Imaging system to measure dynamic activation of PI3K in live hypothalamic neurons. (A) Outline of experimental procedure. CMV prom, cytomegalovirus promoter. (B and C) Low- (B) and high-magnification (C) images illustrating adenoviral expression of the PI3K reporter in a Tg.PomcCre⁺ mouse. (D) Dose-dependent response to insulin of PI3K reporter membrane translocation in POMC neurons. Representative images from various time frames are shown; 0 minutes indicates either before insulin addition or after extensive wash following a previous treatment. Arrows indicate membrane localization.

Generating PomcCre, AgrpCre transgenic mice, and neuron-specific Stat3 knockout mice. (A) Transgene diagrams in which blue and red boxes represent protein-coding regions for the Pomc and Agrp genes, respectively. (B) Schematic representation of the Rosa26 reporter (top) and Stat3 (bottom). (C) X-gal staining of brain sections through the arcuate nucleus from transgenic R26R⁺ mice. (D) X-gal staining (blue) combined with immunohistochemistry (brown) for α-MSH and Agrp; neuropeptide immunostaining is distributed throughout neuronal cell bodies and also in some terminals; X-gal staining is a localized precipitate within the neuronal cytoplasm.

Differential regulation of PI3K by leptin in POMC and Agrp neurons. Example of PI3K reporter distribution during leptin treatment of a POMC neuron (A) and leptin withdrawal from an Agrp neuron (B). Upper and lower panels show the raw image and corresponding cell membrane edges, respectively. (C) Distribution of edge pixels as a function of time under the indicated conditions. (D) Cumulative distribution of the earliest time at which responsive POMC (n = 19) or Agrp (n = 18) neurons first manifest reporter translocation. (E) Percentage of all neurons examined that were responsive to leptin addition (POMC, n = 29) or withdrawal (Agrp, n = 21); each point represents a 2-minute window.

PI3K activation in POMC neurons does not require Stat3 function. (A) Representative Stat3-deficient POMC neuron exposed to 100 nM leptin, in which membrane localization of PI3K reporter protein occurs within several minutes of hormone addition. Arrows indicate membrane localization. (B) Percentage of POMC neurons responsive to leptin in the presence (+/+) or absence (–/–) of Stat3. (C) Unifying mechanism for leptin modulation of key arcuate nucleus neurons in which PI3K activity is a mediator and/or marker of neuronal activation and neuropeptide release in both Agrp (pink) and POMC (green) neurons. The effects of insulin on PI3K activity are direct in both neuronal subtypes, but the effects of leptin on PI3K activity in Agrp neurons require synaptic transmission from POMC or other (gray) inhibitory presynaptic neurons. IR, insulin receptor; LepR, leptin receptor.

PI3K regulation by insulin in Agrp neurons and effects of synaptic inhibitors in POMC and Agrp neurons. (A) In Agrp neurons, leptin withdrawal has the same effect as insulin addition; both treatments stimulate membrane localization of the PI3K reporter protein. The example shown represents the same neuron in which a slice preincubated with 100 nM leptin for 60 minutes was then perfused with aCSF; during this time the PI3K reporter protein exhibited initial membrane localization followed by a return to the cytoplasm by 58 minutes. Addition of 100 nM insulin then triggered membrane localization again. In other experiments, 100 nM insulin was observed to cause PI3K activation in Agrp neurons that had not previously been exposed to leptin. Arrows indicate membrane localization. (B) Example of the same experiment as in A but carried out in the presence of 1 μM TTX (which blocked the previous response to leptin withdrawal). (C and D) Percentage of POMC (C) and Agrp (D) neurons responsive to leptin with (aCSF) or without (low Ca⁺⁺ or TTX) synaptic transmission. *P < 0.05; **P < 0.01 as determined by χ² analysis. (E and F) Percentage of POMC (E) and Agrp (F) neurons responsive to insulin with (aCSF) or without (low Ca⁺⁺) synaptic transmission.