- Email: [email protected]
Sleep deprivation alters phosphorylated CREB levels in the amygdala: Relationship with performance in a fear conditioning task
Behavioural Brain Research 236 (2013) 221–224
Contents lists available at SciVerse ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
Sleep deprivation alters phosphorylated CREB levels in the amygdala: Relationship with performance in a fear conditioning task Nadine Pinho a , Karin Monteiro Moreira a , Debora Cristina Hipolide a,∗ , Rita Sinigaglia-Coimbra b , Tatiana Lima Ferreira c , José N. Nobrega d , Sergio Tufik a , Maria Gabriela Menezes Oliveira a a
Departamento de Psicobiologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil Centro de Microscopia Eletrônica, Laboratório de Fisiopatologia Clínica e Experimental, Universidade Federal de São Paulo, São Paulo, SP, Brazil c Mathematics, Computation and Cognition Center, Universidade Federal do ABC, Santo Andre, SP, Brazil d Research Imaging Centre, Center for Addiction and Mental Health, Toronto, Canada b
h i g h l i g h t s We investigated pCREB expression in the amygdala and fear memory after sleep deprivation. Sleep deprivation reduced pCREB expression in the central amygdaloid nucleus. Sleep deprivation impaired fear memory retention. Twenty-four hours of sleep recovery restored pCREB expression. Twenty-four hours of sleep recovery prevented memory deficit.
a r t i c l e
i n f o
Article history: Received 9 January 2012 Received in revised form 21 August 2012 Accepted 28 August 2012 Available online 4 September 2012 Keywords: Memory Sleep CREB Amygdala Contextual fear conditioning
a b s t r a c t We investigated the relationship between deficits in fear memory induced by sleep deprivation and pCREB expression in the basal and central nuclei of the amygdala. Sleep deprivation reduced pCREB expression in the central nucleus compared to control or sleep recovered groups, and in the basal nucleus only compared to sleep recovered group. Moreover, 24 h of sleep recovery prior to training prevented changes in both pCREB expression and performance. © 2012 Elsevier B.V. All rights reserved.
Several studies indicate that sleep deprivation (SD) causes impairment in cognitive processes, including emotional memory, in humans and experimental animals [1,2]. In animals, SD causes deleterious effects on emotional long-term memory as evaluated by experimental paradigms such as context and tone fear conditioning, inhibitory avoidance and two-way active avoidance [3–5]. Consolidation of long-term fear memories is generally agreed to be dependent on gene transcription and protein synthesis in the amygdala [6]. A critical step in this process is the phosphorylation of CREB (cAMP responsive element binding protein) at the serine
∗ Corresponding author at: Universidade Federal de São Paulo, Rua Napoleão de Barros, 925 Vila Clementino, SP, 04024-002 São Paulo, Brazil. Tel.: +55 11 2149 0155; fax: +55 11 5572 5092. E-mail address: [email protected] (D.C. Hipolide). 0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2012.08.043
133 site and long-term fear memory can be facilitated by CREB over-expression in the basolateral amygdala [6]. Josselyn’s group showed that CREB influences the probability that specific amygdala neurons will participate in fear memory encoding and that the selective ablation of these neurons (with high CREB levels) is sufficient to erase fear memory [6]. Moreover, fear memory retrieval has been shown to induce CREB phosphorylation in the basolateral amygdala [7]. The fact that SD alters pCREB levels in many cerebral areas [8–10] suggests that alterations in CREB phosphorylation could play a role in the memory deficits induced by sleep loss. In general, studies evaluating mechanisms of memory deficits after SD have assessed changes in the hippocampus. The amygdala, a key structure in emotional learning and memory, has not been found by Vecsey et al. [9] to be affected by SD. However, in that study the amygdala was not probed with an aversive conditioning task.
