TASK1 and TASK3 Are Coexpressed With ASIC1 in the Ventrolateral Medulla and Contribute to Central Chemoreception in Rats

Abstract

The ventrolateral medulla (VLM), including the lateral paragigantocellular nucleus (LPGi) and rostral VLM (RVLM), is commonly considered to be a chemosensitive region. However, the specific mechanism of chemoreception in the VLM remains elusive. Acid-sensing ion channels (ASICs), a family of voltage-independent proton-gated cation channels, can be activated by an external pH decrease to cause Na⁺ entry and induce neuronal excitability. TWIK-related acid-sensitive potassium channels (TASKs) are members of another group of pH-sensitive channels; in contrast to AISICs, they can be stimulated by pH increases and are inhibited by pH decreases in the physiological range. Our previous study demonstrated that ASICs take part in chemoreception. The aims of this study are to explore whether TASKs participate in the acid sensitivity of neurons in the VLM, thereby cooperating with ASICs. Our research demonstrated that TASKs, including TASK1 and TASK3, are colocalized with ASIC1 in VLM neurons. Blocking TASKs by microinjection of the non-selective TASK antagonist bupivacaine (BUP), specific TASK1 antagonist anandamide (AEA) or specific TASK3 antagonist ruthenium red (RR) into the VLM increased the integrated phrenic nerve discharge (iPND), shortened the inspiratory time (Ti) and enhanced the respiratory drive (iPND/Ti). In addition, microinjection of artificial cerebrospinal fluid (ACSF) at a pH of 7.0 or 6.5 prolonged Ti, increased iPND and enhanced respiratory drive, which were inhibited by the ASIC antagonist amiloride (AMI). By contrast, microinjection of alkaline ACSF decreased iPND and respiratory drive, which were inhibited by AEA. Taken together, our data suggest that TASK1 and TASK3 are coexpressed with ASIC1 in the VLM. Moreover, TASK1 and TASK3 contribute to the central regulation of breathing by coordinating with each other to perceive local pH changes; these results indicate a novel chemosensitive mechanism of the VLM.

Keywords: TASK1; TASK3; chemoreception; pH-sensitive; ventrolateral medulla.

Figure 1

The location of TWIK-related acid-sensitive potassium channel 1 and 3 (TASK1 and 3)-positive cells in the ventrolateral medulla (VLM) of SD rats. (A) TASK1-positive cells were located in the VLM of rats. (B,C) represent high-power visual fields of the area shown in (A), showing a detailed view of TASK1-positive cells in the VLM. (D) TASK3-positive cells in the VLM of rats. (E,F) represent high-power visual fields of the area shown in (D), showing a detailed view of TASK3-positive cells in the VLM. (G) Coronal diagram of the rat medulla; the area shown in black represents the distribution of TASK1 and TASK3. Cell morphology is indicated by the arrows shown in (C,F). (H–I) Negative control. Scale bar: 200 μm (A,D,H,I); 80 μm (B,E) and 20 μm (C,F).

Figure 2

TASK1 and TASK3 are expressed and colocalized in VLM neurons. (A,B) Representative confocal photomicrograph showing the colocalization of TASK1-ir and TASK3-ir (green) with neurofilament-ir (red) in the VLM. (C) Representative confocal photomicrograph showing the colocalization of TASK1 (red) and TASK3 (green) in the VLM. Original magnification, 200×.

Figure 3

Acid-sensing ion channel 1 (ASIC1), TASK1 and TASK3 are expressed and colocalized in the VLM neurons of adult rats. (A) Representative confocal photomicrographs showing the colocalization of ASIC1-ir (green), TASK1-ir (red) and neurofilament-H (blue) in the VLM. (B) Representative high power visual field of the area shown in (A). (C) Representative confocal photomicrographs showing the colocalization of ASIC1-ir (green), TASK3-ir (red) and neurofilament-H (blue) in the VLM. (D) Representative high power visual field of the area shown in (C). (A–C) Scale bar = 200 μm; (B,D) scale bar = 20 μm.

Figure 4

The non-selective TASKs antagonist, bupivacaine (BUP) stimulated respiration of rats. (A) The phrenic nerve discharge (PND) was recorded from the same animal. The unilateral microinjection of 200 μM BUP into the VLM increased PND and integrated PND (iPND). The microinjection of artificial cerebrospinal fluid (ACSF; pH 7.4) served as a control. (B) Inspiratory time (Ti) was decreased by BUP injection. (C) Group data showing the effects of BUP on iPND. (D) Responses of the respiratory drive. Note that BUP stimulated respiration (*p < 0.05, **p < 0.01 relative to control; n = 6). (E) Histological staining with neutral red: the sky blue spot indicates the injection site in the VLM, and the injection plot was confirmed by comparison with the Bregma −12.72 mm coronal diagram in the Paxinos and Watson stereotaxic atlas of the rat brain. In the diagram, red arrowheads represent the injection points and dots signify injection points out of VLM.

Figure 5

TASK1 antagonist, anandamide (AEA) and TASK3 antagonist, ruthenium red (RR) stimulated the respiration of rats. (A,B) The PND was recorded from the same animal. The unilateral microinjection of 100 μM AEA and 10 μM RR into the VLM increased PND and iPND. The microinjection of ethanol and ACSF (pH 7.4) served as a control. (C–F) Ti was decreased by AEA and RR injection. (D–G) Group data showing the effects of AEA and RR on iPND. (E–H) Responses of the respiratory drive. Note that AEA and RR stimulated respiration, *p < 0.05, **p < 0.01, ***p < 0.001 relative to control; n = 5.

Figure 6

ASIC antagonists attenuated the stimulatory effect of acidification on respiration in rat VLM. (A–C) The unilateral microinjection of 0.1 μL ACSF (pH 7.4, 7.0 and 6.5) into the VLM increased raw PND (A) and iPND (B,C). AMI pre-treatment (100 μM) blocked this effect. The microinjection of ACSF (pH 7.4) served as a control. (D–F) Statistical data showing the effects of alkalization and AMI on Ti (D), iPND (E) and respiratory drive (F). *p < 0.05, **p < 0.01, ***p < 0.001, n = 6.

Figure 7

TASK1 antagonists attenuated the subdued effect of alkalization on respiration in the rat VLM. (A–C) The unilateral microinjection of 0.1 μL ACSF (pH 8.0) into the VLM decreased raw PND (A) and iPND (B,C). TASK1 antagonist AEA pre-treatment (100 μM) blocked this effect. The microinjection of ACSF (pH 7.4) served as a control. (D–F) Statistical data showing the effects of different levels of alkalization and AEA on Ti (D), iPND (E) and respiratory drive (F). *p < 0.05, n = 6.