The results of this study revealed that sleep debt caused by having just a few hours of sleep for 5 days (3 h 29 min/day of sleep restriction compared with the SC session) increased the activity of the left amygdala in response to a fear facial image. In contrast, a happy facial image did not change the activity. Functional connectivity analysis demonstrated that the levels of FCamg-vACC
(left amygdala-vACC functional connectivity) were lower in participants with higher degrees of sleep debt (ΔSWS2 h
and Δδ2 h
). The most important and novel finding in this paper is that declines in FCamg-vACC
were correlated with left amygdala activation and subjective mood deterioration (higher STAI-S and POMS scores). These findings strongly suggest that downregulation of the amygdala by the vACC and subsequent activation of the amygdala in response to negative emotional stimuli are involved in intensified physiological and psychological responses 
and mood deterioration 
due to unpleasant emotional stimuli during sleep debt.
This interpretation is supported by a series of studies. The amygdala is thought to play an important role in the expression of negative emotions 
. Facial expressions of fear were found to activate the amygdala even in healthy individuals with normal sleep 
, and such activation is reportedly more prominent in individuals with depression and anxiety disorders 
. Moreover, the amygdala has a strong functional and anatomical connection with the mPFC region 
, and the strength of this functional connection is correlated with the degree of subjective emotional suppression and reappraisal of negative affect. 
According to previous studies 
, overnight total sleep deprivation diminishes the functional connectivity between the amygdala and the mPFC. In the present study, even a short-term, continuous and accumulating sleep debt that can occur in everyday life clearly resulted in reduced functional connectivity between the amygdala and the mPFC, and more specifically the vACC.
More importantly, subjective mood changes (increased anxiety) following short sleep were correlated with diminished FCamg-vACC
, but not with the change in the activation in the amygdala (Δamygdala activation) itself. This may indicate that diminished synchronization between the amygdala and the vACC plays a more important role than the extent of the event-related local activation in the amygdala, for stabilizing increased anxiety evoked by an unpleasant emotional stimulus. Some models of amygdala functionality 
suggest that the magnitude of the local activity in the amygdala does not play a direct role in modulating the mood states of individuals, but the functional connectivity between the amygdala and the ventral mPFC correlates with STAI state score during resting state fMRI 
. In our functional connectivity analysis, the main event-related hemodynamic response covaries with the seed-related (amygdala) activity in the GLM model of functional connectivity analysis; namely, main event-related immediate reactivity was regressed in our functional connectivity results (see Friston et al., 1997 
for the detailed process). Therefore, FCamg-vACC
in this study does not include simple event-related ‘co-activation’ between two regions, but finer event-unrelated synchronization between two regions beyond the local activities in two regions. This is why the local amygdala activity and the functional connectivity did not correlate with the psychological measurements in the same way; namely, the sustained anxiety (mood) state correlated with the FCamg-vACC
, but not with the event-related local reactivity of the amygdala. This interpretation is supported by the diminished functional connectivity between the amygdala and the vACC or ventral mPFC, regardless of the amygdala's activity, in individuals with social anxiety disorder, in those with the s allele of the serotonin transporter gene and who thus have a high risk of depression, and in those with schizophrenia 
Although it has been shown that positive emotional stimuli also activate the amygdala 
, happy facial expressions did not significantly alter amygdala activity during sleep debt in the present study. This suggests that functional changes in the amygdala and FCamg-vACC
during sleep debt become more apparent when negative emotional stimuli are presented. Overnight total sleep deprivation reportedly induces activation of the amygdala even in response to images associated with positive emotions 
. It is reasonable to assume that this discrepancy is due to differences in the sleep conditions, such as total sleep deprivation in the former study and 5-day sleep restriction in the present one. It is possible that overnight sleep deprivation, more than accumulating sleep debt, affects the expression of positive emotion. In fact, overnight sleep deprivation elicits an antidepressant effect in patients with depression, as well as mood activation in healthy individuals 
. Such effect and mood activation might be related to enhanced amygdala activation to positive emotional stimulus, as observed in a previous study. This notion is supported by the findings of a study in which antidepressant treatment enhanced amygdala activation in response to happy faces in patients with depression 
. However, no similar phenomena have been reported in individuals with consecutive nights of sleep loss.
