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. 2017 Aug 20;38(1):219–232. doi: 10.1007/s10571-017-0534-9

Cellular Localization of Acid-Sensing Ion Channel 1 in Rat Nucleus Tractus Solitarii

Li-Hsien Lin 1, Susan Jones 1, William T Talman 1,2,✉
PMCID: PMC11482015  PMID: 28825196

Abstract

By determining its cellular localization in the nucleus tractus solitarii (NTS), we sought anatomical support for a putative physiological role for acid-sensing ion channel Type 1 (ASIC1) in chemosensitivity. Further, we sought to determine the effect of a lesion that produces gliosis in the area. In rats, we studied ASIC1 expression in control tissue with that in tissue with gliosis, which is associated with acidosis, after saporin lesions. We hypothesized that saporin would increase ASIC1 expression in areas of gliosis. Using fluorescent immunohistochemistry and confocal microscopy, we found that cells and processes containing ASIC1-immunoreactivity (IR) were present in the NTS, the dorsal motor nucleus of vagus, and the area postrema. In control tissue, ASIC1-IR predominantly colocalized with IR for the astrocyte marker, glial fibrillary acidic protein (GFAP), or the microglial marker, integrin αM (OX42). The subpostremal NTS was the only NTS region where neurons, identified by protein gene product 9.5 (PGP9.5), contained ASIC1-IR. ASIC1-IR increased significantly (157 ± 8.6% of control, p < 0.001) in the NTS seven days after microinjection of saporin. As we reported previously, GFAP-IR was decreased in the center of the saporin injection site, but GFAP-IR was increased in the surrounding areas where OX42-IR, indicative of activated microglia, was also increased. The over-expressed ASIC1-IR colocalized with GFAP-IR and OX42-IR in those reactive astrocytes and microglia. Our results support the hypothesis that ASIC1 would be increased in activated microglia and in reactive astrocytes after injection of saporin into the NTS.

Keywords: Astrocyte, Confocal microscopy, Immunofluorescent histochemistry, Microglia

Introduction

Acid-sensing ion channels (ASICs) are voltage-insensitive, proton-gated cation channels that are members of the degenerin/epithelial sodium channel family. As chemoreceptors they are activated by extracellular acidosis as may occur with inflammation, ischemic stroke, traumatic brain injury, and epileptic seizures (Baron and Lingueglia 2015; Lingueglia 2007; Waldmann 2001); but some are also implicated in mechano-transduction (Chung et al. 2010; Lu et al. 2009). Four genes (ACCN1-ACCN4) encode seven types of ASIC subunits (1a, 1b1, 1b2, 2a, 2b, 3, and 4), which consist of three homomeric or heteromeric subunits. Each type of ASIC subunit consists of two transmembrane domains (TM1 and TM2), a large, cysteine-rich extracellular loop and short intracellular N- and C-termini (Chu et al. 2014).

ASICs are widely distributed in the peripheral nervous system (PNS) and central nervous system (CNS) (Deval and Lingueglia 2015). A physiological role for ASICs in the PNS, for example in pain signaling, is well established (Chen et al. 1998; Waldmann 2001; Wu et al. 2004). Numerous studies have also supported their having a role in the CNS in such functions as synaptic plasticity, learning and memory, and fear conditioning (Bianchi and Driscoll 2002; Kreple et al. 2014; Petroff et al. 2008; Ziemann et al. 2009). At a cellular level, ASICs may play a role in excitatory neurotransmission in the CNS, possibly through their responsiveness to local proton concentrations resulting from synaptic transmission and release of glutamate and other neurotransmitters (Vick and Askwith 2015). In the ASIC family, ASIC type 1 (ASIC1) is the most broadly expressed channel in the CNS (Alvarez et al. 2003). Its potential physiological importance is emphasized by recognition that ASIC1 channels have been implicated in neurological diseases such as multiple sclerosis, Parkinson’s and Huntington’s diseases, seizure disorder, and depression (Arun et al. 2013; Chu et al. 2014; Coryell et al. 2009).

