R. Alberto Travagli

Electrophysiological and morphological heterogeneity of rat dorsal vagal neurones which project to specific areas of the gastrointestinal tract.

1 The electrophysiological properties of rat dorsal motor nucleus of the vagus (DMV) neurones (n= 162) were examined using whole cell patch clamp recordings from brainstem slices. Recordings were made from DMV neurones whose projections to the gastrointestinal tract had been identified by previously applying fluorescent retrograde tracers to the gastric fundus, corpus or antrum/pylorus, or to the duodenum or caecum. 2 The neuronal groups were markedly heterogeneous with respect to several electrophysiological properties. For example, neurones which projected to the fundus had a higher input resistance (400 ± 25 MΩ), a smaller and shorter after‐hyperpolarization (16.7 ± 0.49 mV and 63.5 ± 3.9 ms) and a higher frequency of action potential firing (19.3 ± 1.4 action potentials s−1) following injection of depolarizing current (270 pA) when compared with caecum‐projecting neurones (302 ± 22 MΩ; 23.5 ± 0.87 mV and 81.1 ± 5.3 ms; 9.7 ± 1.1 action potentials s−1; P < 0.05 for each parameter). Differences between neuronal groups were also apparent with respect to the distribution of several voltage‐dependent potassium currents. Inward rectification was present only in caecum‐projecting neurones, for example. 3 Neurones (n= 82) were filled with the intracellular stain Neurobiotin allowing post‐fixation morphological reconstruction. Neurones projecting to the caecum had the largest cell volume (5238 ± 535 μm3), soma area (489 ± 46 μm2) and soma diameter (24.6 ± 1.24 μm) as well as the largest number of dendritic branch segments (23 ± 2). 4 In summary, these results suggest that DMV neurones are heterogeneous with respect to some electrophysiological as well as some morphological properties and can be divided into subgroups according to their gastrointestinal projections.

In vitro and in vivo analysis of the Effects of corticotropin releasing factor on rat dorsal vagal complex

In vivo and in vitro electrophysiological experiments were performed on the rat dorsal vagal complex (DVC, i.e. nucleus of the tractus solitarius, NTS, and dorsal motor nucleus of the vagus, DMV) to examine the effects of corticotropin releasing hormone (CRF) on the central components of the vago‐vagal reflex control of gastric function. When applied to gastrointestinal projecting DMV neurones, CRF (10‐300 nm) induced a concentration‐dependent membrane depolarization, an increase in action potential firing rate and decrease in amplitude of the action potential afterhyperpolarization (P < 0.05). Pretreatment with the non‐selective CRF antagonist, astressin (0.5‐1 μM) or the selective CRF2 receptor antagonist, astressin 2B (500 nm) attenuated the CRF‐induced increase in firing rate but did not alter basal discharge rate. CRF (30‐300 nm) increased the amplitude of excitatory postsynaptic currents (EPSCs) evoked by stimulation of the NTS (P < 0.05). An alteration in the paired pulse ratio indicated the EPSCs increase occurred due to actions at presynaptic sites. In the in vivo anaesthetized rat preparation, bilateral microinjections (20 fmol in 20 nl for each site) of CRF in the DVC decreased gastric motility in rats pretreated with the muscarinic agonist, bethanecol (P < 0.05). The effects of CRF were abolished by systemic administration of the NOS inhibitor, L‐NAME, or by bilateral vagotomy. We concluded that CRF had both a direct and an indirect excitatory effect on DMV neurones via activation of CRF2 receptors and the decrease in gastric motility observed following microinjection of CRF in the DVC is due to the activation of an inhibitory non‐adrenergic non‐cholinergic input to the gastrointestinal tract.

Glucose effects on gastric motility and tone evoked from the rat dorsal vagal complex

1 To examine the effects of glucose on the central components of the vago‐vagal reflex control of gastric function, we performed both in vivo and in vitro experiments on neurones in the medial nucleus of the tractus solitarius (mNTS) and in the dorsal motor nucleus of the vagus (DMV). 2 In the in vivo anaesthetized rat preparation, unilateral microinjection of d‐glucose (10 or 50 mm (60 nl)−1) in mNTS produced inhibition of gastric motility and an increase in intragastric pressure. d‐glucose had no effect in the DMV. 3 In the in vitro rat brainstem slice preparation, whole‐cell recordings of DMV neurones showed that increasing the glucose concentration of the perfusion solution from 5 mm to 15 or 30 mm produced outward currents of 35 ± 5 pA (n= 7) and 51 ± 10 pA (n= 11), respectively. These were blocked by tetrodotoxin and picrotoxin, indicating that glucose was acting indirectly to cause the release of GABA. Decreasing the glucose concentration of the perfusing solution by one‐half produced an inward current of 36 ± 5 pA (n= 7). 4 Stimulation of the NTS evoked inhibitory postsynaptic currents (IPSCs) in DMV neurones. The amplitude of the evoked IPSCs was positively correlated with glucose concentration. Perfusion with the ATP‐sensitive K+ (KATP) channel opener diazoxide mimicked the effect of reduced glucose, while perfusion with the KATP channel blocker glibenclamide mimicked the effects of increased glucose. 5 Our data indicate that glucose had no direct excitatory effect on DMV neurones, but DMV neurones appear to be affected by an action of glucose on cell bodies of mNTS neurones via effects on an ATP‐sensitive potassium channel.

