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

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.

Vanilloid, purinergic, and CCK receptors activate glutamate release on single neurons of the nucleus tractus solitarius centralis.

Baroreceptor inputs to nucleus of the tractus solitarius medialis (mNTS) neurons can be differentiated, among other features, by their response to vanilloid or purinergic agonists, active only on C- or A-fibers, respectively. A major aim of this study was to examine whether neurons of NTS centralis (cNTS), a subnucleus dominated by esophageal inputs, exhibit a similar dichotomy. Since it has been suggested that cholecystokinin (CCK), exerts its gastrointestinal (GI)-related effects via paracrine activation of vagal afferent C-fibers, we tested whether CCK-sensitive fibers impinging upon cNTS neurons are responsive to vanilloid but not purinergic agonists. Using whole cell patch-clamp recordings from cNTS, we recorded miniature excitatory postsynaptic currents (mEPSCs) to test the effects of the vanilloid agonist capsaicin, the purinergic agonist α,ß-methylene-ATP (α,ß-Met-ATP), and/or CCK-octapeptide (CCK-8s). α,ß-Met-ATP, capsaicin; and CCK-8s increased EPSC frequency in 37, 71, and 46% of cNTS neurons, respectively. Approximately 30% of cNTS neurons were responsive to both CCK-8s and α,ß-Met-ATP, to CCK-8s and capsaicin, or to α,ß-Met-ATP and capsaicin, while 32% of neurons were responsive to all three agonists. All neurons responding to either α,ß-Met-ATP or CCK-8s were also responsive to capsaicin. Perivagal capsaicin, which is supposed to induce a selective degeneration of C-fibers, decreased the number of cNTS neurons responding to capsaicin or CCK-8s but not those responding to α,ß-Met-ATP. In summary, GI inputs to cNTS neurons cannot be distinguished on the basis of their selective responses to α,ß-Met-ATP or capsaicin. Our data also indicate that CCK-8s increases glutamate release from purinergic and vanilloid responsive fibers impinging on cNTS neurons.

Glucose increases synaptic transmission from vagal afferent central nerve terminals via modulation of 5-HT3 receptors.

Acute hyperglycemia has profound effects on vagally mediated gastrointestinal functions. We have reported recently that the release of glutamate from the central terminals of vagal afferent neurons is correlated directly with the extracellular glucose concentration. The present study was designed to test the hypothesis that 5-HT(3) receptors present on vagal afferent nerve terminals are involved in this glucose-dependent modulation of glutamatergic synaptic transmission. Whole-cell patch-clamp recordings were made from neurons of the nucleus tractus solitarius (NTS) in thin rat brainstem slices. Spontaneous and evoked glutamate release was decreased in a concentration-dependent manner by the 5-HT(3) receptor selective antagonist, ondansetron. Alterations in the extracellular glucose concentration induced parallel shifts in the ondansetron-mediated inhibition of glutamate release. The changes in excitatory synaptic transmission induced by extracellular glucose concentration were mimicked by the serotonin uptake inhibitor, fenfluramine. These data suggest that glucose alters excitatory synaptic transmission within the rat brainstem via actions on tonically active 5-HT(3) receptors, and the number of 5-HT(3) receptors on vagal afferent nerve terminals is positively correlated with the extracellular glucose concentration. These data indicate that the 5-HT(3) receptors present on synaptic connections between vagal afferent nerve terminals and NTS neurons are a strong candidate for consideration as one of the sites where glucose acts to modulate vagovagal reflexes.

Presynaptic melanocortin-4 receptors on vagal afferent fibers modulate the excitability of rat nucleus tractus solitarius neurons.

