Fragmentation of Rapid Eye Movement and Nonrapid Eye Movement Sleep without Total Sleep Loss Impairs Hippocampus-Dependent Fear Memory Consolidation

Abstract

Study objectives: Sleep is important for consolidation of hippocampus-dependent memories. It is hypothesized that the temporal sequence of nonrapid eye movement (NREM) sleep and rapid eye movement (REM) sleep is critical for the weakening of nonadaptive memories and the subsequent transfer of memories temporarily stored in the hippocampus to more permanent memories in the neocortex. A great body of evidence supporting this hypothesis relies on behavioral, pharmacological, neural, and/or genetic manipulations that induce sleep deprivation or stage-specific sleep deprivation.

Methods: We exploit an experimental model of circadian desynchrony in which intact animals are not deprived of any sleep stage but show fragmentation of REM and NREM sleep within nonfragmented sleep bouts. We test the hypothesis that the shortening of NREM and REM sleep durations post-training will impair memory consolidation irrespective of total sleep duration.

Results: When circadian-desynchronized animals are trained in a hippocampus-dependent contextual fear-conditioning task they show normal short-term memory but impaired long-term memory consolidation. This impairment in memory consolidation is positively associated with the post-training fragmentation of REM and NREM sleep but is not significantly associated with the fragmentation of total sleep or the total amount of delta activity. We also show that the sleep stage fragmentation resulting from circadian desynchrony has no effect on hippocampus-dependent spatial memory and no effect on hippocampus-independent cued fear-conditioning memory.

Conclusions: Our findings in an intact animal model, in which sleep deprivation is not a confounding factor, support the hypothesis that the stereotypic sequence and duration of sleep stages play a specific role in long-term hippocampus-dependent fear memory consolidation.

Keywords: NREM sleep fragmentation; REM sleep fragmentation; circadian desynchrony; memory consolidation.

Figure 1

Schematic representation of the training and testing protocols used in the preset study. See Methods for a detailed description of each paradigm.

Figure 2

Sleep under LD22 forced desynchrony. Left panel, Representative locomotor activity records of rats housed under LD24 and LD22 conditions. Locomotor activity (infrared beam interruptions) is plotted as black marks for each 48-h period (double-plotted actogram), with days inserted vertically. The same actogram is shown twice. On the right actogram, white and black horizontal bars indicate light and dark phases of the LD cycle, respectively. Gray shading indicates the times of darkness on successive days. Red line in right LD22 actogram marks the onset of the locomotor activity rhythm that is not entrained by the LD cycle. Periodogram analysis (below each actogram) reveals that the only statistically significant periodicity in the LD24 animal corresponds to a period of 24 h, whereas two periodicities are statistically significant in the LD22 animal (periods indicated on top of periodogram). Y-axes on periodogram represent the % variance explained by oscillations with each specific period. Right panel, Hypnograms indicate vigilance states in 10-min bins for a 24-h period in LD24 animals and for a 22-h period in LD22 animals. Bars above hypnograms represent the LD schedule and gray shaded areas within the plots represent dark phases. For the LD24 hypnogram, (A) represents the light phase and the (B) the dark phase. For the LD22 hymnograms, blue horizontal overlay bar represents aligned phase—(C) dark aligned phase and (D) light aligned phase—and red horizontal overlay bar represents misaligned phase—(E) dark misaligned phase and (F) light misaligned phase. LD, light-dark.

Figure 3

LD22 forced desynchrony induces nonrapid eye movement (NREM) and rapid eye movement (REM) sleep fragmentation without fragmentation of total sleep or deprivation of any specific sleep stage. (A) Percentage of time spent in each sleep stage in LD24 control animals, LD22 aligned animals or LD22 misaligned animals. One-way analysis of variance: Wake (F(2,10) = 1.17, P = 0.35); total sleep (F(2,10) = 1.25, P = 0.33); NREM sleep (F(2,10) = 1.34, P = 0.31); REM sleep (F(2,10) = 0.63, P = 0.55). (B) Survival analysis of wake, total sleep, NREM sleep, and REM sleep. See Table 1 for two-way analysis of variance results. When the effect of group or the interaction was significant, Tukey multiple comparison tests were done (P = 0.05): *LD22 misaligned differs from LD24; ^#LD22 misaligned differs from both LD22 aligned and LD24. n = 7 (LD24); n = 3 (LD22 aligned and misaligned). LD, light-dark.

