The obese gene product leptin is an important signaling protein that regulates food intake and body weight via activation of the hypothalamic leptin receptor (Ob-Rb; Jacob et al., 1997). However, there is growing evidence that Ob-Rb is also expressed in CNS regions, not directly associated with energy homeostasis (Mercer et al., 1996; Hakansson et al., 1998). In the hippocampus, an area of the brain involved in learning and memory, we have found that leptin facilitates the induction of synaptic plasticity. Leptin converts short-term potentiation of synaptic transmission induced by primed burst stimulation of the Schaffer collateral commissural pathway into long-term potentiation. The mechanism underlying this effect involves facilitation of NMDA receptor function because leptin rapidly enhances NMDA-induced increases in intracellular Ca(2+) levels ([Ca(2+)](i)) and facilitates NMDA, but not AMPA, receptor-mediated synaptic transmission. The signaling mechanism underlying these effects involves activation of phosphoinositide 3-kinase, mitogen-activated protein kinase, and Src tyrosine kinases. These data indicate that a novel action of leptin in the CNS is to facilitate hippocampal synaptic plasticity via enhanced NMDA receptor-mediated Ca(2+) influx. Impairment of this process may contribute to the cognitive deficits associated with diabetes mellitus.

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...

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