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![Expression of TASK channel transcripts in midline raphe cell groups. In situ hybridization was performed in parallel on sections of rat brainstem using [ 33 P]-labeled RNA probes complementary to TASK-1 and TASK-3. Film autoradiographs from coronal sections of midbrain show moderate levels of TASK-1 expression ( A ) and high levels of TASK-3 expression ( B ) in the RDo. In transverse sections from the medulla oblongata, fi lm autoradiographs depict expression of TASK-1 ( C ) and TASK-3 ( D ) in the caudal raphe cell groups ROb and RPa. Strong labeling in the hypoglossal motor nuclei ( XII ) is also apparent. TASK-1 is expressed at lower levels in raphe cell groups than in motoneu- rons, whereas levels of TASK-3 are more comparable in those two cell types. It also appeared that TASK-1 was expressed at lower levels than TASK-3, although comparisons between probes should be made with caution. Autoradiographs of sagittal rat brain sections from control ex- periments performed with antisense ( E , F ) and sense ( G , H ) probes to TASK-1 ( E , G ) and TASK-3 ( F , H ) illustrate differential but overlapping brain distribution of TASK channel transcripts and low levels of back- ground labeling. Scale bar: A – D , 1 mm; E – H , 2.5 mm.](profile/Douglas-Bayliss/publication/11511827/figure/fig1/AS:282078773039104@1444264228992/Expression-of-TASK-channel-transcripts-in-midline-raphe-cell-groups-In-situ.png)
Expression of TASK channel transcripts in midline raphe cell groups. In situ hybridization was performed in parallel on sections of rat brainstem using [ 33 P]-labeled RNA probes complementary to TASK-1 and TASK-3. Film autoradiographs from coronal sections of midbrain show moderate levels of TASK-1 expression ( A ) and high levels of TASK-3 expression ( B ) in the RDo. In transverse sections from the medulla oblongata, fi lm autoradiographs depict expression of TASK-1 ( C ) and TASK-3 ( D ) in the caudal raphe cell groups ROb and RPa. Strong labeling in the hypoglossal motor nuclei ( XII ) is also apparent. TASK-1 is expressed at lower levels in raphe cell groups than in motoneu- rons, whereas levels of TASK-3 are more comparable in those two cell types. It also appeared that TASK-1 was expressed at lower levels than TASK-3, although comparisons between probes should be made with caution. Autoradiographs of sagittal rat brain sections from control ex- periments performed with antisense ( E , F ) and sense ( G , H ) probes to TASK-1 ( E , G ) and TASK-3 ( F , H ) illustrate differential but overlapping brain distribution of TASK channel transcripts and low levels of back- ground labeling. Scale bar: A – D , 1 mm; E – H , 2.5 mm.
Source publication
The recently described two-pore-domain K+ channels, TASK-1 and TASK-3, generate currents with a unique set of properties; specifically, the channels produce instantaneous open-rectifier (i.e., "leak") K+ currents that are modulated by extracellular pH and by clinically useful anesthetics. In this study, we used histochemical and in vitro electrophy...
Contexts in source publication
Context 1
... resting membrane potential and input resistance of neurons are key determinants of neuronal excitability; these intrinsic prop- erties are determined, in large part, by constitutive activity of K ϩ selective channels. In recent years, a novel gene family of back- ground K ϩ channels has been identified, members of which have characteristics that are distinctly different from most other K ϩ channels and consistent with a role in setting membrane potential and input resistance (e.g., weak voltage dependence, extremely fast kinetics, etc.) (for review, see Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001; Patel et al., 2001). Although these so-called KCNK channels generally show some degree of background activity at normal resting potentials, they are nevertheless also subject to modulation by numerous factors. For example, channel activity is influenced by prevailing physi- cochemical conditions such as temperature, intracellular or extra- cellular pH, oxygen tension, and membrane stretch and can be modulated by neurotransmitters, bioactive lipids, and anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001; Patel et al., 2001). Thus, the intrinsic properties conferred by these channels are not fixed, and their modulation can have dramatic effects on neuronal excitability. The TASK subgroup of KCNK channels currently comprises five members (Kim and Gnatenco, 2001; Patel and Honore, 2001) that can be further subdivided by sequence homology and functional properties. Included in one group are TASK-2 and TASK- 4/TALK-2 (Reyes et al., 1998; Gray et al., 2000; Decher et al., 2001; Girard et al., 2001), neither of which is strongly expressed in the brain (Girard et al., 2001; Talley et al., 2001). The other group includes TASK-1, TASK-3, and TASK-5. Of these, TASK-5 is distinct in that it has a very restricted CNS distribution (E. M. Talley and D. A. Bayliss, unpublished observations) and, to this point, has not been found to make a functional channel (Kim and Gnatenco, 2001; Vega-Saenz et al., 2001). In contrast, TASK-1 and TASK-3 are widely expressed throughout the brain, with overlapping distributions in many regions (Talley et al., 2000, 2001; Vega-Saenz et al., 2001). Moreover, they present a constellation of functional properties that is unique among all K ϩ channels cloned to date. They generate essentially instantaneous, non-inactivating currents that have a weakly rectifying I–V relationship in asymmetric K ϩ that is predicted by constant field considerations [i.e., “open” or Goldman–Hodgkin– Katz (GHK) rectification]. Furthermore, they are regulated by extracellular pH in the physiological range, with pK for channel inhibition by protons of ϳ 7.4 for TASK-1 and ϳ 6.7 for TASK-3, and they are activated by clinically appropriate concentrations of inhalation anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001). Thus, TASK-1 and TASK-3 represent neuronal “leak” K ϩ channels that are sensitive to both extracellular pH and anesthetics. Understanding the neurobiological consequences that follow from the distinct modulatory potential intrinsic to TASK channels will depend, at least in part, on identifying the neurons that functionally express the channels. Here, we show that serotoner- gic dorsal and caudal raphe neurons express TASK-1 and TASK-3 transcripts and a pH- and anesthetic-sensitive K ϩ con- ductance. In those cells, pH-dependent inhibition of TASK chan- nels may contribute to ventilatory and arousal re fl exes associated with extracellular acidosis; on the other hand, activation of raphe neuronal TASK channels by anesthetics could play a role in their immobilizing and sleep-inducing effects. We determined sites of TASK channel expression in midline structures of the rat brainstem using in situ hybridization with [ 33 P]-labeled antisense RNA probes. Images from these experi- ments are shown in Figure 1, together with control data from sagittal sections of rat brain hybridized with sense and antisense probes (Fig. 1 E – H ), which illustrate the low level of background labeling obtained in these experiments. In fi lm autoradiographs of coronal sections through the RDo, TASK-1 mRNA was de- tected at moderate levels (Fig. 1 A ), whereas TASK-3 expression was found at high levels (Fig. 1 B ). In more caudal brainstem sections, fi lm autoradiographs reveal expression of TASK-1 (Fig. 1 C ) and TASK-3 (Fig. 1 D ) along the midline, in structures corresponding to ROb and RPa. TASK-1 was expressed at much lower levels in raphe neurons than in the hypoglossal motoneu- rons that are apparent in the same sections, whereas expression of TASK-3 was more similar in the two cell groups, although at slightly higher levels in motoneurons. In addition, if one assumes similar hybridization ef fi ciencies for the two probes, it appeared that TASK-3 was expressed at higher levels than TASK-1 in both raphe neurons and motoneurons. The major serotonergic cell groups in the brain are coextensive with midline brainstem raphe nuclei, although a substantial pro- portion of raphe cells are not serotonergic (Jacobs and Azmitia, 1992). To determine whether TASK channel transcripts are ex- pressed in serotonergic raphe neurons, we used nonradioactive in situ hybridization with digoxigenin-labeled RNA probes for TASK channels combined with immunohistochemical localiza- tion of tryptophan hydroxylase, the rate-limiting enzyme in sero- tonin biosynthesis. In control experiments, prominent alkaline phosphatase reaction product was observed in motoneurons from tissue incubated with digoxigenin-labeled TASK channel anti- sense riboprobes but completely absent in control tissue incu- bated with sense probes (Fig. 2). Moreover, the distribution of TASK-expressing brainstem neurons obtained using nonradioac- tive probes conformed to previous in situ hybridization examina- tions (Talley et al., 2000, 2001), again with stronger labeling for TASK-3 than for TASK-1. In addition, the localization of TPH immunoreactivity was exactly as expected for serotonergic neu- rons (i.e., strong labeling in midline raphe nuclei) (Jacobs and Azmitia, 1992). Combined labeling for TASK channel transcripts and TPH in the midbrain dorsal raphe nucleus is shown in Figure 3. Low- power photomicrographs reveal labeling in RDo for TASK-1 (Fig. 3 A ) and TASK-3 mRNA (Fig. 3 E ), with high correspon- dence to the region of TPH immunoreactivity (Fig. 3 B , F ). At higher magni fi cation, numerous individual cells were evident that contain TASK channel transcripts (Fig. 3 C , G ) and TPH immu- noreactivity (Fig. 3 D , H ), and it was clear that, in most cases, TASK mRNA was coexpressed in the TPH-IR neurons (see arrows ), indicating that serotonergic RDo neurons express TASK channel transcripts. Labeling for TASK was seen in a few nonse- rotonergic neurons ( arrowheads ), but TPH-IR cells that did not contain TASK mRNA were encountered only infrequently. Co- localization of TASK channel mRNA in serotonergic neurons was not limited to the dorsal raphe but was also evident along the midline of the caudal brainstem within the ROb and RPa (Fig. 4). High-power photomicrographs show expression of TASK-1 in TPH-IR cells of ROb (Fig. 4 A , B ) and TASK-3 in TPH-IR cells of RPa (Fig. 4 C , D ); it is again clear that hybridization signal for TASK mRNA was found in many TPH-IR cells ( arrows ) and in some apparently nonserotonergic cells ( arrowheads ). To quantify these results, we mapped the distribution of TASK channel transcripts within serotonergic raphe neurons. Sections were examined at levels that contain RDo, RMg, ROb, RPa, and the parapyramidal areas; these maps were used to determine the percentage of TPH - IR neurons in which TASK-1 or TASK-3 transcripts were colocalized (Table 1). Combining data obtained from all raphe nuclei, cell counts revealed that the majority of the TPH-IR cells contained TASK-1 (73%) or TASK-3 (81%) mRNA. A similar percentage of TPH-IR neurons within the RDo contained transcripts for TASK-1 or TASK-3. In the me- dulla, it appeared that a higher percentage of TPH-IR cells contained TASK-3 than TASK-1, although this may partly re fl ect dif fi culties with detection of TASK-1 as a result of its apparently lower expression. TASK-1 and TASK-3 channels generate instantaneous, open- recti fi er K ϩ currents that are sensitive to extracellular pH and to volatile anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001). Given that TASK-1 and TASK-3 transcripts are expressed in serotonergic raphe neurons, we tested whether those cells exhibit pH- and anesthetic-sensitive currents with the properties expected of TASK channels. To this end, whole-cell voltage-clamp recordings were obtained in brainstem slices from neurons in RDo (Figs. 5, 6). All cells included in this study generated an inwardly rectifying K ϩ current in response to bath application of 5-HT, a response characteristic of serotoner- gic raphe neurons (Jacobs and Azmitia, 1992; Penington et al., 1993; Bayliss et al., 1997). In the representative cell of Figure 5 A , this effect is evident as a 5-HT-induced outward shift in current at the holding potential of Ϫ 60 mV. In these 5-HT-responsive, and thus presumably serotonergic RDo ...
