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The Journal of Neuroscience, February 15, 2002, 22(4):1256-1265
Serotonergic Raphe Neurons Express TASK Channel Transcripts and a
TASK-Like pH- and Halothane-Sensitive K+ Conductance
Christopher P.
Washburn*,
Jay E.
Sirois*,
Edmund M.
Talley,
Patrice G.
Guyenet, and
Douglas A.
Bayliss
Department of Pharmacology, University of Virginia,
Charlottesville, Virginia 22908
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ABSTRACT |
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
electrophysiological approaches to determine that TASK channels are
expressed in serotonergic raphe neurons and to show that they confer a
pH and anesthetic sensitivity to these neurons. By combining in
situ hybridization for TASK-1 or TASK-3 with
immunohistochemical localization of tryptophan hydroxylase, we found
that a majority of serotonergic neurons in both dorsal and caudal raphe
cell groups contain TASK channel transcripts (~70-90%). Whole-cell
voltage-clamp recordings were obtained from raphe cells that responded
to 5-HT in a manner characteristic of serotonergic neurons (i.e., with activation of an inwardly rectifying K+ current). In
those cells, we isolated an endogenous K+
conductance that had properties expected of TASK channel currents; raphe neurons expressed a joint pH- and halothane-sensitive
open-rectifier K+ current. The pH sensitivity of
this current (pK ~7.0) was intermediate between that of TASK-1 and
TASK-3, consistent with functional expression of both channel types.
Together, these data indicate that TASK-1 and TASK-3 are expressed and
functional in serotonergic raphe neurons. The pH-dependent inhibition
of TASK channels in raphe neurons may contribute to ventilatory and
arousal reflexes associated with extracellular acidosis; on the other
hand, activation of raphe neuronal TASK channels by volatile
anesthetics could play a role in their immobilizing and
sedative-hypnotic effects.
Key words:
rat; KCNK; acidosis; anesthetic; hybridization; 5-HT
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INTRODUCTION |
The resting membrane potential and
input resistance of neurons are key determinants of neuronal
excitability; these intrinsic properties are determined, in large part,
by constitutive activity of K+ selective
channels. In recent years, a novel gene family of background K+ channels has been identified, members
of which have characteristics that are distinctly different from most
other K+ 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 physicochemical conditions such as temperature,
intracellular or extracellular 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+
channels cloned to date. They generate essentially instantaneous, non-inactivating currents that have a weakly rectifying I-V
relationship in asymmetric K+ 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 ~7.4 for TASK-1 and ~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+ 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 serotonergic dorsal and caudal raphe neurons express
TASK-1 and TASK-3 transcripts and a pH- and anesthetic-sensitive
K+ conductance. In those cells,
pH-dependent inhibition of TASK channels may contribute to ventilatory
and arousal reflexes 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.
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MATERIALS AND METHODS |
Histochemistry
Preparation of histological specimens. For in
situ hybridization using radiolabeled cRNA probes, adult rats
(Wistar and Sprague Dawley; 200-350 gm) were anesthetized with
ketamine and xylazine (200 and 14 mg/kg, i.m.) and decapitated, and
brains were removed, blocked, and frozen on dry ice. Coronal sections
(10 µm) were cut through the brainstem in a cryostat, thaw-mounted
onto charged slides (Superfrost Plus; Fisher Scientific, Houston, TX),
and stored at  80°C. For non-isotopic hybridization and combined
immunohistochemistry, four adult Sprague Dawley rats were anesthetized
with pentobarbital (60 mg/kg, i.p.) and perfused transcardially with
200 ml of PBS, pH 7.4, followed by 4% phosphate-buffered
paraformaldehyde (0.1 M PB; pH 7.4). The
brainstem was removed, post-fixed overnight with the same fixative at
4°C, and then sectioned in the coronal plane (30 µm) on a
vibratome. Sections were stored at 20°C in a cryoprotectant
solution [30% RNase free sucrose, 30% ethylene glycol, and 1%
polyvinylpyrrolidone (PVP-40) in 100 mM sodium phosphate buffer, pH 7.4] (Watson et al., 1986).