ASIC1 is expressed in axons, axon terminals, and cell bodies in the brain and spinal cord (Deval and Lingueglia 2015; Lin et al. 2014a). RT-PCR studies have revealed abundant ASIC1 expression in the nucleus tractus solitarii (NTS) (Huda et al. 2012), a medullary nucleus that plays an important role in cardiovascular, respiratory, and gustatory regulation (Feldman and Ellenberger 1988; Guyenet 2014; Lin 2009). Previous studies have shown that acidification can excite NTS neurons (Dean et al. 1989) and that NTS neurons respond to mild extracellular acidification with transient depolarization and activation of “ASIC-like” inward currents (Huda et al. 2012). These observations have suggested that ASIC1 may play a role in cardiorespiratory control in chemosensitive neurons in this important area as has also been suggested in the lateral hypothalamus (Huda et al. 2012). However, there have been no anatomical studies to show the presence of ASIC1 in the NTS or the type of cells that express ASIC1 in the NTS; thus, though a role of ASIC in chemoresponsiveness of NTS neurons has been established, the fundamental anatomical support for that putative physiological relevance has not been established. Therefore, we performed immunofluorescent histochemistry combined with confocal microscopy to study the distribution of ASIC1 in rat NTS. We also performed multi-labeled immunofluorescent staining to examine the types of cells that express ASIC1 in rat NTS by using the neuronal marker, protein gene product 9.5 (PGP9.5), the astrocytic marker, glial fibrillary acidic protein (GFAP), and the microglial marker, integrin αM (OX42). Previously, we have shown that saporin microinjected into the NTS caused damage to astrocytes and inflammation in the NTS (Lin et al. 2013). Because astrocyte damage and inflammation may lead to acidosis and result in ASIC1 activation, we also tested the hypothesis that saporin would increase ASIC1 expression in the NTS and examined the types of cells in which altered ASIC1 expression might be seen.

Materials and Methods

Animals and Injections

All procedures conformed to standards established in the Guide for Care and Use of Laboratory Animals (National Academy Press, Washington, D.C. 2011). The Institutional Animal Care and Use Committees of the University of Iowa and Department of Veterans Affairs Medical Center, Iowa City reviewed and approved all protocols. Both institutions are accredited by AAALAC, International. All efforts were made to minimize the number of animals used and to avoid their experiencing pain or distress.

For immunofluorescent staining of rat NTS, we euthanized and perfused adult male Sprague–Dawley rats (Harlan; 280–330 g, n = 6) under pentobarbital (50 mg/kg) anesthesia according to procedures described in our earlier publications (Lin et al. 2007; Lin and Talman 2005; Lin et al. 2011). The brain was then removed, post-fixed in 4% paraformaldehyde for 2 h, and then cryo-protected for 2 days in 30% sucrose in phosphate buffered saline (PBS) at 4 °C. Frozen 20-μm coronal sections were cut with a cryostat and mounted on Colorfrost Plus microscope slides (Fisher Scientific, PA, USA). Brain stem sections that contained different rostral/caudal levels of NTS were processed for immunofluorescent staining as will be described later.

For injections of saporin, adult male Sprague–Dawley rats (275–340 g, n = 5) were anesthetized with isoflurane (5% induction and 1.5–2.0% maintenance) delivered in 100% O2 (2 L/min) by a nasal mask. The dorsal surface of the brain stem was exposed as previously described (Talman 1989), and a glass micropipette filled with saporin was stereotactically placed (0.4 mm rostral to the calamus scriptorius, 0.5 mm from the midline, and 0.5 mm below the surface of the brain stem) unilaterally into an area that encompassed the dorsolateral and medial subnuclei of the NTS at the level of the area postrema. The diameter of the glass micropipette was 20–25 microns. Pressure injections (individual increments of 25–50 nl to a combined total of 100 nl containing 3 ng of saporin) were made over 15 min under the control of a WPI PV800 pneumatic ejection system. The pipette was left in place for 15 additional minutes to limit efflux of injectate from the pipette track. Surgical wounds were closed, hemostasis was assured, the animal was treated with buprenorphine (0.05 mg/kg), and anesthesia was stopped. After recovery from anesthesia, the animal was returned to the animal care facility until it was brought to the laboratory to be euthanized 7 days later. Rats that received no injection (n = 6) were used as controls.

