Baptista V, Browning KN, Travagli RA. Effects of cholecystokinin-8s in the nucleus tractus solitarius of vagally deafferented rats. Am J Physiol Regul Integr Comp Physiol 292: R1092-R1100, 2007. First published November 22, 2006; doi:10.1152/ajpregu.00517.2006.-We have shown recently that cholecystokinin octapeptide (CCK-8s) increases glutamate release from nerve terminals onto neurons of the nucleus tractus solitarius pars centralis (cNTS). The effects of CCK on gastrointestinal-related functions have, however, been attributed almost exclusively to its paracrine action on vagal afferent fibers. Because it has been reported that systemic or perivagal capsaicin pretreatment abolishes the effects of CCK, the aim of the present work was to investigate the response of cNTS neurons to CCK-8s in vagally deafferented rats. In surgically deafferented rats, intraperitoneal administration of 1 or 3 g/kg CCK-8s increased c-Fos expression in cNTS neurons (139 and 251% of control, respectively), suggesting that CCK-8s' effects are partially independent of vagal afferent fibers. Using whole cell patch-clamp techniques in thin brain stem slices, we observed that CCK-8s increased the frequency of spontaneous and miniature excitatory postsynaptic currents in 43% of the cNTS neurons via a presynaptic mechanism. In slices from deafferented rats, the percentage of cNTS neurons receiving glutamatergic inputs responding to CCK-8s decreased by ϳ50%, further suggesting that central terminals of vagal afferent fibers are not the sole site for the action of CCK-8s in the brain stem. Taken together, our data suggest that the sites of action of CCK-8s include the brain stem, and in cNTS, the actions of CCK-8s are not restricted to vagal central terminals but that nonvagal synapses are also involved. brain stem; electrophysiology; c-fos CHOLECYSTOKININ, WHICH IS released from intestinal cells following ingestion of nutrients (34,37,47), has profound effects on gastrointestinal functions. In fact, it is well known that cholecystokinin, and, in particular, the cleaved octapeptide cholecystokinin-8s (CCK-8s), induces vagally mediated gastric relaxation, increases pancreatic exocrine secretion, and induces short-term satiety (36,38,41,42,48,62).Many studies have proposed that the vagally mediated effects of CCK-8s are almost exclusively due to a paracrine action of CCK-8s on peripheral, capsaicin-sensitive vagal afferent fibers (8,14,41). This postulate stems mainly from the observations that C-fiber ablation induced by perivagal capsaicin treatment greatly attenuates, or abolishes altogether, the effects of systemic administration of CCK-8s on gastric motility, gastric acid secretion, and pancreatic exocrine secretion (32,33,41,58). One must keep in mind, however, that the localized perineural application involves the use of very high concentrations of capsaicin, usually 33 mM (i.e., 1% solution) (9,23,25,41,56,63,67). Such a high concentration of capsaicin raises the possibility that its observed effects are due to the toxic effects of massiv...

Cholecystokinin (CCK) is released from enteroendocrine cells after ingestion of nutrients and induces multiple effects along the gastrointestinal tract, including gastric relaxation and short-term satiety. We used whole cell patch-clamp and immunohistochemical techniques in rat brain stem slices to characterize the effects of CCK. In 45% of the neurons of nucleus tractus solitarius subnucleus centralis (cNTS), perfusion with the sulfated form of CCK (CCK-8s) increased the frequency of spontaneous excitatory currents (sEPSCs) in a concentration-dependent manner (1-300 nM). The threshold for the CCK-8s excitatory effect was 1 nM, the EC(50) was 20 nM, and E(max) was 100 nM. The excitatory effects of CCK-8s were still present when the slices were preincubated with tetrodotoxin or bicuculline or when the recordings were conducted with Cs(+) electrodes. Pretreatment with the CCK-A receptor antagonist, lorglumide (1 microM), antagonized the effects of CCK-8s, whereas perfusion with the CCK-B preferring agonist CCK-8 nonsulfated (CCK-ns, 1 microM) did not affect the frequency of sEPSCs. Similarly, pretreatment with the CCK-B receptor antagonist, triglumide (1 microM), did not prevent the actions of CCK-8s. Although the majority (i.e., 76%) of CCK-8s unresponsive cNTS neurons had a bipolar somata shape and were TH-IR negative, no differences were found in either the morphological or the neurochemical phenotype of cNTS neurons responsive to CCK-8s. Our results suggest that the excitatory effects of CCK-8s on terminals impinging on a subpopulation of cNTS neurons are mediated by CCK-A receptors; these responsive neurons, however, do not have morphological or neurochemical characteristics that automatically distinguish them from nonresponsive neurons.