Preparation of hybridization probes. Hybridization probes
were prepared as labeled sense and antisense RNA by in vitro
transcription from cDNA templates by using SP6 or T7 RNA polymerase.
The coding region of rat TASK-1 (obtained from A. T. Gray,
University of California, San Francisco, CA; GenBank accession number
AF031384) (Leonoudakis et al., 1998) was ligated into pcDNA3
(Invitrogen, San Diego, CA) using EcoRI and ApaI
and linearized with HindIII for transcription using SP6 RNA
polymerase to generate antisense probes; the corresponding sense probes
were produced by linearizing with EcoRI and transcribing
with T7 RNA polymerase. The coding region of rat TASK-3 was prepared by
PCR using TASK-3 cDNA as a template (obtained from D. Kim, The Chicago
Medical School, Chicago, IL; GenBank accession number AF192366) (Y. Kim
et al., 2000) and ligated into pcDNA3 using BamHI and
EcoRI. Antisense probes were generated by cutting the
resulting construct with HindIII and transcribing with SP6;
the cognate sense probes were made by linearizing with EcoRV
and transcribing with T7. Transcription was performed in the presence
of [ -33P]UTP for preparation of
radioactive probes; radiolabeled RNA was separated from unincorporated
nucleotides by ethanol precipitation. For nonradioactive in
situ hybridization, the Riboprobe System kit (Promega, Madison,
WI) was used to incorporate digoxigenin-11-UTP (Boehringer Mannheim,
Indianapolis, IN) into in vitro transcripts. Probes were
purified with ProbeQuant G-50 Micro Columns (Amersham Biosciences,
Piscataway, NJ), and the amount of digoxigenin-11-UTP incorporation was
estimated from a dot blot with a dilution series of riboprobe and
control RNA (standards from Boehringer Mannheim). Riboprobes were added
directly to the prehybridization solution (see below) at concentrations
that were similar in spot density to the control RNA (~20-100
ng/µl).
Hybridization with [33P]-labeled
probes. Radioactive in situ hybridization was performed
as described previously (Talley et al., 2001). Slide-mounted sections
were fixed briefly (5 min) in 4% paraformaldehyde, rinsed repeatedly
in PBS, treated successively with glycine (0.2% in PBS) and acetic
anhydride (0.25% in 0.1 M triethanolamine, 0.9%
saline, pH 8), and dehydrated in a graded series of ethanols and
chloroform. Hybridization was performed overnight at 60°C in a buffer
of 50% formamide, 4× SSC (1× SSC: 150 mM NaCl
and 15 mM sodium citrate, pH 7), 1× Denhardt's
solution (0.02% each of Ficoll, polyvinylpyrrolidone, and bovine serum albumin), 10% dextran sulfate, 100 mM DTT, 250 µg/ml yeast tRNA, and 0.5 mg/ml salmon testes DNA. After
hybridization, slides were washed through two changes of 4× SSC,
treated with RNase A (50 µg/ml), washed again through two changes
each of 2× SSC and 0.5× SSC, and finally subjected to a
high-stringency wash of 0.1× SSC. Each of these washes lasted 20-30
min. They were performed at 37°C and included 10 mM sodium thiosulphate, with the exception of the
high-stringency wash, which was performed at 55°C without sodium thiosulphate.
