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. 2007 Aug;35(4):559-72.
doi: 10.1016/j.mcn.2007.05.001. Epub 2007 May 10.

Leptin promotes rapid dynamic changes in hippocampal dendritic morphology

Dervla O'Malley  1 , Neil MacDonald, Sarah Mizielinska, Christopher N Connolly, Andrew J Irving, Jenni Harvey
Affiliations

Leptin promotes rapid dynamic changes in hippocampal dendritic morphology

Dervla O'Malley et al. Mol Cell Neurosci. 2007 Aug.

Abstract

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.

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Figures

Fig. 1
Fig. 1
Leptin increases the density of dendritic filopodia in hippocampal neurons. (A) Confocal images of actin staining in hippocampal neurons (6–8 DIC) labeled with Alexa 488-conjugated phalloidin. (Ai) Control neurons display relatively few actin-rich protrusions or growth cones. Exposure of neurons to leptin (50 nM) for 30 min stimulated an increase in the number of filopodia (ii) and growth cones (iii) extending from processes (indicated by arrows). (B) Confocal images of hippocampal neurons (9DIC) dual labeled with Alexa 488-conjugated phalloidin (green) and the somatodendritic marker, MAP2 (red). Leptin (50 nM; 30 min) increased the number of filopodia protruding from dendritic (MAP2-positive) processes (ii) compared to control (i). (C) Histogram illustrating the pooled data of the mean number of dendritic filopodia in control and leptin treated neurons. Leptin stimulates circa a 3-fold increase in the density of filopodia. (D) Histogram illustrating the pooled data of the mean number of dendritic filopodia after 10 min, 20 min, 30 min, 3 h and 18 h exposures to leptin (50 nM). The leptin-induced increase in filopodial density is apparent after only 10 min exposure to leptin and reaches a peak after 3 h. (F) Representative confocal images (i–iii) of leptin receptor (ObR; i) and synapsin-1 (ii) immunoreactivity in 9-day-old hippocampal cultures. The merged image (iii) shows that leptin receptor labeling is highly localized to synapses.
Fig. 2
Fig. 2
Leptin rapidly increases the number and motility of dendritic filopodia. (A) Confocal images of a hippocampal neuron (11 DIC) transiently transfected with cytosolic EGFP. The region of interest was magnified and the images obtained at various time points (3–30 min) are shown in panel Aii. Under control conditions there are a few filopodia extending from dendrites and these had limited motility. Leptin (50 nM) treatment stimulated a rapid increase in number and motility of filopodia extending from the highlighted dendrite (arrow). (B) Histogram of the pooled data illustrating the relative change in the density and motility of filopodia following exposure to leptin (50 nM). *P < 0.05.
Fig. 3
Fig. 3
Leptin stimulates an increase in neurite length. (A) Confocal images of a hippocampal neuron (11 DIC) transiently transfected with cytosolic EGFP. The region of interest in panel Ai is magnified and displayed in panel Aii. In control conditions (in the absence of leptin), there is little or no change in the length of the neurite. However, application of leptin (50 nM) to the same neuron rapidly stimulated elongation of the same process. (B) Pooled data illustrating the mean increase in the relative length of protruding neurites following leptin treatment. *P < 0.05.
Fig. 4
Fig. 4
Synaptic activation of NMDA receptors is required for leptin-induced neurite growth. (A–C) Confocal images of hippocampal neurons (12 DIC) labeled with Alexa 488-conjugated phalloidin. The zoomed regions of interest (white box) are depicted either below (A) or alongside (B, C) the parent image. (A) Prior exposure of neurons to TTX (500 nM; 30 min) attenuated the leptin-induced increase in filopodial density (Aii) compared to control (Ai). (Bi) Incubation with the NMDA receptor antagonist, D-APV (50 μM; 30 min), did not alter the filopodial density per se. However, prior exposure to D-APV attenuated the leptin-induced increase in the density of dendritic filopodia (Bii). Treatment with the NR2B-selective NMDA receptor antagonist, ifenprodil (10 μM; 30 min), had no effect on the number of dendritic filopodia per se (Ci) nor did it affect the ability of leptin to increase the density of filopodia (Cii). (D) Histogram illustrating the pooled data of the mean number of filopodia in control conditions and in neurons treated with leptin (50 nM; 30 min) alone and in the combined presence of NBQX (2 μM; 30 min), D-APV (50 μM; 30 min), ifenprodil (10 μM; 30 min) and TTX (500 nM; 30 min). *, ** and *** denote P < 0.05, P < 0.01 and P < 0.001, respectively.
Fig. 5
Fig. 5
Leptin does not promote the translocation of NR2A subunits into dendritic filopodia. (A) Expression of NR2A and NR2B NMDA receptor subunits in hippocampal cultures. Confocal images of ObR immunolabeling, together with either NR2A (i; 9 DIC) or NR2B (ii; 7DIC) labeling in hippocampal neurons. High levels of NR2A and NR2B are expressed in hippocampal neurons at this stage of development. (B) Confocal images of Alexa phalloidin staining (green), NR2A immunolabeling (red) and the merged images in hippocampal neurons (8 DIC) in control conditions and following exposure to leptin (50 nM; 30 min). Leptin induced an increase in the density of dendritic filopodia; however, this was not associated with any change in the distribution of NR2A staining.
Fig. 6
Fig. 6
Leptin increases the density of filopodia via a MAPK-, but not PI 3-kinase-, driven pathway. (A, B) Confocal images of F-actin staining in hippocampal neurons (10–11 DIC) labeled with Alexa 488-conjugated phalloidin. The magnified regions of interest (white boxes) are illustrated below the parent images. (Ai) Incubation with wortmannin (50 nM; 1 h) had no effect on the density of filopodia per se, and it did not affect the ability of leptin to increase the number of filopodia. In contrast, prior incubation with PD98059 (10 μM; 1 h) attenuated leptin-induced increase in filopodial density (Bii). The density of filopodia was not altered in neurons exposed to PD98059 (Bi). (C) Histogram of pooled data illustrating the mean number of filopodia in control conditions and in neurons exposed to leptin (50 nM; 30 min) both alone and in the presence of wortmannin (WM; 50 nM), LY294002 (LY; 10 μM), PD98059 (PD; 10 μM) and U0126 (UO; 1 μM).
Fig. 7
Fig. 7
Leptin increases the density of hippocampal synapses. (A) Confocal images of hippocampal neurons (7 DIC) labeled with an anti-synapsin 1 antibody. The magnified regions of interest (white box) and corresponding intensity profile are illustrated to the right of the parent images. Incubation with leptin (50 nM; 30 min) stimulated an increase in the number and intensity of synapsin-1 puncta (Aii) compared to control (Ai). (B) Histogram of the pooled data of the relative intensity of staining in neuronal processes in control conditions and following application of leptin (50 nM; 30 min). (C) Confocal images of hippocampal neurons (14 DIC) dual labeled with Alexa 488-conjugated phalloidin (green) and an anti-synapsin 1 antibody (red). Under control conditions, the neurons display tightly intertwined processes with synapsin 1 labeling. Application of leptin (50 nM; 30 min) to these neurons increased the number and intensity of synapsin 1 puncta as well as the density of spines (arrows).
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