262 R.Hagewoud et al. RESULTS (a) 50 Experiment 1:freezing behaviour and CORT response upon re-exposure to shock context 40 As expected,rats that were sleep-deprived for 6 h immediately following training (SDT)showed reduced freezing upon re- 8 exposure to the shock context 24 h after training compared 30 with control animals(T)(17.3±3.9%versus33.6±6.4%， with n=10 and n=9,respectively,in the two groups; 20 117=2.194,P=0.046:Fig.1a). Re-exposure to the shock context induced a clear CORT 10 response in all animals (repeated-measures ANOvA,time effect: F3.42 =73.26,P <0.01).However,this response was reduced significantly in SDT animals compared to T animals (treat- 0 SDT ment x time interaction:F3.42 =3.42,P<0.05;see Fig.Ib). Post-hoc t-tests showed that the plasma levels of CORT in (b)251 the SDT animals were significantly lower than those of the -O-T T animals at t 30 and 60 min (P<0.05 in both cases). ◆SDT Due to missing samples,three animals (one T and two SDT) 20 ★ were excluded from the CORT analysis. 15 Experiment 2:neuronal activation upon re-exposure to shock 骂 context 10 Again,rats that were sleep-deprived for 6 h immediately following training (SDT)showed reduced freezing upon re- 5 exposure to the shock box 24 h after training compared with ● trained,non-sleep-deprived control animals(T)(22.5+3.4% versus 35.2+5.0%,with n 10 and n =9,respectively; 117=2.147,P=0.047. 10 2030 40 50 鸟 A representative picture of pCREB expression in the Time(min) hippocampus is shown in Fig.2a.In the hippocampal sections Figure 1.Effect of brief sleep deprivation(SD)following training on that were collected I h after testing for contextual fear,the freezing behaviour and corticosterone (CORT)response upon re- optical density of pCREB immunoreactivity was affected by exposure to the shock context.(a)Twenty-four hours after training all the experimental treatment in the CAl area(F223 12.665, animals were tested for contextual fear during a 5-min test phase. Animals sleep-deprived for 6 h immediately following training(SDT. P<0.01),but not in the CA3 area (F2.23 1.051,P>0.3) n=10)displayed significantly less freezing behaviour in response to and DG (F2.23 =0.295,P>0.3)(Fig.2b-d).Trained rats the shock context than control animals (T,n 9).(b)CORT response that were re-exposed to the shock context (T)had a significant upon contextual fear-testing in trained animals (T,n=8)and animals increase in pCREB expression in the CAl area compared to subjected to 6 h of SD(SDT,n=8)immediately after training in the HCC animals (P<0.01).This increase was still visible in the contextual fear conditioning paradigm.Plasma CORT levels were animals subjected to brief SD immediately following training significantly lower in SDT animals compared to T animals at t=30 and 60 min.Data are expressed as mean standard error of the (SDT versus HCC,P 0.05),but it was significantly smaller mean.*P 0.05 than in the trained,non-sleep-deprived control animals(SDT versus T,P 0.05). A representative picture of pCREB expression in the increase did not reach statistical significance when compared amygdala is shown in Fig.3a.Due to missing sections,two to the HCC group (P>0.4 for both subregions). animals (one T and one SDT animal)were excluded from analysis.ANOvA revealed a significant main treatment effect for DISCUSSION the number of pCREB-positive cells in the BLA as well as in the CeN(F2.21=3.901,P<0.05andf2.21=3.528, In the present study we confirm the negative effects of brief P 0.05,respectively)(Fig.3b,c).Specifically,the number SD on the consolidation of contextual fear memory. of pCREB-positive cells in the BLA and CeN was higher in We show that 6 h of SD immediately following a mild foot animals subjected to contextual fear-testing (T)compared with shock leads to a reduced freezing response upon re-exposure animals in the HCC group (post-hoc Tukey test,P<0.05 for to the shock box the next day.In addition to this attenuated both regions).Although the number of pCREB immunoreac- behavioural response,the SD animals also displayed a redu- tive cells was also elevated slightly in the SDT group,this ced neuroendocrine activation,as shown by an attenuated 2010 European Sleep Research Society,J.Sleep Res..20,259-266RESULTS Experiment 1: freezing behaviour and CORT response upon re-exposure to shock context As