Non-isotopic in situ hybridization combined with
immunohistochemistry. Sections were removed from
cryoprotectant solution, rinsed in sterile saline, and placed
free-floating into prehybridization mixture at room temperature for 30 min and then at 37°C for 1 hr. The prehybridization mixture consisted
of 0.6 M NaCl, 0.1 M
Tris-Cl, pH 7.5, 0.002 M EDTA, 0.05% NaPPi, 0.5 mg/ml yeast total RNA, 0.05 mg/ml yeast tRNA, 1× Denhardt's BSA, 50%
formamide, 10% dextran sulfate, 0.05 mg/ml oligo-dA, 10 µM of the four deoxynucleoside triphosphates,
0.5 mg/ml herring sperm DNA, and 10 mM DTT in an aqueous solution. Digoxigenin-labeled probes were added directly to the
prehybridization solution, and sections were incubated at 55-60°C
for 16-20 hr. Sections were rinsed through decreasing concentrations
of salt solutions, treated with RNase A at 37°C, and then subjected
to a final high-stringency wash (0.1× SSC at 55°C for 60 min).
Sections were processed for immunochemical detection of digoxigenin
with a sheep polyclonal anti-digoxigenin antibody conjugated to
alkaline phosphatase (Boehringer Mannheim). After incubation in a
blocking solution of 10% goat serum for 30 min, sections were
incubated in anti-digoxigenin antibody for 16-20 hr at 4°C. After
rinsing, the alkaline phosphatase was reacted with
nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate,
4-toluidine salt in a foil-wrapped container. The reaction was
monitored periodically under a dissecting microscope and quenched with
three 10 min rinses in 0.1 M Tris and 1 mM
EDTA, pH 8.5.
After in situ hybridization, sections were processed for
immunohistochemical detection of tryptophan hydroxylase (TPH). At the
conclusion of the quenching steps (see above), free-floating sections
were rinsed in Tris-buffered saline, pH 7.4, and incubated with primary
antibody against TPH (1:1000, mouse monoclonal; Sigma, St. Louis, MO)
for 16-20 hr at 4°C in a solution containing 0.1% Triton X-100,
10% goat serum, and 100 mM Tris-buffered saline. Sections were incubated with biotinylated goat-anti-mouse IgG3 (1:200;
Vector Laboratories, Burlingame, CA) and then with avidin-conjugated Cy3 (1:1000; Jackson ImmunoResearch, West Grove, PA), each for 45 min
at room temperature with intervening rinses. Sections were mounted onto
microscope slides, air dried, and coverslipped with VectaShield (Vector Laboratories).
Control experiments. The probes used for TASK channel
in situ hybridization (Sirois et al., 2000; Talley et al.,
2000, 2001) and the antibody used for detection of tryptophan
hydroxylase (Bayliss et al., 1997) have been characterized extensively.
Additional controls performed in the current context included the use
of corresponding sense probes, which gave uniformly low levels of nonspecific (background) labeling (see Figs. 1, 2). The patterns of
labeling for TASK-1 and TASK-3 obtained with radioactive and non-isotopic cRNA probes were identical. In addition, the distribution of TPH immunoreactivity was exactly as expected for serotonergic neurons (i.e., strong labeling in midline raphe nuclei) (Jacobs and
Azmitia, 1992), with no labeling evident in control tissue in which
either the primary or secondary antibody was omitted (data not shown).
Brain mapping, quantification, and imaging. All slides from
isotopic in situ hybridization experiments were exposed to
film (Hyperfilm  -MAX; Amersham Biosciences) for the same length of time (5 d). The resulting autoradiograms were mounted on a light box
and imaged using a video camera (CCD-72; Dage-MTI, Michigan City, IN).
The same camera settings were used to generate images from each of the
different probes. Sections from combined in situ hybridization and immunohistochemistry experiments were photographed with color slide film (Fuji Provia 1600; Fujifilm, Tokyo, Japan) or
black and white film (Kodak TMAX 100; Eastman Kodak, Rochester, NY). A
Nikon (Tokyo, Japan) film scanner at 3000 dpi resolution was used to
digitize images for processing in Adobe Photoshop (Adobe Systems, San
Jose, CA). Locations of TPH-immunoreactive (IR) neurons that contain
TASK-1 or TASK-3 mRNA were mapped, and cell counts were obtained by
using Neurolucida (MicroBrightField, Colchester, VT) and Canvas
software (Deneba Software, Miami, FL) in sections containing raphe
dorsalis (RDo), magnus (RMg), obscurus (ROb), pallidus (RPa), and the
parapyramidal region. Sections were located relative to bregma using
landmarks from the atlas of Paxinos and Watson (1997).
