months old BDNF+/ and wild-type animals showed significantly lower BDNF levels than the 2 months old wild-type mice in all brain areas investigated. 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323
et al., 2007). In this context it has been shown recently that a single infusion of BDNF in the IL is sufficient to induce extinction learning even without any extinction training (Peters et al., 2010). These authors suggested that the required BDNF for this extinction learning originates from hippocampal neurons, and these findings are in accordance with data from Heldt et al. (2007), who observed impaired extinction learning in a brain area specific knock-out of BDNF in the hippocampus. Interestingly, in addition to the extinction memory deficit, we observed no within-session extinction in the 7 months old BDNF+/ mice, indicating that also the acquisition of extinction memory might depend on BDNF. This observation is in line with results from Karpova et al. (2011) who also observed no distinctive within session extinction in untreated 3 months old BDNF+/ mice (Karpova et al., 2011). Since local interference with BDNF signaling in the BLA leaves the acquisition of extinction memories intact (Chhatwal et al., 2006), it seems reasonable to assume that there might be additional BDNF-dependent processes involved in the acquisition of extinction memories that are located outside the amygdala. Promising brain areas where such acquisition-related BDNF-dependent processes might take place could be the IL-mPFC and the hippocampus (see above). Since we observed an age-dependent reduction of BDNF-levels in both hippocampus and mPFC it remains unclear whether the observed deficit in the acquisition of fear extinction memories in older BDNF+/ mice is due to reduced BDNF levels in the hippocampus, mPFC or maybe both. Peters and colleagues demonstrated that individual animals with deficits in extinction learning displayed reduced BDNF levels in the hippocampus, but not in the amygdala or the IL-mPFC (Peters et al., 2010), suggesting an important role of hippocampal BDNF levels. It should be noted that our tissue preparation samples of the mPFC consist of both, the IL and the prelimbic (PL) sub-areas, thus making it impossible to differentiate whether the age-dependent decline of BDNF protein occurs only in the IL, the PL, or in both structures simultaneously. In a study by Choi and colleagues it has been shown that a specific knockdown of BDNF in the PL causes deficits in fear learning but not in fear extinction learning (Choi et al., 2010). However, in the present study we analyzed the levels of BDNF protein only in three brain areas that have been demonstrated to be crucially involved in fear extinction learning. Since BDNF+/ mice exhibit a chronic reduction of BDNF protein throughout the brain, it cannot be excluded that reduced BDNF levels also in other brain areas contribute to the observed extinction learning deficit. Importantly, we did not observe a generalization of fear in the older BDNF+/ mice, indicated by low freezing before onset of the
CS (habituation period) in the memory test. Furthermore, we observed no differences in spontaneous motor activity in the open field test. This observation is in line with previous results, demonstrating no altered spontaneous motor activity in 1–6 months old BDNF+/ mice (Endres & Lessmann, 2012). Hence it can be concluded that the lack of extinction learning in older BDNF+/ mice is not due to reduced activity of these mice. Our results demonstrate an age-dependent decline of BDNF protein in BDNF+/ mice (2 vs. 7 months) in three different brain areas that are all important in mediating fear extinction learning. These observations support previous studies demonstrating an agedependent decline of BDNF protein levels in wild-type animals (for review see von Bohlen & Halbach, 2010), as well as in BDNF+/ mice (Boger et al., 2011; Carreton et al., 2012). In addition our results support the observation that age-dependent changes in BDNF expression are differently regulated in different brain areas (e.g. Silhol et al., 2005). In general we observed a 50% reduction of BDNF protein in the hippocampus, amygdala and mPFC compared to wild-type mice, being also in line with previous studies (e.g. Abidin et al., 2006; Hill & van den Buuse, 2011; Kolbeck, Bartke, Eberle, & Barde, 1999). Interestingly, this overall reduction of BDNF levels to 50% seems to be still sufficient to mediate proper
Fig. 3. Distance traveled in the open field experiment of 7 months old BDNF+/ and wild-type mice. Animals of different genotypes showed no significant difference in activity during the experiment.