Esophageal sensory afferent inputs terminate principally in the central subnucleus of the tractus solitarius (cNTS). Neurons of the cNTS comprise two major neurochemical subpopulations. One contains neurons that are nitric oxide synthase (NOS) immunoreactive (-IR) while the other comprises neurons that are tyrosine hydroxylase (TH)-IR. We have shown recently that TH-IR neurons are involved in esophageal-distention induced gastric relaxation. We used whole cell patch clamp techniques in rat brainstem slices combined with immunohistochemical and morphological reconstructions to characterize cNTS neurons. Postrecording reconstruction of cNTS neurons revealed two morphological neuronal subtypes; one group of cells (41 out of 131 neurons, i.e., 31%) had a multipolar soma, while the other group (87 out of 131 neurons, i.e., 66%) had a bipolar soma. Of the 43 cells in which we conducted a neurochemical examination, 15 displayed TH-IR (9 with bipolar morphology, 6 with multipolar morphology) while the remaining 28 neurons did not display TH-IR (18 with bipolar morphology, 10 with multipolar morphology). Even though the range of electrophysiological properties varied significantly, morphological or neurochemical distinctions did not reveal characteristics peculiar to the subgroups. Spontaneous excitatory postsynaptic currents (sEPSC) recorded in cNTS neurons had a frequency of 1.5 ± 0.15 events s -1 and an amplitude of 27 ± 1.2 pA (Vh = -50 mV) and were abolished by pretreatment with 30 μM AP-5 and 10 μM CNQX, indicating the involvement of both NMDA and non-NMDA receptors. Some cNTS neurons also received a GABAergic input that was abolished by perfusion with 30-50 μM bicuculline. In conclusion, our data show that despite the heterogeneity of morphological and neurochemical membrane properties, the electrophysiological characteristics of cNTS neurons are not a distinguishing feature.

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Browning KN, Wan S, Baptista V, Travagli RA. Vanilloid, purinergic, and CCK receptors activate glutamate release on single neurons of the nucleus tractus solitarius centralis. Am J Physiol Regul Integr Comp Physiol 301: R394 -R401, 2011. First published May 4, 2011 doi:10.1152/ajpregu.00054.2011.-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 ␣,␤-methylene-ATP (␣,␤-Met-ATP), and/or CCK-octapeptide (CCK-8s). ␣,␤-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 ␣,␤-Met-ATP, to CCK-8s and capsaicin, or to ␣,␤-Met-ATP and capsaicin, while 32% of neurons were responsive to all three agonists. All neurons responding to either ␣,␤-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 ␣,␤-Met-ATP. In summary, GI inputs to cNTS neurons cannot be distinguished on the basis of their selective responses to ␣,␤-Met-ATP or capsaicin. Our data also indicate that CCK-8s increases glutamate release from purinergic and vanilloid responsive fibers impinging on cNTS neurons. brainstem; vagus; electrophysiology VAGAL AFFERENT (SENSORY) FIBERS convey a vast amount of information about the physiological state of the thoracic and abdominal viscera to the central nervous system, specifically, to the nucleus of the tractus solitarius (NTS). Vagal afferent fibers are organized in the NTS in an overlapping topographical manner. Sensory afferents from the stomach and intestine, for example, terminate in the subnuclei commissuralis and medialis, inputs from the stomach terminate in the subnuclei medialis and gelatinosus, while inputs from the esophagus terminate in the subnucleus centralis (49). Vagal afferent fibers relay this visceral information by a mixture of myelinated (A-type) and unmyelinated (C-type) axons. These two types of afferent fibers display distinct physiological and functional characteristics, including different sensory modalities and conduction velocities and may regulate autonomic homeostatic and regulatory reflexes di...

