260 R.Hagewoud et al. fear (Graves et al.,2003:Hagewoud et al.,2010c:Vecsey et al.. provided ad libitum.Animals were maintained on a 12-h 2009).When animals subjected to 5 or 6 h of SD following light/12-h dark cycle with lights on at 08:00 h.Light intensity training are re-exposed to the shock environment the next day. in the light phase was approximately 45 lux.All procedures they exhibit less freezing behaviour compared with trained. described in the present study were approved by the Animal non-sleep-deprived control animals.Importantly,memory Experiment Committee of the University of Groningen in consolidation for cued fear is not affected (Graves et al.. compliance with Dutch law and regulations. 2003;Vecsey et al.,2009).This demonstrates that SD has a negative effect on memory consolidation,particularly when it Experimental set-up involves the hippocampus.Thus,by affecting hippocampus function and interfering with the processing of contextual This study consisted of two experiments in order to examine information,SD leads to a weaker association between the whether a reduced freezing response during testing for contex- context and shock.which then leads to an attenuated fear tual fear memory in animals subjected to 6 h of SD immedi- response when the rats are re-exposed to this context later on. ately following training in the contextual fear conditioning The hippocampus and amygdala are not only important for paradigm is accompanied by:(1)a reduced neuroendocrine the formation of contextual fear memory,but are also involved response,assessed by measuring plasma levels of CORT:and in the retrieval and expression of these memories (Fanselow, (2)a reduced neuronal activation in relevant brain areas,as 2000:Fendt and Fanselow.1999).The recall of contextual fear examined by immunoreactivity for activated CREB.Both memories is associated with increased phosphorylation and experiments contained a non-sleep-deprived trained group activation of the transcription factor 3,5'-cyclic AMP (T,n=9)and a group subjected to 6 h of SD immediately response-element binding protein(CREB)in both the hippo- following training (SDT,n=10).In addition,Experiment 2 campus and amygdala (Mamiya et al.,2009).Therefore,one contained a home cage control (HCC)group (n=7). might expect that the attenuated fear response found in animals that were sleep-deprived after training is associated Contextual fear conditioning with an attenuated expression of phosphorylated CREB (pCREB)in these brain areas compared with trained,non- One week prior to the start of the fear conditioning experiments sleep-deprived control animals.However,such changes in all animals were handled daily for 2 min.Contextual fear regional brain activity have not yet been reported. conditioning was performed in a black plexiglass chamber Moreover,while freezing behaviour is the most commonly (40 x 40 x 40 cm),which was located in a separate experimen- used read-out of fear in most of the studies mentioned above.it tal room.During training an animal was placed in the chamber might be expected that an SD-induced weakening of condi- and exposed to the conditioning context for 3 min followed by a tioned contextual fear can also be measured on a physiological mild electric foot shock (0.7 mA,2 s)delivered through the level,particularly in the degree of activation of neuroendocrine stainless steel grid floor.The animal was removed from the stress systems such as the hypothalamic-pituitary-adrenal chamber and returned to its home cage 30 s after the shock. (HPA)axis. The chamber was cleaned thoroughly with 70%ethanol The first aim of the present study was to assess in rats between subjects.Twenty-four hours later the animal was whether brief SD following exposure to a mild foot shock leads placed in the same chamber for 5 min without receiving a not only to an attenuated freezing response,but also to a shock.Contextual memory was tested by assessing freezing reduced neuroendocrine response upon testing for contextual behaviour,defined as complete lack of movement except for fear memory the next day.Specifically.we hypothesized that in respiration.Behaviour was recorded on videotapes,which was SD animals the reduced freezing response upon re-exposure to analysed afterwards by an experimenter who was blind for the shock box is accompanied by an attenuated adrenal treatment of the animals.The amount of time the animals corticosterone (CORT)response.In the second part of our displayed freezing behaviour was expressed as a percentage of study we tested whether the reduced freezing response in SD total test time. animals is associated with a reduced activation of brain areas known to be involved in the retrieval and expression of fear memory,particularly a reduced expression of pCREB in the Sleep deprivation hippocampus and amygdala. Animals were sleep-deprived for 6 h immediately following training.SD was accomplished by mild stimulation,which involved tapping on the cage,gently shaking the cage or.when METHODS this was not sufficient to keep the animals awake,disturbing the sleeping nest (Hagewoud et al.,2010a:Van der Borght Animals and housing conditions et al..2006).Previous studies have shown that this procedure The experiments were performed with adult male Wistar rats is effective in keeping rodents awake for several hours,as (Harlan,Horst,the Netherlands),weighing 300-350 g at the established by electroencephalic recordings (Meerlo et al., start of the experiments.Animals were housed individually and 2001),without being a major stressor (Hagewoud et al., a layer of sawdust served as bedding.Food and water were 2010a,b,c) 2010 European Sleep Research Society,J.Sleep Res..20,259-266fear (Graves et al., 2003; Hagewoud et al., 