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fear learning and fear extinction in younger BDNF+/ mice (compare Endres & Lessmann, 2012), whereas a further much smaller decline during aging might provoke the observed learning deficit. This observation could indicate that there might be a specific threshold of BDNF that is required for proper learning of these tasks, which is somewhere below 50% of endogenous WT levels. To test this possibility it would be an interesting issue to analyze and compare the BDNF levels of trained animals and correlate this with the behavioral performance. Notably, previous studies in rats and mice have shown that fear extinction training has different consequences in very young as compared to adult animals, which was suggested to result by the formation of perineuronal nets (e.g. Gogolla, Caroni, Luthi, & Herry, 2009; Kim & Richardson, 2007; Quirk et al., 2010). However, these changes in learning mechanisms already occurred between postnatal day 16 and 23 in mice (Gogolla et al., 2009). Since the young adults in the present study were already 2 months of age it can be excluded that such a developmental change in learning mechanisms might underlie the age-dependent learning deficit in our BDNF+/ mice. The present investigation is the first study analyzing fear extinction learning in heterozygous BDNF-knockout mice in detail. We observed an age-dependent deficit in extinction learning of these mice, supporting the importance of endogenous BDNF also in fear extinction learning. This age-dependent learning impairment in older BDNF+/ mice seems to be a result from a decline of BDNF protein levels in the hippocampus, mPFC and amygdala with ongoing age, thus impairing the formation of new extinction memory traces. Our results suggest that BDNF+/ mice are a suitable model to further analyze the influence of reduced BDNF levels on the formation of extinction memory. Further, these results suggest that impaired fear extinction learning might be affected similarly in humans with reduced levels of endogenously released BDNF, such as suggested for carriers of the Val66Met polymorphism, or upon reduced BDNF expression at older ages.
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This study was supported by the Deutsche Forschungsgemeinschaft, SFB 779/B6. The authors would like to thank Dr. Tanja Brigadski for establishing the BDNF ELISA, and Evelyn Friedl, Sybille Natho, Colette Obst, Margit Schmidt, Stephanie Holze and Angela Jahn for excellent technical assistance.
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Neurobiology of Learning and Memory journal homepage: www.elsevier.com/locate/ynlme
Impaired fear extinction learning in adult heterozygous BDNF knock-out mice
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Laura Psotta, Volkmar Lessmann 1, Thomas Endres 1,⇑ Institute of Physiology, Medical Faculty, Otto-von-Guericke-University Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany
a r t i c l e
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Article history: Received 29 October 2012 Revised 25 March 2013 Accepted 25 March 2013 Available online xxxx Keywords: Brain-derived neurotrophic factor Fear extinction Aging Fear conditioning
a b s t r a c t Brain-derived neurotrophic factor (BDNF) is a crucial regulator of neuroplasticity, which underlies learning and memory processes in different brain areas. To investigate the role of BDNF in the extinction of amygdala-dependent cued fear memories, we analyzed fear extinction learning in heterozygous BDNF knock-out mice, which possess a reduction of endogenous BDNF protein levels to 50% of wild-type animals. Since BDNF expression has been shown to decline with aging of animals, we tested the performance in extinction learning of these mice at 2 months (young adults) and 7 months (older adults) of age. The present study shows that older adult heterozygous BDNF knock-out mice, which have a chronic 50% lack of BDNF, also possess a deficit in the acquisition of extinction memory, while extinction learning remains unaffected in young adult heterozygous BDNF knock-out mice. This deficit in extinction learning is accompanied by reduction of BDNF protein in the hippocampus, amygdala and the prefrontal cortex. Ó 2013 Elsevier Inc. All rights reserved.
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Studying the processes underlying acquisition and modulation of fear memories is probably one of the most important research areas in the field of behavioral neuroscience. Anxiety disorders such as phobias and posttraumatic stress disorders (PTSDs), are among the most commonly diagnosed mental health problems (Breslau, Peterson, Poisson, Schultz, & Lucia, 2004), and there is overwhelming evidence that patients suffering these diseases are impaired either in the learning and storage of fear memory or in fear extinction learning (Jovanovic & Ressler, 2010; Mahan & Ressler, 2012). The classical Pavlovian fear conditioning paradigm is an adequate model to analyze the mechanisms of fear memory. Pairing an unconditioned stimulus (US), e.g. an aversive electric foot shock, with an initially neutral, ineffectual stimulus, e.g. a tone, results in a learning process that the neutral stimulus is a predictor for the occurrence of the US and thus the previously neutral stimulus is turned into a conditioned stimulus (CS). However, if the CS is presented repeatedly without the US, the conditioned response diminishes again until almost no remaining fear response can be observed. This phenomenon is called extinction of fear memory and is supposed to be a form of new learning that results in an inhibition of the original fear memory trace, since the original fear behavior can return after a delay of time (spontaneous recovery), or if the CS is presented in a different context than the extinction context (i.e., renewal; for reviews see e.g. Maren, 2011; Quirk & Mueller, 2008).