Context 2
... resting membrane potential and input resistance of neurons are key determinants of neuronal excitability; these intrinsic prop- erties are determined, in large part, by constitutive activity of K ϩ selective channels. In recent years, a novel gene family of back- ground K ϩ channels has been identified, members of which have characteristics that are distinctly different from most other K ϩ channels and consistent with a role in setting membrane potential and input resistance (e.g., weak voltage dependence, extremely fast kinetics, etc.) (for review, see Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001; Patel et al., 2001). Although these so-called KCNK channels generally show some degree of background activity at normal resting potentials, they are nevertheless also subject to modulation by numerous factors. For example, channel activity is influenced by prevailing physi- cochemical conditions such as temperature, intracellular or extra- cellular pH, oxygen tension, and membrane stretch and can be modulated by neurotransmitters, bioactive lipids, and anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001; Patel et al., 2001). Thus, the intrinsic properties conferred by these channels are not fixed, and their modulation can have dramatic effects on neuronal excitability. The TASK subgroup of KCNK channels currently comprises five members (Kim and Gnatenco, 2001; Patel and Honore, 2001) that can be further subdivided by sequence homology and functional properties. Included in one group are TASK-2 and TASK- 4/TALK-2 (Reyes et al., 1998; Gray et al., 2000; Decher et al., 2001; Girard et al., 2001), neither of which is strongly expressed in the brain (Girard et al., 2001; Talley et al., 2001). The other group includes TASK-1, TASK-3, and TASK-5. Of these, TASK-5 is distinct in that it has a very restricted CNS distribution (E. M. Talley and D. A. Bayliss, unpublished observations) and, to this point, has not been found to make a functional channel (Kim and Gnatenco, 2001; Vega-Saenz et al., 2001). In contrast, TASK-1 and TASK-3 are widely expressed throughout the brain, with overlapping distributions in many regions (Talley et al., 2000, 2001; Vega-Saenz et al., 2001). Moreover, they present a constellation of functional properties that is unique among all K ϩ channels cloned to date. They generate essentially instantaneous, non-inactivating currents that have a weakly rectifying I–V relationship in asymmetric K ϩ that is predicted by constant field considerations [i.e., “open” or Goldman–Hodgkin– Katz (GHK) rectification]. Furthermore, they are regulated by extracellular pH in the physiological range, with pK for channel inhibition by protons of ϳ 7.4 for TASK-1 and ϳ 6.7 for TASK-3, and they are activated by clinically appropriate concentrations of inhalation anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001). Thus, TASK-1 and TASK-3 represent neuronal “leak” K ϩ channels that are sensitive to both extracellular pH and anesthetics. Understanding the neurobiological consequences that follow from the distinct modulatory potential intrinsic to TASK channels will depend, at least in part, on identifying the neurons that functionally express the channels. Here, we show that serotoner- gic dorsal and caudal raphe neurons express TASK-1 and TASK-3 transcripts and a pH- and anesthetic-sensitive K ϩ con- ductance. In those cells, pH-dependent inhibition of TASK chan- nels may contribute to ventilatory and arousal re fl exes associated with extracellular acidosis; on the other hand, activation of raphe neuronal TASK channels by anesthetics could play a role in their immobilizing and sleep-inducing effects. We determined sites of TASK channel expression in midline structures of the rat brainstem using in situ hybridization with [ 33 P]-labeled antisense RNA probes. Images from these experi- ments are shown in Figure 1, together with control data from sagittal sections of rat brain hybridized with sense and antisense probes (Fig. 1 E – H ), which illustrate the low level of background labeling obtained in these experiments. In fi lm autoradiographs of coronal sections through the RDo, TASK-1 mRNA was de- tected at moderate levels (Fig. 1 A ), whereas TASK-3 expression was found at high levels (Fig. 1 B ). In more caudal brainstem sections, fi lm autoradiographs reveal expression of TASK-1 (Fig. 1 C ) and TASK-3 (Fig. 1 D ) along the midline, in structures corresponding to ROb and RPa. TASK-1 was expressed at much lower levels in raphe neurons than in the hypoglossal motoneu- rons that are apparent in the same sections, whereas expression of TASK-3 was more similar in the two cell groups, although at slightly higher levels in motoneurons. In addition, if one assumes similar hybridization ef fi ciencies for the two probes, it appeared that TASK-3 was expressed at higher levels than TASK-1 in both raphe neurons and motoneurons. The major serotonergic cell groups in the brain are coextensive with midline brainstem raphe nuclei, although a substantial pro- portion of raphe cells are not serotonergic (Jacobs and Azmitia, 1992). To determine whether TASK channel transcripts are ex- pressed in serotonergic raphe neurons, we used nonradioactive in situ hybridization with digoxigenin-labeled RNA probes for TASK channels combined with immunohistochemical localiza- tion of tryptophan hydroxylase, the rate-limiting enzyme in sero- tonin biosynthesis. In control experiments, prominent alkaline phosphatase reaction product was observed in motoneurons from tissue incubated with digoxigenin-labeled TASK channel anti- sense riboprobes but completely absent in control tissue incu- bated with sense probes (Fig. 2). Moreover, the distribution of TASK-expressing brainstem neurons obtained using nonradioac- tive probes conformed to previous in situ hybridization examina- tions (Talley et al., 2000, 2001), again with stronger labeling for TASK-3 than for TASK-1. In addition, the localization of TPH immunoreactivity was exactly as expected for serotonergic neu- rons (i.e., strong labeling in midline raphe nuclei) (Jacobs and Azmitia, 1992). Combined labeling for TASK channel transcripts and TPH in the midbrain dorsal raphe nucleus is shown in Figure 3. Low- power photomicrographs reveal labeling in RDo for TASK-1 (Fig. 3 A ) and TASK-3 mRNA (Fig. 3 E ), with high correspon- dence to the region of TPH immunoreactivity (Fig. 3 B , F ). At higher magni fi cation, numerous individual cells were evident that contain TASK channel transcripts (Fig. 3 C , G ) and TPH immu- noreactivity (Fig. 3 D , H ), and it was clear that, in most cases, TASK mRNA was coexpressed in the TPH-IR neurons (see arrows ), indicating that serotonergic RDo neurons express TASK channel transcripts. Labeling for TASK was seen in a few nonse- rotonergic neurons ( arrowheads ), but TPH-IR cells that did not contain TASK mRNA were encountered only infrequently. Co- localization of TASK channel mRNA in serotonergic neurons was not limited to the dorsal raphe but was also evident along the midline of the caudal brainstem within the ROb and RPa (Fig. 4). High-power photomicrographs show expression of TASK-1 in TPH-IR cells of ROb (Fig. 4 A , B ) and TASK-3 in TPH-IR cells of RPa (Fig. 4 C , D ); it is again clear that hybridization signal for TASK mRNA was found in many TPH-IR cells ( arrows ) and in some apparently nonserotonergic cells ( arrowheads ). To quantify these results, we mapped the distribution of TASK channel transcripts within serotonergic raphe neurons. Sections were examined at levels that contain RDo, RMg, ROb, RPa, and the parapyramidal areas; these maps were used to determine the percentage of TPH - IR neurons in which TASK-1 or TASK-3 transcripts were colocalized (Table 1). Combining data obtained from all raphe nuclei, cell counts revealed that the majority of the TPH-IR cells contained TASK-1 (73%) or TASK-3 (81%) mRNA. A similar percentage of TPH-IR neurons within the RDo contained transcripts for TASK-1 or TASK-3. In the me- dulla, it appeared that a higher percentage of TPH-IR cells contained TASK-3 than TASK-1, although this may partly re fl ect dif fi culties with detection of TASK-1 as a result of its apparently lower expression. TASK-1 and TASK-3 channels generate instantaneous, open- recti fi er K ϩ currents that are sensitive to extracellular pH and to volatile anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001). Given that TASK-1 and TASK-3 transcripts are expressed in serotonergic raphe neurons, we tested whether those cells exhibit pH- and anesthetic-sensitive currents with the properties expected of TASK channels. To this end, whole-cell voltage-clamp recordings were obtained in brainstem slices from neurons in RDo (Figs. 5, 6). All cells included in this study generated an inwardly rectifying K ϩ current in response to bath application of 5-HT, a response characteristic of serotoner- gic raphe neurons (Jacobs and ...