Baptista, Vander and Wamberto Antonio Varanda. Glycine binding site of the synaptic NMDA receptor in subpostremal NTS neurons. J Neurophysiol 94: 147-152, 2005; doi:10.1152/jn.00927.2004. The nucleus of the tractus solitarius (NTS) plays an important role in the control of several autonomic reflex functions and has glutamate and GABA as main neurotransmitters. In this work, we used patch-clamp recordings in transverse slice preparations from rats to study whether the glycine binding site of the N-methyl-D-aspartate (NMDA) receptor is saturated or not in neurons of the subpostremal NTS. Except at hyperpolarized voltages and close to the reversal potential, glycine potentiated the NMDA responses in a concentration-dependent manner. The total charge transferred by glutamatergic currents was enhanced by glycine (500 M; from 28 Ϯ 13 to 42 Ϯ 18 pC at ϩ50 mV, n ϭ 7, P Ͻ 0.05). Glycine increased the conductance of the postsynaptic membrane, without altering its reversal potential, both in the presence (from 2.4 Ϯ 0.06 to 3.4 Ϯ 0.09 nS; n ϭ 7) and absence (from 3.1 Ϯ 0.06 to 4.4 Ϯ 0.10 nS; n ϭ 8) of Mg 2ϩ in the bathing solution. D-serine, in the presence of strychnine, also increased the amplitude of the NMDA component (by 68 Ϯ 19%, P Ͻ 0.05, n ϭ 5). The membrane potential was hyperpolarized (16 Ϯ 6 mV, n ϭ 8) by glycine, suggesting the presence of inhibitory glycinergic receptors. Our results indicate that the glycine site of the NMDA receptor in neurons of the subpostremal NTS is not saturated and that glycine may act as a modulator of the NMDA transmission in this nucleus.

The nucleus tractus solitarius (NTS) plays an important role in the control of autonomic reflex functions. Glutamate, acting on N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic receptors, is the major neurotransmitter in this nucleus, and the relative contribution of each receptor to signal transmission is unclear. We have examined NMDA excitatory postsynaptic currents (NMDA-EPSCs) in the subpostremal NTS using the whole cell patch clamp technique on a transverse brainstem slice preparation. The NMDA-EPSCs were evoked by stimulation of the solitary tract over a range of membrane potentials. The NMDA-EPSCs, isolated pharmacologically, presented the characteristic outward rectification and were completely blocked by 50 µM DL-2-amino-5-phosphonopentanoic acid. The I-V relationship of the NMDA response shows that current, with a mean (± SEM) amplitude of -41.2 ± 5.5 pA, is present even at a holding potential of -60 mV, suggesting that the NMDA receptors are weakly blocked by extracellular Mg 2+ at near resting membrane potentials. This weak block can also be inferred from the value of 0.67 ± 0.17 for parameter δ obtained from a fit of the Woodhull equation to the I-V relationship. The maximal inward current measured on the I-V relationship was at -38.7 ± 4.2 mV. The decay phase of the NMDA currents was fitted with one exponential function with a decay time constant of 239 ± 51 and 418 ± 80 ms at a holding potential of -60 and +50 mV, respectively, which became slower with depolarization (e-fold per 145 mV).

From a Cartesian perspective of rational analysis, the electric potential difference across the cell membrane is one of the fundamental concepts for the study of physiology. Unfortunately, undergraduate students often struggle to understand the genesis of this energy gradient, which makes the teaching activity a hard task for the instructor. The topic of bioelectrogenesis encompasses multidisciplinary concepts, involves several mechanisms, and is a dynamic process, i.e., it never turns off during the lifetime of the cell. Therefore, to improve the transmission and acquisition of knowledge in this field, I present an alternative didactic model. The design of the model assumes that it is possible to build, in a series of sequential steps, an assembly of proteins within the membrane of an isolated cell in a simulated electrophysiology experiment. Initially, no proteins are inserted in the membrane and the cell is at a baseline energy state; the extracellular and intracellular fluids are at thermodynamic equilibrium. Students are guided through a sequence of four steps that add key membrane transport proteins to the model cell. The model is simple at the start and becomes progressively more complex, finally producing transmembrane chemical and electrical gradients. I believe that this didactic approach helps instructors with a more efficient tool for the teaching of the mechanisms of resting membrane potential while helping students avoid common difficulties that may be encountered when learning this topic.