222
N. Pinho et al. / Behavioural Brain Research 236 (2013) 221–224
In non-sleep deprived animals aversive challenges increased levels of phosphorylated CREB (pCREB) in several brain areas including the basolateral and central amygdaloid nuclei [11]. Recently, Hagewoud et al. [12] observed that impaired fear memory retrieval after SD is associated with a reduction in the normal pCREB increase in the amygdala after training in a contextual fear conditioning task. The purpose of the present study was to investigate the effects of different durations of SD on the acquisition of contextual fear conditioning and on pCREB levels in the basal and central nuclei of the amygdala. We were also interested in verifying whether sleep recovery might prevent SD-induced changes in behavior and in pCREB levels in the amygdala. Wistar male rats, 3–4 months old (340–510 g of body weight), bred and raised in the animal facility of the Department of Psychobiology of Universidade Federal de São Paulo, were used. Animals were maintained under controlled temperature (23 ± 2 ◦ C) and 12:12-h light–dark cycle (lights on at 7:00 a.m.) conditions. Rat chow and tap water were provided ad libitum. The procedures were conducted following the Ethical Committee and in accord with international rules for animal use and care. Rats were sleep deprived by the multiple platform method [13]. Prior to the sleep deprivation period, all animals had been submitted to 2 days sessions of habituation to the water tank environment for 1 h each day. Animals in the control group were subjected to the same habituation procedure daily until the time of behavioral testing, so that all animals experienced the same environmental conditions prior to the behavioral procedure. The behavioral test was conducted as described in Dametto et al. [3] with minor modifications. In the first experiment animals were sleep deprived for 24, 48 and 72 h (SD group) while control animals remained in their home cages in the same room where the sleep deprivation procedure took place (control cage – CC). Immediately following the sleep deprivation period, animals were trained in the contextual fear conditioning (CFC) task. For the training session the animals were individually placed in the conditioning apparatus and 2 min later five footshocks (0.6 mA, 1 s) were delivered, 30 s apart from each other. Thirty seconds after the last footshock, the animals were removed from the apparatus and returned to their home cages. The test session was performed 24 h after training. Each animal was placed in the same training context and no footshock was delivered. Freezing time – defined as complete immobility of the animal, with the absence of vibrissae movements and sniffing – was recorded continuously for 5 min by an observer blind to the animal group condition. Results are shown in Fig. 1A. Following a one-way ANOVA [F(3,44) = 3.18; p = 0.033], post hoc tests revealed that the 72 h SD group had significantly less freezing behavior than the control group (p < 0.05). A separate set of animals was subjected to the sleep deprivation procedure as described above to verify whether 24 h of recovery sleep before training could prevent the CFC memory impairment. One day before the SD group began the sleep deprivation protocol another group of animals also began a 72 h of sleep deprivation period at the end of which they were allowed to sleep for 24 h in their home cages (sleep recovery – SR). All three groups (CC, SD and SR) were then subjected to CFC training and testing as described for Experiment 1. Fig. 1B shows the effects of sleep recovery on CFC. Post hoc tests after a one-way ANOVA [F(2,33) = 10.58; p < 0.01] showed that the 72 h SD group had significantly less freezing behavior than CC and SR groups (p < 0.05). The third experiment was conducted to evaluate CREB and pCREB levels in the basal and central nuclei of the amygdala after CFC in CC, SD and SR groups. Immediately after the end of the sleep deprivation period or after 24 h of pre-training recovery sleep, animals were trained in the CFC as described above. Three hours after CFC training, the rats were anesthetized by i.p. injection of 90 mg/kg ketamine and 10 mg/kg of xylazine and transcardially perfused
Fig. 1. Effects of pre-training sleep deprivation (24, 48 and 72 h) (A) and 72 h pre-training sleep deprivation followed by 24 h of sleep recovery (B) on freezing response in a contextual fear conditioning test. Data are presented as freezing time (mean ± S.E.M.) during 5 min test. Number of animals per group: 12; *p < 0.05 when compared to control group; #p < 0.05 when compared to sleep recovery group.
with saline 0.9% for 30 s followed by 150 ml of paraformaldehyde (PFA 4% in 0.1 M sodium phosphate buffer, pH 7.2). The time point for pCREB and CREB assessment was chosen based on Stanciu [11]. Brains were collected, cryoprotected in 40% sucrose-phosphate buffer for 3 days, then frozen at −20 ◦ C for 24 h and then sliced coronally into 30 m sections (cryostat), from the beginning to the end of the amygdala, at every fifth section. Two consecutive sections were collected for CREB and pCREB immunohistochemistry respectively. One series of sections was processed using anti-CREB monoclonal rabbit IgG at 1:1500 dilution (Cell Signaling, Beverly, MA) and the next series with anti-pCREB polyclonal rabbit IgG at 1:500 dilution (Cell Signaling, Beverly, MA). Amygdala nuclei (basal and central) were identified according to Paxinos and Watson [14]. The analyzed sections typically extended from bregma to −1.8 mm to −3.14 mm (central nucleus) and from bregma to −2.12 mm to −3.30 mm (basal nucleus). Slides were viewed and photographed at 100× magnification on a light microscope connected to an Olympus DP712 camera. Six photomicrographs were taken of each nucleus in each cerebral hemisphere and positive CREB and pCREB cells were counted using the NIH Image J program. Three 40 m × 40 m windows were overlapped on the photomicrographs for delimitation of the fields of cellular counting with two lines of inclusion and two lines of exclusion. A small sampling window was placed on the central part of these nuclei since CREB and pCREB were homogenously expressed throughout central and basal amygdala nucleus in the sections. Every immunoreactive cell within the 40 m × 40 m area (including inclusion lines) was visually counted. The total area analyzed for each nucleus was 4800 m2 on each hemisphere.