Context 3
... resting membrane potential and input resistance of neurons are key determinants of neuronal excitability; these intrinsic prop- erties are determined, in large part, by constitutive activity of K ϩ selective channels. In recent years, a novel gene family of back- ground K ϩ channels has been identified, members of which have characteristics that are distinctly different from most other K ϩ channels and consistent with a role in setting membrane potential and input resistance (e.g., weak voltage dependence, extremely fast kinetics, etc.) (for review, see Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001; Patel et al., 2001). Although these so-called KCNK channels generally show some degree of background activity at normal resting potentials, they are nevertheless also subject to modulation by numerous factors. For example, channel activity is influenced by prevailing physi- cochemical conditions such as temperature, intracellular or extra- cellular pH, oxygen tension, and membrane stretch and can be modulated by neurotransmitters, bioactive lipids, and anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001; Patel et al., 2001). Thus, the intrinsic properties conferred by these channels are not fixed, and their modulation can have dramatic effects on neuronal excitability. The TASK subgroup of KCNK channels currently comprises five members (Kim and Gnatenco, 2001; Patel and Honore, 2001) that can be further subdivided by sequence homology and functional properties. Included in one group are TASK-2 and TASK- 4/TALK-2 (Reyes et al., 1998; Gray et al., 2000; Decher et al., 2001; Girard et al., 2001), neither of which is strongly expressed in the brain (Girard et al., 2001; Talley et al., 2001). The other group includes TASK-1, TASK-3, and TASK-5. Of these, TASK-5 is distinct in that it has a very restricted CNS distribution (E. M. Talley and D. A. Bayliss, unpublished observations) and, to this point, has not been found to make a functional channel (Kim and Gnatenco, 2001; Vega-Saenz et al., 2001). In contrast, TASK-1 and TASK-3 are widely expressed throughout the brain, with overlapping distributions in many regions (Talley et al., 2000, 2001; Vega-Saenz et al., 2001). Moreover, they present a constellation of functional properties that is unique among all K ϩ channels cloned to date. They generate essentially instantaneous, non-inactivating currents that have a weakly rectifying I–V relationship in asymmetric K ϩ that is predicted by constant field considerations [i.e., “open” or Goldman–Hodgkin– Katz (GHK) rectification]. Furthermore, they are regulated by extracellular pH in the physiological range, with pK for channel inhibition by protons of ϳ 7.4 for TASK-1 and ϳ 6.7 for TASK-3, and they are activated by clinically appropriate concentrations of inhalation anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001). Thus, TASK-1 and TASK-3 represent neuronal “leak” K ϩ channels that are sensitive to both extracellular pH and anesthetics. Understanding the neurobiological consequences that follow from the distinct modulatory potential intrinsic to TASK channels will depend, at least in part, on identifying the neurons that functionally express the channels. Here, we show that serotoner- gic dorsal and caudal raphe neurons express TASK-1 and TASK-3 transcripts and a pH- and anesthetic-sensitive K ϩ con- ductance. In those cells, pH-dependent inhibition of TASK chan- nels may contribute to ventilatory and arousal re fl exes associated with extracellular acidosis; on the other hand, activation of raphe neuronal TASK channels by anesthetics could play a role in their immobilizing and sleep-inducing effects. We determined sites of TASK channel expression in midline structures of the rat brainstem using in situ hybridization with [ 33 P]-labeled antisense RNA probes. Images from these experi- ments are shown in Figure 1, together with control data from sagittal sections of rat brain hybridized with sense and antisense probes (Fig. 1 E – H ), which illustrate the low level of background labeling obtained in these experiments. In fi lm autoradiographs of coronal sections through the RDo, TASK-1 mRNA was de- tected at moderate levels (Fig. 1 A ), whereas TASK-3 expression was found at high levels (Fig. 1 B ). In more caudal brainstem sections, fi lm autoradiographs reveal expression of TASK-1 (Fig. 1 C ) and TASK-3 (Fig. 1 D ) along the midline, in structures corresponding to ROb and RPa. TASK-1 was expressed at much lower levels in raphe neurons than in the hypoglossal motoneu- rons that are apparent in the same sections, whereas expression of TASK-3 was more similar in the two cell groups, although at slightly higher levels in motoneurons. In addition, if one assumes similar hybridization ef fi ciencies for the two probes, it appeared that TASK-3 was expressed at higher levels than TASK-1 in both raphe neurons and motoneurons. The major serotonergic cell groups in the brain are coextensive with midline brainstem raphe nuclei, although a substantial pro- portion of raphe cells are not serotonergic (Jacobs and Azmitia, 1992). To determine whether TASK channel transcripts are ex- pressed in serotonergic raphe neurons, we used nonradioactive in situ hybridization with digoxigenin-labeled RNA probes for TASK channels combined with immunohistochemical localiza- tion of tryptophan hydroxylase, the rate-limiting enzyme in sero- tonin biosynthesis. In control experiments, prominent alkaline phosphatase reaction product was observed in motoneurons from tissue incubated with digoxigenin-labeled TASK channel anti- sense riboprobes but completely absent in control tissue incu- bated with sense probes (Fig. 2). Moreover, the distribution of TASK-expressing brainstem neurons obtained using nonradioac- tive probes conformed to previous in situ hybridization examina- tions (Talley et al., 2000, 2001), again with stronger labeling for TASK-3 than for TASK-1. In addition, the localization of TPH immunoreactivity was exactly as expected for serotonergic neu- rons (i.e., strong labeling in midline raphe nuclei) (Jacobs and Azmitia, 1992). Combined labeling for TASK channel transcripts and TPH in the midbrain dorsal raphe nucleus is shown in Figure 3. Low- power photomicrographs reveal labeling in RDo for TASK-1 (Fig. 3 A ) and TASK-3 mRNA (Fig. 3 E ), with high correspon- dence to the region of TPH immunoreactivity (Fig. 3 B , F ). At higher magni fi cation, numerous individual cells were evident that contain TASK channel transcripts (Fig. 3 C , G ) and TPH immu- noreactivity (Fig. 3 D , H ), and it was clear that, in most cases, TASK mRNA was coexpressed in the TPH-IR neurons (see arrows ), indicating that serotonergic RDo neurons express TASK channel transcripts. Labeling for TASK was seen in a few nonse- rotonergic neurons ( arrowheads ), but TPH-IR cells that did not contain TASK mRNA were encountered only infrequently. Co- localization of TASK channel mRNA in serotonergic neurons was not limited to the dorsal raphe but was also evident along the midline of the caudal brainstem within the ROb and RPa (Fig. 4). High-power photomicrographs show expression of TASK-1 in TPH-IR cells of ROb (Fig. 4 A , B ) and TASK-3 in TPH-IR cells of RPa (Fig. 4 C , D ); it is again clear that hybridization signal for TASK mRNA was found in many TPH-IR cells ( arrows ) and in some apparently nonserotonergic cells ( arrowheads ). To quantify these results, we mapped the distribution of TASK channel transcripts within serotonergic raphe neurons. Sections were examined at levels that contain RDo, RMg, ROb, RPa, and the parapyramidal areas; these maps were used to determine the percentage of TPH - IR neurons in which TASK-1 or TASK-3 transcripts were colocalized (Table 1). Combining data obtained from all raphe nuclei, cell counts revealed that the majority of the TPH-IR cells contained TASK-1 (73%) or TASK-3 (81%) mRNA. A similar percentage of TPH-IR neurons within the RDo contained transcripts for TASK-1 or TASK-3. In the me- dulla, it appeared that a higher percentage of TPH-IR cells contained TASK-3 than TASK-1, although this may partly re fl ect dif fi culties with detection of TASK-1 as a result of its apparently lower expression. TASK-1 and TASK-3 channels generate instantaneous, open- recti fi er K ϩ currents that are sensitive to extracellular pH and to volatile anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001). Given that TASK-1 and TASK-3 transcripts are expressed in serotonergic raphe neurons, we tested whether those cells exhibit pH- and anesthetic-sensitive currents with the properties expected of TASK channels. To this end, whole-cell voltage-clamp recordings were obtained in brainstem slices from neurons in RDo (Figs. 5, 6). All cells included in this study generated an inwardly rectifying K ϩ current in response to bath application of 5-HT, a response characteristic of serotoner- gic raphe neurons (Jacobs and Azmitia, 1992; Penington et al., 1993; Bayliss et al., 1997). In the representative cell of Figure 5 A , this effect is evident as a 5-HT-induced outward shift in current at the holding potential of Ϫ 60 mV. In these 5-HT-responsive, and thus presumably serotonergic RDo cells, we characterized currents that were sensitive to bath acidi fi cation and to halothane, ...