Immunofluorescent Histochemistry

Antibody Characterization

Anti-ASIC1 antibody (Santa Cruz Biotechnology Cat# sc-28756 RRID:AB_2222808): an epitope corresponding to amino acids 505–574 mapping at the C-terminus of ASIC1 of human origin was used as immunogen for the antibody (information provided by Santa Cruz Biotechnology), which was made in rabbit. This antibody recognizes both ASIC1a and ASIC1b isoforms. Its specificity has been confirmed with Western blot analysis to recognize a single band protein of predicted molecular weight (about 70 kDa), which is consistent with published data (Alvarez et al. 2003) and in our earlier publication (Lin et al. 2014b). There was no immunoreactivity (IR) for ASIC1 when this antibody was omitted from the incubation solution (Fig. 1) as a negative control.

Fig. 1.

Fig. 1

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Immunoreactivity (IR) was not found in the NTS or adjacent areas when ASIC1 antibody was not present (a). ASIC1-IR was observed (b) when ASIC1 antibody was included in the incubation mixture. Scale bar = 100 µm

Glia fibrillary acidic protein (GFAP, an astrocyte marker): anti-GFAP antibodies were purchased from Sigma (Cat# G3893 RRID:AB_477010; 1:100 dilution, made in mouse) and Santa Cruz Biotechnology (Cat# sc-6170 RRID:AB_641021; 1:50 dilution, made in goat). Anti-GFAP from Sigma has been shown to stain specifically astrocytes as demonstrated in previous publications (Lathia et al. 2008; Poesen et al. 2008). Anti-GFAP antibody from Santa Cruz Biotechnology was also shown to stain specifically for astrocytes (El et al. 2012; Farioli-Vecchioli et al. 2012). We did not note any IR for GFAP when either GFAP antibody had been omitted from the incubation solution.

Protein gene product (PGP9.5, a neuronal marker): PGP9.5 antibodies were purchased from Millipore (Cat# AB5898, RRID:AB_92122; 1:100 dilution, made in Guinea pig). This antibody has been used to stain neurons specifically (Lin 2013; Lin et al. 2013; Miki et al. 2015). There was no IR for PGP9.5 when this antibody was not included in the incubation solution.

Integrin αM (OX42, a microglia marker): OX42 antibody was purchased from AbD Serotec (Cat# MCA275R RRID:AB_321302, 1:100 dilution, made in mouse). This antibody has been used successfully in staining microglia (Backes et al. 2016; Zheng et al. 2015). We did not observe any OX42-IR when this antibody was omitted from the incubation solution.

Immunofluorescent Staining

Procedures similar to those described in our previous publications (Lin et al. 2007; Lin and Talman 2005, 2006) were used for immunofluorescent staining of brain stem sections. Immunofluorescent histochemistry for ASIC1 was performed with or without biotin–streptavidin amplification. Sections were incubated in rabbit anti-ASIC1 antibody 1:100 dilution with biotin–streptavidin amplification, 1:20 dilution without biotin–streptavidin amplification in 10% donkey normal serum for 24 h in a humid chamber at 25 °C. For immunofluorescent staining of ASIC1 with biotin–streptavidin amplification, sections were washed with PBS after being incubated with primary antibody. They were then incubated with biotin-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Labs Cat# 711-065-152, RRID:AB_2340593, 1:200 dilution) in PBS for 20–24 h at 4 °C. They were washed again and incubated with Alexa Fluor 488®-conjugated streptavidin (Jackson ImmunoResearch Labs Cat# 016-540-084 RRID:AB_2337249, 1:200 dilution) in PBS for 20–24 h at 4 °C. For immunofluorescent staining of ASIC1 without biotin–streptavidin amplification, sections were incubated with Alexa Fluor 488®-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Labs Cat# 711-545-152 RRID:AB_2313584,1:200 dilution) in PBS for 20–24 h at 4 °C after they had been incubated with primary ASIC1 antibody and washed. Stained sections were cover-slipped with Prolong Gold Anti-fade Reagents (Invitrogen-Molecular Probes, RRID not available) after the final washes with PBS and then were examined with a confocal microscope (see below). Negative controls consisted of tissue processed in the absence of primary antibodies.

