Sleep deprivation and memory consolidation 261 Permanent heart cannulations,blood sampling and CORT assay with PBS the sections were incubated at room temperature for 3 h with biotinylated goat anti-rabbit immunoglobin G To assess conditioned HPA axis responses in Experiment 1. (IgG)(1 500:Jackson Immunoresearch Laboratories)in 1% permanent heart cathethers were used that permitted frequent NGS,0.1%Triton X-100 in PBS.After 5x 5 min rinsing blood sampling in unrestrained,freely moving rats (Steffens, with PBS.sections were incubated for 1.5 h at room 1969).The animals were provided with a polyethylene catheter temperature with avidin-biotin complex (1:500.ABC Elite in the right atrium of the heart.The catheter was inserted kit;Vector Laboratories,Burlingame,CA,USA),0.1% through the right jugular vein and externalized on the top of Triton X-100 in PBS.After this step the sections were rinsed the skull.The whole procedure was performed under isoflu- overnight in PBS at 4C.After rinsing the sections rane/O2 inhalation anaesthesia.Animals had at least 2 weeks 6 x 10 min with PBS.labelled cells were visualized with of recovery before the start of the experiment.During this diaminobenzidine (DAB,0.7 mg mL-in milli q water; period,animals were habituated to handling and blood Sigma-Aldrich.Steinheim.Germany)with 0.1%H2O,as a sampling procedures. reaction initiator.The reaction was stopped by rinsing with On the second day of the experiment,when animals were re- PBS. exposed to the shock context for contextual fear-testing,blood For each subject,three sections were selected at approxi- samples were taken at baseline,5 min.30 and 60 min.The mately bregma-2.80 to-3.60 mm for the dorsal hippocampus baseline sample was collected at least 1 h before testing.The and two sections were selected for the amygdala at bregma 5-min sample was taken in the shock box at the end of the -2.12 to -3.30 mm (Paxinos and Watson.1998).In the re-exposure.Blood samples were collected in precooled plastic amygdala,immunopositive cells were counted bilaterally in centrifuge tubes containing 0.01%ethylenediamine tetraacetic each section at a 50x magnification using a computerized acid (EDTA)as anticoagulant and antioxidant.Blood was image analysis system (Quantimet 550:Leica.Cambridge, centrifuged at 4C for 15 min at 2600g and plasma was UK).For the basolateral amygdala (BLA),a fixed sample stored at-80C until further processing.CORT levels were window of 0.37 mm2 was used and for the central nucleus of determined by radioimmunoassay (MP Biomedicals,Orange- the amygdala (CeN)the sample window was 0.20 mm. burg,NY,USA). A threshold was set that marked all cells to be included in the counting.In the hippocampus,the cell layers were densely Assessment of regional pCREB expression packed with pCREB immunopositive cells,which made it difficult to distinguish and count individual cells.Instead, In Experiment 2 we tested whether a reduced conditioned optical densities (OD)were measured for the granular cell freezing response in animals that were sleep-deprived after the layer of the dentate gyrus (DG)and for the pyramidal cell initial shock exposure would be associated with changes in layer of cornu ammonis (CA)areas I and 3 of the dorsal regional brain activation by performing immunohistochemis- hippocampus using a 50x magnification.The OD is expressed try for phosphorylation of the transcription factor CREB.One in arbitrary units corresponding to grey levels using the hour after testing for contextual fear memory,rats were Quantimet image analysis system (Leica).To correct for sacrificed for brain collection.A group of HCC rats was variability in background staining among sections,the back- sacrificed in parallel.Under deep pentobarbital anaesthesia, ground labelling was measured in the stratum radiatum and rats were perfused transcardially with 150 mL 0.9%NaCl,1E extracted from the OD of the area of interest.The experi- per mL heparin,followed by 300 mL 4%paraformaldehyde menter was blind to the treatment of individual animals during for fixation.Brains were collected,postfixated for 24 h in 4% all cell counting and OD measurements.Data on pCREB paraformaldehyde,rinsed for I day in 0.01 M phosphate- immunoreactivity are expressed as percentage of the mean buffered saline (PBS.pH 7.4)and then transferred to a 30% value of the HCC group sucrose in PBS cryoprotectant overnight at 4 C.Brains were stored at-80 C until further processing. Statistical analysis Thirty um coronal sections containing the amygdala and hippocampus were collected and stored in PBS containing Behavioural data were analysed using an independent-samples 0.1%sodium azide.The brain sections were rinsed 3 x 5 min 1-test.The CORT responses were analysed using a repeated- in PBS,followed by 30 min in 0.3%H2O2 in PBS.After measures analysis of variance (ANOvA)with a between-subject 4 x 5 min rinsing in PBS,sections were preincubated at room factor 'treatment'(T or SDT)and a within-subject factor 'time temperature for 30 min in 5%normal goat serum (NGS: (at baseline,5,30 and 60 min).A post-hoc t-test was used to Jackson Immunoresearch Laboratories,West Grove,PA. establish at which time-points the treatment groups differed. USA),0.1%Triton X-100 in PBS to block non-specific pCREB immunoreactivity was analysed using a one-way ANovA binding of immunoreagents.Subsequently,sections were with a between-subjects factor 'treatment'(HCC.T or SDT). incubated for 2 h at room temperature followed by overnight Post-hoc comparisons were made using a Tukey test.In all incubation at 4C with rabbit polyclonal anti-p-CREB cases P 0.05 was considered significant.All data in the text antibody (1:2000:Upstate,Temecula,CA,USA)in 0.3% and figures are expressed as mean+standard error of the Triton X-100.1%NGS in PBS.After rinsing 4x 10 min mean (SEM). 