Characterization of neurons of the nucleus tractus solitarius pars centralis

Abstract

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.

Presynaptic Melanocortin-4 Receptors on Vagal Afferent Fibers Modulate the Excitability of Rat Nucleus Tractus Solitarius Neurons

“…Because the major source of glutamatergic input to neurons of the NTS originates from vagal afferent fibers (Champagnat et al, 1986;Mifflin and Felder, 1990;Andresen and Kunze, 1994;Smith et al, 1998;Hornby, 2001;Jean, 2001;Baptista et al, 2005b;Travagli et al, 2006), we hypothesized that the modulation of sEPSCs by ␣MSH and MTII may target vagal afferent (sensory) terminals preferentially. We thus conducted a series of experiments in animals in which the vagal afferent terminals were surgically removed.…”

“…At the conclusion of electrophysiological recording, the position of the neuron was noted before Neurobiotin was injected by passing positive current pulses into the NTS neuron (1 s duration, 0.5 Hz for 20 min); the brainstem slice was then fixed overnight at 4°C in Zamboni's solution [1.6% (w/v) para-formaldehyde, 19 mM KH 2 PO 4 , and 100 mM Na 2 HPO 4 in 240 ml of saturated picric acid-1600 ml of H 2 O; adjusted to pH 7.4 with HCl]. The fixative was cleared from the slice with multiple washes of PBS (in mM: 115 NaCl, 75 Na 2 HPO 4 ⅐7H 2 O, 7.5 KH 2 PO 4 , 0.15% Triton X-100, and 0.1% bovine serum albumin), and the injected neurobiotin was visualized using a cobalt-nickel enhancement of the Avidin D-horseradish peroxidase (0.05% diaminobenzidine in PBS containing 0.5% gelatin supplemented with 0.025% CoCl 2 and 0.02% NiNH 4 SO 4 ) technique as described previously (Browning et al, 1999(Browning et al, , 2005Martinez-Pena y Valenzuela et al, 2004;Baptista et al, 2005b;Wan et al, 2007).…”

“…Data analysis was performed as described previously (Browning et al, 1999(Browning et al, , 2005Martinez-Pena y Valenzuela et al, 2004;Baptista et al, 2005b;Wan et al, 2007). Micrograph of representative neurons were taken using a Spot Insight camera and software (Diagnostic Instruments, Sterling Heights, MI) connected to a Nikon (Tokyo, Japan) E400 microscope; the contrast and brightness of the micrographs were digitally enhanced with Adobe software (Adobe Systems).…”

“…Vagal afferent fibers use glutamate as their main transmitter onto NTS neurons (Andresen and Yang, 1990;Smith et al, 1998;Jean, 2001;Baptista et al, 2005b;Travagli et al, 2006) and intraperitoneal administration of satiety agents (e.g., CCK, glucagon-like peptide-1, or cocaine-and amphetamine-regulated transcript) increases c-Fos expression in NTS (Rinaman et al, 1993;Zittel et al, 1999;Sayegh and Ritter, 2000;Zheng et al, 2002b;Cano et al, 2003;Gulley et al, 2005;Arora and Anubhuti, 2006;Berthoud et al, 2006), including the POMC neurons of the caudal NTS (Appleyard et al, 2005). This c-Fos expression is likely caused by increased glutamate release, because administration of glutamatergic antagonists decreases satiety (Covasa et al, 2004a,b;Zheng et al, 1999Zheng et al, , 2002a.…”

Effects of cholecystokinin-8s in the nucleus tractus solitarius of vagally deafferented rats

“…The methods of slicing the brain stem and the identification of cNTS neurons have been described previously (3,4,59). Briefly, 25-to 35-day-old Sprague-Dawley rats of either sex were anesthetized with isoflurane (abolition of the foot pinch withdrawal reflex) before being killed by severing the blood vessels in the chest.…”