Our previously described (Lin et al. 2008, 2011) procedures were used for multiple-label immunofluorescent staining for ASIC1, GFAP, and protein gene product PGP9.5; for ASIC1 and integrin αM (OX42); and for ASIC1, GFAP, and OX42. We used anti-GFAP antibodies from different species to avoid mixing primary antibodies from the same species for multiple-label immunofluorescent staining (see below) so that false colocalization could be prevented. Each antibody produced the same staining pattern for its respective protein in the NTS. Sections were incubated in 10% donkey serum with a mixture of primary antibodies that were made in different species. After washing, sections were incubated in a mixture of donkey fluorophore-conjugated secondary antibodies (Fluor 488®-conjugated donkey anti-rabbit IgG, Jackson ImmunoResearch Labs Cat# 711-545-152, RRID:AB_2313584 or Rhodamine Red X or RRX-conjugated donkey anti-goat IgG, Jackson ImmunoResearch Labs Cat# 705-295-003 RRID:AB_2340422 or RRX-conjugated donkey anti-mouse IgG, Jackson ImmunoResearch Labs Cat# 715-295-150 RRID:AB_2340831 or Alexa Fluor 647®-conjugated donkey anti-mouse IgG, Jackson ImmunoResearch Labs Cat# 715-606-151 RRID:AB_2340866 or Alexa Fluor 647®-conjugated donkey anti-Guinea Pig IgG, ImmunoResearch Labs Cat# 706-605-148; all in 1:200 dilution, against species from which the primary antibodies were made) for multiple labeling. They were then washed and cover-slipped as described above.

Confocal Laser Scanning Microscopy

As previously described (Lin et al. 2007; Lin and Talman 2006), we analyzed stained sections with a Zeiss LSM 710 confocal laser scanning microscope. Sections labeled with multiple antibodies were scanned sequentially in different channels to separate labels. Images from different channels were each assigned a pseudo-color and then were superimposed. Confocal images were obtained and processed with software provided with the Zeiss LSM 710. Adobe Photoshop image editing software (Adobe Photoshop CS4; RRID:SCR_002078) was used as we switched between channels on the monitor to determine if a structure was labeled by one or more antibodies. Another image editing program, Microsoft PowerPoint (2010, RRID not available), was used to create montages.

Image Analysis

We quantified ASIC1-IR changes in the NTS from sections that were stained without biotin–streptavidin amplification using the NIH ImageJ software, a public domain program available from the National Institutes of Health (ImageJ, RRID:SCR_003070; http://rsb.info.nih.gov/ij/). For AISC1-IR, we analyzed all IR observed in cells and processes in the NTS. IR was measured as gray value in arbitrary units and was adjusted for differences in NTS size among sections. We used 2–3 sections of the NTS within 200 μm of the center of the injection site from all rats for analysis. Corresponding levels from untreated rats were used as controls. Two-tailed T test was used to determine if ASIC1-IR in the NTS was significantly altered by saporin injection.

Results

ASIC1 in Control NTS

As stated earlier, there was no IR for ASIC1 when primary antibody had been omitted from the incubation solution, a negative control (Fig. 1). In contrast, ASIC-IR was observed in the NTS and adjacent areas when ASIC1 antibody was present (Fig. 1). ASIC1-IR positive processes were seen at all four levels of the NTS (Fig. 2a–d). We also observed ASIC1-IR positive processes in the dorsal motor nucleus of vagus (DMV) (Fig. 2b, d). The strongest ASIC1-IR was present in the area postrema (AP) where cells and processes containing a high level of ASIC1-IR were seen (Fig. 2c). We observed few cells that contained AISC1-IR in the NTS except in the subpostremal NTS that is located adjacent to the AP (Fig. 2c).