Figure 4

Training during LD22 misaligned phases leads to impaired long-term memory consolidation of hippocampus-dependent contextual fear conditioning. (A) Post-shock freezing during training. Whereas immediate-shocked control animals (LD24 IS) do not freeze during training, LD24, LD22 aligned, and LD22 misaligned animals show normal freezing responses. One-way analysis of variance: F(3,24) = 4.51, P = 0.012; *P = 0.05, **P = 0.01 different from LD24 IS, Dunnett multiple comparisons test. (B) Freezing during short-term memory test. LD22 aligned and misaligned animals show similar short-term (15 min post-training) memory consolidation. Two-tailed Student t test: t(22) = 0.83, P = 0.41. (C) Training during the light phase of LD24 and LD22 aligned days, but not during the light phase of LD22 misaligned days, leads to higher test freezing responses than training with an immediate-shock. Bottom of x-axis indicates training and testing conditions; all animals were trained during the light phase; one-way analysis of variance: F(4,32) = 5.75, P = 0.0013. (D) Training during the dark phase of LD24 but not during the dark phase of LD22 misaligned days, leads to higher test freezing responses than training with an immediate shock. Bottom of x-axis indicates training and testing conditions; all animals were trained during the dark phase; one-way analysis of variance: F(3,24) = 4.05, P = 0.0184. *P = 0.05, **P = 0.01, ***P = 0.001 different from LD24 immediate shock, Dunnett multiple comparisons test. Number of animals is indicated in brackets.

Figure 5

Training under LD22 forced desynchrony conditions has no effect on hippocampus-dependent spatial memory consolidation. (A) Responses during the Morris water maze training trials. Top panel: Latency to reach the hidden platform during successive training trials for LD24, LD22 aligned, and LD22 misaligned animals. Two-way analysis of variance (ANOVA): Trial (F(15,315) = 9.08, P < 0.0001); group (F(2,21) = 0.56, P = 0.58); interaction (F(30,315) = 0.94, P = 0.56). Bottom panel: Training trial number on which rats reached the hidden platform by their own. One-way ANOVA: F(2,21) = 3.58, P = 0.046. *P = 0.05 different from LD24, Dunnett multiple comparisons test. (B) Responses during the probe test. Top panel: Latency for the first crossing in the original platform location. One-way ANOVA: F(3,32) = 5.67, P = 0.0031. Center panel: Number of crossings over the original platform location. One-way ANOVA: F_(3,32) = 10.99, P < 0.0001. Bottom panel: Total duration on the original location of the platform. One-way ANOVA: F(3,32) = 7.15, P = 0.0008. *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001 different from LD24 untrained, Dunnett multiple comparisons test. n = 8 for LD24, aligned and misaligned; n = 12 for LD24 untrained. LD, light-dark.

Figure 6

Training under LD22 forced desynchrony conditions has no effect on long-term memory consolidation of hippocampus-independent cued fear conditioning. (A) Post-tone freezing scores during long-term memory test from animals trained under LD24, LD22 aligned, and LD22 misaligned conditions. One-way ANOVA: F(3,32) = 7.80, P = 0.0005. (B) Number of fecal boli during test trial. One-way ANOVA: F(3,32) = 5.35, P = 0.0042. *P = 0.05, ***P = 0.001 different from LD24 immediate shock, Dunnett multiple comparisons test. n = 7 (LD24); n = 9 (LD24 IS); n = 10 (aligned); n = 10 (misaligned). LD, light-dark.

Figure 7

Rapid eye movement (REM) and nonrapid eye movement (NREM) sleep fragmentation, but not total sleep fragmentation, are associated with impaired memory consolidation in hippocampus-depended contextual fear conditioning. For each sleep stage, the fragmentation index is drawn from the exponential decay coefficient of the survival curve for that stage in each animal. Correlation coefficient (R²) and associated P value for the regression analysis are shown in each case (n = 7).