The nucleus tractus solitarius (NTS) integrates visceral sensory signals with information from the forebrain to control homeostatic functions, including food intake. Melanocortin 3/4 receptor (MC3/4R) ligands administered directly to the caudal brainstem powerfully modulate meal size but not frequency, suggesting the enhancement of visceral satiety signals. Using whole-cell recordings from rat brainstem slices, we examined the effects of melanocortin ligands, alpha-melanocyte-stimulating hormone (alphaMSH) and melanotan II (MTII), on EPSC in NTS neurons. Thirty-two percent of NTS neurons responded to perfusion with MTII or alphaMSH with either an increase (24%) or a decrease (8%) in the frequency, but not amplitude, of spontaneous EPSCs; the effects of MTII were abolished by pretreatment with SHU9119. After surgical vagal deafferentation, only four of 34 (9%) NTS neurons responded to MTII with an increase in EPSC frequency. When EPSCs were evoked by electrical stimulation of the tractus solitarius in Krebs' solution with 2.4 mm Ca(2+)(e), alphaMSH and MTII increased the amplitude in six of the 28 neurons tested, decreased amplitude in 14 with no effect in the remaining eight neurons. In four of six neurons unresponsive to MTII, decreasing Ca(2+)(e) levels to 1.5 mM uncovered an excitatory effect of MTII on EPSC amplitude. Reverse transcription-PCR analysis revealed the presence of MC4R, but not MC3R, in nodose ganglia. These results show that MC4R signaling leads mainly to presynaptic modulation of glutamatergic synaptic transmission and suggest that melanocortinergic-induced decrease of food intake may occur via enhancement of vagal afferent satiation signals from the gastrointestinal tract.

D-glucose modulates synaptic transmission from the central terminals of vagal afferent fibers.

Experimental evidence suggests that glucose modulates gastric functions via vagally mediated effects. It is unclear whether glucose affects only peripheral vagal nerve activity or whether glucose also modulates vagal circuitry at the level of the brain stem. This study used whole cell patch-clamp recordings from neurons of the nucleus of the tractus solitarius (NTS) to assess whether acute variations in glucose modulates vagal brain stem neurocircuitry. Increasing D-glucose concentration induced a postsynaptic response in 40% of neurons; neither the response type (inward vs. outward current) nor response magnitude was altered in the presence of tetrodotoxin suggesting direct effects on the NTS neuronal membrane. In contrast, reducing d-glucose concentration induced a postsynaptic response (inward or outward current) in 54% of NTS neurons; tetrodotoxin abolished these responses, suggesting indirect sites of action. The frequency, but not amplitude, of spontaneous and miniature excitatory postsynaptic currents (EPSCs) was correlated with d-glucose concentration in 79% of neurons tested (n = 48). Prior surgical afferent rhizotomy abolished the ability of D-glucose to modulate spontaneous EPSC frequency, suggesting presynaptic actions at vagal afferent nerve terminals to modulate glutamatergic synaptic transmission. In experiments in which EPSCs were evoked via electrical stimulation of the tractus solitarius, EPSC amplitude correlated with D-glucose concentration. These effects were not mimicked by L-glucose, suggesting the involvement of glucose metabolism, not uptake, in the nerve terminal. These data suggest that the synaptic connections between vagal afferent nerve terminals and NTS neurons are a strong candidate for consideration as one of the sites where glucose-evoked changes in vagovagal reflexes occurs.

Glucagon-like peptide-1 modulates synaptic transmission to identified pancreas-projecting vagal motoneurons.

Using a brainstem slice preparation, we aimed to study the pre- and postsynaptic effects of glucagon-like peptide-1 (GLP-1) on synaptic transmission to identified pancreas-projecting vagal motoneurons. Following blockade of GABAergic mediated currents with bicuculline, perfusion with 100 nM GLP-1 increased both amplitude and frequency of excitatory postsynaptic currents (EPSCs) in 21 of 52 neurons. Perfusion with the GLP-1 selective agonist exendin-4 (100 nM), also increased the frequency of spontaneous EPSCs, while pretreatment with the GLP-1 selective antagonist, exendin 9-39, prevented the effects of GLP-1. In the presence of kynurenic acid to block ionotropic glutamatergic currents, perfusion with GLP-1 increased the frequency of inhibitory postsynaptic currents (IPSCs) in 28 of 74 neurons; in 14 of these responsive neurons, GLP-1 also increased IPSC amplitude, indicating actions at both pre- and postsynaptic sites. Perfusion with exendin-4 increased the frequency of spontaneous IPSCs, while pretreatment with exendin 9-39 prevented the effects of GLP-1. These results suggest that GLP-1 modulates both excitatory and inhibitory synaptic inputs to pancreas-projecting vagal motoneurons.

