Leptin inhibits epileptiform‐like activity in rat hippocampal neurones via PI 3‐kinase‐driven activation of BK channels

Abstract

Journal of Physiologyleptin activates BK channels in hippocampal neurones (Shanley et al. 2002), we hypothesised that leptin, via BK channel stimulation, could modulate aberrant synaptic activity in hippocampal neurones. In this study we show, using hippocampal slices and cultured neurones, that leptin inhibits epileptiform-like activity via PI 3-kinasedriven BK channel activation. This process represents a novel mechanism for controlling hippocampal excitability. Some of these data have been published previously in abstract form (Shanley et al. 2000). METHODS MaterialsRecombinant human leptin (Sigma, St Louis, MO, USA) prepared in 0.01-0.02 % bovine serum albumin as a carrier was used in all experiments. LY 294002, wortmannin, (Calbiochem, La Jolla, CA, USA); tetrodotoxin, PD 98059 (Tocris Cookson, Baldwin, MO, USA); NS-1619 (Biomol); nifedipine, D-APV, diazoxide, glipizide (Sigma); and iberiotoxin and charybdotoxin (Alomone Labs, Israel) were all obtained commercially. Cell cultureCultures of hippocampal neurones were prepared using standard procedures as described previously (Irving & Collingridge, 1998), but were maintained in serum replacement medium (SR2, Sigma). In brief, rat pups 1-3 days old were killed by cervical dislocation and hippocampi removed. The hippocampi were washed in standard Hepes-buffered saline (HBS) comprising (mM): NaCl 135; KCl 5; CaCl 2 1; MgCl 2 1; Hepes 10; D-glucose 25; at pH 7.4. The hippocampi were then treated with a mixture of protease type XIV and type X (both at 0.5 mg ml _1 ; Sigma) for 25 min at room temperature. Dissociated cells were plated onto sterile culture dishes, pretreated with poly-L-lysine (20 mg ml _1 for 1-2 h). Cultures were maintained in a humidified atmosphere of 5 % CO 2 at 37°C for up to 2 weeks. ImmunocytochemistryA goat polyclonal antibody directed against the C-terminal domain of the leptin receptor (Santa Cruz Biotechnology; Hakansson et al. 1998) was used. All immunocytochemical procedures were carried out in HBS. Prior to labelling, hippocampal cultures were fixed with 4 % paraformaldehyde and permeabilised with 0.1 % triton X-100. Cells were then exposed to 10 % blocking milk for 15 min. Hippocampal cultures were incubated with the leptin receptor antibody overnight at 4°C. Immunostaining was visualised by the addition of an Alexa 488-conjugated donkey anti-goat secondary antibody (Molecular Probes). In dual labelling experiments, monoclonal markers for MAP2, GAP43 and synapsin 1 (all from Sigma) were visualised with a Cy3-conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch). In the absence of primary antibody, no labelling was observed following incubation with any of the secondary antibodies. In control experiments, leptin receptor immunoreactivity was blocked by prior incubation of primary antibody with control peptide (200 mg ml _1 ). A laser scanning confocal imaging system (Bio-Rad Microradiance or Zeiss LSM 510) was used for image acquistion. Laser lines of 488 and 543 nm were used to excite Alexa 488 and Cy3, respectivel...

Down-regulation of BK channel expression in the pilocarpine model of temporal lobe epilepsy

“…In this study, spontaneous bursting activity in the cerebral cortical neurons of the El mouse, which shows a high susceptibility to convulsions, was also completely inhibited by IbTX (Jin et al, 2000). However, insulin and leptin that inhibit epileptiform-like bursting activities in rat hippocampal neurons might involve in activation of BK channels (O'Malley et al, 2003;Shanley et al, 2002). Interestingly, BK channel knockout mice do no apparently exhibit seizures but cerebellar-related neurological problems (i.e.…”

Leptin regulation of hippocampal synaptic function in health and disease

“…In accordance with the high levels of leptin receptor expression detected at hippocampal synapses [6], evidence is growing that leptin is a potent modulator of hippocampal excitatory synaptic function [11][12][13][14][15]. Indeed, studies in obese leptin-insensitive rodents (Zucker fa/fa rats; db/db mice) have identified deficits in hippocampal long-term potentiation (LTP) and long-term depression (LTD) as well as spatial memory [16,17].…”

“…Thus, leptin results in a persistent increase in excitatory synaptic transmission (leptin-induced LTP) in adult (12-16 week old) hippocampal slices [12,29], an effect requiring leptin receptor activation as robust leptin-induced LTP was observed in Zucker lean, but not leptin-insensitive, Zucker fa/fa, rats [12]. In hippocampal neurons, leptin receptors are expressed at both presynaptic and postsynaptic sites [6], and consequently leptin-induced LTP could potentially be expressed at either locus. However, no significant changes in paired pulse facilitation ratio (PPR) and coefficient of variation (CV) accompany the leptin-driven increase in synaptic efficacy indicating a postsynaptic expression mechanism.…”

“…The central actions of leptin are not restricted to the neural control of feeding behaviour, as leptin can also influence various developmental processes in the immature brain. Indeed, in support of extra-hypothalamic targets, leptin receptors are widely distributed throughout the central nervous system (CNS), with high levels of expression detected in the hippocampus and cerebellum in particular [4][5][6]. Leptin receptor expression in the hypothalamus is altered by changes in the circulating levels of leptin [7].…”

A Tale of Two Hormones: Role of Leptin and Insulin in Hippocampal Synaptic Function

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