N. Pinho et al. / Behavioural Brain Research 236 (2013) 221–224
223
Fig. 2. Number of CREB-positive cells (A and B) and pCREB:CREB ratio (C and D) in basal (left column) and central nuclei (right column) of the amygdala after 72 h of paradoxical sleep deprivation (PSD 72 h) and sleep recovery (24 h of sleep recovery after 72 h of paradoxical sleep deprivation) followed by contextual fear conditioning training. Representative brain section (CREB-immunoreactivity) showing size (scale bar, 200 m) and location of the central (CE) and basal (BA) nuclei of the amygdala (E). Data are presented as mean ± S.E.M. Number of animals per group: 9 with exception of group PSD 72 h (n = 8). *p < 0.05 when compared to control group and # when compared to sleep recovery group.
The number of CREB immunoreactive cells and the ratio pCREB:CREB were analyzed by one-way ANOVA with group (CC, SD and SR) as factor. When necessary, the analysis was followed by the Newman–Keuls significant difference test, with the level of significance set at p < 0.05. Fig. 2 shows the effects of sleep deprivation and 24 h of sleep recovery prior to training on CREB (A and B) and pCREB:CREB ratio immunoreactivity (C and D). The number of CREB-immunoreactive cells exhibited no change after sleep deprivation or sleep recovery either in the basal (Fig. 2A) or central nuclei (Fig. 2B). Fig. 2C and D shows pCREB levels in basal and central nuclei of amygdala, respectively. One-way ANOVAs indicated significant differences in pCREB:CREB in the basal [F(2,23) = 3.69; p = 0.040] and central nuclei [F(2,23) = 4.18; p = 0.028]. Post hoc tests revealed that in the basal nucleus animals deprived of sleep for 72 h had significantly less pCREB:CREB immunoreactive cells than animals in the 24 h of pre-training recovery sleep group (p < 0.05). In the central nucleus the sleep deprived animals had significantly less pCREB:CREB-immunoreactive cells than the control and the 24 h of pre-training recovery sleep groups (p < 0.05). The number of CREB and pCREB-immunoreactive cells was also analyzed in the dorsal striatum as a control for possible widespread pCREB effects after SD. For this brain area ANOVA did not show differences for CREB [F(2,23) = 1.08; p = 0.35] or pCREB [F(2,23) = 0.47; p = 0.63]. In the present study it was observed that 72 h, but not 24 or 48 h, of SD disrupted performance in the CFC task. Similarly, a previous study in our laboratory had shown that 24 h of SD did not impair either tone fear conditioning or inhibitory avoidance, two other tasks that are frequently used to evaluate emotional memory [15]. Indeed, the impaired performance of 72 h of SD prior to CFC training was prevented by 24 h of sleep recovery before training. It was also
found that 72 h of SD reduced the number of pCREB-positive cells in the central amygdaloid nucleus and that 24 h of sleep recovery was sufficient to restore pCREB to control levels. The same pattern of results was observed in the basal amygdala, where pCREB levels were reduced in SD rats compared to the animals that had 24 h of pre-training recovery sleep, although the reduction did not reach statistical difference when compared to controls. The amygdala is generally agreed to have a key role in emotional memory processes. In a well accepted conceptualization of the neural substrates of fear conditioning, the basal amygdala is thought to receive contextual information from the hippocampus and sensory information from the thalamus and then integrate these inputs. The basal amygdala has direct projections to the central amygdaloid nucleus, which is essential for integration of behavioral, autonomic and hormonal fear conditioning responses [16]. Moreover, CREB phosphorylation seems to play a role in amydala-mediated memory processes as suggested by the observation of increased pCREB levels in central amygdaloid nucleus after contextual fear conditioning training [11]. It seems reasonable to suppose that the reduction in CREB phosphorylation observed in sleep-deprived animals may be one of the factors that could explain the deleterious effects of sleep loss on cognitive tasks. When pre-training SD was followed by 24 h of sleep recovery, both the impairment in CFC performance and the reduction in CREB phosphorylation were prevented. Machado et al. [17] noted that after 4 days of SD a complete recovery of the sleep/wake cycle occurs within 24 h. Recent studies have shown that pCREB changes in cortical areas are related to sleep/wake states [8]. Vecsey et al. [9] found significant pCREB alterations in hippocampus but not in the basolateral amygdala complex after 5 h of total sleep deprivation. In the present study 72 h of SD changed pCREB:CREB ratios in two amygdala
224
N. Pinho et al. / Behavioural Brain Research 236 (2013) 221–224
nuclei. It should be noted however that in the present study sleepdeprived animals were also exposed to an aversive memory task which is known to increase in pCREB levels in the amygdala. Thus while sleep deprivation per se may not affect pCREB levels in the amygdala [9], when this structure is recruited by emotional memory processing sleep deprivation may prevent the increases in pCREB levels. In our study, no changes in non-phosphorylated CREB immunoreactivity were detected. This suggests that the observed pCREB:CREB alterations are not due to a change in the total amount of nuclear CREB, but may indeed reflect changes in phosphorylation processes. In addition we analyzed the number of CREB and pCREBimmunoreactive cells in dorsal striatum, a structure not involved in the CFC [18], as a control for possible widespread pCREB effects. While sleep deprivation in other test conditions seems to affect CREB expression in the dorsal striatum [19], we did not observe differences in CREB and pCREB levels after SD and recovery in striatal neurons. In short, we show that 72 h of SD prior to training impairs contextual fear conditioning and that this impairment is accompanied by changes in immunoreactive pCREB in the amygdala. Moreover, 24 h of sleep recovery prevented alterations in aversive learning behavior and in amygdaloid pCREB levels. Conflict of interest statement This was not an industry supported study. The authors have indicated no financial conflicts of interest. Acknowledgments This work was conducted in the Department of Psychobiology of the Universidade Federal de Sao Paulo, Sao Paulo, Brazil. Supported by AFIP, FAPESP. References [1] Walker MP. Cognitive consequences of sleep and sleep loss. Sleep Medicine 2008;9(Suppl. 1):S29–34. [2] Diekelmann S, Wilhelm I, Born J. The whats and whens of sleep-dependent memory consolidation. Sleep Medicine Reviews 2009;13:309–21 [Epub 2009 February 28].