Fig. 2.

Fig. 2

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Confocal images of ASIC1 immunofluorescent staining at four rostral to caudal levels of rat NTS (delineated by dotted lines): rostral (a), intermediate (b), subpostremal (c), and caudal (d). ASIC1-immunoreactivity (IR) is seen mostly in process-like structures (arrows in a–d) in the NTS and dorsal motor nucleus of vagus (DMV). Many cells in the area postrema (AP, panel C) contained ASIC1-IR, but only a few cells (arrowheads in c) in the subpostremal area of the NTS contained ASIC1-IR (panel C). Scale bar = 50 µm

We next performed immunostaining for multiple peptides to determine if structures that contained ASIC1-IR also contained markers for astrocytes, neurons, or microglia. The distribution of GFAP-IR, ASIC1-IR, and PGP9.5 in the same NTS tissue at the level of AP is shown in Fig. 3a–c, respectively. The merged confocal image (Fig. 3d) showed that many ASIC1-IR-containing processes colocalized with GFAP-IR and, therefore, were of astrocytic origin as shown to better advantage with higher magnification (Fig. 4). These double-labeled processes were present at all four levels of the NTS. Most ASIC1-IR-containing processes did not contain PGP9.5-IR and were, therefore, not likely to be neurons. Some of the cells in the subpostremal area of the NTS contained both ASIC1-IR and PGP9.5-IR and were, therefore, neurons (Fig. 4). Similarly, in the adjacent AP, many cells that contained ASIC1-IR also contained PGP9.5-IR (Fig. 5). In contrast, many processes in the AP stained only for ASIC1-IR while a few processes stained only for GFAP-IR. It was rare to see processes that stained for both ASIC1-IR and GFAP-IR in the AP.

Fig. 3.

Fig. 3

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Confocal images with a 20-µm-thick optical section showing the NTS at the level of AP with staining for glial fibrillary acidic protein (GFAP), which labels astrocytes (a), ASIC1 (b), and PGP9.5, a neuronal marker (c). A merged image is shown in d. Processes that contain ASIC1-IR in the NTS often contain GFAP-IR. A few cells that contain both ASIC1-IR and PGP9.5-IR are present in the subpostremal NTS. The boxed area in d is shown in a higher magnification in Fig. 4. Gr gracilis nucleus, cc central canal. Other abbreviations are as in Fig. 1. Scale bar = 100 µm

Fig. 4.

Fig. 4

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Higher magnification of the boxed area in Fig. 3. In this 5-µm optical section, colocalization of ASIC1-IR and GFAP-IR in processes (white arrows in a, b, and d) in the NTS indicates that ASIC1-IR is present in NTS astrocytes. In contrast, colocalization of both ASIC1-IR and PGP9.5-IR (arrow heads in b, c, and d) indicates the presence of ASIC1 in neurons. Scale bar = 25 µm

Fig. 5.

Fig. 5

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We observed colocalization of PGP9.5-IR (c) and ASIC1-IR (b) in many cells of the AP (arrow heads in b, c, and the merged image d). However, structures that are positive for GFAP-IR alone (white arrows in a and d) or ASIC1-IR alone (pink arrows in b and d) are also present in the AP. Scale bar = 25 µm

We performed double labeling of ASIC1 and OX42, a marker for microglia to further examine if ASIC1-IR-containing structures in the NTS were microglia. As shown in Fig. 6, we observed some ASIC1-IR-containing processes that also contained OX42 and were thus of microglial origin. In NTS, there were ASIC1-IR processes that did not stain for OX42-IR. For example, cells that stained for ASIC1-IR in the subpostremal area did not stain for OX42. In contrast, many processes that stained for ASIC1-IR in the AP also contained OX42-IR (Fig. 6) and thus were microglial processes.