Characterization of the in vitro effects of 5-hydroxytryptamine (5-HT) on identified neurones of the rat dorsal motor nucleus of the vagus (DMV).

Whole cell patch clamp techniques were used on thin brainstem slices to investigate the effects of 5‐hydroxytryptamine (5‐HT) on gastrointestinal‐projecting dorsal motor nucleus of the vagus (DMV) neurones. Neurones were identified as projecting to the stomach (n=122) or intestine (n=84) if they contained the fluorescent tracer Dil after it had been applied to the gastric fundus, corpus or antrum/pylorus or to the duodenum or caecum. A higher proportion of intestinal neurones (69%) than gastric neurones (47%) responded to 5‐HT with a concentration‐dependent inward current which was antagonized fully by the 5‐HT2A receptor antagonist ketanserin (1 μM). Stimulation of the nucleus tractus solitarius (NTS) induced inhibitory synaptic currents that were reduced in amplitude by application of the 5‐HT1A receptor agonist 8‐OHDPAT (1 μM) or the 5‐HT1A/1B receptor agonist TFMPP (1 μM) in 61% and 52% of gastric‐ and intestinal‐projecting neurones, respectively. 5‐HT also significantly reduced the frequency but not the amplitude of spontaneous inhibitory currents. These data show that 5‐HT excites directly a larger proportion of intestinal projecting neurones than gastric‐projecting neurones, as well as inhibiting synaptic transmission from the NTS to the DMV. These data imply that the response to DMV neurones to 5‐HT may be determined and classified by their specific projections.

Central Nervous System Control of Gastrointestinal Motility and Secretion and Modulation of Gastrointestinal Functions

Although the gastrointestinal (GI) tract possesses intrinsic neural plexuses that allow a significant degree of autonomy over GI functions, the central nervous system (CNS) provides extrinsic neural inputs that regulate, modulate, and control these functions. While the intestines are capable of functioning in the absence of extrinsic inputs, the stomach and esophagus are much more dependent upon extrinsic neural inputs, particularly from parasympathetic and sympathetic pathways. The sympathetic nervous system exerts a predominantly inhibitory effect upon GI muscle and provides a tonic inhibitory influence over mucosal secretion while, at the same time, regulates GI blood flow via neurally mediated vasoconstriction. The parasympathetic nervous system, in contrast, exerts both excitatory and inhibitory control over gastric and intestinal tone and motility. Although GI functions are controlled by the autonomic nervous system and occur, by and large, independently of conscious perception, it is clear that the higher CNS centers influence homeostatic control as well as cognitive and behavioral functions. This review will describe the basic neural circuitry of extrinsic inputs to the GI tract as well as the major CNS nuclei that innervate and modulate the activity of these pathways. The role of CNS-centered reflexes in the regulation of GI functions will be discussed as will modulation of these reflexes under both physiological and pathophysiological conditions. Finally, future directions within the field will be discussed in terms of important questions that remain to be resolved and advances in technology that may help provide these answers.

Vagally mediated effects of glucagon-like peptide 1: in vitro and in vivo gastric actions

