Article Figures & Data
Figures
- Figure 1.
Colocalization of the POMC–DsRed fluorophore and immunoreactivity for ACTH. The Distribution of POMC neurons is indicated by the presence of DsRed in coronal (A) and sagittal (B; a medial slice cut in the sagittal plane) brain slices from POMC–DsRed transgenic mice (leftmost images). ACTH-immunoreactivity visualized with a FITC-conjugated secondary antibody (middle, green). Right, Merged images show extensive overlap between POMC–DsRed and ACTH immunoreactivity. Rostral (R), caudal (C), dorsal (D), and ventral (V) orientations are indicated in the bottom left.
- Figure 2.
Specificity of the ACTH antibody. A, ACTH-IR in a brain slice containing the arcuate nucleus from a wild-type C57BL/6J mouse visualized with diaminobenzidine and imaged with bright-field microscopy. B, Preabsorption of the ACTH antibody with ACTH 1-39 peptide eliminates immunoreactivity. C, ACTH-IR in the arcuate nucleus of a wild-type mouse and a POMC knock-out mouse (D). Immunofluorescent detection of ACTH (E) and α-MSH (F) in the same brain section shows overlap of the two immunoreactive peptides (G), although α-MSH-IR is predominately in fibers. R, Rostral; C, caudal; D, dorsal; V, ventral.
- Figure 3.
Distribution of GAD-positive and GAD-negative POMC neurons. A, Low-power image of a sagittal brain slice containing the arcuate nucleus taken from a POMC–DsRed;GAD67–gfp double-transgenic mouse showing DsRed (left), GAD67–gfp (middle), and a merged image (right). Arrows indicate some of the double-labeled cells. B, Larger image of the area outlined by the box in A (right). C, Larger image of a sagittal section from a double-transgenic mouse. The circle indicates a region that is often observed to contain primarily GAD67–gfp-negative POMC–DsRed neurons. D, A coronal section shows double-labeled cells distributed throughout the mediolateral extent of the arcuate nucleus. Orientation for sagittal sections is indicated in the top right. R, Rostral; C, caudal; D, dorsal; V, ventral.
- Figure 4.
ACTH-IR in POMC–DsRed neurons with or without GAD67–gfp. Brain sections from POMC–DsRed;GAD67–gfp double-transgenic mice were processed for ACTH-IR. DsRed (A) and gfp (B) colocalized in some neurons (open arrows), whereas a portion of POMC–DsRed neurons did not express GAD67–gfp (filled arrows). DsRed neurons displayed ACTH-IR (C) regardless of the presence or absence of GAD67–gfp. E–G, An enlarged image shows a cell positive for both POMC–DsRed and GAD67–gfp (open arrow) and a POMC–DsRed cell without GAD67–gfp (filled arrow). Both cells display ACTH-IR (F, G). R, Rostral; C, caudal; D, dorsal; V, ventral.
- Figure 5.
Presynaptic regulation of GABAergic and non-GABAergic POMC neurons. Spontaneous IPSCs in POMC neurons not expressing GAD67–gfp (A) and in GABAergic POMC neurons (B) at baseline (left traces) and after a 3–5 min application of ME (right traces). ME-induced inhibition of sIPSCs is summarized in D (n = 7–10). C, Frequency of basal sIPSCs and sEPSCs in POMC neurons without GAD (n = 10) and with GAD67–gfp (n = 7). Error bars represent SEM.
- Figure 6.
Postsynaptic regulation and properties of GABAergic and non-GABAergic POMC neurons. A, Perfusion of ME (10 μm) caused an outward current in both GAD67–gfp-positive and GAD67–gfp-negative POMC neurons, although the average magnitude of the current was larger in POMC neurons without GAD (n = 8–10). The recording shown in A is a representative trace made in a GABAergic POMC neuron. B, Input resistance in GAD-negative (gray bars; n = 14) and GAD67–gfp-expressing (black bars; n = 15) POMC neurons. C, Capacitance measurements in GAD-negative (n = 12) and GAD67–gfp-expressing (n = 15) POMC neurons. *p < 0.05. Error bars represent SEM.
- Figure 7.
Inhibitory and excitatory POMC neurons. Representative images of a GAD67–gfp-positive POMC–DsRed neuron (A) and a POMC–DsRed neuron that does not express the GAD67–gfp transgene (B) in primary neuron cultures. C, Depolarization evokes the release of GABA from a GAD67–gfp-expressing POMC neuron as indicated by the autaptic IPSC that is blocked by the GABAA receptor antagonist bicuculline. D, Autaptic glutamate release is evoked from cultured POMC neurons that do not express GAD67. The current resulting from glutamate release is blocked by the AMPA receptor blocker DNQX. C and D are representative traces from the GABAergic (n = 10) and glutamatergic (n = 8) cells that displayed autaptic currents.
