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. 2012 Oct 1;590(19):4917-32.
doi: 10.1113/jphysiol.2012.234187. Epub 2012 Jul 9.

Regulation of hippocampus-dependent memory by the zinc finger protein Zbtb20 in mature CA1 neurons

Anjing Ren  1 , Huan Zhang, Zhifang Xie, Xianhua Ma, Wenli Ji, David Z Z He, Wenjun Yuan, Yu-Qiang Ding, Xiao-Hui Zhang, Weiping J Zhang
Affiliations

Regulation of hippocampus-dependent memory by the zinc finger protein Zbtb20 in mature CA1 neurons

Anjing Ren et al. J Physiol. .

Abstract

The mammalian hippocampus harbours neural circuitry that is crucial for associative learning and memory. The mechanisms that underlie the development and regulation of this complex circuitry are not fully understood. Our previous study established an essential role for the zinc finger protein Zbtb20 in the specification of CA1 field identity in the developing hippocampus. Here, we show that conditionally deleting Zbtb20 specifically in mature CA1 pyramidal neurons impaired hippocampus-dependent memory formation, without affecting hippocampal architecture or the survival, identity and basal excitatory synaptic activity of CA1 pyramidal neurons. We demonstrate that mature CA1-specific Zbtb20 knockout mice exhibited reductions in long-term potentiation (LTP) and NMDA receptor (NMDAR)-mediated excitatory post-synaptic currents. Furthermore, we show that activity-induced phosphorylation of ERK and CREB is impaired in the hippocampal CA1 of Zbtb20 mutant mice. Collectively, these results indicate that Zbtb20 in mature CA1 plays an important role in LTP and memory by regulating NMDAR activity, and activation of ERK and CREB.