⇑ Corresponding author. Fax: +49 0 391 67 15819. 1
E-mail address: [email protected] (T. Endres). These authors shared senior authorship.
Brain-derived neurotrophic factor (BDNF) is a protein which is known to play a critical role in neuronal differentiation and survival (Davey & Davies, 1998; Lewin & Barde, 1996). Furthermore, BDNF is also involved in the induction and expression of activitydependent synaptic plasticity (like e.g. long-term potentiation) thus regulating processes involved in learning and memory formation (see e.g. Bekinschtein et al., 2008; Cowansage, LeDoux, & Monfils, 2010; Gottmann, Mittmann, & Lessmann, 2009). BDNF also plays a critical role in the acquisition and consolidation of amygdala dependent fear memory (Endres & Lessmann, 2012; Ou & Gean, 2006; Ou, Yeh, & Gean, 2010; Rattiner, Davis, French, & Ressler, 2004; Rattiner, Davis, & Ressler, 2005) and very recently, it has been shown that BDNF is also involved in the extinction of fear memories (for review see: Andero & Ressler, 2012). Fear extinction training induces a strong increase in BDNF mRNA in the infralimbic medial prefrontal cortex (IL; Bredy et al., 2007) and BDNF-infusion in the IL facilitated extinction learning even without extinction training (Peters, Dieppa-Perea, Melendez, & Quirk, 2010). Furthermore, a region-specific genetic knock-down of BDNF in the hippocampus impairs fear extinction (Heldt, Stanek, Chhatwal, & Ressler, 2007). Chhatwal and colleagues showed that extinction training leads to an increase of BDNF mRNA within the basolateral amygdala (BLA), and that interfering with BDNF signaling by overexpressing non-functional TrkB receptors (tTrkB) in the amygdala, prevents the retention of extinction memory, but not the within-session extinction, suggesting a role of BDNF in the consolidation of extinction memory (Chhatwal, Stanek-Rattiner, Davis, & Ressler, 2006). Furthermore, Soliman et al. (2010) showed impaired fear extinction learning in mice that carry a heterozygous or homozygous mutation in the BDNF gene (Val/Met and Met/Met) which affects BDNF secretion (Egan et al.,
1074-7427/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nlm.2013.03.003
Please cite this article in press as: Psotta, L., et al. Impaired fear extinction learning in adult heterozygous BDNF knock-out mice. Neurobiology of Learning and Memory (2013), http://dx.doi.org/10.1016/j.nlm.2013.03.003
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2003). In addition, systemic application of the TrkB-receptor agonist 7,8-Dihydroxyflavone was able to foster fear extinction learning (Andero et al., 2010). In conclusion, the above mentioned studies suggest that BDNF is an important regulator of fear extinction learning of amygdala-dependent cued fear memories. Given the known physiological decline of endogenous BDNF protein expression with aging (Boger et al., 2011; Carreton et al., 2012; Silhol, Bonnichon, Rage, & Tapia-Arancibia, 2005) we here focused on the role that diminished endogenous BDNF levels exert on fear extinction during early aging. In a recent study we could show, that heterozygous BDNF knock-out mice (BDNF+/ mice), which retain roughly 50% of BDNF protein levels compared to their wild-type littermates, display an age-dependent deficit in fear learning (Endres & Lessmann, 2012): if the mice were 3 months of age or beyond, the ability to consolidate fear memories was impaired. Based on these results we hypothesized that these mice might exhibit also an age-dependent deficit in fear extinction learning. Several studies have shown that the protein levels of BDNF in the brain decreases with increasing age (see e.g. Boger et al., 2011; von Bohlen & Halbach, 2010). Thus, in young BDNF+/ mice the level of BDNF might still be adequate to form new extinction memory traces, whereas during aging the quantity of BDNF might fall below a critical threshold, so that no extinction memory can be formed. In order to test this, we analyzed fear extinction learning in young adult (2 months) and older adult (7 months) BDNF+/ mice and their wild-type littermates. In addition, we quantified BDNF protein levels in brain areas which are well known to be essential for fear extinction learning, i.e. basolateral amygdala, hippocampus and the mPFC, using an enzyme-linked immunosorbent assay (ELISA). To test our hypothesis, we used 2 or 7 months old male BDNF+/ mice (Korte et al., 1995), which were backcrossed to a C57BL/6J genetic background (Charles River, Sulzfeld, Germany) for more than 10 generations. Wild-type littermates (WT) served as controls. The experimental group sizes were between eight and twelve mice per genotype. Mice were group-housed (2–4 per cage) in a temperature controlled room (22 °C) with a 12 h