Sleep-dependent gene expression in the hippocampus and prefrontal cortex following long-term potentiation

Abstract

The activity-dependent transcription factor zif268 is re-activated in sleep following hippocampal long-term potentiation (LTP). However, the activation of secondary genes, possibly involved in modifying local synaptic strengths and ultimately stabilizing memory traces during sleep, has not yet been studied. Here, we investigated changes in hippocampal and cortical gene expression at a time point subsequent to the previously reported initial zif268 re-activation during sleep. Rats underwent unilateral hippocampal LTP and were assigned to SLEEP or AWAKE groups. Eighty minutes after a long rapid-eye-movement sleep (REMS) episode (or an equivalent amount of time for awake group) animals had their hippocampi dissected and processed for gene microarray hybridization. Prefrontal and parietal cortices were also collected for qRT-PCR analysis. The microarray analysis identified 28 up-regulated genes in the hippocampus: 11 genes were enhanced in the LTPed hemisphere of sleep animals; 13 genes were enhanced after sleep, regardless of hemisphere; and 4 genes were enhanced in LTPed hemisphere, regardless of behavioral state. qRT-PCR analysis confirmed the up-regulation of aif-1 and sc-65 during sleep. Moreover, we observed a down-regulation of the purinergic receptor, P2Y4R in the LTP hemisphere of awake animals and a trend for the protein kinase, CaMKI to be up-regulated in the LTP hemisphere of sleep animals. In the prefrontal cortex, we showed a significant LTP-dependent down-regulation of gluR1 and spinophilin specifically during sleep. Zif268 was down-regulated in sleep regardless of the hemisphere. No changes in gene expression were observed in the parietal cortex. Our findings indicate that a set of synaptic plasticity-related genes have their expression modulated during sleep following LTP, which can reflect biochemical events associated with reshaping of synaptic connections in sleep following learning.

Figure 1. Experimental design

Stimulating and recording electrodes were implanted bilaterally in the mPP and DG for stimulation and recording of fEPSPs, respectively. After recovery from surgery, the animals underwent 5 days of habituation and handling, during which baseline recordings were performed for both hemispheres. Following this, HFS (2 series of 10 trains at 400 Hz, 5 min apart) was applied to the mPP unilaterally in order to induce LTP in the DG. The contralateral hemisphere was not tetanized. LTP was assessed for 15 min in each hemisphere after HFS. The animals were kept awake for 3 h and were either allowed to sleep (SLEEP group) or kept awake (AWAKE or AW group). After a long REMS episode (>120 s), they were kept awake for an additional 80 min. The AWAKE animals were kept awake for a comparative amount of time. All animals were then decapitated and the prefrontal cortex, hippocampus and parietal cortex were dissected from the LTP and control (ipsi- and contralateral) hemispheres for microarray and qRT-PCR analysis. Trunk blood samples from each animal were also collected for CORT measurements.

Figure 2. QRT-PCR analysis of hippocampal gene expression

A, Sleep-LTP interaction. CaMKI and P2Y4R showed a significant interaction between LTP and behavioral state (F_1,6=6.79 p<0.05 and F_1,6=5.92 p<0.05, respectively). CaMKI showed a trend towards lower expression in the LTP hemisphere of AWAKE animals and to be upregulated in the LTP hemisphere of SLEEP animals, compared to the control hemisphere. P2Y4R was significantly less expressed in the LTP hemisphere of AWAKE animals (50%), although it did not change in SLEEP animals. B, Sleep effect. Aif-1 and sc-65 were confirmed to be upregulated during sleep: aif-1 (F_1,6=10.96 p<0.02; 50%) and sc-65 (F_1,6=18.19 p<0.01; 35%). In addition, the transcription factors, zif268 and dbp showed a trend towards upregulation during sleep (F_1,6=3.93 p=0.09; 36% and F_1,6=4.04 p=0.09; 44%, respectively) (supporting the microarray results). C, Other - genes selected based on functional relevance or expression patterns, which did not meet microarray screening criteria (see Material and Methods). J01878 showed a trend towards up-regulation in the LTP hemisphere in SLEEP group (62%). Data represent mean (± SEM) percent change, compared to AWAKE-Control samples, following β-actin normalization. Two-way ANOVA and Welch t-test: #, p<0.01; *, p<0.05.

Figure 3. QRT-PCR analysis of gene expression in the hippocampus, prefrontal cortex (PFC) and parietal cortex (PC) of AWAKE (n=4) and REMS (n=4) animals following unilateral hippocampal LTP

A, Sleep effect. In the hippocampus, the qRT-PCR results were consistent with the microarray findings. We did not observe significant group effects for any of the 6 genes analyzed. Zif268 showed a non-significant up-regulation trend during sleep in the hippocampus. In the PFC, sleep induced down-regulation of zif268 (−40%), gluR1 (−61%) and spinophilin (−21%). The parietal cortex showed a non-significant trend for gluR1 (−47%) to be down-regulated during sleep. B, Sleep-LTP effect. In the hippocampus, we did not observe any significant gene expression changes during sleep in the LTP hemisphere. In the PFC, however, gluR1 (−25%) and spinophilin (−21%) were specifically down-regulated in the LTP hemisphere during sleep. No significant gene expression changes were observed in the PC. Data are presented as the mean (± SEM) ΔCt after β-actin normalization. Two-way ANOVA and Welch t-test: #, p<0.01; *, p<0.05.