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 muM AP-5 and 10 muM 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 muM 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.

Fig. 1

(A) Current clamp recording trace showing a single action potential evoked upon injection of DC. Holding potential = –60 mV. (B) Voltage clamp recording trace showing the afterhyperpolarization current evoked upon stepping the membrane from –60 to +10 mV. (C) Current clamp recording trace showing the action potentials frequency response to injection of 15 and 240 pA DC. Holding potential = –60 mV. Traces from panels A–C are from the same cNTS neuron. (D) Graphic summarizing the action potentials frequency response to injection of graded intensities of DC (15–240 pA; N = 32). Holding potential = –60 mV.

Fig. 2

(A) Schematic diagram depicting the localization of cNTS neurons according to their soma shape (Bipolar = square; multipolar = circle). For purposes of clarity, not all the neurons have been reported in the graphic. Note that there is no discrete localization of any particular type of cNTS neuron. TS = tractus solitarius; AP = area postrema; DMV = dorsal motor nucleus of the vagus; cc = central canal; IV Ventr. = fourth ventricle. (B) Computer-aided reconstructions of two cNTS neurons. Note the distinctive multipolar (left) and bipolar (right) soma shape.

Fig. 3

Green, FITC filters = tyrosine hydroxylase (TH-IR); red, TRITC filters = Neurobiotin-filled neuron; yellow = merged image with TH-IR and Neurobiotin co-localization. Micrographs depicting two cNTS neurons that were filled with Neurobiotin® following electrophysiological recordings and postfixation immunoreactivity. The neuron depicted in panels A–D did not contain TH-IR; conversely, the neuron depicted in panels E–H co-localized with TH-IR. Panels A and E = low magnification micrograph showing the localization of the Neurobiotin®-labeled cNTS neuron with respect to the tractus solitarius (TS), the dorsal motor nucleus of the vagus (DMV), and neurons in the A2 area (TH-IR positive neurons). Panels B and F = high magnification micrograph (TRITC filters) showing the same neurons as above. Note that the cNTS cell in panel B has a bipolar soma shape, while the cNTS neuron in panel F has a multipolar soma shape. Panels C and G = high magnification micrograph showing the same area as above but taken with FITC filters to visualize TH-IR positive neurons. Panels D and H = merged high magnification micrograph of panels B and C (D) and panels F and G (H). Note that in panel D, the Neurobiotin-filled neuron does not co-localize TH-IR and Neurobiotin®, while in panel H, the Neurobiotin®-filled cNTS neuron co-localizes with TH-IR. Scale bar = 20 μM.

Fig 4

(A) Representative traces showing spontaneous excitatory postsynaptic currents (sEPSC) in the presence of 30 μM bicuculline (V_hold = –70 mV, a–b, and –30 mV, c–e). Note that at V_hold = –70 mV, perfusion with the non-NMDA antagonist CNQX (10 μM) abolished completely the sEPSC (b). Conversely, at V_hold = –30 mV, perfusion with both CNQX and the NMDA antagonist AP-5 (30 μM) abolished sEPSCs (d–e). (B) Representative averaged sEPSCs recorded at V_hold = –30 mV showing that perfusion with AP-5 decreased the area under the curve (change transferred) and perfusion with AP-5 and CNQX completely abolished the sEPSCs. Traces in control and in the presence of AP-5 were normalized, conversely, the trace in presence of AP-5 and CNQX was not normalized. (C) Summary graphic depicting the charge transferred at different holding potentials. *P < 0.05 vs. control.