Electrophysiology
General preparation. Whole-cell recordings were
performed in vitro using brainstem slices, essentially as
described previously (Sirois et al., 2000; Talley et al., 2000).
Briefly, rats [Sprague Dawley, postnatal day 1 (P1) to P9 for caudal
raphe and P15-P29 for dorsal raphe] were anesthetized either by
chilling on ice (<P5) or with ketamine-xylazine (as above), and the
brainstem was removed after decapitation. Transverse slices (200 µm)
were cut with a microslicer (DSK-1000; Dosaka) in an ice-cold solution containing (in mM): 260 sucrose, 3 KCl, 5 MgCl2, 1 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, and 1 kynurenic acid. Slices
were incubated for ~1 hr at 37°C in a solution consisting of (in
mM): 130 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose. Slices were maintained in
this incubation solution at room temperature (22-25°C) for periods
up to 6 hr. Cutting and incubation solutions were bubbled continuously
with 95% O2 and 5%
CO2.
Recording. Slices were submerged in a chamber mounted on a
fixed-stage microscope (Axioskop FS; Zeiss, Thornwood, NY), perfused continuously (~2 ml/min), and visualized using differential
interference contrast optics. Dorsal and caudal raphe neurons were
identified visually by their location ventral to the aqueduct or along
the midline, respectively, and characterized electrophysiologically by
their response to serotonin (5-hydroxytryptamine, 5-HT) (Jacobs and
Azmitia, 1992; Penington et al., 1993; Bayliss et al., 1997). Electrical recordings were performed at room temperature in a bath
solution composed of (in mM): 130 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 10 HEPES, and 10 glucose, with pH adjusted using either NaOH or HCl. For voltage-clamp
recordings, bath solutions contained tetrodotoxin (0.75-1
µM); for current-clamp recordings, slices were
perfused in a bath solution containing bicuculline (10 µM), strychnine (30 µM), and CNQX (10 µM).
Patch electrodes were pulled from borosilicate glass capillaries
(Warner Instruments, Hamden, CT) on a two-stage puller (Sutter Instruments, Novato, CA) to a DC resistance of ~3-5 M when filled with internal solution containing (in mM): 120 KCH3SO3, 4 NaCl2, 1 MgCl2, 0.5 CaCl2, 10 HEPES, 10 EGTA, 3 Mg-ATP, and 0.3 GTP-Tris, pH 7.2; electrode tips were coated with Sylgard 184 (Dow
Corning, Midland, MI). 5-HT was applied at bath concentrations ranging from 2 to 100 µM. To augment 5-HT current amplitude in
caudal raphe neurons, the concentration of KCl was increased to 6 mM during 5-HT application in some experiments (Penington
et al., 1993). Osmolarity was maintained by reducing the concentration of NaCl correspondingly. All solutions were bubbled with a room air gas
mixture (21%O2/balance N2)
and perfused at ~2 ml/min; solutions containing halothane were
equilibrated using a calibrated vaporizer (Ohmeda, Helsinki, Finland)
and covered tightly with parafilm to prevent loss of anesthetic to the
atmosphere. Aqueous concentrations of anesthetic solutions were
determined by gas chromatographic analysis from samples collected at
the point of presentation to the preparation (Sirois et al., 2000).
Data acquisition and analysis. Voltage commands were applied
via an Axopatch 200B patch-clamp amplifier and digitized with a
Digidata 1200 analog-to-digital converter (Axon Instruments, Union
City, CA). Cells were held at 60 mV, and membrane currents in
response to a hyperpolarizing ramp (60 to 130 mV) were recorded at
a constant interval (0.1 Hz). Current-voltage relationships were
determined at steady state for various treatment protocols by
hyperpolarizing the cell in 10 mV steps (to 130 mV). Currents were
filtered at 2 kHz with a four-pole, low-pass Bessel filter. Series
resistance was typically <20 M and was compensated by 65-70%. A
liquid junction potential of 10 mV was corrected offline. Data
analysis was performed using the pClamp suite of programs (Axon
Instruments). Statistical analysis of data were performed using
Student's t tests; significance was accepted if
p < 0.05.