light–dark cycle (lights on at 7 a.m.) with food and water access ad libitum. All experimental procedures were performed in accordance with the ethical guidelines for the use of animals in experiments and were approved by the local animal care committee (Landesverwaltungsamt SachsenAnhalt, IPHY/G/01-872/08). For the fear conditioning experiments we used an automated setup that assessed the activity of the animals via an array of infra-red light beams (Fear conditioning system, TSE systems GmbH, Bad Homburg, Germany) which was located in a separate testing room. For the 2 months old mice we used an 8 kHz sine tone (30 s, 75 dB, SPL) as CS, that was paired three times with a co-terminating scrambled foot shock (1 s, 0.7 mA). Since we knew from our previous experiments that this protocol was not able to induce fear in BDNF+/ mice, which are older than 3 months of age (Endres & Lessmann, 2012), we first established a fear conditioning protocol that could compensate this learning deficit in older BDNF+/ mice. It turned out that a protocol applying five CS–US pairings with a shock intensity of 0.4 mA was able to induce a comparable amount of conditioned fear in older BDNF+/ mice and their wildtype littermates, as observed with three pairings in the younger animals. In addition, we verified that the two conditioning protocols induced a comparable amount of conditioned fear and a similar extinction of fear in young WT mice (F6,133 = 1.6, p > 0.05, data not shown). On the first day of the experiment, animals were fear conditioned according to the above mentioned protocols and 24 h later the animals underwent extinction training. The extinction training was identical for both age groups and was performed in a different context than the fear conditioning. It consisted of 28 CS presentations with a consistent inter-stimulus interval of 5 s. On day three we tested the animals for extinction memory by exposing them to
the extinction context where they received three CS presentations after a habituation phase of 180 s. To rule out that older BDNF+/ animals show a reduced exploratory activity which might interfere with the analysis of freezing behavior as an indicator of fear behavior, we performed open field experiments with both genotypes. The animals were exposed to an open field arena (30 30 45 cm) under dim light conditions for 10 min. The distance traveled by the animals was recorded and analyzed by a video tracking system (Any-maze, Stoelting Co). In order to compare the baseline BDNF protein levels in the extinction learning relevant brain areas between the two genotypes as well as between the different age groups, we used a second set of animals (n = 4–8 per age/genotype), which were sacrificed and the brains were removed and sliced using a brain matrix (AgnTho’s AB, Lidingö, Sweden). Probes of the amygdala, the hippocampus and the mPFC were taken with a soft tissue biopsy puncher (Zivic instruments, Pittsburgh PA, USA) and stored at 80 °C. BDNF protein levels were measured with the BDNF Quantikine ELISA kit (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s instruction. During the first three presentations of the CS in the extinction session, young BDNF+/ mice as well as their wild-type littermates showed a robust and comparable amount of conditioned fear behavior in response to the CS. With ongoing CS presentations the freezing behavior declined (Fig. 1A, phase: F5,84 = 5.21, p = 0.0003) and there were no differences between the two genotypes (genotype: F1,84 = 0.07, p = 0.79; genotype phase: F5,84 = 0.62, p = 0.68), indicating successful fear learning as well as successful within-session extinction regardless of the genotype (Fig. 1A). In the 7 months old animals we observed also a comparable extent of conditioned freezing in both genotypes during the first three CS presentations, indicating successful fear learning even in the adult BDNF+/ mice. However, with ongoing CS presentations the BDNF+/ mice did not show a reduction in freezing behavior contrary to their wild-type littermates, indicating no within-session extinction in these animals (Fig. 1C). An ANOVA revealed no significant general effect of the number of CS presentation (phase: F5,126 = 1.59, p = 0.16), indicating such an unsuccessful extinction learning. But we observed a significant effect of the factor genotype (F1,126 = 18.64, p < 0.0001), indicating a different extinction performance between WT and BDNF+/ mice. However, we did not see a significant interaction between these two factors (genotype x phase: F5,126 = 1.26, p = 0.28). Twenty-four hours after the extinction training, we re-exposed the animals to the CS in the extinction context in order to test their extinction memory. Consistent with our results