Context 4
... resting membrane potential and input resistance of neurons are key determinants of neuronal excitability; these intrinsic prop- erties are determined, in large part, by constitutive activity of K ϩ selective channels. In recent years, a novel gene family of back- ground K ϩ channels has been identified, members of which have characteristics that are distinctly different from most other K ϩ channels and consistent with a role in setting membrane potential and input resistance (e.g., weak voltage dependence, extremely fast kinetics, etc.) (for review, see Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001; Patel et al., 2001). Although these so-called KCNK channels generally show some degree of background activity at normal resting potentials, they are nevertheless also subject to modulation by numerous factors. For example, channel activity is influenced by prevailing physi- cochemical conditions such as temperature, intracellular or extra- cellular pH, oxygen tension, and membrane stretch and can be modulated by neurotransmitters, bioactive lipids, and anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001; Patel et al., 2001). Thus, the intrinsic properties conferred by these channels are not fixed, and their modulation can have dramatic effects on neuronal excitability. The TASK subgroup of KCNK channels currently comprises five members (Kim and Gnatenco, 2001; Patel and Honore, 2001) that can be further subdivided by sequence homology and functional properties. Included in one group are TASK-2 and TASK- 4/TALK-2 (Reyes et al., 1998; Gray et al., 2000; Decher et al., 2001; Girard et al., 2001), neither of which is strongly expressed in the brain (Girard et al., 2001; Talley et al., 2001). The other group includes TASK-1, TASK-3, and TASK-5. Of these, TASK-5 is distinct in that it has a very restricted CNS distribution (E. M. Talley and D. A. Bayliss, unpublished observations) and, to this point, has not been found to make a functional channel (Kim and Gnatenco, 2001; Vega-Saenz et al., 2001). In contrast, TASK-1 and TASK-3 are widely expressed throughout the brain, with overlapping distributions in many regions (Talley et al., 2000, 2001; Vega-Saenz et al., 2001). Moreover, they present a constellation of functional properties that is unique among all K ϩ channels cloned to date. They generate essentially instantaneous, non-inactivating currents that have a weakly rectifying I–V relationship in asymmetric K ϩ that is predicted by constant field considerations [i.e., “open” or Goldman–Hodgkin– Katz (GHK) rectification]. Furthermore, they are regulated by extracellular pH in the physiological range, with pK for channel inhibition by protons of ϳ 7.4 for TASK-1 and ϳ 6.7 for TASK-3, and they are activated by clinically appropriate concentrations of inhalation anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001). Thus, TASK-1 and TASK-3 represent neuronal “leak” K ϩ channels that are sensitive to both extracellular pH and anesthetics. Understanding the neurobiological consequences that follow from the distinct modulatory potential intrinsic to TASK channels will depend, at least in part, on identifying the neurons that functionally express the channels. Here, we show that serotoner- gic dorsal and caudal raphe neurons express TASK-1 and TASK-3 transcripts and a pH- and anesthetic-sensitive K ϩ con- ductance. In those cells, pH-dependent inhibition of TASK chan- nels may contribute to ventilatory and arousal re fl exes associated with extracellular acidosis; on the other hand, activation of raphe neuronal TASK channels by anesthetics could play a role in their immobilizing and sleep-inducing effects. We determined sites of TASK channel expression in midline structures of the rat brainstem using in situ hybridization with [ 33 P]-labeled antisense RNA probes. Images from these experi- ments are shown in Figure 1, together with control data from sagittal sections of rat brain hybridized with sense and antisense probes (Fig. 1 E – H ), which illustrate the low level of background labeling obtained in these experiments. In fi lm autoradiographs of coronal sections through the RDo, TASK-1 mRNA was de- tected at moderate levels (Fig. 1 A ), whereas TASK-3 expression was found at high levels (Fig. 1 B ). In more caudal brainstem sections, fi lm autoradiographs reveal expression of TASK-1 (Fig. 1 C ) and TASK-3 (Fig. 1 D ) along the midline, in structures corresponding to ROb and RPa. TASK-1 was expressed at much lower levels in raphe neurons than in the hypoglossal motoneu- rons that are apparent in the same sections, whereas expression of TASK-3 was more similar in the two cell groups, although at slightly higher levels in motoneurons. In addition, if one assumes similar hybridization ef fi ciencies for the two probes, it appeared that TASK-3 was expressed at higher levels than TASK-1 in both raphe neurons and motoneurons. The major serotonergic cell groups in the brain are coextensive with midline brainstem raphe nuclei, although a substantial pro- portion of raphe cells are not serotonergic (Jacobs and Azmitia, 1992). To determine whether TASK channel transcripts are ex- pressed in serotonergic raphe neurons, we used nonradioactive in situ hybridization with digoxigenin-labeled RNA probes for TASK channels combined with immunohistochemical localiza- tion of tryptophan hydroxylase, the rate-limiting enzyme in sero- tonin biosynthesis. In control experiments, prominent alkaline phosphatase reaction product was observed in motoneurons from tissue incubated with digoxigenin-labeled TASK channel anti- sense riboprobes but completely absent in control tissue incu- bated with sense probes (Fig. 2). Moreover, the distribution of TASK-expressing brainstem neurons obtained using nonradioac- tive probes conformed to previous in situ hybridization examina- tions (Talley et al., 2000, 2001), again with stronger labeling for TASK-3 than for TASK-1. In addition, the localization of TPH immunoreactivity was exactly as expected for serotonergic neu- rons (i.e., strong labeling in midline raphe nuclei) (Jacobs and Azmitia, 1992). Combined labeling for TASK channel transcripts and TPH in the midbrain dorsal raphe nucleus is shown in Figure 3. Low- power photomicrographs reveal labeling in RDo for TASK-1 (Fig. 3 A ) and TASK-3 mRNA (Fig. 3 E ), with high correspon- dence to the region of TPH immunoreactivity (Fig. 3 B , F ). At higher magni fi cation, numerous individual cells were evident that contain TASK channel transcripts (Fig. 3 C , G ) and TPH immu- noreactivity (Fig. 3 D , H ), and it was clear that, in most cases, TASK mRNA was coexpressed in the TPH-IR neurons (see arrows ), indicating that serotonergic RDo neurons express TASK channel transcripts. Labeling for TASK was seen in a few nonse- rotonergic neurons ( arrowheads ), but TPH-IR cells that did not contain TASK mRNA were encountered only infrequently. Co- localization of TASK channel mRNA in serotonergic neurons was not limited to the dorsal raphe but was also evident along the midline of the caudal brainstem within the ROb and RPa (Fig. 4). High-power photomicrographs show expression of TASK-1 in TPH-IR cells of ROb (Fig. 4 A , B ) and TASK-3 in TPH-IR cells of RPa (Fig. 4 C , D ); it is again clear that hybridization signal for TASK mRNA was found in many TPH-IR cells ( arrows ) and in some apparently nonserotonergic cells ( arrowheads ). To quantify these results, we mapped the distribution of TASK channel transcripts within serotonergic raphe neurons. Sections were examined at levels that contain RDo, RMg, ROb, RPa, and the parapyramidal areas; these maps were used to determine the percentage of TPH - IR neurons in which TASK-1 or TASK-3 transcripts were colocalized (Table 1). Combining data obtained from all raphe nuclei, cell counts revealed that the majority of the TPH-IR cells contained TASK-1 (73%) or TASK-3 (81%) mRNA. A similar percentage of TPH-IR neurons within the RDo contained transcripts for TASK-1 or TASK-3. In the me- dulla, it appeared that a higher percentage of TPH-IR cells contained TASK-3 than TASK-1, although this may partly re fl ect dif fi culties with detection of TASK-1 as a result of its apparently lower expression. TASK-1 and TASK-3 channels generate instantaneous, open- recti fi er K ϩ currents that are sensitive to extracellular pH and to volatile anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001). Given that TASK-1 and TASK-3 transcripts are expressed in serotonergic raphe neurons, we tested whether those cells exhibit pH- and anesthetic-sensitive currents with the properties expected of TASK channels. To this end, whole-cell voltage-clamp recordings were obtained in brainstem slices from neurons in RDo (Figs. 5, 6). All cells included in this study generated an inwardly rectifying K ϩ ...