2010c; Vecsey et al., 2009). When animals subjected to 5 or 6 h of SD following training are re-exposed to the shock environment the next day, they exhibit less freezing behaviour compared with trained, non-sleep-deprived control animals. Importantly, memory consolidation for cued fear is not affected (Graves et al., 2003; Vecsey et al., 2009). This demonstrates that SD has a negative effect on memory consolidation, particularly when it involves the hippocampus. Thus, by affecting hippocampus function and interfering with the processing of contextual information, SD leads to a weaker association between the context and shock, which then leads to an attenuated fear response when the rats are re-exposed to this context later on. The hippocampus and amygdala are not only important for the formation of contextual fear memory, but are also involved in the retrieval and expression of these memories (Fanselow, 2000; Fendt and Fanselow, 1999). The recall of contextual fear memories is associated with increased phosphorylation and activation of the transcription factor 3¢,5¢-cyclic AMP response-element binding protein (CREB) in both the hippo￾campus and amygdala (Mamiya et al., 2009). Therefore, one might expect that the attenuated fear response found in animals that were sleep-deprived after training is associated with an attenuated expression of phosphorylated CREB (pCREB) in these brain areas compared with trained, non￾sleep-deprived control animals. However, such changes in regional brain activity have not yet been reported. Moreover, while freezing behaviour is the most commonly used read-out of fear in most of the studies mentioned above, it might be expected that an SD-induced weakening of condi￾tioned contextual fear can also be measured on a physiological level, particularly in the degree of activation of neuroendocrine stress systems such as the hypothalamic–pituitary–adrenal (HPA) axis. The first aim of the present study was to assess in rats whether brief SD following exposure to a mild foot shock leads not only to an attenuated freezing response, but also to a reduced neuroendocrine response upon testing for contextual fear memory the next day. Specifically, we hypothesized that in SD animals the reduced freezing response upon re-exposure to the shock box is accompanied by an attenuated adrenal corticosterone (CORT) response. In the second part of our study we tested whether the reduced freezing response in SD animals is associated with a reduced activation of brain areas known to be involved in the retrieval and expression of fear memory, particularly a reduced expression of pCREB in the hippocampus and amygdala. METHODS Animals and housing conditions The experiments were performed with adult male Wistar rats (Harlan, Horst, the Netherlands), weighing 300–350 g at the start of the experiments. Animals were housed individually and a layer of sawdust served as bedding. Food and water were provided ad libitum. Animals were maintained on a 12-h light ⁄ 12-h dark cycle with lights on at 08:00 h. Light intensity in the light phase was approximately 45 lux. All procedures described in the present study were approved by the Animal Experiment Committee of the University of Groningen in compliance with Dutch law and regulations. Experimental set-up This study consisted of two experiments in order to examine whether a reduced freezing response during testing for contex￾tual fear memory in animals subjected to 6 h of SD immedi￾ately following training in the contextual fear conditioning paradigm is accompanied by: (1) a reduced neuroendocrine response, assessed by measuring plasma levels of CORT; and (2) a reduced neuronal activation in relevant brain areas, as examined by immunoreactivity for activated CREB. Both experiments contained a non-sleep-deprived trained group (T, n = 9) and a group subjected to 6 h of SD immediately following training (SDT, n = 10). In addition, Experiment 2 contained a home cage control (HCC) group (n = 7). Contextual fear conditioning One week prior to the start of the fear conditioning experiments all animals were handled daily for 2 min. Contextual fear conditioning was performed in a black plexiglass chamber (40 · 40 · 40 cm), which was located in a separate experimen￾tal room. During training an animal was placed in the chamber and exposed to the conditioning context for 3 min followed by a mild electric foot shock (0.7 mA, 2 s) delivered through the stainless steel grid floor. The animal was removed from the chamber and returned to its home cage 30 s after the shock. The chamber was cleaned thoroughly with 70% ethanol between subjects. Twenty-four hours later the animal was placed in the same chamber for 5 min without receiving a shock. Contextual memory was tested by assessing freezing behaviour, defined as complete lack of movement except for respiration. Behaviour was recorded on videotapes, which was analysed afterwards by an experimenter who was blind for treatment of the animals. The amount of time the animals displayed freezing behaviour was expressed as a percentage of total test time. Sleep deprivation Animals were sleep-deprived for 6 h immediately following training. SD was accomplished by mild stimulation, which involved tapping on the cage, gently shaking the cage or, when this was not sufficient to keep the animals awake, disturbing the sleeping nest (Hagewoud et al., 2010a; Van der Borght et al., 2006). Previous studies have shown that this procedure is effective in keeping rodents awake for several hours, as established by electroencephalic recordings (Meerlo et al., 2001), without being a major stressor (Hagewoud et al., 2010a,b,c). 260 R. Hagewoud et al. 2010 European Sleep Research Society, J. Sleep Res., 20, 259–266