for the within-session extinction, we observed successful extinction memory in young animals for both genotypes, since BDNF+/ as well as the wild-type mice showed significantly less freezing in response to the CS compared to the beginning of the extinction training (paired t-test comparison, p’s < 0.05) (Fig. 1B). In contrast to the results for young animals but in accordance with our observation regarding the within-session extinction, in the 7 months old animals we observed a successful extinction memory only in wild-type mice (p < 0.05) whereas the BDNF+/ mice still showed a high amount of freezing behavior in response to the CS, which was not different from the freezing behavior at the beginning of the extinction training (p > 0.05). This result further demonstrates the impairment in fear extinction learning in 7 months old BDNF+/ mice. Importantly, the increased freezing in response to the CS in the 7 months old BDNF+/ mice is not due to any generalization of fear in these animals since we observed a similar low amount of freezing during the habituation period of the extinction memory test as in their wild-type littermates (Fig. 1D, p > 0.05). Although we verified before that our stronger conditioning protocol used for the older animals induced similar fear learning and
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Fig. 1. Fear extinction learning in 2 (A and B) and 7 (C and D) months old BDNF+/ and wild-type mice. (A) 2 months old animals showed no difference in extinction learning between the two genotypes, during the extinction training. Both genotypes showed a reduction of freezing behavior within the training session. (B) In the extinction memory test that was performed 24 h after the extinction training the 2 months old animals showed intact extinction memory and no elevated freezing behavior during the habituation phase of the memory test. (C) In contrast to the young animals, 7 months old BDNF+/ mice were not able to extinguish the cued fear memory, since they showed no reduction in freezing behavior during the extinction training. (D) In the extinction memory test, the 7 months old BDNF+/ mice did not show reduced cued fear behavior. In addition, they showed no elevated freezing during the habituation of the memory test, thus excluding a generalized fear behavior. (Indicates significant difference between BDNF+/ and wild- type mice, # indicates significant difference in comparison to the freezing behavior expressed at the beginning of the extinction training (CS1-3)).
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fear extinction in young WT mice, it might still be possible that the stronger fear conditioning protocol induced a different, more extinction-resistant type of fear learning in BDNF+/ mice. To address this issue, we performed an additional series of experiments, in which we applied the stronger fear conditioning protocol also to 2 months old BDNF+/ mice and observed the same results as seen in response to the weaker fear conditioning protocol: both genotypes showed intact within-session extinction learning (ANOVA: phase: F5,60 = 6.46, p < 0.0001, genotype: F1,60 = 1.16, p = 0.29; genotype phase: F5,60 = 1.01, p = 0.40) as well as a comparable extinction memory (p = 0.23). These results show that 2 months old animals, conditioned with the stronger protocol are able to form reliable fear extinction learning. Another reason that could account for the increased freezing behavior observed in older BDNF+/ mice during the extinction training is a potential generally reduced agility in these animals. To rule this out, we performed an open field test, which clearly showed that there are no differences in spontaneous activity behavior between the two genotypes (p = 0.13, t-test comparison) (Fig. 3). To test whether the age dependent decline in extinction learning is accompanied by reduced expression of BDNF protein, we performed ELISA measurements in different brain areas. The BDNF protein quantification showed a clear overall decline of protein concentration in BDNF+/ mice within the analyzed brain areas (Fig. 2; genotype: F1,58 = 87.7, p < 0.0001). Furthermore the BDNF levels generally decreased with age (age: F1,58 = 26.9, p < 0.0001) and there was a clear difference in BDNF protein levels between the analyzed brain areas (area: F2,58 = 16.6, p < 0.0001). Probably