Context 5
... resting membrane potential and input resistance of neurons are key determinants of neuronal excitability; these intrinsic prop- erties are determined, in large part, by constitutive activity of K ϩ selective channels. In recent years, a novel gene family of back- ground K ϩ channels has been identified, members of which have characteristics that are distinctly different from most other K ϩ channels and consistent with a role in setting membrane potential and input resistance (e.g., weak voltage dependence, extremely fast kinetics, etc.) (for review, see Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001; Patel et al., 2001). Although these so-called KCNK channels generally show some degree of background activity at normal resting potentials, they are nevertheless also subject to modulation by numerous factors. For example, channel activity is influenced by prevailing physi- cochemical conditions such as temperature, intracellular or extra- cellular pH, oxygen tension, and membrane stretch and can be modulated by neurotransmitters, bioactive lipids, and anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001; Patel et al., 2001). Thus, the intrinsic properties conferred by these channels are not fixed, and their modulation can have dramatic effects on neuronal excitability. The TASK subgroup of KCNK channels currently comprises five members (Kim and Gnatenco, 2001; Patel and Honore, 2001) that can be further subdivided by sequence homology and functional properties. Included in one group are TASK-2 and TASK- 4/TALK-2 (Reyes et al., 1998; Gray et al., 2000; Decher et al., 2001; Girard et al., 2001), neither of which is strongly expressed in the brain (Girard et al., 2001; Talley et al., 2001). The other group includes TASK-1, TASK-3, and TASK-5. Of these, TASK-5 is distinct in that it has a very restricted CNS distribution (E. M. Talley and D. A. Bayliss, unpublished observations) and, to this point, has not been found to make a functional channel (Kim and Gnatenco, 2001; Vega-Saenz et al., 2001). In contrast, TASK-1 and TASK-3 are widely expressed throughout the brain, with overlapping distributions in many regions (Talley et al., 2000, 2001; Vega-Saenz et al., 2001). Moreover, they present a constellation of functional properties that is unique among all K ϩ channels cloned to date. They generate essentially instantaneous, non-inactivating currents that have a weakly rectifying I–V relationship in asymmetric K ϩ that is predicted by constant field considerations [i.e., “open” or Goldman–Hodgkin– Katz (GHK) rectification]. Furthermore, they are regulated by extracellular pH in the physiological range, with pK for channel inhibition by protons of ϳ 7.4 for TASK-1 and ϳ 6.7 for TASK-3, and they are activated by clinically appropriate concentrations of inhalation anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001). Thus, TASK-1 and TASK-3 represent neuronal “leak” K ϩ channels that are sensitive to both extracellular pH and anesthetics. Understanding the neurobiological consequences that follow from the distinct modulatory potential intrinsic to TASK channels will depend, at least in part, on identifying the neurons that functionally express the channels. Here, we show that serotoner- gic dorsal and caudal raphe neurons express TASK-1 and TASK-3 transcripts and a pH- and anesthetic-sensitive K ϩ con- ductance. In those cells, pH-dependent inhibition of TASK chan- nels may contribute to ventilatory and arousal re fl exes associated with extracellular acidosis; on the other hand, activation of raphe neuronal TASK channels by anesthetics could play a role in their immobilizing and sleep-inducing effects. We determined sites of TASK channel expression in midline structures of the rat brainstem using in situ hybridization with [ 33 P]-labeled antisense RNA probes. Images from these experi- ments are shown in Figure 1, together with control data from sagittal sections of rat brain hybridized with sense and antisense probes (Fig. 1 E – H ), which illustrate the low level of background labeling obtained in these experiments. In fi lm autoradiographs of coronal sections through the RDo, TASK-1 mRNA was de- tected at moderate levels (Fig. 1 A ), whereas TASK-3 expression was found at high levels (Fig. 1 B ). In more caudal brainstem sections, fi lm autoradiographs reveal expression of TASK-1 (Fig. 1 C ) and TASK-3 (Fig. 1 D ) along the midline, in structures corresponding to ROb and RPa. TASK-1 was expressed at much lower levels in raphe neurons than in the hypoglossal motoneu- rons that are apparent in the same sections, whereas expression of TASK-3 was more similar in the two cell groups, although at slightly higher levels in motoneurons. In addition, if one assumes similar hybridization ef fi ciencies for the two probes, it appeared that TASK-3 was expressed at higher levels than TASK-1 in both raphe neurons and motoneurons. The major serotonergic cell groups in the brain are coextensive with midline brainstem raphe nuclei, although a substantial pro- portion of raphe cells are not serotonergic (Jacobs and Azmitia, 1992). To determine whether TASK channel transcripts are ex- pressed in serotonergic raphe neurons, we used nonradioactive in situ hybridization with digoxigenin-labeled RNA probes for TASK channels combined with immunohistochemical localiza- tion of tryptophan hydroxylase, the rate-limiting enzyme in sero- tonin biosynthesis. In control experiments, prominent alkaline phosphatase reaction product was observed in motoneurons from tissue incubated with digoxigenin-labeled TASK channel anti- sense riboprobes but completely absent in control tissue incu- bated with sense probes (Fig. 2). Moreover, the distribution of TASK-expressing brainstem neurons obtained using nonradioac- tive probes conformed to previous in situ hybridization examina- tions (Talley et al., 2000, 2001), again with stronger labeling for TASK-3 than for TASK-1. In addition, the localization of TPH immunoreactivity was exactly as expected for serotonergic neu- rons (i.e., strong labeling in midline raphe nuclei) (Jacobs and Azmitia, 1992). Combined labeling for TASK channel transcripts and TPH in the midbrain dorsal raphe nucleus is shown in Figure 3. Low- power photomicrographs reveal labeling in RDo for TASK-1 (Fig. 3 A ) and TASK-3 mRNA (Fig. 3 E ), with high correspon- dence to the region of TPH immunoreactivity (Fig. 3 B , F ). At higher magni fi cation, numerous individual cells were evident that contain TASK channel transcripts (Fig. 3 C , G ) and TPH immu- noreactivity (Fig. 3 D , H ), and it was clear that, in most cases, TASK mRNA was coexpressed in the TPH-IR neurons (see arrows ), indicating that serotonergic RDo neurons express TASK channel transcripts. Labeling for TASK was seen in a few nonse- rotonergic neurons ( arrowheads ), but TPH-IR cells that did not contain TASK mRNA were encountered only infrequently. Co- localization of TASK channel mRNA in serotonergic neurons was not limited to the dorsal raphe but was also evident along the midline of the caudal brainstem within the ROb and RPa (Fig. 4). High-power photomicrographs show expression of TASK-1 in TPH-IR cells of ROb (Fig. 4 A , B ) and TASK-3 in TPH-IR cells of RPa (Fig. 4 C , D ); it is again clear that hybridization signal for TASK mRNA was found in many TPH-IR cells ( arrows ) and in some apparently nonserotonergic cells ( arrowheads ). To quantify these results, we mapped the distribution of TASK channel transcripts within serotonergic raphe neurons. Sections were examined at levels that contain RDo, RMg, ROb, RPa, and the parapyramidal areas; these maps were used to determine the percentage of TPH - IR neurons in which TASK-1 or TASK-3 transcripts were colocalized (Table 1). Combining data obtained from all raphe nuclei, cell counts revealed that the majority of the TPH-IR cells contained TASK-1 (73%) or TASK-3 (81%) mRNA. A similar percentage of TPH-IR neurons within the RDo contained transcripts for TASK-1 or TASK-3. In the me- dulla, it appeared that a higher percentage of TPH-IR cells contained TASK-3 than TASK-1, although this may partly re fl ect dif fi culties with detection of TASK-1 as a result of its apparently lower expression. TASK-1 and TASK-3 channels generate instantaneous, open- recti fi er K ϩ currents that are sensitive to extracellular pH and to volatile anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001). Given that TASK-1 and TASK-3 transcripts are expressed in serotonergic raphe neurons, we tested whether those cells exhibit pH- and anesthetic-sensitive currents with the properties expected of TASK channels. To this end, whole-cell voltage-clamp recordings were obtained in brainstem slices from neurons in RDo (Figs. 5, 6). All cells included in this study generated an inwardly rectifying K ϩ current in response to bath application of 5-HT, a response characteristic of serotoner- gic raphe neurons (Jacobs and Azmitia, 1992; Penington et al., 1993; Bayliss et al., 1997). In the representative cell of Figure 5 A , this effect is evident as a 5-HT-induced outward shift in current at the holding potential of Ϫ 60 mV. ...