Fig. 6.

Fig. 6

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ASIC1-IR (a and d) colocalizes with IR of the microglia marker OX42 (b and e) in the NTS (a–c) and AP (d–f). Arrows indicate processes that are double labeled for ASIC1-IR and OX42-IR. Panels c and f show the merged confocal images for NTS and AP, respectively. Scale bar = 25 µm

ASIC1 in the NTS After Treatment with Saporin

Seven days after injection of saporin into the NTS, ASIC1-IR was significantly increased (157 ± 8.6% of control, p < 0.001) in the NTS (Fig. 7). We also noted increased ASIC1-IR in areas adjacent to the treated NTS, such as the AP, the gracilis nucleus (Gr), and the DMV (Fig. 7). Although we saw a dramatic decrease in GFAP-IR in the NTS after the injection, as we have reported (Lin et al. 2013), GFAP-IR was increased, as we have also reported, (Lin et al. 2013) in areas that surrounded the GFAP-IR depleted area. In areas of increased GFAP-IR, cell bodies of astrocytes were swollen and their processes thickened (Fig. 8), indicative of astrogliosis (Sofroniew 2009). There was also increased OX42-IR in the injected NTS, consistent with our previous observation (Lin et al. 2013). Cell bodies of these microglia were enlarged (Figs. 8, 9) as occurs in the activated state (Graeber 2010). Merged confocal images showed prominent colocalization of ASIC1-IR and GFAP-IR in this area (Fig. 7). A high magnification of tissue stained for ASIC1, OX42, and GFAP in this area revealed that increased ASIC1-IR not only colocalized with increased GFAP-IR (Fig. 8) in some cells and processes, but also it colocalized with increased OX42-IR (Fig. 8) in other cells and processes. In the area where GFAP-IR was almost depleted, the increased ASIC1-IR was found to be colocalized mainly with increased OX42-IR (Fig. 9), although a few processes that were double labeled with both GFAP-IR and ASIC1-IR were observed (Fig. 9). As reported earlier (Lin et al. 2013), we did not see a change in PGP9.5-IR after saporin injection, nor did we see an increase in ASIC1-IR in neurons labeled with PGP9.5-IR.

Fig. 7.

Fig. 7

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Compared to the control rat (a) ASIC1-IR is significantly (p < 0.001) increased in the NTS after saporin injection (b). This increase is also seen in adjacent areas such as the Gr and DMV (b). As reported in our previous publication (Lin et al. 2013), there is a dramatic decrease in GFAP-IR in the saporin injected area (d), as compared to that of the control side (c). Note, however, that GFAP-IR increases in areas surrounding the area of depletion. An example of such an area is indicated by double arrows in d. Merged images of a and c, b and d are shown in e and f, respectively. Colocalization of ASIC-IR and GFAP-IR (double arrows in f) is very prominent in these areas where both ASIC1 and GFAP are up-regulated. Abbreviations are as in Figs. 1 and 2. Scale bar = 100 µm

Fig. 8.

Fig. 8

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Peripheral to the injected site, where increased ASIC1-IR and GFAP-IR is observed, increased ASIC1-IR colocalizes with increased GFAP-IR (arrows in a, b, and d). The increased ASIC1-IR also colocalizes with increased OX42-IR (arrowheads in b, c, and d). GFAP-IR and OX42-IR do not co-localize. Scale bar = 25 µm

Fig. 9.

Fig. 9

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In the center of the injection site, where increased ASIC1-IR and decreased GFAP-IR are seen, increased ASIC1-IR mostly colocalizes with OX42-IR (arrowheads in b, c, and d). Only a few processes are positive for both ASCI-IR and GFAP-IR (arrows in a, b, and d). Scale bar = 25 µm

Discussion

This paper makes the following observations that have not previously been reported. First, processes that were positive for ASIC1-IR were present throughout the rostral to caudal levels of the NTS, DMV, and AP in rat. Second, these processes colocalized with the astrocytic marker, GFAP, or with the microglial marker, OX42. Third, ASIC1-IR-containing neurons were sparse in the NTS but were most prevalent in the subpostremal region of the nucleus. Fourth, injection of saporin into the NTS led to over-expression of ASIC1-IR in reactive astrocytes and microglia.

