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...

Recent studies have implicated the hormone leptin in synaptic plasticity associated with neuronal development and learning and memory. Indeed, leptin facilitates hippocampal long-term potentiation and leptin-insensitive rodents display impaired hippocampal synaptic plasticity suggesting a role for endogenous leptin. Structural changes are also thought to underlie activity-dependent synaptic plasticity and this may be regulated by specific growth factors. As leptin is reported to have neurotrophic actions, we have examined the effects of leptin on the morphology and filopodial outgrowth in hippocampal neurons. Here, we demonstrate that leptin rapidly enhances the motility and density of dendritic filopodia and subsequently increases the density of hippocampal synapses. This process is dependent on the synaptic activation of NR2A-containing NMDA receptors and is mediated by the MAPK (ERK) signaling pathway. As dendritic morphogenesis is associated with activity-dependent changes in synaptic strength, the rapid structural remodeling of dendrites by leptin has important implications for its role in regulating hippocampal synaptic plasticity and neuronal development.

Stress and anxiety are important causal and exacerbating factors in functional gastro-intestinal (GI) disorders such as irritable bowel syndrome. Stress affects GI motility, faecal transit and visceral pain sensitivity. Additionally, permeability and function of the gut epithelium, which acts as a barrier between the external environment and the body's internal milieu is altered by stress. However, the effects of an enhanced stress response on colonic morphology require further investigation. We have used two animal models of stress and anxiety, the maternally separated (MS) and Wistar Kyoto (WKY) rats to examine colonic morphology. These rats exhibit increased anxiety behaviours, visceral hypersensitivity and increased stress-induced defecation in the open field arena. At a morphological level, increased mucus secretion and an associated elevation in the number of mucosal goblet cells was observed in the high anxiety rats. Additionally, the mucosal layer was flattened in MS and WKY rats, a finding indicative of mild mucosal damage. Furthermore, the muscular layer of the distal colon in these animals was thickened, an observation that may have implications for faecal transit and visceral pain perception. This study provides evidence of altered colonic function and morphology in two animal models with a heightened response to stress.