Vagally mediated, nonparacrine effects of cholecystokinin-8s on rat pancreatic exocrine secretion.

Cholecystokinin (CCK) has been proposed to act in a vagally dependent manner to increase pancreatic exocrine secretion via actions exclusively at peripheral vagal afferent fibers. Recent evidence, however, suggests the CCK-8s may also affect brain stem structures directly. We used an in vivo preparation with the aims of 1) investigating whether the actions of intraduodenal casein perfusion to increase pancreatic protein secretion also involved direct actions of CCK at the level of the brain stem and, if so, 2) determining whether, in the absence of vagal afferent inputs, CCK-8s applied to the dorsal vagal complex (DVC) can also modulate pancreatic exocrine secretion (PES). Sprague-Dawley rats (250-400 g) were anesthetized and the common bile-pancreatic duct was cannulated to collect PES. Both vagal deafferentation and pretreatment with the CCK-A antagonist lorglumide on the floor of the fourth ventricle decreased the casein-induced increase in PES output. CCK-8s microinjection (450 pmol) in the DVC significantly increased PES; the increase was larger when CCK-8s was injected in the left side of the DVC. Protein secretion returned to baseline levels within 30 min. Microinjection of CCK-8s increased PES (although to a lower extent) also in rats that underwent complete vagal deafferentation. These data indicate that, as well as activating peripheral vagal afferents, CCK-8s increases pancreatic exocrine secretion via an action in the DVC. Our data suggest that the CCK-8s-induced increases in PES are due mainly to a paracrine effect of CCK; however, a relevant portion of the effects of CCK is due also to an effect of the peptide on brain stem vagal circuits.

Cholecystokinin-8s excites identified rat pancreatic-projecting vagal motoneurons.

It is known that cholecystokinin (CCK) acts in a paracrine fashion to increase pancreatic exocrine secretion via vagal circuits. Recent evidence, however, suggests that CCK-8s actions are not restricted to afferent vagal fibers, but also affect brain stem structures directly. Within the brain stem, preganglionic neurons of the dorsal motor nucleus of the vagus (DMV) send efferent fibers to subdiaphragmatic viscera, including the pancreas. Our aims were to investigate whether DMV neurons responded to exogenously applied CCK-8s and, if so, the mechanism of action. Using whole cell patch-clamp recordings we show that perfusion with CCK-8s induced a concentration-dependent excitation in approximately 60% of identified pancreas-projecting DMV neurons. The depolarization was significantly reduced by tetrodotoxin, suggesting both direct (on the DMV membrane) and indirect (on local synaptic circuits) effects. Indeed, CCK-8s increased the frequency of miniature excitatory currents onto DMV neurons. The CCK-A antagonist, lorglumide, prevented the CCK-8s-mediated excitation whereas the CCK-B preferring agonist, CCK-nonsulfated, had no effect, suggesting the involvement of CCK-A receptors only. In voltage clamp, the CCK-8s-induced inward current reversed at -106 +/- 3 mV and the input resistance increased by 150 +/- 15%, suggesting an effect mediated by the closure of a potassium conductance. Indeed, CCK-8s reduced both the amplitude and the time constant of decay of a calcium-dependent potassium conductance. When tested with pancreatic polypeptide (which reduces pancreatic exocrine secretion), cells that responded to CCK-8s with an excitation were, instead, inhibited by pancreatic polypeptide. These data indicate that CCK-8s may control pancreas-exocrine secretion also via an effect on pancreas-projecting DMV neurons.