Role of the vagus in the reduced pancreatic exocrine function in copper-deficient rats

Abstract

Copper plays an essential role in the function and development of the central nervous system and exocrine pancreas. Dietary copper limitation is known to result in noninflammatory atrophy of pancreatic acinar tissue. Our recent studies have suggested that vagal motoneurons regulate pancreatic exocrine secretion (PES) by activating selective subpopulations of neurons within vagovagal reflexive neurocircuits. We used a combination of in vivo, in vitro, and immunohistochemistry techniques in a rat model of copper deficiency to investigate the effects of a copper-deficient diet on the neural pathways controlling PES. Duodenal infusions of Ensure or casein, as well as microinjections of sulfated CCK-8, into the dorsal vagal complex resulted in an attenuated stimulation of PES in copper-deficient animals compared with controls. Immunohistochemistry of brain stem slices revealed that copper deficiency reduced the number of tyrosine hydroxylase-immunoreactive, but not neuronal nitric oxide synthase- or choline acetyltransferase-immunoreactive, neurons in the dorsal motor nucleus of the vagus (DMV). Moreover, a copper-deficient diet reduced the number of large (>11 neurons), but not small, intrapancreatic ganglia. Electrophysiological recordings showed that DMV neurons from copper-deficient rats are less responsive to CCK-8 or pancreatic polypeptide than are DMV neurons from control rats. Our results demonstrate that copper deficiency decreases efferent vagal outflow to the exocrine pancreas. These data indicate that the combined selective loss of acinar pancreatic tissue and the decreased excitability of efferent vagal neurons induce a deficit in the vagal modulation of PES.

Fig. 1.

Copper deficiency induces acinar degeneration and loss of large intrapancreatic ganglia. A and E: photomicrographs of pancreata from age-matched ∼60-day-old control (A) and copper-deficient (E) animals. Note extensive degeneration of acinar tissue in the pancreas from the copper-deficient animal. Scale bars, 5 mm. B and F: schematic drawings of a pancreas from a control (B) and a copper-deficient (F) animal showing distribution of intrapancreatic ganglia. Copper deficiency caused a selective reduction in intrapancreatic ganglia containing ≥11 neurons in the tail of the pancreas. Symbols represent intrapancreatic ganglia containing a defined number of neurons: ▲, 1–5 neurons/ganglion; ■, 6–10 neurons/ganglion; ◆, 11–15 neurons/ganglion; ▼, 15–20 neurons/ganglion; ∗, >20 neurons/ganglion. C and G: low-magnification photomicrographs of hematoxylin-eosin-stained pancreata from age-matched ∼45-day-old control (C) and copper-deficient (G) animals. Note degeneration of acinar tissue in the pancreas from the copper-deficient animal. Scale bars, 5 mm. D and H: high-magnification photomicrographs from pancreata shown in C and G. Note degradation of acinar tissue in the pancreas from the copper-deficient animal (H) compared with intact tissue from the control animal (D). Scale bar, 0.5 mm.

Fig. 2.

Copper-deficient diet attenuates increase in pancreatic exocrine secretion (PES) stimulated by intraduodenal infusions of Ensure and casein or microinjections of sulfated CCK-8 (CCK-8s) into the dorsal motor nucleus of the vagus (DMV). A: time course of the effect of Ensure on PES in control and copper-deficient rats (n = 4 for both). Infusion started at time 0; horizontal bar shows duration of Ensure infusion. Intraduodenal Ensure infusion significantly increased PES in control, but not copper-deficient, rats. B: time course of the effect of casein on PES in control (n = 6) and copper-deficient (n = 4) animals. Intraduodenal casein infusion significantly increased PES in control, but not copper-deficient, rats. C: effect of Ensure infusion on total protein output in control and copper-deficient animals (n = 4 for both). Note significantly reduced peak PES in copper-deficient rats compared with control animals. D: effect of casein infusion on total protein output in control (n = 6) and copper-deficient (n = 4) animals. Note significantly reduced peak PES in copper-deficient rats compared with control animals. E: effect of CCK-8s (450 pmol in 60 nl) microinjection into the dorsal vagal complex (DVC) on peak PES. *P < 0.05. F: schematic drawing of the DVC showing location of CCK-8s injection sites. All injections were made on the left side, but injections from 2 experimental groups are plotted on different sides of the schematic diagram for clarity. Each symbol [▫ (control, n = 5) and ● (copper-deficient, n = 9)] represents 1 injection site. NTS, nucleus tractus solitarius; AP, area postrema; 12, hypoglossal nucleus; sol, tractus solitarius; cc, central canal.