Context 6
... resting membrane potential and input resistance of neurons are key determinants of neuronal excitability; these intrinsic prop- erties are determined, in large part, by constitutive activity of K ϩ selective channels. In recent years, a novel gene family of back- ground K ϩ channels has been identified, members of which have characteristics that are distinctly different from most other K ϩ channels and consistent with a role in setting membrane potential and input resistance (e.g., weak voltage dependence, extremely fast kinetics, etc.) (for review, see Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001; Patel et al., 2001). Although these so-called KCNK channels generally show some degree of background activity at normal resting potentials, they are nevertheless also subject to modulation by numerous factors. For example, channel activity is influenced by prevailing physi- cochemical conditions such as temperature, intracellular or extra- cellular pH, oxygen tension, and membrane stretch and can be modulated by neurotransmitters, bioactive lipids, and anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001; Patel et al., 2001). Thus, the intrinsic properties conferred by these channels are not fixed, and their modulation can have dramatic effects on neuronal excitability. The TASK subgroup of KCNK channels currently comprises five members (Kim and Gnatenco, 2001; Patel and Honore, 2001) that can be further subdivided by sequence homology and functional properties. Included in one group are TASK-2 and TASK- 4/TALK-2 (Reyes et al., 1998; Gray et al., 2000; Decher et al., 2001; Girard et al., 2001), neither of which is strongly expressed in the brain (Girard et al., 2001; Talley et al., 2001). The other group includes TASK-1, TASK-3, and TASK-5. Of these, TASK-5 is distinct in that it has a very restricted CNS distribution (E. M. Talley and D. A. Bayliss, unpublished observations) and, to this point, has not been found to make a functional channel (Kim and Gnatenco, 2001; Vega-Saenz et al., 2001). In contrast, TASK-1 and TASK-3 are widely expressed throughout the brain, with overlapping distributions in many regions (Talley et al., 2000, 2001; Vega-Saenz et al., 2001). Moreover, they present a constellation of functional properties that is unique among all K ϩ channels cloned to date. They generate essentially instantaneous, non-inactivating currents that have a weakly rectifying I–V relationship in asymmetric K ϩ that is predicted by constant field considerations [i.e., “open” or Goldman–Hodgkin– Katz (GHK) rectification]. Furthermore, they are regulated by extracellular pH in the physiological range, with pK for channel inhibition by protons of ϳ 7.4 for TASK-1 and ϳ 6.7 for TASK-3, and they are activated by clinically appropriate concentrations of inhalation anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001). Thus, TASK-1 and TASK-3 represent neuronal “leak” K ϩ channels that are sensitive to both extracellular pH and anesthetics. Understanding the neurobiological consequences that follow from the distinct modulatory potential intrinsic to TASK channels will depend, at least in part, on identifying the neurons that functionally express the channels. Here, we show that serotoner- gic dorsal and caudal raphe neurons express TASK-1 and TASK-3 transcripts and a pH- and anesthetic-sensitive K ϩ con- ductance. In those cells, pH-dependent inhibition of TASK chan- nels may contribute to ventilatory and arousal re fl exes associated with extracellular acidosis; on the other hand, activation of raphe neuronal TASK channels by anesthetics could play a role in their immobilizing and sleep-inducing effects. We determined sites of TASK channel expression in midline structures of the rat brainstem using in situ hybridization with [ 33 P]-labeled antisense RNA probes. Images from these experi- ments are shown in Figure 1, together with control data from sagittal sections of rat brain hybridized with sense and antisense probes (Fig. 1 E – H ), which illustrate the low level of background labeling obtained in these experiments. In fi lm autoradiographs of coronal sections through the RDo, TASK-1 mRNA was de- tected at moderate levels (Fig. 1 A ), whereas TASK-3 expression was found at high levels (Fig. 1 B ). In more caudal brainstem sections, fi lm autoradiographs reveal expression of TASK-1 (Fig. 1 C ) and TASK-3 (Fig. 1 D ) along the midline, in structures corresponding to ROb and RPa. TASK-1 was expressed at much lower levels in raphe neurons than in the hypoglossal motoneu- rons that are apparent in the same sections, whereas expression of TASK-3 was more similar in the two cell groups, although at slightly higher levels in motoneurons. In addition, if one assumes similar hybridization ef fi ciencies for the two probes, it appeared that TASK-3 was expressed at higher levels than TASK-1 in both raphe neurons and motoneurons. The major serotonergic cell groups in the brain are coextensive with midline brainstem raphe nuclei, although a substantial pro- portion of raphe cells are not serotonergic (Jacobs and Azmitia, 1992). To determine whether TASK channel transcripts are ex- pressed in serotonergic raphe neurons, we used nonradioactive in situ hybridization with digoxigenin-labeled RNA probes for TASK channels combined with immunohistochemical localiza- tion of tryptophan hydroxylase, the rate-limiting enzyme in sero- tonin biosynthesis. In control experiments, prominent alkaline phosphatase reaction product was observed in motoneurons from tissue incubated with digoxigenin-labeled TASK channel anti- sense riboprobes but completely absent in control tissue incu- bated with sense probes (Fig. 2). Moreover, the distribution of TASK-expressing brainstem neurons obtained using nonradioac- tive probes conformed to previous in situ hybridization examina- tions (Talley et al., 2000, 2001), again with stronger labeling for TASK-3 than for TASK-1. In addition, the localization of TPH immunoreactivity was exactly as expected for serotonergic neu- rons (i.e., strong labeling in midline raphe nuclei) (Jacobs and Azmitia, 1992). Combined labeling for TASK channel transcripts and TPH in the midbrain dorsal raphe nucleus is shown in Figure 3. Low- power photomicrographs reveal labeling in RDo for TASK-1 (Fig. 3 A ) and TASK-3 mRNA (Fig. 3 E ), with high correspon- dence to the region of TPH immunoreactivity (Fig. 3 B , F ). At higher magni fi cation, numerous individual cells were evident that contain TASK channel transcripts (Fig. 3 C , G ) and TPH immu- noreactivity (Fig. 3 D , H ), and it was clear that, in most cases, TASK mRNA was coexpressed in the TPH-IR neurons (see arrows ), indicating that serotonergic RDo neurons express TASK channel transcripts. Labeling for TASK was seen in a few nonse- rotonergic neurons ( arrowheads ), but TPH-IR cells that did not contain TASK mRNA were encountered only infrequently. Co- localization of TASK channel mRNA in serotonergic neurons was not limited to the dorsal raphe but was also evident along the midline of the caudal brainstem within the ROb and RPa (Fig. 4). High-power photomicrographs show expression of TASK-1 in TPH-IR cells of ROb (Fig. 4 A , B ) and TASK-3 in TPH-IR cells of RPa (Fig. 4 C , D ); it is again clear that hybridization signal for TASK mRNA was found in many TPH-IR cells ( arrows ) and in some apparently nonserotonergic cells ( arrowheads ). To quantify these results, we mapped the distribution of TASK channel transcripts within serotonergic raphe neurons. Sections were examined at levels that contain RDo, RMg, ROb, RPa, and the parapyramidal areas; these maps were used to determine the percentage of TPH - IR neurons in which TASK-1 or TASK-3 transcripts were colocalized (Table 1). Combining data obtained from all raphe nuclei, cell counts revealed that the majority of the TPH-IR cells contained TASK-1 (73%) or TASK-3 (81%) mRNA. A similar percentage of TPH-IR neurons within the RDo contained transcripts for TASK-1 or TASK-3. In the me- dulla, it appeared that a higher percentage of TPH-IR cells contained TASK-3 than TASK-1, although this may partly re fl ect dif fi culties with detection of TASK-1 as a result of its apparently lower expression. TASK-1 and TASK-3 channels generate instantaneous, open- recti fi er K ϩ currents that are sensitive to extracellular pH and to volatile anesthetics (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001). Given that TASK-1 and TASK-3 transcripts are expressed in serotonergic raphe neurons, we tested whether those cells exhibit pH- and anesthetic-sensitive currents with the properties expected of TASK channels. To this end, whole-cell voltage-clamp recordings were obtained in brainstem slices from neurons in RDo (Figs. 5, 6). All cells included in this study generated an inwardly rectifying K ϩ current in response to bath application of 5-HT, a response characteristic of serotoner- gic raphe neurons (Jacobs and Azmitia, 1992; Penington et al., 1993; Bayliss et al., 1997). In the representative cell of Figure 5 A , this effect is evident as a 5-HT-induced outward shift in current at the holding potential of Ϫ 60 mV. In these 5-HT-responsive, and thus presumably serotonergic RDo cells, we characterized currents that were sensitive to bath acidi fi cation and to halothane, following the protocol ...
Citations
... When considering AP's refractory period shown in figure 1, it is difficult not to consider the magnitude of the effect of the size of the ion channels, especially on the K 2P channels. This type of channel has been identified in many sites throughout the nervous system and has been shown to exhibit unique electrophysiological and pharmacological properties [35,37,38]. The main role of these channels is the rapid and efficient passage of K + ions, giving rise to the refractory period between one AP and another. ...
... The difference is that the energy needed for closure is much lower; therefore, the differences between the two occurrences, as shown in figures 3 and 6, are smaller than the one presented in figure 1. Applying this force using a nanoindentor on the neuron would affect different phases of the AP (figures 1-3 and 6). However, the main differences occur in the periods before the AP and at the end of the repolarization phase [38][39][40][41]. Interestingly, the main channels that exhibit high activity during these AP stages are the leak channels, the K 2P . ...
