Neuronal connections of the central amygdalar nucleus with refeeding-activated brain areas in rats

Abstract

Following fasting, satiety is accompanied by neuronal activation in brain areas including the central amygdalar nucleus (CEA). Since CEA is known to inhibit food intake, we hypothesized that CEA contributes to the termination of meal during refeeding. To better understand the organization of this satiety-related circuit, the interconnections of the CEA with refeeding-activated neuronal groups were elucidated using retrograde (cholera toxin-β subunit, CTB) and anterograde (phaseolus vulgaris leucoagglutinin, PHA-L) tracers in male rats. C-Fos-immunoreactivity was used as marker of neuronal activation. The refeeding-activated input of the CEA primarily originated from the paraventricular thalamic, parasubthalamic and parabrachial nuclei. Few CTB-c-Fos double-labeled neurons were detected in the prefrontal cortex, lateral hypothalamic area, nucleus of the solitary tract (NTS) and the bed nuclei of the stria terminalis (BNST). Only few refeeding-activated proopiomelanocortin-producing neurons of the arcuate nucleus projected to the CEA. Anterograde tract tracing revealed a high density of PHAL-labeled axons contacted with refeeding-activated neurons in the BNST, lateral hypothalamic area, parasubthalamic, paraventricular thalamic and parabrachial nuclei and NTS; a low density of labeled axons was found in the paraventricular hypothalamic nucleus. Chemogenetic activation of the medial CEA (CEAm) inhibited food intake during the first hour of refeeding, while activation of lateral CEA had no effect. These data demonstrate the existence of reciprocal connections between the CEA and distinct refeeding-activated hypothalamic, thalamic and brainstem nuclei, suggesting the importance of short feedback loops in the regulation of satiety and importance of the CEAm in the regulation of food intake during refeeding.

Keywords: Anterograde tract tracing; Connectivity map; Parabrachial nucleus; Parasubthalamic nucleus; Retrograde tract tracing; Satiety; c-Fos.

Figure 1

Localization of hSyn-hM3D(Gq)-mCherry AAV injection sites. The animals shown on (A-F) were used to study the effects of CEAm activation, while animals shown in (G–J) were used in experiments studying the effects of the activation of CEAlc. Scale bar = 500μm

Figure 2

Effect of chemogenetic activation of CEA subnuclei on c-Fos expression in this nucleus. Saline treatment did not cause neuronal activation in the CEA of rats injected with hM3D(Gq)-mCherry expressing AAV in the CEAm (A) or CEAlc (C). C-Fos-immunoreactivity is labeled with green color, while the red fluorescence of the mCherry labels the injection site. CNO treatment induced marked neuronal activation in the CEAm, but did not influence c-Fos expression in the CEAlc in the CEAm injected animals (B). Conversely, CNO treatment induced marked neuronal activation in the CEAlc, but did not influence c-Fos expression in the CEAm in the CEAlc injected animals (D). Scale bar =500μm

Figure 3

Effect of chemogenetic activation of the CEA subnuclei on food intake during refeeding. CNO-induced activation of the neurons of CEAm significantly decreased food intake during the first hour of refeeding compared to food intake of the same animal after saline injection (A; p=0.007). The color lines label the differences of food intake of the same animal after saline and CNO injection. In the second hour, the animals ate significantly more food when the CEAm was activated by CNO (B; p=0.006). Chemogenetic activation of CEAlc did not cause a significant change of food intake. First hour food intake is shown in (C) (p= 0.13). Second hour food intake is shown in (D) (p= 0.55). M1–6= rats with CEAm injection; L1–4= rats with CEAlc injection.

Figure 4

Representative images of CTB and PHA-L injection sites in the CEA. A: Core of CTB injection site deposited into the CEA. B: Core of PHA-L injection site centered in the CEA. Double-labeling immunohistochemistry for CTB (green; A1, A2), PHA-L (green; B1,B2) and c-Fos (red; A3, B3) was performed to reveal the injection sites. Areas adjacent to the CEA are not activated by refeeding. st= stria terminalis Scale bars: 500μm.