[3] Dametto M, Suchecki D, Bueno OF, Moreira KM, Tufik S, Oliveira MG. Social stress does not interact with paradoxical sleep deprivation-induced memory impairment. Behavioural Brain Research 2002;129:171–8. [4] Moreira KM, Ferreira TL, Hipolide DC, Fornari RV, Tufik S, Oliveira MG. Modafinil prevents inhibitory avoidance memory deficit induced by sleep deprivation in rats. Sleep 2010;33(7):990–3. [5] Datta S, Mavanji V, Ulloor J, Patterson EH. Activation of phasic pontine-wave generator prevents rapid eye movement sleep deprivation-induced learning impairment in the rat: a mechanism for sleep-dependent plasticity. Journal of Neuroscience 2004;24(6):1416–27. [6] Josselyn SA. Continuing the search for the engram: examining the mechanism of fear memories. Journal of Psychiatry and Neuroscience 2010;35: 221–8. [7] Hall J, Thomas KL, Everitt BJ. Fear memory retrieval induces CREB phosphorylation and Fos expression within the amygdala. European Journal of Neuroscience 2001;13:1453–8. [8] Cirelli C, Tononi G. Differential expression of plasticity-related genes in waking and sleep and their regulation by the noradrenergic system. Journal of Neuroscience 2000;20:9187–94. [9] Vecsey CG, Baillie GS, Jaganath D, Havekes R, Daniels A, Wimmer M, et al. Sleep deprivation impairs cAMP signalling in the hippocampus. Nature 2009;461:1122–5. [10] Xu ZQ, Gao CY, Fang CQ, Zhou HD, Jiang XJ. The mechanism and characterization of learning and memory impairment in sleep-deprived mice. Cell Biochemistry and Biophysics 2010;58:137–40. [11] Stanciu M, Radulovic J, Spiess J. Phosphorylated cAMP response element binding protein in the mouse brain after fear conditioning: relationship to Fos production. Brain Research Molecular Brain Research 2001;94: 15–24. [12] Hagewoud R, Bultsma LJ, Barf RP, Koolhaas JM, Meerlo P. Sleep deprivation impairs contextual fear conditioning and attenuates subsequent behavioural, endocrine and neuronal responses. Journal of Sleep Research 2011;20(2):259–66. [13] Suchecki D, Tufik S. Social stability attenuates the stress in the modified multiple platform method for paradoxical sleep deprivation in the rat. Physiology and Behavior 2000;68:309–16. [14] Paxinos G, Watson C, editors. The rat brain in stereotaxic coordinates. New York: Academic Press; 1986. [15] Bueno OF, Lobo LL, Oliveira MG, Gugliano EB, Pomarico AC, Tufik S. Dissociated paradoxical sleep deprivation effects on inhibitory avoidance and conditioned fear. Physiology and Behavior 1994;56(4):775–9. [16] LeDoux JE. Emotion circuits in the brain. Annual Review of Neuroscience 2000;23:155–84. [17] Machado RB, Hipólide DC, Benedito-Silva AA, Tufik S. Sleep deprivation induced by the modified multiple platform technique: quantification of sleep loss and recovery. Brain Research 2004;1004:45–51. [18] Ferreira TL, Shammah-Lagnado SJ, Bueno OF, Moreira KM, Fornari RV, Oliveira MG. The indirect amygdala-dorsal striatum pathway mediates conditioned freezing: insights on emotional memory networks. Neuroscience 2008;153(1):84–94. [19] Hagewoud R, Havekes R, Tiba PA, Novati A, Hogenelst K, Weinreder P, et al. Coping with sleep deprivation: shifts in regional brain activity and learning strategy. Sleep 2010;33(11):1465–73.