Glucagon‐like peptide‐1 (GLP‐1) is a neuropeptide released following meal ingestion that, among other effects, decreases gastric tone and motility. The central targets and mechanism of action of GLP‐1 on gastric neurocircuits have not, however, been fully investigated. A high density of GLP‐1 containing neurones and receptors are present in brainstem vagal circuits, suggesting that the gastroinhibition may be vagally mediated. We aimed to investigate: (1) the response of identified gastric‐projecting neurones of the dorsal motor nucleus of the vagus (DMV) to GLP‐1 and its analogues; (2) the effects of brainstem application of GLP‐1 on gastric tone; and (3) the vagal pathway utilized by GLP‐1 to induce gastroinhibition. We conducted our experiments using whole‐cell recordings from identified gastric‐projecting DMV neurones and microinjection in the dorsal vagal complex (DVC) of anaesthetized rats while monitoring gastric tone. Perfusion with GLP‐1 induced a concentration‐dependent excitation of a subpopulation of gastric‐projecting DMV neurones. The GLP‐1 effects were mimicked by exendin‐4 and antagonized by exendin‐9–39. In an anaesthetized rat preparation, application of exendin‐4 to the DVC decreased gastric tone in a concentration‐dependent manner. The gastroinhibitory effects of exendin‐4 were unaffected by systemic pretreatment with the pro‐motility muscarinic agonist bethanechol, but were abolished by systemic administration of the nitric oxide synthase (NOS) inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME), or by bilateral vagotomy. Our data indicate that GLP‐1 activates selective receptors to excite DMV neurones mainly and that the gastroinhibition observed following application of GLP‐1 in the DVC is due to the activation of an inhibitory non‐adrenergic, non‐cholinergic input to the stomach.

μ-Opioid Receptor Trafficking on Inhibitory Synapses in the Rat Brainstem

Whole-cell recordings were made from identified gastric-projecting rat dorsal motor nucleus of the vagus (DMV) neurons. The amplitude of evoked IPSCs (eIPSCs) was unaffected by perfusion with met-enkephalin (ME) or by μ-, δ-, or κ-opioid receptor selective agonists, namely d-Ala2-N-Me-Phe4-Glycol5-enkephalin (DAMGO), cyclic [d-Pen2-d-Pen5]-enkephalin, or trans-3,4-dichloro-N-methyl-N-[2-(1-pyrolytinil)-cyclohexyl]-benzeneacetamide methane sulfonate (U50,488), respectively. Brief incubation with the adenylate cyclase activator forskolin or the nonhydrolysable cAMP analog 8-bromo-cAMP, thyrotropin releasing hormone, or cholecystokinin revealed the ability of ME and DAMGO to inhibit IPSC amplitude; this inhibition was prevented by pretreatment with the μ-opioid receptor (MOR1) selective antagonist d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2. Conversely, incubation with the adenylate cyclase inhibitor dideoxyadenosine, with the protein kinase A (PKA) inhibitor N-[2-(p-Bromocinnamyl-amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89), or with the Golgi-disturbing agent brefeldin A, blocked the ability of forskolin to facilitate the inhibitory actions of ME. Immunocytochemical experiments revealed that under control conditions, MOR1 immunoreactivity (MOR1-IR) was colocalized with glutamic acid decarboxylase (GAD)-IR in profiles apposing DMV neurons only after stimulation of the cAMP-PKA pathway. Pretreatment with H89 or brefeldin A or incubation at 4°C prevented the forskolin-mediated insertion of MOR1 on GAD-IR-positive profiles. These results suggest that the cAMP-PKA pathway regulates trafficking of μ-opioid receptors into the cell surface of GABAergic nerve terminals. By consequence, the inhibitory actions of opioid peptides in the dorsal vagal complex may depend on the state of activation of brainstem vagal circuits.

The peptide TRH uncovers the presence of presynaptic 5‐HT1A receptors via activation of a second messenger pathway in the rat dorsal vagal complex

1 It is well recognized that brainstem microinjections of 5‐hydroxytryptamine (serotonin, 5‐HT) and thyrotropin‐releasing hormone (TRH) act synergistically to stimulate gastric function in vivo. Previous in vitro experiments have shown that this synergism does not occur at the level of the dorsal motor nucleus of the vagus (DMV) motoneurone. 2 In order to determine the mechanism of this action, whole cell patch clamp recordings were made from identified gastric‐projecting rat DMV neurones to investigate the effects of 5‐HT and TRH on GABAergic inhibitory postsynaptic currents (IPSCs) evoked by stimulation of the nucleus of the tractus solitarius (NTS). 3 5‐HT (30 μM) decreased IPSC amplitude by 26 ± 2.5 % in approximately 43 % of DMV neurones. In the remaining neurones in which 5‐HT had no effect on IPSC amplitude, exposure to TRH (1 μM) uncovered the ability of subsequent applications of 5‐HT to decrease IPSC amplitude by 28 ± 3 %. Such TRH‐induced 5‐HT responses were prevented by the 5‐HT1A antagonist NAN‐190 (1 μM) and mimicked by the 5‐HT1A agonist 8‐OH‐DPAT (1 μM). 4 Increasing cAMP levels using the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 10 μM), the non‐hydrolysable cAMP analogue 8‐bromo‐cAMP (1 mM), or the adenylate cyclase activator forskolin (10 μM), like TRH, uncovered the ability of 5‐HT to decrease evoked IPSC amplitude (17 ± 2.2 %, 28.5 ± 5.3 % and 30 ± 4.8 %, respectively), in neurones previously unresponsive to 5‐HT. Conversely, the adenylate cyclase inhibitor, dideoxyadenosine (10 μM) and the protein kinase A inhibitor, Rp‐cAMP (10 μM), blocked the ability of TRH to uncover the presynaptic inhibitory actions of 5‐HT. 5 These results suggest that activation of presynaptic TRH receptors initiates an intracellular signalling cascade that raises the levels of cAMP sufficient to uncover previously silent 5‐HT1A receptors on presynaptic nerve terminals within the dorsal vagal complex.