Figure 5

Schematic representation of the core of CTB and PHAL injection sites within the CEA in 3 or 4 anteroposterior levels, respectively. The injection sites were mapped to planes of the Paxinos rat brain atlas (Paxinos and Watson 1998). Case numbers and the pattern demonstrating the boundaries of each injection core are indicated above the images.

Figure 6

Schematic drawings illustrate the brain regions where the refeeding-activated (c-Fos-positive, black) neurons that projects to the CEA (CTB-immunoreactive perikarya, brown) are located. These double-labeled neurons are present in the prelimbic area (PrL; A, B), agranular insular area, posterior part (AIp; C, D), visceral area (VISC; D), supplementary somatosensory area (SSs; D, F), primary somatosensory cortex, barrel field (SSp-bfd; D), bed nuclei of the stria terminalis (BST; E, G), perifornical nucleus (PeF; H, I), piriform area (PIR; J), lateral hypothalamic area (LH; K), nucleus of the solitary tract (NTS; L), ventrolateral division of the periaqueductal gray (PAGvl; M, P), paraventricular thalamic nucleus (PVT; N), parasubthalamic nucleus (PSTN; N, Q), arcuate hypothalamic nucleus (ARC; N, R), PBN (O, S). Representative images illustrate the distribution of the double labeled neurons in these areas. Arrows indicate examples of double-labeled neurons. Scale bar = 500μm in the schematic drawing and 20μm in the photomicrograph.

Figure 6

Schematic drawings illustrate the brain regions where the refeeding-activated (c-Fos-positive, black) neurons that projects to the CEA (CTB-immunoreactive perikarya, brown) are located. These double-labeled neurons are present in the prelimbic area (PrL; A, B), agranular insular area, posterior part (AIp; C, D), visceral area (VISC; D), supplementary somatosensory area (SSs; D, F), primary somatosensory cortex, barrel field (SSp-bfd; D), bed nuclei of the stria terminalis (BST; E, G), perifornical nucleus (PeF; H, I), piriform area (PIR; J), lateral hypothalamic area (LH; K), nucleus of the solitary tract (NTS; L), ventrolateral division of the periaqueductal gray (PAGvl; M, P), paraventricular thalamic nucleus (PVT; N), parasubthalamic nucleus (PSTN; N, Q), arcuate hypothalamic nucleus (ARC; N, R), PBN (O, S). Representative images illustrate the distribution of the double labeled neurons in these areas. Arrows indicate examples of double-labeled neurons. Scale bar = 500μm in the schematic drawing and 20μm in the photomicrograph.

Figure 7

Connections between the POMC neurons of the arcuate hypothalamic nucleus and the CEA. Confocal microscopic image (A) of triple-immunolabeled section indicates the presence of CTB (green) in a refeeding-activated (c-Fos-positive, red), POMC-producing (blue) perikarion in the arcuate hypothalamic nucleus of brain previously injected with CTB into the CEA. Arrow points to a triple-labeled neuron. The image was made by projecting 7 consecutive 1.8 μm thick confocal optical slices into one plane. To facilitate identification of triple-labeled cells, CTB, c-Fos and POMC-labeling of the same cells are shown in separate images in the insets. Note that the vast majority of the c-Fos-containing POMC neurons do not contain CTB-immunoreactivity. Confocal image of a triple-immunolabeled section (B) shows POMC-IR (blue) boutons on refeeding-activated neurons (c-Fos-IR, red) in the parasubthalamic nucleus (PSTN) that projects to the CEA (CTB-IR, green). Arrowheads point to POMC-IR varicosities on the surface of double-labeled, c-Fos-and POMC-IR neurons. The cells in squares are shown in higher magnification in the insets. B3 shows the c-Fos-IR nucleus of the neuron illustrated in B2. The POMC-IR varicosities contacting the perikaryon and the dendrite of this double-labeled neuron are pointed by arrows. B3 also illustrates a refeeding-activated and CEA-projecting neuron contacted by a POMC-IR varicosity (arrow). Note that primary antibodies against c-Fos and POMC were produced in rabbit, thus immunolabeling was performed sequentially, which prevented cross-reaction in the cytoplasm, but not in the cell nuclei. Scale bars: 20μm.