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RESULTS |
Expression of TASK channel transcripts in midline brainstem neurons
of rat
We determined sites of TASK channel expression in midline
structures of the rat brainstem using in situ hybridization
with [33P]-labeled antisense RNA probes.
Images from these experiments are shown in Figure
1, together with control data from
sagittal sections of rat brain hybridized with sense and antisense
probes (Fig. 1E-H), which illustrate the low
level of background labeling obtained in these experiments. In film
autoradiographs of coronal sections through the RDo, TASK-1 mRNA was
detected at moderate levels (Fig. 1A), whereas TASK-3
expression was found at high levels (Fig. 1B). In
more caudal brainstem sections, film autoradiographs reveal expression
of TASK-1 (Fig. 1C) and TASK-3 (Fig. 1D)
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 motoneurons 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 efficiencies for the two probes, it appeared that
TASK-3 was expressed at higher levels than TASK-1 in both raphe neurons
and motoneurons.
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Figure 1.
Expression of TASK channel transcripts in midline
raphe cell groups. In situ hybridization was performed
in parallel on sections of rat brainstem using
[33P]-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, film 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 motoneurons, 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 experiments 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 background
labeling. Scale bar: A-D, 1 mm; E-H,
2.5 mm.
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Expression of TASK channel transcripts in brainstem
serotonergic neurons
The major serotonergic cell groups in the brain are coextensive
with midline brainstem raphe nuclei, although a substantial proportion
of raphe cells are not serotonergic (Jacobs and Azmitia, 1992). To
determine whether TASK channel transcripts are expressed in
serotonergic raphe neurons, we used nonradioactive in situ hybridization with digoxigenin-labeled RNA probes for TASK channels combined with immunohistochemical localization of tryptophan
hydroxylase, the rate-limiting enzyme in serotonin biosynthesis. In
control experiments, prominent alkaline phosphatase reaction product
was observed in motoneurons from tissue incubated with
digoxigenin-labeled TASK channel antisense riboprobes but completely
absent in control tissue incubated with sense probes (Fig.
2). Moreover, the distribution of
TASK-expressing brainstem neurons obtained using nonradioactive probes
conformed to previous in situ hybridization examinations (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 neurons (i.e., strong labeling
in midline raphe nuclei) (Jacobs and Azmitia, 1992).
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Figure 2.
Specificity of TASK-3 and TASK-1 labeling using
nonradioactive cRNA probes. In situ hybridization with
digoxigenin-labeled sense and antisense RNA probes for TASK-1 and
TASK-3. Note that high levels of TASK-1 (A) and
TASK-3 (B) expression were detected in
hypoglossal motoneurons using antisense probes; as expected, there was
no labeling in motoneurons in adjacent sections incubated with the
corresponding sense probes (C, D). Scale
bar, 50 µm.
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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. 3A) and TASK-3 mRNA (Fig.
3E), with high correspondence to the region of TPH
immunoreactivity (Fig.
3B,F). At higher
magnification, numerous individual cells were evident that contain TASK
channel transcripts (Fig. 3C,G) and TPH
immunoreactivity (Fig.
3D,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
nonserotonergic neurons (arrowheads), but TPH-IR cells that
did not contain TASK mRNA were encountered only infrequently. Colocalization 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.
4A,B) and TASK-3 in TPH-IR cells of
RPa (Fig. 4C,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).
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Figure 3.