Unexpectedly, the presentation of emotional facial images under the non-conscious condition did not elicit changes in the amygdala activation after sleep restriction in the present study. A previous study showed that responses to masked priming are enhanced after a 2-day partial sleep deprivation 
. Moreover, overnight total sleep deprivation strengthened the functional connectivity of amygdala with the sub-cortical region (midbrain) while diminishing the functional connectivity with the mPFC 
. Based on these findings, we had expected to observe certain functional alteration in not only conscious processing through the cortical pathway, but also non-conscious processing through the subcortical pathway. However, no such alteration was observed in our participants. Any changes in non-conscious processing might be observable by adjusting the length of non-conscious image presentation, the intensity of the facial image stimuli, or increasing the sample size.
Interestingly, the decline in FCamg-vACC
was correlated with the increase in %SWS and δ wave power in the early period of the sleep, but not with subjective sleepiness. Because the increase in SWS and δ wave power is thought to be a sensitive indicator of the sleep homeostatic process 
, it is possible that FCamg-vACC
is associated with the neural basis that enhances homeostatic sleep pressure following short hours of sleep. It is also thought that the increase in homeostatic sleep pressure is caused by the accumulation of sleep promoting substances in brain, such as prostaglandin D2 and adenosine 
. For example, adenosine receptor antagonists have been shown to reduce sleep time by increasing wake hours and reduce slow wave activity during sleep 
. Recent studies have hypothesized that adenosine, accumulated in the synaptic cleft because of consecutive synaptic transmission, binds to the A1 receptor to suppress synaptic transmission 
. It is possible that synaptic suppression by a sleep-promoting substance is involved in the attenuation of FCamg-vACC
during sleep debt. Functional connectivity of the BOLD signal reportedly reflects a synaptic anatomical pathway 
; therefore, it is expected that synaptic suppression by adenosine causes a decline in functional connectivity. This hypothesis is supported by a previous study which reported that a lack of sleep leads to attenuated resting state functional connectivity 
. Moreover, a positron emission tomography (PET) study of human subjects revealed increased adenosine binding to the A1 receptor in regions including the vACC during sleep deprivation 
. The use of PET with fMRI to elucidate the effects of sleep-promoting substances such as adenosine, including the effect of adenosine on the dynamics of neural activity in the brain, will likely elucidate the neural basis that alters the mechanism of emotion regulation.
Some limitations need to be taken into account when interpreting the present findings. First, participants in this study spent the first 3 days at home. Even though their sleep schedule was enforced by the use of the actigraph and mail alerts, their actual sleep time (8 h 5 min and 4 h 36 min in the SC and SD session, respectively) was longer than the scheduled sleep time (8 and 4 h in the SC and SD session, respectively). Despite the minor increase in sleep time, the SD session had a sleep loss of 3 h 30 min compared with the SC session. This level of sleep loss is rather frequently experienced in everyday life; however, the presence of sleep debt was confirmed on the last day of the session by the increase in %SWS and δ wave power. Because sleep requirements vary among individuals, the same sleep schedule may result in large individual differences in sleep debt 
. In other words, even if everyone were to be placed strictly on the same sleep schedule, the effects of sleep debt on brain activity and subjective moods would vary widely among individuals. If sleep can be restricted based on individual sleep requirements, functional changes in the amygdala and FCamg-vACC
might be identified more accurately.
The results of this study indicate that a short-term sleep loss, which is often experienced in everyday life, can aggravate subjective mood including anxiety, and the mechanism appears to involve functional alteration of the amygdala and FCamg-vACC
. Long work hours, night-owl lifestyles, and an increase in shift work are the major contributors to sleep loss and thus the risk for depression 
. Therefore, ensuring adequate sleep is an important lifestyle factor that deserves more attention in terms of managing mental health, including depression.