Neuropeptide Y and peptide YY inhibit excitatory synaptic transmission in the rat dorsal motor nucleus of the vagus

Pancreatic polypeptides (PPs) such as neuropeptide Y (NPY) and peptide YY (PYY) exert profound, vagally mediated effects on gastrointestinal (GI) motility and secretion. Whole‐cell patch clamp recordings were made from brainstem slices containing identified GI‐projecting rat dorsal motor nucleus of the vagus (DMV) neurons to determine the mechanism of action of PPs. Electrical stimulation of nucleus tractus solitarii (NTS) induced excitatory postsynaptic currents (EPSCs) that were reduced in a concentration‐dependent manner by NPY and PYY (both at 0.1–300 nm) in 65 % of the neurons. An increase in the paired‐pulse ratio without changes in the postsynaptic membrane input resistance or EPSC rise and decay time suggested that the effects of PPs on EPSCs were due to actions at presynaptic receptors. The Y1 and Y2 receptor selective agonists [Leu31,Pro34]NPY and NPY(3–36) (both at 100 nm) mimicked the inhibition of NPY and PYY on the EPSC amplitude. The effects of 100 nm NPY, but not PYY, were antagonized partially by the Y1 receptor selective antagonist BIBP3226 (0.1 μm). In addition, the inhibition of the EPSC amplitude induced by NPY, but not PYY, was attenuated partially by pretreatment with the α2 adrenoceptor antagonist yohimbine (10 μm), and occluded partially by the α2 adrenoceptor agonist UK14,304 (10 μm) as well as by pretreatment with reserpine. Pretreatment with a combination of BIBP3226 and yohimbine almost completely antagonized the NPY‐mediated effects on EPSCs. Contrary to the inhibition of EPSCs, perfusion with PPs had no effect on the amplitude of inhibitory postsynaptic currents (IPSCs) and a minimal effect on a minority of DMV neurons. Differences in the receptor subtypes utilized and in the mechanism of action of NPY and PYY may indicate functional differences in their roles within the circuitry of the dorsal vagal complex (DVC).

Functional Organization of Presynaptic Metabotropic Glutamate Receptors in Vagal Brainstem Circuits

We demonstrated previously that, by suppressing cAMP levels, metabotropic glutamate receptors (mGluRs) play a crucial role in opioid receptor trafficking on GABAergic nerve terminals within gastric brainstem vagal circuits. Using whole-cell patch-clamp recordings, we aimed to correlate the influence of sensory vagal afferent fibers with the functional organization of mGluRs on the synaptic connections between the nucleus tractus solitarius and dorsal motor nucleus of the vagus. Group II mGluRs were identified on both excitatory and inhibitory synapses; the receptor-selective agonist APDC [(2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate] induced a concentration-dependent decrease in glutamatergic and GABAergic synaptic transmission (EC50, ∼20 μm for both). The group II mGluRs were activated tonically on GABAergic, but not glutamatergic synapses, as the receptor-selective antagonist (2S)-α-ethylglutamic acid (EGLU; 200 μm) modulated GABA currents only. After selective vagal deafferentation, EGLU was without effect, suggesting that vagal afferent (sensory) fibers are the source of this tonic input. Conversely, group III mGluRs, although not activated tonically, were present on excitatory, but not inhibitory, synapses; in fact, the receptor-selective agonist l-AP-4 [l-(+)-2-amino-4-phosphonbutyric acid] induced a concentration-dependent decrease in glutamatergic synaptic transmission (EC50, ∼2 μm) but had no effect on GABAergic synaptic transmission. Together with our previous results on receptor trafficking, these data suggest that visceral information plays a fundamental role in shaping the response of homeostatic brainstem circuits that receive inputs from higher integrative neuronal centers.