Information transfer in the nervous system is traditionally understood by the transmission of action potentials along neuronal dendrites, with ion channels in the membrane as the basic unit operator for their creation and propagation. We present here a new model for the multiphysics behaviour of ion channels and the action potential dynamics in nervous and other signal-transmitting systems. This model is based on the long-term suppression of an action potential as a response to mechanical input. While other models focus on electrical aspects of the action potential, an increasing body of experiments highlights its electro-mechanical nature and points in particular towards an alteration of the action potential when subjected to a mechanical input. Here, we propose a new phenomenological framework able to capture the mechanical aspect of ion channel dynamics and the resulting effect on the overall electrophysiology of the membrane. The model is introduced here through a set of coupled differential equations that describe the system while agreeing with the general findings of the experiments that support an electro-mechanical model. It also confirms that transient quasi-static mechanical loads reversibly affect the amplitude and rate of change of neuronal action potentials, which are smaller and slower under indentation loading conditions. Changes after the loading release are also reversible, albeit on a different time scale.
... When considering the AP's refractory period shown in Figure 1, it is difficult not to consider the magnitude of the effect of size of the ion channels, especially on the K2P channels. This type of channel has been identified in many sites throughout the nervous system and has been shown to exhibit unique electrophysiological and pharmacological properties [35] [37] [38]. ...
... However, the main differences occur in the periods before the AP and at the end of the repolarization phase [38][39][40][41]. Interestingly, the main channels that exhibit high activity during these AP stages are the leak channels, the K2P channelsthe smallest and most susceptible channels to pressure effects. ...
Information transfer in the nervous system is traditionally understood by means of transmission of action potentials along neuronal dendrites, with ion channels in the membrane as the basic unit operator for their creation and propagation. We present here a new model for the multiphysics behavior of ion channels and the action potential dynamics in nervous and other signal-transmitting systems. This model builds on the notion of long-term memory-like action potential suppression as a response to mechanical input. While other models focus on the electrical aspects of the action potential, an increasing body of experiments has highlighted its electro-mechanical nature, and, in particular, point towards an alteration of the action potential when subjected to a mechanical input. Here, we propose a new phenomenological framework able to capture the mechanical memory-like dynamics of ion channels and the resulting effect on the overall electrophysiology of the membrane. The model is introduced through a set of coupled differential equations that describe the system while agreeing with the general findings of those experiments. It also confirms that transient quasi-static mechanical loads reversibly affect the amplitude and rate of change of the neuronal action potentials, which are smaller and slower upon indentation loading conditions. Changes after the loading release are also reversible albeit in a different time scale.
... Leak K + -currents are often formed by the K2P channel subunits (Patel & Honore, 2001). Among them, the TASK channels are widely expressed in the brainstem and mediate a major component of g Leak , as well as the CO 2 /[H + ] sensitivity, of the respiratory neurones and motoneurones (Bayliss et al. 2001;Washburn et al. 2002;Lesage, 2003;Washburn et al. 2003;Berg et al. 2004;Koizumi et al. 2010;Wang et al. 2013;Bayliss et al. 2015). However, expression of TASK1, TASK2 and TASK3 mRNA was not observed in pF L expiratory neurones, supporting our conclusion that they are not chemosensitive. ...
Active expiration is essential for increasing pulmonary ventilation during high chemical drive (hypercapnia). The lateral parafacial (pFL) region, which contains expiratory neurones, drives abdominal muscles during active expiration in response to hypercapnia. However, the electrophysiological properties and synaptic mechanisms determining the activity of pFL expiratory neurones, as well as the specific conditions for their emergence, are not fully understood. Using whole cell electrophysiology and single cell quantitative RT‐PCR techniques, we describe the intrinsic electrophysiological properties, the phenotype and the respiratory‐related synaptic inputs to the pFL expiratory neurones, as well as the mechanisms for the expression of their expiratory activity under conditions of hypercapnia‐induced active expiration, using in situ preparations of juvenile rats. We also evaluated whether these neurones possess intrinsic CO2/[H⁺] sensitivity and burst generating properties. GABAergic and glycinergic inhibition during inspiration and expiration suppressed the activity of glutamatergic pFL expiratory neurones in normocapnia. In hypercapnia, these neurones escape glycinergic inhibition and generate burst discharges at the end of expiration. Evidence for the contribution of post‐inhibitory rebound, CaV3.2 isoform of T‐type Ca²⁺ channels and intracellular [Ca²⁺] is presented. Neither intrinsic bursting properties, mediated by persistent Na⁺ current, nor CO2/[H⁺] sensitivity or expression of CO2/[H⁺] sensitive ion channels/receptors (TASK or GPR4) were observed. On the other hand, hyperpolarisation‐activated cyclic nucleotide–gated and twik‐related K⁺ leak channels were recorded. Post‐synaptic disinhibition and the intrinsic electrophysiological properties of glutamatergic neurones play important roles in the generation of the expiratory oscillations in the pFL region during hypercapnia in rats. image
Key points
Hypercapnia induces active expiration in rats and the recruitment of a specific population of expiratory neurones in the lateral parafacial (pFL) region.
Post‐synaptic GABAergic and glycinergic inhibition both suppress the activity of glutamatergic pFL neurones during inspiratory and expiratory phases in normocapnia.
Hypercapnia reduces glycinergic inhibition during expiration leading to burst generation by pFL neurones; evidence for a contribution of post‐inhibitory rebound, voltage‐gated Ca²⁺ channels and intracellular [Ca²⁺] is presented.
pFL glutamatergic expiratory neurones are neither intrinsic burster neurones, nor CO2/[H⁺] sensors, and do not express CO2/[H⁺] sensitive ion channels or receptors.
Post‐synaptic disinhibition and the intrinsic electrophysiological properties of glutamatergic neurones both play important roles in the generation of the expiratory oscillations in the pFL region during hypercapnia in rats.
... TASK-1 may be most abundant in the cerebellum, while TASK-3 may be more abundant in the hippocampus, cortex, cerebellum, and certain nuclei, such as the paraventricular nuclei of the thalamus, locus coeruleus, and the dorsal raphe [11]. TASK electrophysiology has been studied in cerebellar granule neurons of rats and serotonergic raphe neurons of mice [69,70]. Knocking out TASK-1 from mice did not alter resting membrane potential of cerebellar granule neurons or the properties of their action potentials [71]. ...
Two-pore-domain potassium (K2P) channels are widespread in the nervous system and play a critical role in maintaining membrane potential in neurons and glia. They have been implicated in many stress-relevant neurological disorders, including pain, sleep disorder, epilepsy, ischemia, and depression. K2P channels give rise to leaky K+ currents, which stabilize cellular membrane potential and regulate cellular excitability. A range of natural and chemical effectors, including temperature, pressure, pH, phospholipids, and intracellular signaling molecules, substantially modulate the activity of K2P channels. In this review, we summarize the contribution of K2P channels to neuronal excitability and to potassium homeostasis in glia. We describe recently discovered functions of K2P channels in glia, such as astrocytic passive conductance and glutamate release, microglial surveillance, and myelin generation by oligodendrocytes. We also discuss the potential role of glial K2P channels in neurological disorders. In the end, we discuss current limitations in K2P channel researches and suggest directions for future studies.
... The pH sensitivities are used to differentiate TASK1 from TASK3. 5 The pK value of TASK1 ranges from pH 7.3 to 7.5 and that of TASK3 ranges from pH 6.5 to 6.7. 4 Our previous study demonstrated that TASK1 and TASK3 are coexpressed with acid-sensing ion channel 1 (ASIC1) in ventrolateral medulla (VLM) and contribute to the central regulation of breathing by coordinating with each other to perceive local pH change. 6 However, the role of TASKs in the respiratory chemoreflex remains to be explored. ...
TWIK‐related acid‐sensitive potassium channels (TASKs)‐like current was recorded in orexin neurons in the lateral hypothalamus (LH), which are essential in respiratory chemoreflex. However, the specific mechanism responsible for the pH‐sensitivity remains elusive. Thus, we hypothesized that TASKs contribute to respiratory chemoreflex. In the present study, we found that TASK1 and TASK3 were expressed in orexin neurons. Blocking TASKs or microinjecting acid artificial cerebrospinal fluid (ACSF) in the LH stimulated breathing. In contrast, alkaline ACSF inhibited breathing, which was attenuated by blocking TASK1. Damage of orexin neurons attenuated the stimulatory effect on respiration caused by microinjection of acid ACSF (at a pH of 6.5) or TASKs antagonists. The orexinA‐positive fiber and orexin type 1 receptor (OX1R) neurons were located in the nucleus tractus solitarius (NTS). The exciting effect of acidosis in the LH on respiration was inhibited by blocking OX1R of the NTS. Taken together, we conclude that orexin neurons sense the extracellular pH change through TASKs and regulate respiration by projecting to the NTS.