due to similar changes in all of the different brain areas and genotypes the interaction of these three factors was not significant (genotype age area: F2,58 = 1.4, p = 0.21). In summary these data demonstrate that there is a decline of BDNF protein levels with age in all brain areas investigated in BDNF+/ and wild-type mice, which might underlie the observed learning deficit in adult BDNF+/ mice. In conclusion our results clearly demonstrate that in young adult (2 months) BDNF+/ mice fear extinction learning is still intact, whereas we observed a strong impairment in extinction learning in older adult (7 months) BDNF+/ mice. These older mice neither showed any within-session extinction nor any extinction memory in the memory retrieval test. These results support the notion that BDNF is critically involved in the formation of extinction memories. Furthermore, we could demonstrate that the learning deficit is accompanied by reduction of BDNF protein in three different brain areas which are relevant for extinction learning (Fig. 2), further stressing the impact of reduced levels of endogenous BDNF on fear extinction learning. Our results are in line with a recent study from Soliman and colleagues which demonstrated that mice and humans, carrying the single nucleotide Val66Met polymorphism in the BDNF gene, which leads to a reduced BDNF release, exhibit deficits in fear extinction learning (Soliman et al., 2010). Further evidence for a role of BDNF expression in extinction learning is provided by a study showing that extinction of conditioned fear results in histone acetylation in the BDNF gene promoter region in the prefrontal cortex, resulting in a simultaneous increase in BDNF mRNA expression in the prefrontal cortex (Bredy
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Fig. 2. BDNF protein concentration measured by ELISA in 2 and 7 months old BDNF+/ and wild-type mice in the hippocampus, amygdala and mPFC. BDNF+/ mice showed a significantly lower of BDNF protein level in all brain areas analyzed, and at both ages, compared to wild-type mice. The BDNF protein concentration in the hippocampus, amygdala and mPFC of 2 months old BDNF+/ and wild-type mice was significantly higher than the respective BDNF level in the 7 months mice. Seven months old BDNF+/ and wild-type animals showed significantly lower BDNF levels than the 2 months old wild-type mice in all brain areas investigated. 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323
et al., 2007). In this context it has been shown recently that a single infusion of BDNF in the IL is sufficient to induce extinction learning even without any extinction training (Peters et al., 2010). These authors suggested that the required BDNF for this extinction learning originates from hippocampal neurons, and these findings are in accordance with data from Heldt et al. (2007), who observed impaired extinction learning in a brain area specific knock-out of BDNF in the hippocampus. Interestingly, in addition to the extinction memory deficit, we observed no within-session extinction in the 7 months old BDNF+/ mice, indicating that also the acquisition of extinction memory might depend on BDNF. This observation is in line with results from Karpova et al. (2011) who also observed no distinctive within session extinction in untreated 3 months old BDNF+/ mice (Karpova et al., 2011). Since local interference with BDNF signaling in the BLA leaves the acquisition of extinction memories intact (Chhatwal et al., 2006), it seems reasonable to assume that there might be additional BDNF-dependent processes involved in the acquisition of extinction memories that are located outside the amygdala. Promising brain areas where such acquisition-related BDNF-dependent processes might take place could be the IL-mPFC and the hippocampus (see above). Since we observed an age-dependent reduction of BDNF-levels in both hippocampus and mPFC it remains unclear whether the observed deficit in the acquisition of fear extinction memories in older BDNF+/ mice is due to reduced BDNF levels in the hippocampus, mPFC or maybe both. Peters and colleagues demonstrated that individual animals with deficits in extinction learning displayed reduced BDNF levels in the hippocampus, but not in the amygdala or the IL-mPFC (Peters et al., 2010), suggesting an important role of hippocampal BDNF levels. It should be noted that our tissue preparation samples of the mPFC consist of both, the IL and the prelimbic (PL) sub-areas, thus making it impossible to differentiate whether the age-dependent decline of BDNF protein occurs only in the IL, the PL, or in both structures simultaneously. In a study by Choi and colleagues it has been shown that a specific knockdown of BDNF in the PL causes deficits in fear learning but not in fear extinction learning (Choi et al., 2010). However, in the present study we analyzed the levels of BDNF protein only in three brain areas that have been demonstrated to be crucially involved in fear extinction learning. Since BDNF+/ mice exhibit a chronic reduction of BDNF protein throughout the brain, it cannot be excluded that reduced BDNF levels also in other brain areas contribute to the observed extinction learning deficit. Importantly, we did not observe a generalization of fear in the older BDNF+/ mice, indicated by low freezing before onset of the