TASK-1 and TASK-3 transcripts are expressed in
TPH-IR neurons of the dorsal raphe. In situ
hybridization with digoxigenin-labeled RNA probes complementary to
TASK-1 and TASK-3 was combined with immunohistochemistry for TPH on
coronal sections of rat midbrain at the level of RDo. Low-powered
bright-field images show labeling for TASK-1 (A)
and TASK-3 (E); fluorescence photomicrographs of
the same sections reveal immunostaining for TPH (B,
F). Note the strong correspondence in
localization of TASK-expressing and TPH-immunoreactive (i.e.,
serotonergic) cells. High-magnification bright-field images show
individual TASK-1-labeled neurons (C) and
TASK-3-labeled neurons (G); immunofluorescence
photomicrographs identify TPH-IR neurons in the same sections
(D, H). It is clear that most
serotonergic neurons express TASK channels (arrows);
some TASK-expressing neurons that are not noticeably TPH-IR were also
apparent (see arrowheads in C and
G). Scale bars: A, 100 µm;
C, 25 µm. Sections in A-D were taken
from a slightly different rostrocaudal level than those in
E-H.
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Figure 4.
TASK-1 and TASK-3 transcripts are colocalized in
serotonergic neurons of the caudal raphe. In situ
hybridization with digoxigenin-labeled RNA probes complementary to
TASK-1 and TASK-3 was combined with immunohistochemistry for TPH on
coronal sections of rat brainstem. A, C,
TASK-1-expressing neurons of ROb and TASK-3-expressing neurons of RPa
are shown in bright-field photomicrographs. B,
D, Immunofluorescence photomicrographs of the
corresponding sections shows the location of TPH-IR neurons. Many
serotonergic neurons contain TASK channel transcripts
(arrows); TASK channel expression was also observed in
TPH-IR negative neurons (arrowheads). Scale bar, 25 µm.
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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 medulla, it appeared that a higher percentage
of TPH-IR cells contained TASK-3 than TASK-1, although this may partly
reflect difficulties with detection of TASK-1 as a result of its
apparently lower expression.
Dorsal raphe neurons express a pH- and
halothane-sensitive K+ conductance with
properties of TASK channels
TASK-1 and TASK-3 channels generate instantaneous, open-rectifier
K+ 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+ current in response to bath application
of 5-HT, a response characteristic of serotonergic raphe neurons
(Jacobs and Azmitia, 1992; Penington et al., 1993; Bayliss et al.,
1997). In the representative cell of Figure 5A, this effect
is evident as a 5-HT-induced outward shift in current at the holding
potential of 60 mV.
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Figure 5.
Dorsal raphe neurons express a pH- and
halothane-sensitive K+ conductance with properties
of TASK channels. Dorsal raphe neurons were recorded under whole-cell
voltage clamp in slices of rat midbrain. A, As expected
for serotonergic dorsal raphe neurons, 5-HT (100 µM)
evoked an outward shift in membrane current at holding potential of
60 mV. Extracellular acidification (from pH 7.3 to pH 6.0) evoked an
inward shift in holding current. After wash into control pH 7.3 solution, halothane (1.25 mM) evoked an outward shift in
current, and, in the presence of halothane, the current shift induced
by reacidifying the bath solution was enhanced in amplitude.
B, Examples of current responses to incrementing voltage
steps ( 10 mV) from 60 mV that were used to construct
I-V relationships (taken at times corresponding to
those indicated in A); note that currents were
essentially completely activated before the end of the capacitive
transient (i.e., they were instantaneous) and were non-inactivating.
C, Averaged I-V relationships of
pH-sensitive currents (diamonds) were derived by
subtracting currents recorded in the acidified bath
(b) from those obtained under control conditions
(a); the pH-sensitive current in the presence of
halothane (squares) was likewise derived by subtracting
currents recorded before (c) and after
(d) bath acidification during continued exposure
to halothane (±SEM; n = 14). D,
Subtracting pH-sensitive currents obtained in the presence of halothane
from those obtained under control conditions yielded the averaged
I-V relationship of the joint halothane- and
pH-sensitive component of dorsal raphe current (±SEM;
n = 14). Those I-V data were well
fitted to the GHK equation, consistent with involvement of an
open-rectifier TASK-like current.