Contents lists available at SciVerse ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
Sleep deprivation alters phosphorylated CREB levels in the amygdala: Relationship with performance in a fear conditioning task Nadine Pinho a , Karin Monteiro Moreira a , Debora Cristina Hipolide a,∗ , Rita Sinigaglia-Coimbra b , Tatiana Lima Ferreira c , José N. Nobrega d , Sergio Tufik a , Maria Gabriela Menezes Oliveira a a
Departamento de Psicobiologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil Centro de Microscopia Eletrônica, Laboratório de Fisiopatologia Clínica e Experimental, Universidade Federal de São Paulo, São Paulo, SP, Brazil c Mathematics, Computation and Cognition Center, Universidade Federal do ABC, Santo Andre, SP, Brazil d Research Imaging Centre, Center for Addiction and Mental Health, Toronto, Canada b
h i g h l i g h t s We investigated pCREB expression in the amygdala and fear memory after sleep deprivation. Sleep deprivation reduced pCREB expression in the central amygdaloid nucleus. Sleep deprivation impaired fear memory retention. Twenty-four hours of sleep recovery restored pCREB expression. Twenty-four hours of sleep recovery prevented memory deficit.
a r t i c l e
i n f o
Article history: Received 9 January 2012 Received in revised form 21 August 2012 Accepted 28 August 2012 Available online 4 September 2012 Keywords: Memory Sleep CREB Amygdala Contextual fear conditioning
a b s t r a c t We investigated the relationship between deficits in fear memory induced by sleep deprivation and pCREB expression in the basal and central nuclei of the amygdala. Sleep deprivation reduced pCREB expression in the central nucleus compared to control or sleep recovered groups, and in the basal nucleus only compared to sleep recovered group. Moreover, 24 h of sleep recovery prior to training prevented changes in both pCREB expression and performance. © 2012 Elsevier B.V. All rights reserved.
Several studies indicate that sleep deprivation (SD) causes impairment in cognitive processes, including emotional memory, in humans and experimental animals [1,2]. In animals, SD causes deleterious effects on emotional long-term memory as evaluated by experimental paradigms such as context and tone fear conditioning, inhibitory avoidance and two-way active avoidance [3–5]. Consolidation of long-term fear memories is generally agreed to be dependent on gene transcription and protein synthesis in the amygdala [6]. A critical step in this process is the phosphorylation of CREB (cAMP responsive element binding protein) at the serine
∗ Corresponding author at: Universidade Federal de São Paulo, Rua Napoleão de Barros, 925 Vila Clementino, SP, 04024-002 São Paulo, Brazil. Tel.: +55 11 2149 0155; fax: +55 11 5572 5092. E-mail address: [email protected] (D.C. Hipolide). 0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2012.08.043
133 site and long-term fear memory can be facilitated by CREB over-expression in the basolateral amygdala [6]. Josselyn’s group showed that CREB influences the probability that specific amygdala neurons will participate in fear memory encoding and that the selective ablation of these neurons (with high CREB levels) is sufficient to erase fear memory [6]. Moreover, fear memory retrieval has been shown to induce CREB phosphorylation in the basolateral amygdala [7]. The fact that SD alters pCREB levels in many cerebral areas [8–10] suggests that alterations in CREB phosphorylation could play a role in the memory deficits induced by sleep loss. In general, studies evaluating mechanisms of memory deficits after SD have assessed changes in the hippocampus. The amygdala, a key structure in emotional learning and memory, has not been found by Vecsey et al. [9] to be affected by SD. However, in that study the amygdala was not probed with an aversive conditioning task.