CS (habituation period) in the memory test. Furthermore, we observed no differences in spontaneous motor activity in the open field test. This observation is in line with previous results, demonstrating no altered spontaneous motor activity in 1–6 months old BDNF+/ mice (Endres & Lessmann, 2012). Hence it can be concluded that the lack of extinction learning in older BDNF+/ mice is not due to reduced activity of these mice. Our results demonstrate an age-dependent decline of BDNF protein in BDNF+/ mice (2 vs. 7 months) in three different brain areas that are all important in mediating fear extinction learning. These observations support previous studies demonstrating an agedependent decline of BDNF protein levels in wild-type animals (for review see von Bohlen & Halbach, 2010), as well as in BDNF+/ mice (Boger et al., 2011; Carreton et al., 2012). In addition our results support the observation that age-dependent changes in BDNF expression are differently regulated in different brain areas (e.g. Silhol et al., 2005). In general we observed a 50% reduction of BDNF protein in the hippocampus, amygdala and mPFC compared to wild-type mice, being also in line with previous studies (e.g. Abidin et al., 2006; Hill & van den Buuse, 2011; Kolbeck, Bartke, Eberle, & Barde, 1999). Interestingly, this overall reduction of BDNF levels to 50% seems to be still sufficient to mediate proper
Fig. 3. Distance traveled in the open field experiment of 7 months old BDNF+/ and wild-type mice. Animals of different genotypes showed no significant difference in activity during the experiment.
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fear learning and fear extinction in younger BDNF+/ mice (compare Endres & Lessmann, 2012), whereas a further much smaller decline during aging might provoke the observed learning deficit. This observation could indicate that there might be a specific threshold of BDNF that is required for proper learning of these tasks, which is somewhere below 50% of endogenous WT levels. To test this possibility it would be an interesting issue to analyze and compare the BDNF levels of trained animals and correlate this with the behavioral performance. Notably, previous studies in rats and mice have shown that fear extinction training has different consequences in very young as compared to adult animals, which was suggested to result by the formation of perineuronal nets (e.g. Gogolla, Caroni, Luthi, & Herry, 2009; Kim & Richardson, 2007; Quirk et al., 2010). However, these changes in learning mechanisms already occurred between postnatal day 16 and 23 in mice (Gogolla et al., 2009). Since the young adults in the present study were already 2 months of age it can be excluded that such a developmental change in learning mechanisms might underlie the age-dependent learning deficit in our BDNF+/ mice. The present investigation is the first study analyzing fear extinction learning in heterozygous BDNF-knockout mice in detail. We observed an age-dependent deficit in extinction learning of these mice, supporting the importance of endogenous BDNF also in fear extinction learning. This age-dependent learning impairment in older BDNF+/ mice seems to be a result from a decline of BDNF protein levels in the hippocampus, mPFC and amygdala with ongoing age, thus impairing the formation of new extinction memory traces. Our results suggest that BDNF+/ mice are a suitable model to further analyze the influence of reduced BDNF levels on the formation of extinction memory. Further, these results suggest that impaired fear extinction learning might be affected similarly in humans with reduced levels of endogenously released BDNF, such as suggested for carriers of the Val66Met polymorphism, or upon reduced BDNF expression at older ages.
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Acknowledgments
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This study was supported by the Deutsche Forschungsgemeinschaft, SFB 779/B6. The authors would like to thank Dr. Tanja Brigadski for establishing the BDNF ELISA, and Evelyn Friedl, Sybille Natho, Colette Obst, Margit Schmidt, Stephanie Holze and Angela Jahn for excellent technical assistance.
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Please cite this article in press as: Psotta, L., et al. Impaired fear extinction learning in adult heterozygous BDNF knock-out mice. Neurobiology of Learning and Memory (2013), http://dx.doi.org/10.1016/j.nlm.2013.03.003
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