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Figure 6.
The pH- and anesthetic-sensitive current in RDo
has a pH sensitivity intermediate between TASK-1 and TASK-3.
A, Voltage-clamp recording in a serotonergic dorsal
raphe neuron. 5-HT (100 µM) induced an outward shift in
membrane current at 60 mV that is characteristic of serotonergic RDo
neurons. Bath alkalization (from pH 7.3 to pH 8.4) evoked an outward
current under control conditions and in the presence of halothane (1.25 mM). B, Averaged data depicting the
I-V relationship of the current evoked by alkalized
solution in halothane; these data were well fitted with the GHK
constant field equation (±SEM; n = 11).
C, To determine the pH sensitivity of currents in RDo
neurons, the extracellular pH was varied between pH 6.0 and pH 8.4 in
the continued presence of halothane (to enhance the amplitude of
pH-sensitive currents). D, For each cell, currents
measured under these pH conditions were normalized to the maximum and
minimum current, and those normalized data were fitted to a logistic
equation that predicted a pK of ~7.0 for the pH- and
halothane-sensitive dorsal raphe current. This value is intermediate
between those measured for cloned rTASK-1 and rTASK-3 (~7.4 and 6.7, respectively).
|
|
In these 5-HT-responsive, and thus presumably serotonergic RDo cells,
we characterized currents that were sensitive to bath acidification and
to halothane, following the protocol illustrated in Figure
5A. Bath acidification (from pH 7.3 to pH 6.0) produced a
small but reproducible inward shift in holding current. Subsequent application of halothane (1.25 mM) at pH 7.3 evoked an outward current, and, in the continued presence of halothane,
bath acidification again induced an inward current shift, but that
current was now enhanced in amplitude. Every RDo neuron tested with
this protocol showed an enhanced pH-sensitive current in the presence
of halothane (n = 14). Averaged data from these
cells revealed that the inward shift in holding current obtained during
bath acidification was 9.9 ± 1.3 pA under control conditions
and 25.6 ± 2.6 pA in the presence of halothane
(p < 0.001); the outward current induced by
halothane itself was 27.7 ± 3.8 pA.
Current responses to hyperpolarizing voltage steps obtained under these
conditions (at points corresponding to those indicated by
lowercase letters in Fig. 5A) are depicted in
Figure 5B. Note that currents were fully activated within
the time needed to charge the membrane and note also the absence of any
significant time-dependent current relaxation in these records.
Subtracting control currents from those obtained in pH 6.0 yielded the
pH-sensitive current; those subtracted currents were obtained in a
group of RDo neurons to determine the averaged I-V
relationship of the pH-sensitive current (Fig. 5C,
diamonds). This averaged I-V relationship was associated with a negative slope, consistent with inhibition of a
K+ conductance. However, it did not
reverse over the voltage range tested, suggesting that additional
conductances likely contribute to pH-sensitive currents in RDo neurons.
A similar subtraction procedure was used to determine the
I-V relationship of the pH-sensitive current obtained in
the presence of halothane (Fig. 5C, squares); under these conditions, the pH-sensitive current was associated with a
more prominent decrease in conductance. As shown in Figure 5D, subtraction of pH-sensitive currents in the presence of
halothane from those in the absence of halothane yielded the component
of pH-sensitive current that was enhanced by halothane. The
I-V relationship of this subtracted current reversed near
EK and was well fitted by the GHK
constant field equation, indicating that like TASK currents, the joint
halothane- and pH-sensitive current in RDo displayed properties
expected of an open rectifier K+ conductance.
