Neuronal activation of brain vagal-regulatory pathways and upper gut enteric plexuses by insulin hypoglycemia

Abstract

Neuronal activation of brain vagal-regulatory nuclei and gastric/duodenal enteric plexuses in response to insulin (2 U/kg, 2 h) hypoglycemia was studied in rats. Insulin hypoglycemia significantly induced Fos expression in the paraventricular nucleus of the hypothalamus, locus coeruleus, dorsal motor nucleus of the vagus (DMN), and nucleus tractus solitarii (NTS), as well as in the gastric/duodenal myenteric/submucosal plexuses. A substantial number of insulin hypoglycemia-activated DMN and NTS neurons were choline acetyltransferase and tyrosine hydroxylase positive, respectively, whereas the activated enteric neurons included NADPH- and vasoactive intestinal peptide neurons. The numbers of Fos-positive cells in each above-named brain nucleus or in the gastric/duodenal myenteric plexus of insulin-treated rats were negatively correlated with serum glucose levels and significantly increased when glucose levels were lower than 80 mg/dl. Acute bilateral cervical vagotomy did not influence insulin hypoglycemia-induced Fos induction in the brain vagal-regulatory nuclei but completely and partially prevented this response in the gastric and duodenal enteric plexuses, respectively. These results revealed that brain-gut neurons regulating vagal outflow to the stomach/duodenum are sensitively responsive to insulin hypoglycemia.

Fig. 1.

Fos expression in the paraventricular nucleus of the hypothalamus (PVN; A), dorsal motor nucleus of the vagus (DMN; B), and nucleus tractus solitarii (NTS; C) and in the myenteric plexus of the corpus (D), antrum (E), and duodenum (F) in control rats and in rats treated with insulin (2 U/kg sc, 2 h). Each column represents the mean ± SE of no. of rats indicated. *P < 0.05 compared with the corresponding values of the control rats.

Fig. 2.

Photomicrographs of the hypothalamic, pontine, and medullary sections showing Fos immunoreactivity in the PVN (A), locus coeruleus (LC; B), and DMN/NTS (C) in control rats and in rats treated with insulin (2 U/kg sc, 2 h). The anatomic locations of the photomicrographs are indicated in the coronal sections (top), adapted from the atlas of Paxinos and Watson (49). Fos-positive nuclei are presented as dark brown staining in the cell nuclei. Scale bars, 100 μm. AP, area postrema; III, third ventricle; cc, central canal.

Fig. 3.

Photomicrographs of the corpus (A), antral (B), and duodenal (C) longitudinal muscle/myenteric plexus (LMMP) whole-mount preparations showing Fos immunoreactivity in myenteric ganglia in control rats and in rats treated with insulin (2 U/kg sc, 2 h). Scale bar, 100 μm.

Fig. 4.

Dose-related decrease of serum glucose levels (A) and the nos. of immunoreactive (IR)-Fos (Fos-IR)-positive neurons in the DMN (B) and corpus myenteric ganglia (C) in rats treated with different doses of insulin as indicated (sc, 2 h). Each column represents the mean ± SE of 4 rats. *P < 0.05 compared with the value of saline-treated group (dose 0).

Fig. 5.

Correlations between serum glucose levels and the nos. of Fos-IR-positive neurons in the PVN (A), DMN (B), NTS (C), AP (D), and LC (E) (per section) and in the myenteric ganglia of the corpus (F), antrum (G), and duodenum (H) (per ganglion) in rats treated with saline (0) or insulin at doses of 0.1, 0.25, 0.5, 1.0, or 2.0 U/kg (sc, 2 h,). r, Correlation coefficient.

Fig. 6.

Photomicrographs of LMMP whole-mount preparations showing Fos-IR and cuprolinic blue double staining in the corpus (A), antral (B), and duodenal (C) myenteric ganglia of insulin (2 U/kg sc, 2 h)-treated rats undergone either sham operation or acute bilateral cervical vagotomy. The neurons are revealed as blue staining in cytoplasm. Fos immunoreactivity was revealed as the brown staining in the cell nuclei. Scale bar, 50 μm.

Fig. 7.

Photomicrographs of brain stem sections showing double immunostaining of Fos-IR and choline acetyltransferase (ChAT)-IR in DMN neurons [A; interaural −4.68 mm (49)] and Fos-IR and tyrosine hydroxylase (TH)-IR in NTS neurons (B; interaural −4.80 mm) of insulin (2 U/kg sc, 2 h)-treated rats. Fos immunoreactivity was detected as a dark blue reaction product in the nuclei, and (ChAT) or (TH) immunoreactivity appeared as a brown reaction product in the cytoplasm. Scale bar, 100 μm.

Fig. 8.

Photomicrographs of the corpus (A), antral (B), and duodenal (C) LMMP whole-mount preparations showing double staining of Fos-IR and NADPH diaphorase (NAPDH-d) in the myenteric ganglia of insulin (2 U/kg sc, 2 h)-treated rats. Fos immunoreactivity was detected as a brown nuclear staining, and NADPH-d appeared as a dark blue reaction product in the cytoplasm. Scale bar, 100 μm.

Fig. 9.

Photomicrographs of the corpus (A) and antral (B) submucosal whole-mount preparations showing double staining of Fos-IR and NADPH-d in the submucosal neurons of insulin (2 U/kg sc, 2 h)-treated rats. Fos immunoreactivity was detected as a brown nuclear staining, and NADPH-d appeared as a dark blue reaction product in the cytoplasm. Scale bar, 100 μm.

Fig. 10.

Photomicrographs of whole-mount preparations showing Fos-IR and vasoactive intestinal peptide (VIP)-IR double staining in the duodenal myenteric (A) and submucosal (B) ganglia of rats treated with insulin (2.0/kg sc, 2 h). Fos immunoreactivity was detected as a dark blue reaction product in the nuclei, and VIP immunoreactivity appeared as a brown staining in the cytoplasm. Scale bar, 100 μm.