There is growing evidence that insulin can modulate CNS function. Indeed in the hippocampus, insulin receptors as well as crucial components of the insulin receptor-signalling pathways are expressed. In the present study, we have used immunocytochemical techniques to determine the functional localisation of insulin receptors in hippocampal cultures and Ca²⁺ imaging techniques to investigate a potential role for insulin in modulating neuronal activity. Neonatal rats were killed by cervical dislocation and hippocampal neurones were cultured as described previously (Irving & Collingridge, 1998). Immunocytochemical labelling was performed on cells fixed in paraformaldahyde (4 %) and permeabilised with Triton X-100 (0.1 %). Cells were labelled overnight with a polycolonal antibody directed against the β-subunit of the insulin receptor. Dual labelling was carried out using antibodies raised against the somato-dendritic marker, MAP2, and the synaptic protein, synapsin-1. For measurement of changes in intracellular Ca²⁺ levels, cells of between 7 and 12 days were loaded with fura-2 AM (6 µM) and changes in fluorescence ratio were measured with a digital epifluoresence imaging system. Comparison of insulin receptor immunoreactivity with MAP2 labelling demonstrated extensive insulin receptor labelling on the soma and dendrites of hippocampal neurones (n = 3). Dual labelling with synapsin 1 also demonstrated punctate insulin receptor labelling associated with synaptic terminals (n = 3). In functional studies, the actions of insulin on synaptically driven spontaneous Ca²⁺ oscillations, generated by perfusion with a glycine-containing Mg²⁺-free standard medium were investigated. Application of insulin (10-50 nM) rapidly and reversibly inhibited the spontaneous Ca²⁺ oscillations by 64.9 ± 4.2 % (mean ± S.E.M.; n = 349). Subsequent application of insulin, 20-30 min later, induced a comparable response (n = 43). As insulin activates ATP-sensitive K⁺ (K_ATP) channels in the hypothalamus (Spanswick et al. 2000), the role of K_ATP channels in the actions of insulin was examined. Incubation with glibenclamide (1 µM) blocked the actions of insulin on Ca²⁺ oscillations by 65.6 ± 2.2 % (n = 69). In conclusion, we have demonstrated insulin receptor immunoreactivity on hippocampal neurones and at synapses. In functional studies, insulin reversibly inhibits synaptically driven Ca²⁺ oscillations, via a process involving K_ATP channel activation. These data suggest that insulin plays an important role in modulating hippocampal synaptic function.We thank The Wellcome Trust for financial support (grant no. 055291).

Irving, A.J. & Collingridge, G.L. (1998). J. Physiol. 511, 747-759. abstract

Spanswick, D., Smith, M.A., Mirshamsi, S., Routh, V.H. & Ashford, M.L.J. (2000). Nat. Neurosci. 3, 757-758.