Permissive Role of Insulin in the Expression of Long-Term Potentiation in the Hippocampus of Immature Rats

Many studies indicate that impairment in insulin signaling leads to learning and memory deficits. However, previous studies failed to establish a clear role of insulin in long-term potentiation (LTP), the best cellular model of memory formation. Here we show that while insulin pretreatment did not affect LTP magnitude in the adult rat hippocampus, it facilitated LTP expression in the immature hippocampus. The tyrosine kinase inhibitor AG-1024 abolished the effect of insulin in young rats, suggesting the involvement of the insulin receptor. On the other hand, increasing extracellular glucose concentration failed to facilitate LTP and application of an insulin-responsive glucose transporter-4 inhibitor did not impair the effect of insulin. These results suggest that the facilitatory action of insulin on LTP is not an indirect effect on glucose homeostasis/utilization. Involvement of the MAPK/ERK pathway, a known downstream pathway of insulin signaling, was revealed by pretreatment with PD98059, which blocked the insulin-mediated LTP facilitation. Consistent with this, high-frequency stimulation induced a significant increase in the level of phosphorylated Erk-2 in insulinReceived: October 15, 2010 Accepted: December 30, 2010 Published online: February 22, 2011 W.H. Yung School of Biomedical Sciences, Faculty of Medicine The Chinese University of Hong Kong Shatin, Hong Kong, SAR (China) Tel. +852 2608 6880, E-Mail whyung @ cuhk.edu.hk © 2011 S. Karger AG, Basel 1424–862X/10/0184–0236$26.00/0 Accessible online at: www.karger.com/nsg Insulin Facilitates LTP Expression Neurosignals 2010;18:236–245 237 memory functions. Insulin also exerts a restorative action on memory impairment in various settings, including drug-induced experimental model of diabetes [17, 18] , models of stress [19] and also in Alzheimer’s disease patients [20] . These findings are consistent with the observation that insulin receptors are especially abundant in the hippocampus in both rats and humans [21–24] and that insulin receptors on pyramidal neurons are upregulated after training in the water maze task [25] . It is widely accepted that long-term synaptic plasticity in the hippocampus, especially long-term potentiation (LTP), is the mechanism that underlies some forms of learning and memory. In the hippocampus, insulin receptor immunoreactivity and insulin binding sites are particularly prominent in the CA1 subfield [26, 27] . However, although a role of insulin in inducing a long-term depression-like phenomenon has been well documented in the hippocampus [28] , the effect of insulin on LTP remains unclear. In fact, it has been shown that insulin does not facilitate high-frequency stimulation (HFS)-induced LTP [29] . Also, although some studies show that in experimental diabetes, LTP is impaired and can be rescued by insulin [17, 30] , there are reports showing that LTP is preserved in this condition [e.g. 31 ]. Thus, the exact relationship between insulin and LTP is still obscure. In this study, we addressed the question of whether insulin application has any effect on LTP expression in the hippocampus of normal, healthy rats. Since it is known that insulin receptor expression in the brain is developmentally regulated, being highest in young age and decreasing with age [1, 32] , we examined and compared the effect of insulin on LTP in both immature and adult rats. Our experiments reveal that insulin plays a critical permissive role in LTP expression in the immature brain via insulin receptors but not through its influence on glucose utilization/homeostasis. This finding suggests that insulin may be an indispensible endogenous factor allowing LTP expression in normal young animals for learning and memory, and provides an explanation for the impaired cognitive function in children with insulin-dependent type 1 diabetes [33] . Materials and Methods All experimental protocols and procedures described were performed in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Experimentation Ethical Committee of the Chinese University of Hong Kong. Efforts were made to minimize the number of animals used and their suffering. Hippocampal Brain Slices Sprague-Dawley rats, provided by the Laboratory Animal Services Center of the Chinese University of Hong Kong, were anesthetized with isoflurane, and then sacrificed by decapitation. The brains were immediately removed and cut into two halves in the sagittal plane and then immersed in ice-cold artificial cerebrospinal fluid (ACSF) of the following composition: 125 m M NaCl, 2.0 m M KCl, 1.2 m M MgSO 4 , 2.5 m M CaCl 2 , 1.2 m M KH 2 PO 4 , 11 m M glucose, and 26 m M NaHCO 3 , which was continuously bubbled with 95% O 2 and 5% CO 2 (pH 7.4; osmolarity 290–310 mosm). The brains were then glued with the lateral side down onto a platform in a chamber filled with oxygenated ice-cold ACSF, and 300mthick parasagittal sections were cut using a vibrating microtome (Integraslice 7550MM, Campden Instruments Ltd., Loughborough, UK). Slices were preincubated in a holding chamber containing oxygenated ACSF at 34 8 1 ° C for at least 1 h. LTP Measurements A planar multielectrode recording setup (MED64 system, Alpha Med Sciences Co., Ltd, Tokyo, Japan) was employed to record the field excitatory postsynaptic potential (fEPSP), and to study LTP. The methodology has been described in details elsewhere [34, 35] . Briefly, hippocampal slices were placed on special probes that were fabricated with 8 ! 8 electrode arrays (20 ! 20  m, made of indium tinoxide and platinum black) and precoated with polyethylenimine (Sigma). The P210A probes (Alpha Med Sciences) with an interelectrode distance of 100  m were routinely used. Correct placement of the electrodes at the CA3-CA1 region was done manually, monitored by a microscope (MIC-D, Olympus Ltd., Japan). To increase the efficiency of the experiments and to minimize the variation in the results arising from differences in incubation times, a maximum of 4 slices were studied simultaneously by means of a splitter provided by the manufacturer. Each slice was submerged in and superfused with oxygenated ACSF at a flow rate of 1.3–1.5 ml/min. fEPSPs were recorded from the dendritic layer of CA1 neurons by choosing an electrode in the Schaffer collateral pathway as the stimulating electrode. Based on the stimulus-response curve, we chose a stimulation intensity that evoked the fEPSP with a magnitude of 30–40% of the maximum response. After allowing a stable baseline of 30 min in which the stimulus was delivered every 60 s, an LTP induction protocol consisting of 1 train of 100-Hz stimuli that lasted for 1 s was applied, and the field potential response after the tetanus was recorded for 1 h. The magnitude of LTP was quantified as percentage change in the average amplitude of the fEPSP taken at 50to 60-min intervals after LTP induction. Drugs were applied to the slices for at least 30 min before the delivery of HFS. Protein Analyses After preparation of hippocampal slices from young rats, the CA1 regions from 5–7 slices were cut and incubated with radioimmunoprecipitation assay lysis buffer including Pimix (1: 200 dilution) and 0.1 M phenylmethylsulfonyl fluoride (1: 100 dilution) for 30 min. After centrifugation at 13,000 rpm for 15 min, the supernatant was measured for the concentration of protein and was then subjected to SDS–PAGE (10% polyacrylamide gel electrophoresis). The protein was transferred to nitrocellulose membrane using an electrophoretic transfer system. The membrane was blocked with Tris-buffered saline mixed with Tween-20 including 5% milk for 1 h followed by overnight incubation at 4 ° C