Figure 3.

5-HT evoked Ca²⁺ transients in PPG neurons are mainly observed in dendrites. (A) Intracellular Ca²⁺ changes in PPG neurons can be monitored using the genetically encoded Ca²⁺ indicator, GCaMP3. Glucagon promoter (Glu)-Cre mice were crossed with mice expressing CAG-promoter-STOP-GCaMP3 in the Rosa26 locus. The Rosa26 locus is active in most cell types. In cells with active glucagon promoter, the bacterial recombinase Cre is produced and excises the STOP sequence flanked by lox sites upstream of the GCaMP3 gene. This process results in cytosolic expression of GCaMP3 in these cells, detected here with an anti-GFP antibody (B; scale bar: 100 μm). (C) PPG neurons respond to both leptin (1 nM, n = 8 somata, left panel) and CCK-8 (200 nM, n = 12 somata, middle panel) with an increase in cytosolic Ca²⁺. The panel on the right shows three pseudocolored cells, two of which (white arrowheads) show increased fluorescence intensity after application of 200 nM CCK-8. The black arrowhead indicates one cell that did not increase [Ca²⁺]_i in the presence of 200 nM CCK-8 (Scale bar: 30 μm). (D) GCaMP3 fluorescence intensity is sensitive to changes in cytosolic Ca²⁺ concentration. For example, bath application of 100 μM glutamate (glut) leads to a strong reversible rise in somatic Ca²⁺ concentration (Scale bar: 20 μm). The panel on the left shows two pseudocolored PPG neurons before, during and after stimulation with 100 μM glutamate. (E) Fluorescence intensity expressed as a fraction of the intensity at the beginning of the experiment. Traces from individual cells are plotted in gray or dark blue, and the average response is shown in red. While virtually every cell responded to 100 μM glutamate with a somatic Ca²⁺ transient, in this example, only one of nine PPG neurons responded to 20 μM 5-HT (shown in the dark blue trace). (F) Representative images showing dendritic GCaMP3 fluorescence intensity before, during and after 20 μM 5-HT. Arrows point to dendrites with transient increases in Ca²⁺ (Scale bar = 50 μm). Transient changes in intracellular Ca²⁺ in PPG dendrites (n = 19) in response to 20 μM 5-HT and 100 μM glutamate are shown in the middle. Measurements from individual regions of interest are shown in gray, the mean trace is shown in red. On the right, quantification of responses showing median AUC during the response to 2 μM, 20 μM, or 200 μM 5-HT (n = 88 dendrites (4 mice)). Friedman test (Chi-square = 60.03, p < 0.0001) followed by post-hoc comparisons revealed the response to 2 μM 5-HT to be significantly different to both 20 μM and 200 μM 5-HT (p < 0.0001 for both).