Although ASIC is found in the cell membrane, ASIC1-IR in NTS cells in this study appeared more diffusely over cells as has been reported by others who showed that ASIC1-IR appeared uniformly over the cell as if in cytoplasm (Alvarez et al. 2003; Ohbuchi et al. 2010; Yu et al. 2015a). This finding is not unique for ASIC in that IR for receptors that are found on the cell membrane also appear more uniformly over cells rather than on the cell’s perimeter alone (Lin et al. 2008).

An earlier electrophysiological study demonstrated a subset of dissociated NTS neurons that were capable of responding to changes in pH within the physiological range of 7.4–7.0 (Huda et al. 2012). The neuronal response was mediated by ASICs (Huda et al. 2012), but the technique used to dissociate NTS neurons did not allow determination of the subnuclear location of responsive neurons. Those observations are consistent with studies showing that neurons within the NTS respond to acidification (Dean and Putnam 2010; Putnam et al. 2004). Using RT-PCR, Huda et al. further reported that the ASIC1 transcript was noted in their RT-PCR products (Huda et al. 2012). Although they did not perform anatomical studies, a two-dimensional model revealed that the neurons from which they recorded were located in a limited area of the NTS, with maximum likelihood in a region dorsal to the central canal (Huda et al. 2012), and consistent with the subpostremal subnucleus of NTS. Those findings support our own immunofluorescent observation that ASIC1-IR positive neurons were present in the subpostremal area of the NTS. The existence of ASIC1-containing neurons in the NTS provides anatomical support for other investigators’ suggestion that ASIC1 may play a role in cardiorespiratory control in chemosensitive neurons in this important region (Huda et al. 2012).

Although ASIC1-IR was essentially confined to NTS neurons of the subpostremal region, it was observed in astrocytes throughout the rat NTS as has been described at other central sites as described below. Not only has ASIC current been recorded in cultured astrocytes, expression of ASIC1 protein has been demonstrated by western blotting and by immunofluorescent staining in astrocytes both in culture and in tissue (Huang et al. 2010). Using a combination of electrophysiology, calcium imaging, and immunocytochemistry, other investigators have demonstrated that ASIC1a is expressed in high density in NG2 glia in rat hippocampus (Lin et al. 2010). Similarly, ASIC1 has also been detected in rabbit retinal glia by Western blotting (Brockway et al. 2002). However, the presence of ASIC1 in glia may not be uniform throughout the CNS in that one study did not find ASIC1 in glia of the cerebellum (Alvarez et al. 2003). At the present time, it is not known what role ASIC1 plays in NTS astrocytes; but others have suggested that astrocytes may be chemosensitive (Gourine and Kasparov 2011; Gourine et al. 2010) and may be involved in neurotransmitter release and glia-neuronal crosstalk (Gundersen et al. 2015).

We also observed ASIC1-IR in microglia throughout the rat NTS. In contrast with descriptions of ASIC1 in astrocytes, fewer studies have reported ASIC1 in microglia. A recent publication did convincingly demonstrate ASIC1 expression in primary cultured rat microglia by quantitative real-time PCR, Western blotting, and immunofluorescence experiments (Yu et al. 2015b). Our finding that ASIC1 is present in NTS microglia in situ is consistent with that report. Microglia are resident immune cells that have a role in the maintenance of synaptic integrity and respond to brain injury or neurodegenerative disease by becoming activated (Graeber 2010). Recent reports have suggested that microglia, which themselves express receptors for neurotransmitters (Pocock and Kettenmann 2007), may participate in central autonomic control (Pocock and Kettenmann 2007) befitting their close anatomical relationship with synapses (Schafer et al. 2013).