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Figures

Figure 1
Figure 1. Lack of Zbtb20 protein expression in CA1 pyramidal cells of the adult CA1-ZB20KO mouse hippocampus
Zbtb20 expression was detected by immunohistochemistry with anti-Zbtb20 antibody 9A10 and visualized with Alexa 594 (red) on coronal forebrain sections from mice at P17 (A), P21 (B), P24 (C), 1 month (D), 2 months (E) and 4 months (F) of age. Zbtb20 expression in CA1 pyramidal cells of mutant mice began to reduce markedly at P24 and became almost undetectable by 2 months of age, but remained unaltered in the CA3 and DG regions at all ages examined. (Scale bars: 200 μm).
Figure 2
Figure 2. Impaired spatial memory of CA1-ZB20KO mice in the Morris water maze and novel object recognition tests
A, mean escape latency during 8 training days. CA1-ZB20KO mice showed normal acquisition of spatial reference memory in the Morris water maze task. B and C, in probe tests conducted 1 day (B) or 10 days (C) after the acquisition phase, CA1-ZB20KO mice spent significantly less time in the target quadrant than control mice (P < 0.05). D, there was no difference in swim speed between control and CA1-ZB20KO mice during the 8 training days and 2 probe tests (P1 and P2). E, exploratory preference in the training session of novel object recognition task. The dotted line represents performance at the chance level (50%). The amount of time spent exploring the two objects was the same for control and CA1-ZB20KO mice. F, memory in control and CA1-ZB20KO mice was measured after training at different time intervals as indicated. CA1-ZB20KO mice exhibited much weaker preference for the novel object than did control mice in retention tests conducted 1 h and 1 day after training. *P < 0.05.
Figure 3
Figure 3. Impaired contextual fear memory but not cued fear memory in CA1-ZB20KO mice
A, C and E, contextual fear conditioning 1 h (A), 1 day (C), and 10 days (E) after training. B, D and F, cued fear conditioning 1 h (B), 1 day (D) and 10 days (F) after training. G, freezing behaviour evoked by foot shocking did not differ significantly between CA1-ZB20KO and control mice during the five post-shock intervals. H, 1 day and 10 days after fear conditioning, freezing behaviour evoked by exposure to the conditioned environment was significantly impaired in CA1-ZB20KO mice compared to control mice. *P < 0.05; **P < 0.01.
Figure 4
Figure 4. CA1-ZB20KO mice show normal density of dendritic spines in CA1 pyramidal neurons
A, representative images of Golgi stained dendrites from the CA1 region of the hippocampus (Scale bar: 5 μm). B, there was no significant difference in the density of dendritic spines in CA1 pyramidal neurons between control and CA1-ZB20KO mice (Control, 27 neurons, 3 mice; CA1-ZB20KO, 27 neurons, 3 mice, P > 0.05).
Figure 5
Figure 5. Normal basal synaptic transmission in CA1-ZB20KO mice
A, input–output plot of synaptic transmission between stimulation strength and corresponding fEPSP slope. B, input–output plot of synaptic transmission between fibre volley and corresponding fEPSP slope. C, plot of synaptic transmission between stimulation strength and presynaptic fibre volley amplitude. D, CA1-ZB20KO and control slices showed no significant difference in PPF of the EPSP at various interpulse intervals. Control, 13 slices, 6 mice; CA1-ZB20KO, 15 slices, 8 mice.
Figure 6
Figure 6. Impaired LTP and NMDAR activity in control and CA1-ZB20KO mouse hippocampal slices
A, LTP elicited by TBS in control slices (Control, 5 slices, 3 mice; Control+TBS, 8 slices, 5 mice). B, LTP elicited by TBS in CA1-ZB20KO slices (CA1-ZB20KO, 7 slices, 4 mice; CA1-ZB20KO+TBS, 8 slices, 6 mice). C, Zbtb20 knockout impairs TBS-induced LTP in CA1-ZB20KO slices (panel C is a composite of A and B). D, mean ΔfEPSP slope measured 60–90 min after induction of LTP in control and CA1-ZB20KO slices (Control, 8 slices, 5 mice; CA1-ZB20KO, 8 slices, 6 mice). E, decreased NMDAR-mediated responses in the hippocampal CA1 of CA1-ZB20KO mice. Lower traces show representative AMPAR-mediated EPSCs recorded at −70 mV in the control solution. Upper traces show representative NMDAR-mediated EPSCs recorded at +40 mV in the presence of 10 μm CNQX, an AMPAR blocker. F, the NMDA/AMPA ratio in CA1 neurons of CA1-ZB20KO mice (34.2 ± 4.0%, 10 neurons, 10 slices, 5 mice) was significantly lower than in control mice (50.8 ± 4.4%, 9 neurons, 9 slices, 5 mice). The dotplots display the actual ratios for each animal in the control and CA1-ZB20KO groups **P < 0.01.
Figure 7
Figure 7. Impaired ERK and CREB phosphorylation in the hippocampal CA1 of CA1-ZB20KO mice
A and C, representative western blots of ERK (A) and CREB (C) protein concentrations and phosphorylation levels in the tissue lysates from hippocampal CA1 of sham shocked and shocked animals. No significant changes in overall ERK or CREB levels were observed between sham shocked and shocked control and CA1-ZB20KO mice. B and D, densitometric analysis of ERK (B) and CREB (D) activation 1 min, 1 h and 24 h after shock (both isoforms of pERK were together analysed). Levels of pERK and pCREB in control and CA1-ZB20KO mice were significantly increased 1 h and 24 h after shock, but this increase was significantly less in CA1-ZB20KO mice at 1 h compared with that in control counterparts. No significant changes in ERK or CREB activation were observed 1 min after shock. #P < 0.05, ##P < 0.01 vs. control mice 1 h after shock, **P < 0.01 vs. sham shocked mice.
Figure 8
Figure 8. Decreased expression of pCREB in the CA1 of hippocampus of CA1-ZB20KO mice
Immunohistochemistry was used to detect pCREB expression on coronal forebrain sections. A and B, pCREB expression was low and not different between sham foot shocked control and CA1-ZB20KO mice. C and D, pCREB expression was significantly increased in the hippocampus 1 h after foot shock. In the CA1 marked by box, pCREB expression in CA1-ZB20KO mice was significantly lower than control mice. C′ and D′, High-magnification views of the boxed areas in C and D, respectively. (Scale bars: 200 μm for A-D; 50 μm for C′ and D′).
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