222
N. Pinho et al. / Behavioural Brain Research 236 (2013) 221–224
In non-sleep deprived animals aversive challenges increased levels of phosphorylated CREB (pCREB) in several brain areas including the basolateral and central amygdaloid nuclei [11]. Recently, Hagewoud et al. [12] observed that impaired fear memory retrieval after SD is associated with a reduction in the normal pCREB increase in the amygdala after training in a contextual fear conditioning task. The purpose of the present study was to investigate the effects of different durations of SD on the acquisition of contextual fear conditioning and on pCREB levels in the basal and central nuclei of the amygdala. We were also interested in verifying whether sleep recovery might prevent SD-induced changes in behavior and in pCREB levels in the amygdala. Wistar male rats, 3–4 months old (340–510 g of body weight), bred and raised in the animal facility of the Department of Psychobiology of Universidade Federal de São Paulo, were used. Animals were maintained under controlled temperature (23 ± 2 ◦ C) and 12:12-h light–dark cycle (lights on at 7:00 a.m.) conditions. Rat chow and tap water were provided ad libitum. The procedures were conducted following the Ethical Committee and in accord with international rules for animal use and care. Rats were sleep deprived by the multiple platform method [13]. Prior to the sleep deprivation period, all animals had been submitted to 2 days sessions of habituation to the water tank environment for 1 h each day. Animals in the control group were subjected to the same habituation procedure daily until the time of behavioral testing, so that all animals experienced the same environmental conditions prior to the behavioral procedure. The behavioral test was conducted as described in Dametto et al. [3] with minor modifications. In the first experiment animals were sleep deprived for 24, 48 and 72 h (SD group) while control animals remained in their home cages in the same room where the sleep deprivation procedure took place (control cage – CC). Immediately following the sleep deprivation period, animals were trained in the contextual fear conditioning (CFC) task. For the training session the animals were individually placed in the conditioning apparatus and 2 min later five footshocks (0.6 mA, 1 s) were delivered, 30 s apart from each other. Thirty seconds after the last footshock, the animals were removed from the apparatus and returned to their home cages. The test session was performed 24 h after training. Each animal was placed in the same training context and no footshock was delivered. Freezing time – defined as complete immobility of the animal, with the absence of vibrissae movements and sniffing – was recorded continuously for 5 min by an observer blind to the animal group condition. Results are shown in Fig. 1A. Following a one-way ANOVA [F(3,44) = 3.18; p = 0.033], post hoc tests revealed that the 72 h SD group had significantly less freezing behavior than the control group (p < 0.05). A separate set of animals was subjected to the sleep deprivation procedure as described above to verify whether 24 h of recovery sleep before training could prevent the CFC memory impairment. One day before the SD group began the sleep deprivation protocol another group of animals also began a 72 h of sleep deprivation period at the end of which they were allowed to sleep for 24 h in their home cages (sleep recovery – SR). All three groups (CC, SD and SR) were then subjected to CFC training and testing as described for Experiment 1. Fig. 1B shows the effects of sleep recovery on CFC. Post hoc tests after a one-way ANOVA [F(2,33) = 10.58; p < 0.01] showed that the 72 h SD group had significantly less freezing behavior than CC and SR groups (p < 0.05). The third experiment was conducted to evaluate CREB and pCREB levels in the basal and central nuclei of the amygdala after CFC in CC, SD and SR groups. Immediately after the end of the sleep deprivation period or after 24 h of pre-training recovery sleep, animals were trained in the CFC as described above. Three hours after CFC training, the rats were anesthetized by i.p. injection of 90 mg/kg ketamine and 10 mg/kg of xylazine and transcardially perfused
Fig. 1. Effects of pre-training sleep deprivation (24, 48 and 72 h) (A) and 72 h pre-training sleep deprivation followed by 24 h of sleep recovery (B) on freezing response in a contextual fear conditioning test. Data are presented as freezing time (mean ± S.E.M.) during 5 min test. Number of animals per group: 12; *p < 0.05 when compared to control group; #p < 0.05 when compared to sleep recovery group.
with saline 0.9% for 30 s followed by 150 ml of paraformaldehyde (PFA 4% in 0.1 M sodium phosphate buffer, pH 7.2). The time point for pCREB and CREB assessment was chosen based on Stanciu [11]. Brains were collected, cryoprotected in 40% sucrose-phosphate buffer for 3 days, then frozen at −20 ◦ C for 24 h and then sliced coronally into 30 m sections (cryostat), from the beginning to the end of the amygdala, at every fifth section. Two consecutive sections were collected for CREB and pCREB immunohistochemistry respectively. One series of sections was processed using anti-CREB monoclonal rabbit IgG at 1:1500 dilution (Cell Signaling, Beverly, MA) and the next series with anti-pCREB polyclonal rabbit IgG at 1:500 dilution (Cell Signaling, Beverly, MA). Amygdala nuclei (basal and central) were identified according to Paxinos and Watson [14]. The analyzed sections typically extended from bregma to −1.8 mm to −3.14 mm (central nucleus) and from bregma to −2.12 mm to −3.30 mm (basal nucleus). Slides were viewed and photographed at 100× magnification on a light microscope connected to an Olympus DP712 camera. Six photomicrographs were taken of each nucleus in each cerebral hemisphere and positive CREB and pCREB cells were counted using the NIH Image J program. Three 40 m × 40 m windows were overlapped on the photomicrographs for delimitation of the fields of cellular counting with two lines of inclusion and two lines of exclusion. A small sampling window was placed on the central part of these nuclei since CREB and pCREB were homogenously expressed throughout central and basal amygdala nucleus in the sections. Every immunoreactive cell within the 40 m × 40 m area (including inclusion lines) was visually counted. The total area analyzed for each nucleus was 4800 m2 on each hemisphere.