Fig. 3.

Copper-deficient diet selectively reduces the number of tyrosine hydroxylase (TH)-immunoreactive (IR) neurons in the DMV. A and B: representative photomicrographs showing TH-IR in the DVC of a control (A) and a copper-deficient (B) rat. Scale bar, 100 μm. C: average number of TH-IR neurons per section in the DMV (n = 12 control and 16 copper-deficient rats). D: average number of TH-IR neurons per section in the NTS (n = 4 control and 6 copper-deficient rats). E: number of choline acetyltransferase (ChAT)-IR neurons in the DMV (n = 4 control and 4 copper-deficient rats). F: number of nitric oxide synthase (NOS)-IR neurons in the DMV (n = 12 control and 10 copper-deficient rats). G: number of NOS-IR neurons in the NTS (n = 4 control and 5 copper-deficient rats). Caudal, sections from the DMV or NTS at levels caudal to AP; intermediate, sections from the DMV or NTS at the level of AP; rostral, sections from the DMV or NTS at levels rostral to AP. Copper-deficient diet reduced the number of TH-positive neurons in the caudal and intermediate, but not rostral, DMV or the NTS. Also, copper-deficient diet did not alter the number of ChAT- or NOS-IR neurons in any part of the DVC. *P < 0.05.

Fig. 4.

Copper-deficient diet alters postsynaptic responses of identified pancreas-projecting DMV neurons to CCK-8s and pancreatic polypeptide (PP). A: representative trace showing increase in firing rate in a pancreas-projecting DMV neuron during CCK-8s application. B: concentration-response curves to CCK-8s in control (n = 4–8 per concentration) and copper-deficient (n = 12–16 per concentration) animals. C: percentage of pancreas-projecting DMV neurons that responded to CCK-8s in control (n = 98 of 164) and copper-deficient (n = 24 of 55) animals. Note decreased percentage of CCK-8s-responding neurons in copper-deficient rats compared with control animals. D: representative trace showing PP-induced hyperpolarization in a pancreas-projecting DMV neuron. E: concentration-response curve to PP. Note significant rightward shift in copper-deficient (n = 5–10 per concentration) compared with control (n = 4–6 per concentration) animals. F: percentage of PP-responsive neurons in control (n = 12 of 13) and copper-deficient (n = 42 of 74) animals. Note decreased percentage of pancreas-projecting neurons that responded to CCK-8s and PP and significant rightward shift in the concentration-response curve to PP in copper-deficient rats.

Fig. 5.

Copper-deficient diet does not alter presynaptic responses of pancreas-projecting DMV neurons to CCK-8s. A and B: representative traces showing CCK-8s-induced increase in spontaneous inhibitory postsynaptic current (sIPSC) frequency in a control (A) and a copper-deficient (B) animal. C: CCK-8s-induced increase in spontaneous excitatory postsynaptic current (sEPSC) frequency in control (n = 8 of 20) and copper-deficient (n = 16 of 36) animals. In both instances, perfusion with CCK-8s increased sEPSC frequency. D: CCK-8s-induced increase in sIPSC frequency in control (n = 9 of 18) and copper-deficient (n = 12 of 34) animals. In both instances, perfusion with CCK-8s increased sIPSC frequency. E and F: percentage of pancreas-projecting neurons in which CCK-8s induced an increase in sEPSC (E) and sIPSC (F) frequency. *P < 0.05.