Figure 8

Schematic drawings and representative images illustrate the distribution of PHA-L-immunoreactive axons in refeeding-activated areas: the bed nuclei of the stria terminalis (BST; A1, A2), paraventricular hypothalamic nucleus, medial parvicellular part (PVHmp; B1, B2), paraventricular hypothalamic nucleus, posterior magnocellular part (PVHpm; B1, B3), lateral hypothalamus (LHA; C1, C2), periaqueductal gray (PAG; D1, D2), parasubthalamic nucleus (PSTN; E1, E2), paraventricular thalamic nucleus (PVT; E1, E3), parabrachial nucleus (PB; F1, F2), nucleus tractus solitarius (NTS; G1, G2),. The indicates a high density of PHA-L fibers, whereas indicates a low density of PHA-L fibers. Confocal images illustrate PHA-L (red) fibers among refeeding-activated neurons. Arrowheads point to PHA-L-immunoreactive (red) contacts on c-Fos (green)/HuCD (blue) double-labeled neurons. Higher magnification of framed areas is shown in the insets. Scale bars: 500μm in the schematic drawings and 20μm in the confocal images.

Figure 8

Schematic drawings and representative images illustrate the distribution of PHA-L-immunoreactive axons in refeeding-activated areas: the bed nuclei of the stria terminalis (BST; A1, A2), paraventricular hypothalamic nucleus, medial parvicellular part (PVHmp; B1, B2), paraventricular hypothalamic nucleus, posterior magnocellular part (PVHpm; B1, B3), lateral hypothalamus (LHA; C1, C2), periaqueductal gray (PAG; D1, D2), parasubthalamic nucleus (PSTN; E1, E2), paraventricular thalamic nucleus (PVT; E1, E3), parabrachial nucleus (PB; F1, F2), nucleus tractus solitarius (NTS; G1, G2),. The indicates a high density of PHA-L fibers, whereas indicates a low density of PHA-L fibers. Confocal images illustrate PHA-L (red) fibers among refeeding-activated neurons. Arrowheads point to PHA-L-immunoreactive (red) contacts on c-Fos (green)/HuCD (blue) double-labeled neurons. Higher magnification of framed areas is shown in the insets. Scale bars: 500μm in the schematic drawings and 20μm in the confocal images.

Figure 9

Schematic illustration of the afferent (A) and efferent (B) connections of the CEA with refeeding-activated brain areas.

Figure 10

Graphs summarizing the effects of the chemogenetic activation of CEAlc and CEAm on the number of c-Fos-IR nuclei in brain regions shown to respond to refeeding by neuronal activation. Control animals (ctr) were also injected with hSyn-hM3D(Gq)-mCherry AAV into the CEAlc or CEAm, but received saline treatment instead of CNO. In the BNST and PSTN, the activation of CEAlc did not influence the number of c-Fos-IR neurons, but activation of CEAm caused a marked increase of the number of c-Fos-IR neurons. In the parabrachial nucleus (PBN), both the activation of the CEAlc and the CEAm induced significant increase of the number of c-Fos-IR neurons, however, the effect of the CEAm was more pronounced. (*=p<0.05, **=p<0.01, ***=p<0.001)

Figure 11

Confocal images of triple-immunolabeled sections show mCherry-containing axons (red) that label the axons of CEA origin; and activated (c-Fos-positive, green), HuCD-producing neurons (blue) in saline (A, D, G)-and CNO (B,E,H)-treated rats in the following areas: BNST (A–C), PSTN (D–F), PBN (G–I). Arrowheads show the activated neurons that receive inputs from mCherry positive fibers. Cells in the white boxes in (C, F and I) are shown in higher magnification in insets. ac= anterior commissure, cp= cerebral peduncle; STh= subthalamic nucleus; scp= superior cerebellar peduncle; Scale bar: 50μm in A,B,D,E,G,H 10μm on C, F, I.