Hydrogen sulfide augments synaptic neurotransmission in the nucleus of the solitary tract

Abstract

Within the brain stem, the nucleus tractus solitarii (NTS) serves as a principal central site for sensory afferent integration from the cardiovascular and respiratory reflexes. Neuronal activity and synaptic transmission in the NTS are highly pliable and subject to neuromodulation. In the central nervous system, hydrogen sulfide (H₂S) is a gasotransmitter generated primarily by the enzyme cystathionine-β-synthase (CBS). We sought to determine the role of H₂S, and its generation by CBS, in NTS excitability. Real-time RT-PCR, immunoblot, and immunohistochemistry analysis identified the presence of CBS in the NTS. Patch-clamp electrophysiology in brain stem slices examined excitatory postsynaptic currents (EPSCs) and membrane properties in monosynaptically driven NTS neurons. Confocal imaging of labeled afferent synaptic terminals in NTS slices monitored intracellular calcium. Exogenous H₂S significantly increased the amplitude of evoked solitary tract (TS)-EPSCs, frequency of miniature (m)EPSCs, and presynaptic terminal calcium fluorescence in the NTS. H₂S did not alter action potential discharge or postsynaptic properties. On the other hand, the CBS inhibitor aminooxyacetate (AOA) significantly reduced the amplitude of TS-EPSCs and presynaptic terminal calcium fluorescence in the NTS without altering postsynaptic properties. Taken together, these data support a presynaptic role for endogenous H₂S in modulation of excitatory neurotransmission in the NTS.

Fig. 1.

Cystathionine-β-synthase (CBS) is located in the nucleus tractus solitarii (NTS). A: real-time RT-PCR analysis demonstrated CBS mRNA (152 bp arrow) in tissue from NTS and cerebellum (CB; used as a positive control; Abe and Kimura 1996). No PCR product was observed in the no template (NT), no primer (NP), and no reverse transcriptase (−RT) lanes. L, 123-bp ladder. NTS and CB samples were run in duplicate. B: immunoblot analysis illustrated that CBS protein was located in the NTS. Twenty micrograms of NTS protein (3rd lane) was matched with HEK cells transfected with CBS (2nd lane) and a rat brain lysate (1st lane). Immunoblots for CBS show bands in each lane at ∼62 kDa arrow. MM, molecular mass.

Fig. 2.

CBS immunoreactivity (IR) in the NTS. Coronal sections (30 μm) were immunohistochemically processed to examine the distribution of CBS within the NTS. As shown in A, CBS-IR was observed throughout the NTS, including in cell bodies. Preabsorption of the primary antibody with the recombinant protein decreased IR (B). Images in A and B were taken at the same exposure time and light histogram characteristics. To determine the caudal to rostral localization of CBS-IR, positively labeled CBS cells were counted bilaterally throughout the NTS (C; n = 3). CBS-IR was found in the caudal (D) and postremal (E) regions of the NTS, with diminished IR in the rostral (F) region. AP, area postrema. Scale bars, 50 μm.

Fig. 3.

CBS is located in glia and nonglia cells. Pseudocolored photomicrographs of CBS (1, green) and glial fibrillary acid protein (GFAP) (2, red) in the NTS are shown. Subsequent merged images are also shown (3) In representative images of the NTS (A and B), CBS and GFAP do not colabel, suggesting that CBS is localized to neurons (white arrows). Scattered throughout the NTS, CBS and GFAP colabeling was observed near blood vessels (yellow arrowheads). Note the cross section of a blood vessel that contains CBS and GFAP-IR in A. Shown in B is a longitudinal section of an NTS blood vessel containing CBS and GFAP. Scale bars, 50 μm.

Fig. 4.