expected, rats that were sleep-deprived for 6 h immediately following training (SDT) showed reduced freezing upon re￾exposure to the shock context 24 h after training compared with control animals (T) (17.3 ± 3.9% versus 33.6 ± 6.4%, with n = 10 and n = 9, respectively, in the two groups; t17 = 2.194, P = 0.046; Fig. 1a). Re-exposure to the shock context induced a clear CORT response in all animals (repeated-measures anova, time effect: F3,42 = 73.26, P < 0.01). However, this response was reduced significantly in SDT animals compared to T animals (treat￾ment · time interaction: F3,42 = 3.42, P < 0.05; see Fig. 1b). Post-hoc t-tests showed that the plasma levels of CORT in the SDT animals were significantly lower than those of the T animals at t = 30 and 60 min (P < 0.05 in both cases). Due to missing samples, three animals (one T and two SDT) were excluded from the CORT analysis. Experiment 2: neuronal activation upon re-exposure to shock context Again, rats that were sleep-deprived for 6 h immediately following training (SDT) showed reduced freezing upon re￾exposure to the shock box 24 h after training compared with trained, non-sleep-deprived control animals (T) (22.5 ± 3.4% versus 35.2 ± 5.0%, with n = 10 and n = 9, respectively; t17 = 2.147, P = 0.047). A representative picture of pCREB expression in the hippocampus is shown in Fig. 2a. In the hippocampal sections that were collected 1 h after testing for contextual fear, the optical density of pCREB immunoreactivity was affected by the experimental treatment in the CA1 area (F2,23 = 12.665, P < 0.01), but not in the CA3 area (F2,23 = 1.051, P > 0.3) and DG (F2,23 = 0.295, P > 0.3) (Fig. 2b–d). Trained rats that were re-exposed to the shock context (T) had a significant increase in pCREB expression in the CA1 area compared to HCC animals (P < 0.01). This increase was still visible in the animals subjected to brief SD immediately following training (SDT versus HCC, P < 0.05), but it was significantly smaller than in the trained, non-sleep-deprived control animals (SDT versus T, P < 0.05). A representative picture of pCREB expression in the amygdala is shown in Fig. 3a. Due to missing sections, two animals (one T and one SDT animal) were excluded from analysis. anova revealed a significant main treatment effect for the number of pCREB-positive cells in the BLA as well as in the CeN (F2,21 = 3.901, P < 0.05 and F2,21 = 3.528, P < 0.05, respectively) (Fig. 3b,c). Specifically, the number of pCREB-positive cells in the BLA and CeN was higher in animals subjected to contextual fear-testing (T) compared with animals in the HCC group (post-hoc Tukey test, P < 0.05 for both regions). Although the number of pCREB immunoreac￾tive cells was also elevated slightly in the SDT group, this increase did not reach statistical significance when compared to the HCC group (P > 0.4 for both subregions). DISCUSSION In the present study we confirm the negative effects of brief SD on the consolidation of contextual fear memory. We show that 6 h of SD immediately following a mild foot shock leads to a reduced freezing response upon re-exposure to the shock box the next day. In addition to this attenuated behavioural response, the SD animals also displayed a redu￾ced neuroendocrine activation, as shown by an attenuated T SDT Freezing (%) 0 10 20 30 40 50 * Time (min) 0 10 20 30 40 50 60 CORT (µg dL–1) 0 5 10 15 20 25 T SDT * * (a) (b) Figure 1. Effect of brief sleep deprivation (SD) following training on freezing behaviour and corticosterone (CORT) response upon re￾exposure to the shock context. (a) Twenty-four hours after training all animals were tested for contextual fear during a 5-min test phase. Animals sleep-deprived for 6 h immediately following training (SDT, n = 10) displayed significantly less freezing behaviour in response to the shock context than control animals (T, n = 9). (b) CORT response upon contextual fear-testing in trained animals (T, n = 8) and animals subjected to 6 h of SD (SDT, n = 8) immediately after training in the contextual fear conditioning paradigm. Plasma CORT levels were significantly lower in SDT animals compared to T animals at t = 30 and 60 min. Data are expressed as mean ± standard error of the mean. *P < 0.05. 262 R. Hagewoud et al. 2010 European Sleep Research Society, J. Sleep Res., 20, 259–266