Our data show intense ASIC1-IR in neurons and microglia of the AP. This observation has not been reported previously, nor is there any indication as to what might be the role for ASIC1 in this known chemotactic area. As one of the circumventricular organs, the AP lies outside the blood–brain barrier and has direct access to circulating cerebrospinal fluid (Leslie 1986). Thus, it is possible that ASIC1 functions as an acid sensor in AP neurons and microglia in monitoring hydrogen ion concentration in the cerebrospinal fluid although past studies have failed to show that the AP responds to hydrogen ion (Hori et al. 1970; Loeschcke et al. 1963). After injection of saporin into the NTS, there was a significant increase in ASIC1-IR that represented over-expression of ASIC1 in reactive astrocytes in the region. Up-regulation of ASIC1 has been found in axons and oligodendrocytes in mice with acute experimental autoimmune encephalomyelitis and in human with active multiple sclerosis (Arun et al. 2013; Vergo et al. 2011). Increased expression of ASIC1 was also noted after traumatic spinal cord injury (Hu et al. 2011). In addition, up-regulation of ASIC1a channel in spinal dorsal horn neurons has been shown to contribute to pain hypersensitivity (Duan et al. 2007). Because blocking ASIC1 with antagonists or antisense oligonucleotide reduced tissue damage and promoted the recovery of neurological function, it has been suggested that ASIC1 may be involved in the cellular mechanisms underlying certain neurological conditions (Arun et al. 2013; Hu et al. 2011; Vergo et al. 2011). Up-regulation of ASIC1 in reactive astrocytes has not been reported previously. We do not know the significance of over-expression of ASIC1 in astrocytes in the area surrounding injury although it is likely that this may relate to tissue damage.

Finally, we report here that ASIC1 is over-expressed in reactive microglia in the NTS after saporin injection. This finding is consistent with an earlier report (Yu et al. 2015b). In lipopolysaccharide-induced inflammation, not only was ASIC1 up-regulated but also ASIC-like current and acid-induced elevation of intracellular calcium were increased. Because an ASIC antagonist and ASIC1 blocker inhibited these responses and reduced the expression of inflammatory cytokines, it was suggested the ASIC1 participates in neuroinflammatory reaction (Yu et al. 2015b). ASIC1 could play a similar role in activated microglia in the NTS as a result of inflammation induced by saporin injection.

Conclusions

Cells and processes that contain ASIC1 are present in the NTS, DMV, and AP. ASIC1 is expressed in astrocytes and microglia throughout the NTS, but the subpostremal NTS is the only NTS area where neurons containing ASIC1 are observed. Up-regulation of ASIC1 is noted in reactive astrocytes and microglia in the NTS after saporin injection. Increased ASIC1-IR in astrocytes in NTS is likely associated with tissue damage caused by saporin injection, while increased ASIC1-IR in microglia likely reflects an inflammatory response to the saporin injection.

Acknowledgements

This work was funded in part by NIH RO1 HL 088090 (to L.H. Lin and W. T. Talman) and in part by a Merit Review from the Department of Veterans Affairs (to W. T. Talman).

Abbreviations

AP

Area postrema

ASIC

Acid-sensing ion channel

ASIC1

Acid-sensing ion channel type1

cc

Central canal

CNS

Central nervous system

DMV

Dorsal motor nucleus of vagus

GFAP

Glial fibrillary acidic protein

Gr

Gracilis nucleus

IR

Immunoreactivity

NTS

Nucleus tractus solitarii

OX42

Integrin αM chain or CD11b (clone OX-42)

PBS

Phosphate buffered saline

PGP9.5

Protein gene product 9.5

PNS

Peripheral nervous system

RRX

Rhodamine red X

RT-PCR

Reverse transcription polymerase chain reaction

Author contributions

LHL and WTT designed experiments; LHL and SJ performed experiments; LHL and WTT analyzed data; LHL and WTT wrote the article. All authors have approved the final article.

Compliance with ethical standards

Conflict of interest

None of the authors has a conflict of interest.

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