Exogenous hydrogen sulfide (H₂S) augments evoked synaptic activity. A: representative traces of solitary tract (TS) excitatory postsynaptic currents (EPSCs) (30 sweeps, averaged) that were augmented during bath application of NaHS (10 μM, 5 min) compared with artificial cerebrospinal fluid (aCSF) control. Cells were voltage clamped at −60 mV. B: group and scatterplot comparison (n = 10) shows that bath application of 10 μM NaHS (filled circles) increased the amplitude of monosynaptic TS-EPSCs compared with aCSF control (open circles; *P < 0.05). Small connected symbols indicate data from individual neurons. Large circles indicate means ± SE. C: 20-Hz TS train produced frequency-dependent depression in synaptic currents in aCSF control and after bath application of NaHS (10 μM). NaHS also augmented the amplitude of TS-EPSC1, -2, -3, -7, -10, -12, and -20 (*P < 0.05). D: representative example (average of 5 sweeps) of 2 consecutive TS-EPSCs during control and NaHS (20 Hz). Note that NaHS augmented both TS-EPSCs, yet the increase in TS-EPSC2 was greater than in TS-EPSC1. E: bath application of NaHS (10 μM, n = 10) significantly increased the paired pulse ratio (PPR; TS-EPSC2/TS-EPSC1) compared with control. *P < 0.05.

Fig. 5.

Exogenous H₂S increases miniature (m)EPSC frequency. A: representative example of mEPSCs during aCSF control (top) and bath application of NaHS (10 μM, bottom). Recordings were made with GABAzine (25 μM) and tetrodotoxin (1 μM) present in the bath. Note the increase in current frequency. Group analysis of mEPSCs (n = 6) showed that bath application of NaHS (10 μM) did not change the amplitude of mEPSCs (B). However, NaHS enhanced the frequency of mEPSCs (C; *P < 0.05).

Fig. 6.

Blockade of endogenous H₂S production reduces synaptic activity. A: representative traces of TS-EPSCs (30 sweeps, averaged) illustrating that bath application of aminooxyacetate (AOA; 1 mM, 5 min) reduced the amplitude of TS-EPSCs. B: group and scatterplot data of neurons prior to (open circles) and after (gray circles) bath application of AOA (1 mM). Small connected symbols indicate data from individual neurons. Large circles indicate means ± SE. Blockade of endogenous H₂S production reduced the amplitude of TS-EPSCs (n = 15; *P < 0.05). C: stimulating the TS at 20 Hz produced frequency-dependent depression of TS-EPSCs in both control and AOA (1 mM; n = 15). Bath application of AOA significantly depressed the 1st and 4th TS-EPSC amplitudes (*P < 0.05) and reduced the amplitude of TS-EPSC9 (+P = 0.08) and TS-EPSC10 (+P = 0.07).

Fig. 7.

H₂S modulates presynaptic calcium concentration. A and D: example of Calcium Green-labeled afferent varicosities in the NTS during stimulation of the TS during aCSF. B: Varicosities shown in A during TS stimulation in the presence of NaHS (10 μM). Yellow arrows and outlines in A and B highlight sensory afferent varicosities that augment fluorescent intensity after NaHS. The outlined varicosities are further plotted in C and demonstrate a significant increase in change in peak fluorescence from baseline (%ΔF/F) on application of NaHS (red) compared with the control stimulation in aCSF (black). E: varicosities shown in D during TS stimulation in the presence of AOA (1 mM). Yellow outlines in D and E demonstrate sensory afferent varicosities that have reduced fluorescent intensity after AOA. The outlined varicosities are plotted in F and demonstrate a significant reduction in %ΔF/F following application of AOA (red) compared with control stimulation in aCSF (black). Black arrow in C and F denotes point of TS stimulation. G: change in fluorescence intensity during time control [aCSF, 103 regions of interest (ROIs)], NaHS (10 μM, 88 ROIs), and AOA (1 mM, 80 ROIs) compared with the 1st initial event. Note that %ΔF/F did not significantly change over time. However, H₂S significantly increased the fluorescence of sensory afferent varicosities, while AOA significantly decreased the fluorescence of sensory afferent varicosities (*P < 0.05). Scale bar, 5 μm.