2010 European Sleep Research Society.J.Sleep Res.,20.259-266Permanent heart cannulations, blood sampling and CORT assay To assess conditioned HPA axis responses in Experiment 1, permanent heart cathethers were used that permitted frequent blood sampling in unrestrained, freely moving rats (Steffens, 1969). The animals were provided with a polyethylene catheter in the right atrium of the heart. The catheter was inserted through the right jugular vein and externalized on the top of the skull. The whole procedure was performed under isoflu￾rane ⁄ O2 inhalation anaesthesia. Animals had at least 2 weeks of recovery before the start of the experiment. During this period, animals were habituated to handling and blood sampling procedures. On the second day of the experiment, when animals were re￾exposed to the shock context for contextual fear-testing, blood samples were taken at baseline, 5 min, 30 and 60 min. The baseline sample was collected at least 1 h before testing. The 5-min sample was taken in the shock box at the end of the re-exposure. Blood samples were collected in precooled plastic centrifuge tubes containing 0.01% ethylenediamine tetraacetic acid (EDTA) as anticoagulant and antioxidant. Blood was centrifuged at 4 C for 15 min at 2600 g and plasma was stored at )80 C until further processing. CORT levels were determined by radioimmunoassay (MP Biomedicals, Orange￾burg, NY, USA). Assessment of regional pCREB expression In Experiment 2 we tested whether a reduced conditioned freezing response in animals that were sleep-deprived after the initial shock exposure would be associated with changes in regional brain activation by performing immunohistochemis￾try for phosphorylation of the transcription factor CREB. One hour after testing for contextual fear memory, rats were sacrificed for brain collection. A group of HCC rats was sacrificed in parallel. Under deep pentobarbital anaesthesia, rats were perfused transcardially with 150 mL 0.9% NaCl, 1E per mL heparin, followed by 300 mL 4% paraformaldehyde for fixation. Brains were collected, postfixated for 24 h in 4% paraformaldehyde, rinsed for 1 day in 0.01 m phosphate￾buffered saline (PBS, pH 7.4) and then transferred to a 30% sucrose in PBS cryoprotectant overnight at 4 C. Brains were stored at )80 C until further processing. Thirty lm coronal sections containing the amygdala and hippocampus were collected and stored in PBS containing 0.1% sodium azide. The brain sections were rinsed 3 · 5 min in PBS, followed by 30 min in 0.3% H2O2 in PBS. After 4 · 5 min rinsing in PBS, sections were preincubated at room temperature for 30 min in 5% normal goat serum (NGS; Jackson Immunoresearch Laboratories, West Grove, PA, USA), 0.1% Triton X-100 in PBS to block non-specific binding of immunoreagents. Subsequently, sections were incubated for 2 h at room temperature followed by overnight incubation at 4 C with rabbit polyclonal anti-p-CREB antibody (1 : 2000; Upstate, Temecula, CA, USA) in 0.3% Triton X-100, 1% NGS in PBS. After rinsing 4 · 10 min with PBS the sections were incubated at room temperature for 3 h with biotinylated goat anti-rabbit immunoglobin G (IgG) (1 : 500; Jackson Immunoresearch Laboratories) in 1% NGS, 0.1% Triton X-100 in PBS. After 5 · 5 min rinsing with PBS, sections were incubated for 1.5 h at room temperature with avidin–biotin complex (1: 500, ABC Elite kit; Vector Laboratories, Burlingame, CA, USA), 0.1% Triton X-100 in PBS. After this step the sections were rinsed overnight in PBS at 4 C. After rinsing the sections 6 · 10 min with PBS, labelled cells were visualized with diaminobenzidine (DAB, 0.7 mg mL)1 in milli q water; Sigma-Aldrich, Steinheim, Germany) with 0.1% H2O2 as a reaction initiator. The reaction was stopped by rinsing with PBS. For each subject, three sections were selected at approxi￾mately bregma )2.80 to )3.60 mm for the dorsal hippocampus and two sections were selected for the amygdala at bregma )2.12 to )3.30 mm (Paxinos and Watson, 1998). In the amygdala, immunopositive cells were counted bilaterally in each section at a 50· magnification using a computerized image analysis system (Quantimet 550; Leica, Cambridge, UK). For the basolateral amygdala (BLA), a fixed sample window of 0.37 mm2 was used and for the central nucleus of the amygdala (CeN) the sample window was 0.20 mm2 . A threshold was set that marked all cells to be included in the counting. In the hippocampus, the cell layers were densely packed with pCREB immunopositive cells, which made it difficult to distinguish and count individual cells. Instead, optical densities (OD) were measured for the granular cell layer of the dentate gyrus (DG) and for the pyramidal cell layer of cornu ammonis (CA) areas 1 and 3 of the dorsal hippocampus using a 50· magnification. The OD is expressed in arbitrary units corresponding to grey levels using the Quantimet image analysis system (Leica). To correct for variability in background staining among sections, the back￾ground labelling was measured in the stratum radiatum and extracted from the OD of the area of interest. The experi￾menter was blind to the treatment of individual animals during all cell counting and OD measurements. Data on pCREB immunoreactivity are expressed as percentage of the mean value of the HCC group. Statistical analysis Behavioural data were analysed using an independent-samples t-test. The CORT responses were analysed using a repeated￾measures analysis of variance (anova) with a between-subject factor treatment (T or SDT) and a within-subject factor time (at baseline, 5, 30 and 60 min). A post-hoc t-test was used to establish at which time-points the treatment groups differed. pCREB immunoreactivity was analysed using a one-way anova with a between-subjects factor treatment (HCC, T or SDT). Post-hoc comparisons were made using a Tukey test. In all cases P < 0.05 was considered significant. All data in the text and figures are expressed as mean ± standard error of the mean (SEM). Sleep deprivation and memory consolidation 261 2010 European Sleep Research Society, J. Sleep Res., 20, 259–266