Expression of the circadian clock gene Period2 in the hippocampus: possible implications for synaptic plasticity and learned behaviour

Abstract

Genes responsible for generating circadian oscillations are expressed in a variety of brain regions not typically associated with circadian timing. The functions of this clock gene expression are largely unknown, and in the present study we sought to explore the role of the Per2 (Period 2) gene in hippocampal physiology and learned behaviour. We found that PER2 protein is highly expressed in hippocampal pyramidal cell layers and that the expression of both protein and mRNA varies with a circadian rhythm. The peaks of these rhythms occur in the late night or early morning and are almost 180° out-of-phase with the expression rhythms measured from the suprachiasmatic nucleus of the same animals. The rhythms in Per2 expression are autonomous as they are present in isolated hippocampal slices maintained in culture. Physiologically, Per2-mutant mice exhibit abnormal long-term potentiation. The underlying mechanism is suggested by the finding that levels of phosphorylated cAMP-response-element-binding protein, but not phosphorylated extracellular-signal-regulated kinase, are reduced in hippocampal tissue from mutant mice. Finally, Per2-mutant mice exhibit deficits in the recall of trace, but not cued, fear conditioning. Taken together, these results provide evidence that hippocampal cells contain an autonomous circadian clock. Furthermore, the clock gene Per2 may play a role in the regulation of long-term potentiation and in the recall of some forms of learned behaviour.

Figure 1. PER2 is rhythmically expressed in the mouse hippocampus

Mice were held in an LD cycle and brains collected at different times in the daily cycle (ZT 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24). Sections of the brains containing the hippocampus and SCN were stained for PER2 expression using IHC. (A) IHC expression was largely localized to the hippocampal regions known as CA1, CA2, CA3 and DG. The patterns of staining varied as a function of time of day, and examples of immunoreactivity in the peak (ZT 2) and trough (ZT 14) of the daily rhythm are shown. The weak, poorly localized staining at ZT 14 is likely to still represent positive immunolabelling. Keeping the IHC conditions constant, there was no positive immunoreactivity in the hippocampus of a Per2-null mutant. Similarly pre-absorption controls lacked this diffuse immunolabelling (Supplementary Figure S1 at http://www.asnneuro.org/an/001/an001e012add.htm). KO, knockout. (B) The number of PER2 immunopositive neurons were counted by observers blinded to the experimental conditions. Levels of PER2 expression in the hippocampus varied as a function of time of day, with peak expression in the late night/early morning and troughs of expression in the late day/early night. (C) In contrast, peak PER2 expression in the SCN was out-of-phase with the hippocampus, with highest expression at the late day/early night. At each time point n = 4. Values are means±S.E.M.

Figure 2. Western blot analysis indicates that hippocampal PER2 expression exhibits a circadian rhythm

Mice were held in DD and wheel-running activity was measured to determine circadian phase. (A) Example of Western blots measuring PER2 expression in whole hippocampal tissue as a function of time of day. Tubulin protein expression was measured as a control for loading and did not vary as a function of time of day (see the Materials and methods section). (B) Levels of PER2 protein expression (normalized to tubulin) varied as a function of time of day in both LD and DD conditions.

Figure 3. A diurnal rhythm in Per2 mRNA in the hippocampus as measured by ISH

Mice were held in DD and wheel-running activity was measured to determine circadian phase. ISH was used to measure Per2 expression in the hippocampus and SCN (n = 3–4 per group). (A) Film images of ISH for Per2 on tissue sections taken from mice sacrificed during the peak and trough of expression. Scale bar = 1 mm. (B) Levels of Per2 expression in the hippocampus varied as a function of time of day, with the strongest hybridization signal in the late night/early morning. (C) Peak Per2 expression in the SCN was out-of-phase with the hippocampus, with the strongest hybridization signal at mid-day. At each time point n = 3–4. Values are means±S.E.M.

Figure 4. Circadian rhythm of PER2::LUC bioluminescence in isolated hippocampus

(A) Organotypical hippocampal slices obtained from PER2::LUC transgenic mice were maintained in culture, and bioluminescence was recorded by imaging (left-hand panel; bioluminescence intensity on a pseudocolour scale) or luminometry (right-hand panel). Rhythmicity of bioluminescence damped over a period of several days. The media was changed immediately before these luminescence measurements began. (B) Recordings of PER2::LUC rhythms from individual cells over a 5-day period. The x-axis label refers to the days in culture.

Figure 5. Per2-mutant mice exhibit abnormal LTP in the hippocampus

(A) LTP measured by stimulating the SC and recording the fEPSP from the CA1 dendritic layer in brain slices from WT (•) and Per2-mutant (○) mice. Data are presented as the fEPSP slope (normalized as a percentage of the baseline). The high-frequency stimulation (3×100 Hz; 1 s duration; 3 min interstimulus intervals) was given at time = 0. Experiments were carried out in the presence of the GABA_A blocker PTX. (B) In contrast, the magnitude of LTP evoked by a weaker stimulation (1×100 Hz) did not significantly vary between WT (•) and Per2-mutant (○) mice.

Figure 6. p-CREB is down-regulated in Per2-mutant mice

(A) Western blot showing hippocampal p-CREB, total CREB, p-ERK (pMAPK) and total ERK (total MAPK) protein expression in WT and Per2-mutant mice (normalized to tubulin). (B) p-CREB and p-ERK protein expression was normalized to total CREB and total ERK expression respectively. These p-CREB/total CREB and p-ERK/total ERK measurements from Per2-mutant mice were compared with data from WT mice. Values are means±S.E.M., *P<0.05.

Figure 7. Per2-mutant mice exhibit severe deficits in the recall of trace-fear conditioning

(A) Levels of trace-fear conditioning recall in Per2-mutant mice tested at 24 h intervals post-training during the day (ZT 6) show severe deficits in their ability to recall learning. (B) This deficit in trace-fear recall persisted in Per2-mutant mice trained and tested during the night (ZT 18). Fear-learning behaviour was measured as the percentage freezing over the total testing time. (C) In contrast, the Per2-mutant mice did not exhibit deficits in recall of cued-fear conditioning. (D) The Per2-mutant mice exhibit deficits in the recall of trace-fear conditioning when held on a 23-h cycle. Values are means±S.E.M., *P<0.05.