N. Pinho et al. / Behavioural Brain Research 236 (2013) 221–224
223
Fig. 2. Number of CREB-positive cells (A and B) and pCREB:CREB ratio (C and D) in basal (left column) and central nuclei (right column) of the amygdala after 72 h of paradoxical sleep deprivation (PSD 72 h) and sleep recovery (24 h of sleep recovery after 72 h of paradoxical sleep deprivation) followed by contextual fear conditioning training. Representative brain section (CREB-immunoreactivity) showing size (scale bar, 200 m) and location of the central (CE) and basal (BA) nuclei of the amygdala (E). Data are presented as mean ± S.E.M. Number of animals per group: 9 with exception of group PSD 72 h (n = 8). *p < 0.05 when compared to control group and # when compared to sleep recovery group.
The number of CREB immunoreactive cells and the ratio pCREB:CREB were analyzed by one-way ANOVA with group (CC, SD and SR) as factor. When necessary, the analysis was followed by the Newman–Keuls significant difference test, with the level of significance set at p < 0.05. Fig. 2 shows the effects of sleep deprivation and 24 h of sleep recovery prior to training on CREB (A and B) and pCREB:CREB ratio immunoreactivity (C and D). The number of CREB-immunoreactive cells exhibited no change after sleep deprivation or sleep recovery either in the basal (Fig. 2A) or central nuclei (Fig. 2B). Fig. 2C and D shows pCREB levels in basal and central nuclei of amygdala, respectively. One-way ANOVAs indicated significant differences in pCREB:CREB in the basal [F(2,23) = 3.69; p = 0.040] and central nuclei [F(2,23) = 4.18; p = 0.028]. Post hoc tests revealed that in the basal nucleus animals deprived of sleep for 72 h had significantly less pCREB:CREB immunoreactive cells than animals in the 24 h of pre-training recovery sleep group (p < 0.05). In the central nucleus the sleep deprived animals had significantly less pCREB:CREB-immunoreactive cells than the control and the 24 h of pre-training recovery sleep groups (p < 0.05). The number of CREB and pCREB-immunoreactive cells was also analyzed in the dorsal striatum as a control for possible widespread pCREB effects after SD. For this brain area ANOVA did not show differences for CREB [F(2,23) = 1.08; p = 0.35] or pCREB [F(2,23) = 0.47; p = 0.63]. In the present study it was observed that 72 h, but not 24 or 48 h, of SD disrupted performance in the CFC task. Similarly, a previous study in our laboratory had shown that 24 h of SD did not impair either tone fear conditioning or inhibitory avoidance, two other tasks that are frequently used to evaluate emotional memory [15]. Indeed, the impaired performance of 72 h of SD prior to CFC training was prevented by 24 h of sleep recovery before training. It was also
found that 72 h of SD reduced the number of pCREB-positive cells in the central amygdaloid nucleus and that 24 h of sleep recovery was sufficient to restore pCREB to control levels. The same pattern of results was observed in the basal amygdala, where pCREB levels were reduced in SD rats compared to the animals that had 24 h of pre-training recovery sleep, although the reduction did not reach statistical difference when compared to controls. The amygdala is generally agreed to have a key role in emotional memory processes. In a well accepted conceptualization of the neural substrates of fear conditioning, the basal amygdala is thought to receive contextual information from the hippocampus and sensory information from the thalamus and then integrate these inputs. The basal amygdala has direct projections to the central amygdaloid nucleus, which is essential for integration of behavioral, autonomic and hormonal fear conditioning responses [16]. Moreover, CREB phosphorylation seems to play a role in amydala-mediated memory processes as suggested by the observation of increased pCREB levels in central amygdaloid nucleus after contextual fear conditioning training [11]. It seems reasonable to suppose that the reduction in CREB phosphorylation observed in sleep-deprived animals may be one of the factors that could explain the deleterious effects of sleep loss on cognitive tasks. When pre-training SD was followed by 24 h of sleep recovery, both the impairment in CFC performance and the reduction in CREB phosphorylation were prevented. Machado et al. [17] noted that after 4 days of SD a complete recovery of the sleep/wake cycle occurs within 24 h. Recent studies have shown that pCREB changes in cortical areas are related to sleep/wake states [8]. Vecsey et al. [9] found significant pCREB alterations in hippocampus but not in the basolateral amygdala complex after 5 h of total sleep deprivation. In the present study 72 h of SD changed pCREB:CREB ratios in two amygdala
224
N. Pinho et al. / Behavioural Brain Research 236 (2013) 221–224
nuclei. It should be noted however that in the present study sleepdeprived animals were also exposed to an aversive memory task which is known to increase in pCREB levels in the amygdala. Thus while sleep deprivation per se may not affect pCREB levels in the amygdala [9], when this structure is recruited by emotional memory processing sleep deprivation may prevent the increases in pCREB levels. In our study, no changes in non-phosphorylated CREB immunoreactivity were detected. This suggests that the observed pCREB:CREB alterations are not due to a change in the total amount of nuclear CREB, but may indeed reflect changes in phosphorylation processes. In addition we analyzed the number of CREB and pCREBimmunoreactive cells in dorsal striatum, a structure not involved in the CFC [18], as a control for possible widespread pCREB effects. While sleep deprivation in other test conditions seems to affect CREB expression in the dorsal striatum [19], we did not observe differences in CREB and pCREB levels after SD and recovery in striatal neurons. In short, we show that 72 h of SD prior to training impairs contextual fear conditioning and that this impairment is accompanied by changes in immunoreactive pCREB in the amygdala. Moreover, 24 h of sleep recovery prevented alterations in aversive learning behavior and in amygdaloid pCREB levels. Conflict of interest statement This was not an industry supported study. The authors have indicated no financial conflicts of interest. Acknowledgments This work was conducted in the Department of Psychobiology of the Universidade Federal de Sao Paulo, Sao Paulo, Brazil. Supported by AFIP, FAPESP. References [1] Walker MP. Cognitive consequences of sleep and sleep loss. Sleep Medicine 2008;9(Suppl. 1):S29–34. [2] Diekelmann S, Wilhelm I, Born J. The whats and whens of sleep-dependent memory consolidation. Sleep Medicine Reviews 2009;13:309–21 [Epub 2009 February 28].