... So far there has been no evidence of a direct sensing of CO 2 ; in contrast CO 2 sensing occurs through measuring of the pH-level (Feldman et al., 2003;Jiang et al., 2005). There are many proteins sensitive to changes in pH, for example low resistance gap junctions (Dean et al., 2001Solomon et al., 2001;Solomon and Dean, 2002), inward rectifier potassium channels (Zhu et al., 2000;Jiang et al., 2001), pH-sensitive membrane ion-transport proteins (Putnam, 2001;Wiemann and Bingmann, 2001), and TASK channels (Bayliss et al., 2001;Washburn et al., 2002). Inhibition of TASK-1 by extracellular acidosis leads to depolarization of the membrane and increases excitability of the cell (Wang et al., 2008). ...
Atrial fibrillation (AF) is the most common sustained arrhythmia with a prevalence of up to 4% and an upwards trend due to demographic changes. It is associated with an increase in mortality and stroke incidences. While stroke risk can be significantly reduced through anticoagulant therapy, adequate treatment of other AF related symptoms remains an unmet medical need in many cases. Two main treatment strategies are available: rate control that modulates ventricular heart rate and prevents tachymyopathy as well as rhythm control that aims to restore and sustain sinus rhythm. Rate control can be achieved through drugs or ablation of the atrioventricular node, rendering the patient pacemaker-dependent. For rhythm control electrical cardioversion and pharmacological cardioversion can be used. While electrical cardioversion requires fasting and sedation of the patient, antiarrhythmic drugs have other limitations. Most antiarrhythmic drugs carry a risk for pro-arrhythmic effects and are contraindicated in patients with structural heart diseases. Furthermore, catheter ablation of pulmonary veins can be performed with its risk of intraprocedural complications and varying success. In recent years TASK-1 has been introduced as a new target for AF therapy. Upregulation of TASK-1 in AF patients contributes to prolongation of the action potential duration. In a porcine model of AF, TASK-1 inhibition by gene therapy or pharmacological compounds induced cardioversion to sinus rhythm. The DOxapram Conversion TO Sinus rhythm (DOCTOS)-Trial will reveal whether doxapram, a potent TASK-1 inhibitor, can be used for acute cardioversion of persistent and paroxysmal AF in patients, potentially leading to a new treatment option for AF.
... Medullary raphe neurons (both serotonergic and non-serotonergic) are sensitive to pH [32][33][34][35][36][37] and are highly likely to contribute to respiratory chemosensitivity. Consistent with this role, raphe neuron processes are in close proximity to blood vessels [38]. ...
... royalsocietypublishing.org/journal/rsfs Interface Focus 11: 20200029 retain the ability to close to CO 2 , and this also depends on the carbamylation motif [35]. All present-day amniotes can trace common ancestry to those that survived the Permo-Triassic catastrophe. ...
Breathing is essential to provide the O 2 required for metabolism and to remove its inevitable CO 2 by-product. The rate and depth of breathing is controlled to regulate the excretion of CO 2 to maintain the pH of arterial blood at physiological values. A widespread consensus is that chemosensory cells in the carotid body and brainstem measure blood and tissue pH and adjust the rate of breathing to ensure its homeostatic regulation. In this review, I shall consider the evidence that underlies this consensus and highlight historical data indicating that direct sensing of CO 2 also plays a significant role in the regulation of breathing. I shall then review work from my laboratory that provides a molecular mechanism for the direct detection of CO 2 via the gap junction protein connexin26 (Cx26) and demonstrates the contribution of this mechanism to the chemosensory regulation of breathing. As there are many pathological mutations of Cx26 in humans, I shall discuss which of these alter the CO 2 sensitivity of Cx26 and the extent to which these mutations could affect human breathing. I finish by discussing the evolution of the CO 2 sensitivity of Cx26 and its link to the evolution of amniotes.
... TASK1 and TASK3 function as homo or heteromeric channels [16,47,51], whereas TASK5 appears to be non-functional in cell lines [52]. The expression of TASK channels is determined at the protein or mRNA level in a wide variety of cells, such as brain neurons [9,103], endocrine cells [32,38,45,78], cardiac myocytes [85], and smooth muscle cells [61,83,98]. An increase or decrease in TASK channel activity is associated with a variety of pathological conditions. ...
... On the other hand, single channel analysis of TASK channels in rat [51] and mouse carotid body glomus cells [100] and human atrial myocytes [85] has revealed that the majority of TASK channel activity is contributed by heteromeric TASK1-TASK3 channels. Furthermore, brain neurons, such as hypoglossal motoneurons [9] and serotonergic neurons in the raphe nucleus [103], have also been suggested to express heteromeric TASK1-TASK3 channels. This difference between PC12 and other cells expressing the heteromeric channel may depend on whether the cell expresses p11 or not. ...
TWIK-related acid-sensitive K+ (TASK) channels contribute to the resting membrane potential in various kinds of cells, such as brain neurons, smooth muscle cells, and endocrine cells. Loss-of-function mutations at multiple sites in the KCNK3 gene encoding for TASK1 channels are one of the causes of pulmonary arterial hypertension in humans, whereas a mutation at only one site is reported for TASK3 channels, resulting in a syndrome of mental retardation, hypotonia, and facial dysmorphism. TASK channels are subject to regulation by G protein-coupled receptors (GPCRs). Two mechanisms have been proposed for the GPCR-mediated inhibition of TASK channels: a change in gating and channel endocytosis. The most feasible mechanism for altered gating is diacylglycerol binding to a site in the C-terminus, which is shared by TASK1 and TASK3. The inhibition of channel function by endocytosis requires the presence of a tyrosine residue subjected to phosphorylation by the non-receptor tyrosine kinase Src and a dileucine motif in the C-terminus of TASK1. Therefore, homomeric TASK1 and heteromeric TASK1-TASK3 channels, but not homomeric TASK3, are internalized by GPCR stimulation. Tyrosine phosphorylation by Src is expected to result in a conformational change in the C-terminus, allowing for AP-2, an adaptor protein for clathrin, to bind to the dileucine motif. It is likely that a raft membrane domain is a platform where TASK1 is located and the signaling molecules protein kinase C, Pyk2, and Src are recruited in sequence in response to GPCR stimulation.
... The reduced sensitivity of 5-HT 1A -autoreceptors and α 2adrenoceptors in mice treated with conjugated-TASK3-siRNA would be causing changes in membrane potential of monoamine neurons after partial TASK3 inactivation. Indeed, a lower number of constitutively active TASK3 channels would increase the resting membrane potential [46], leaving the 5-HT and NE neurons less sensitive to the hyperpolarizing actions of somatodendritic autoreceptors. Alternatively, an intra-membrane interaction between G protein-coupled inwardly rectifying potassium (GIRK) channels associated to monoamine autoreceptors and TASK3 channels could be involved. ...
Current pharmacological treatments for major depressive disorder (MDD) are severely compromised by both slow action and limited efficacy. RNAi strategies have been used to evoke antidepressant-like effects faster than classical drugs. Using small interfering RNA (siRNA), we herein show that TASK3 potassium channel knockdown in monoamine neurons induces antidepressant-like responses in mice. TASK3-siRNAs were conjugated to cell-specific ligands, sertraline (Ser) or reboxetine (Reb), to promote their selective accumulation in serotonin (5-HT) and norepinephrine (NE) neurons, respectively, after intranasal delivery. Following neuronal internalization of conjugated TASK3-siRNAs, reduced TASK3 mRNA and protein levels were found in the brainstem 5-HT and NE cell groups. Moreover, Ser-TASK3-siRNA induced robust antidepressant-like behaviors, enhanced the hippocampal plasticity, and potentiated the fluoxetine-induced increase on extracellular 5-HT. Similar responses, yet of lower magnitude, were detected for Reb-TASK3-siRNA. These findings provide substantial support for TASK3 as a potential target, and RNAi-based strategies as a novel therapeutic approach to treat MDD.
... TASKs are also pH-sensitive, which can be by inhibited by acidification and activated by alkalization. Among the three TASK subunits, TASK1 and TASK3 are widely expressed throughout the brain, including in the VLM and raphe nuclei (Washburn et al., 2002(Washburn et al., , 2003. These subunits are sensitive to extracellular protons with different sensitivities and contribute to the regulation of neuronal excitability (Bayliss et al., 2015). ...
... Their pH sensitivities are used to differentiate TASK1 from TASK3 (Hartness et al., 2001;Czirják and Enyedi, 2002;Washburn et al., 2003). TASKs in serotonergic raphe neurons exert pH and anesthetic sensitivity in vitro (Washburn et al., 2002). Inhibition of the TASKs by extracellular acidosis leads to an increased excitability of brainstem respiratory neurons (Duprat et al., 2007). ...
... However, little is known about the coexpression of ASIC1 and TASKs (1 and 3) in the CNS. In the present study, we first examined the expression of TASK1 and TASK3 by localizing TASK1-and TASK3positive cells in the VLM by immunohistochemistry. TASK1 and TASK3 subunits are colocalized with most serotonergic dorsal and caudal raphe neurons and with noradrenergic cells of the LC Washburn et al., 2002). According to the immunofluorescence results, TASK1 and TASK3 coexpression were detected in the VLM, which indicates that heteromeric TASKs may also be involved in central chemoreception. ...
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.