[3] Dametto M, Suchecki D, Bueno OF, Moreira KM, Tufik S, Oliveira MG. Social stress does not interact with paradoxical sleep deprivation-induced memory impairment. Behavioural Brain Research 2002;129:171–8. [4] Moreira KM, Ferreira TL, Hipolide DC, Fornari RV, Tufik S, Oliveira MG. Modafinil prevents inhibitory avoidance memory deficit induced by sleep deprivation in rats. Sleep 2010;33(7):990–3. [5] Datta S, Mavanji V, Ulloor J, Patterson EH. Activation of phasic pontine-wave generator prevents rapid eye movement sleep deprivation-induced learning impairment in the rat: a mechanism for sleep-dependent plasticity. Journal of Neuroscience 2004;24(6):1416–27. [6] Josselyn SA. Continuing the search for the engram: examining the mechanism of fear memories. Journal of Psychiatry and Neuroscience 2010;35: 221–8. [7] Hall J, Thomas KL, Everitt BJ. Fear memory retrieval induces CREB phosphorylation and Fos expression within the amygdala. European Journal of Neuroscience 2001;13:1453–8. [8] Cirelli C, Tononi G. Differential expression of plasticity-related genes in waking and sleep and their regulation by the noradrenergic system. Journal of Neuroscience 2000;20:9187–94. [9] Vecsey CG, Baillie GS, Jaganath D, Havekes R, Daniels A, Wimmer M, et al. Sleep deprivation impairs cAMP signalling in the hippocampus. Nature 2009;461:1122–5. [10] Xu ZQ, Gao CY, Fang CQ, Zhou HD, Jiang XJ. The mechanism and characterization of learning and memory impairment in sleep-deprived mice. Cell Biochemistry and Biophysics 2010;58:137–40. [11] Stanciu M, Radulovic J, Spiess J. Phosphorylated cAMP response element binding protein in the mouse brain after fear conditioning: relationship to Fos production. Brain Research Molecular Brain Research 2001;94: 15–24. [12] Hagewoud R, Bultsma LJ, Barf RP, Koolhaas JM, Meerlo P. Sleep deprivation impairs contextual fear conditioning and attenuates subsequent behavioural, endocrine and neuronal responses. Journal of Sleep Research 2011;20(2):259–66. [13] Suchecki D, Tufik S. Social stability attenuates the stress in the modified multiple platform method for paradoxical sleep deprivation in the rat. Physiology and Behavior 2000;68:309–16. [14] Paxinos G, Watson C, editors. The rat brain in stereotaxic coordinates. New York: Academic Press; 1986. [15] Bueno OF, Lobo LL, Oliveira MG, Gugliano EB, Pomarico AC, Tufik S. Dissociated paradoxical sleep deprivation effects on inhibitory avoidance and conditioned fear. Physiology and Behavior 1994;56(4):775–9. [16] LeDoux JE. Emotion circuits in the brain. Annual Review of Neuroscience 2000;23:155–84. [17] Machado RB, Hipólide DC, Benedito-Silva AA, Tufik S. Sleep deprivation induced by the modified multiple platform technique: quantification of sleep loss and recovery. Brain Research 2004;1004:45–51. [18] Ferreira TL, Shammah-Lagnado SJ, Bueno OF, Moreira KM, Fornari RV, Oliveira MG. The indirect amygdala-dorsal striatum pathway mediates conditioned freezing: insights on emotional memory networks. Neuroscience 2008;153(1):84–94. [19] Hagewoud R, Havekes R, Tiba PA, Novati A, Hogenelst K, Weinreder P, et al. Coping with sleep deprivation: shifts in regional brain activity and learning strategy. Sleep 2010;33(11):1465–73.