TASK-1 and TASK-3 contribute to the dorsal raphe pH-
and halothane-sensitive K+ current
We found expression of both TASK-1 and TASK-3 channel mRNA in
serotonergic raphe neurons, with apparently higher expression of
TASK-3. Although these TASK channels are each modulated by extracellular pH, they exhibit a different range of sensitivity; TASK-1
is activated strongly by bath alkalization from pH 7.3 (Duprat et al.,
1997; Leonoudakis et al., 1998; Y. Kim et al., 1999; Lopes et al.,
2000; Talley et al., 2000), whereas TASK-3 is nearly fully activated at
pH 7.3, and additional alkalization has little effect on TASK-3
currents (Y. Kim et al., 2000; Rajan et al., 2000; Meadows and Randall,
2001). As shown in Figure 6A, and consistent with a
contribution of TASK-1 to the pH- and anesthetic-sensitive current in
RDo neurons, we found an outward shift in holding current when the pH
of the bath solution was increased from 7.3 to 8.4, both under control
conditions (6.4 ± 3.3 pA) and in the presence of halothane
(16.2 ± 5.8 pA; n = 11). These effects were
associated with an increase in conductance, and I-V
relationships of the current induced by bath alkalization in the
presence of halothane displayed properties consistent with activation
of an open-rectifier K+ conductance (Fig.
6B).
To characterize further the pH sensitivity of the raphe neuronal
current, we varied extracellular pH in the continued presence of
halothane (i.e., under conditions that maximize the contribution of
TASK-like currents to the pH response). As shown in Figure 6C, currents were measured in bath solutions titrated to
four different pH values in the presence of halothane. Currents were normalized, and a pH curve was generated by a logistic fit to the
data (Fig. 6D). The calculated pK for RDo neurons was
~7.0, intermediate between that reported for rTASK-1
(pK ~7.4) (Talley et al., 2000) and rTASK-3 (pK ~6.7) (Y. Kim et
al., 2000), suggesting that both channels contribute to the pH- and
anesthetic-sensitive current in RDo neurons.
The pH- and anesthetic-sensitive current in caudal raphe neurons
has properties of an open-rectifier K+
conductance
In addition to TASK-expressing cells in the RDo, we found that a
high percentage of medullary serotonergic neurons express TASK channel
transcripts. Accumulating evidence indicates that firing activity of
serotonergic caudal raphe neurons is modulated by physiological changes
in pH (Richerson, 1995; Wang and Richerson, 2000; Wang et al., 2001).
Therefore, we tested whether TASK-like currents might also contribute
to the pH sensitivity of medullary raphe neurons.
We characterized pH- and anesthetic-sensitive currents in caudal raphe
neurons using the same protocol used in RDo neurons. Again, our
analysis was limited to those cells that responded to 5-HT with an
increase in an inwardly rectifying K+
current (data not shown) typical of serotonergic neurons (Bayliss et
al., 1997). As exemplified in the records of Figure
7, the response of caudal raphe neurons
to bath acidification and halothane was essentially identical to that
described above for RDo (compare with Fig. 5); acidification produced
an inward shift in holding current that was enhanced in the presence of
halothane. All caudal raphe neurons tested with this protocol displayed
an enhanced pH-sensitive current in the presence of halothane
(n = 7). The current induced by bath acidification
averaged 8.5 ± 2.0 pA under control conditions and 26.0 ± 8.3 pA in the presence of halothane (p < 0.05), which itself induced a 32.5 ± 9.2 pA current in those same
cells. As with RDo neurons, the I-V relationships of pH- and halothane-sensitive current in caudal raphe neurons displayed properties consistent with involvement of an openly rectifying K+ channel, as illustrated in Figure
7B. This suggests that TASK channels contribute also to the
pH sensitivity of serotonergic caudal raphe neurons.
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Figure 7.
Caudal raphe neurons also express a pH- and
halothane-sensitive open-rectifier K+ current.
A, Voltage-clamp recording of membrane current in a
representative caudal raphe cell held at 60 mV. Extracellular
acidification evoked an inward shift in holding current that reversed
when the cell was returned to the control bath solution. Addition of
halothane (1.25 mM) to the bath then induced an outward
current, and, in the continued presence of halothane, the inward shift
in current associated with bath acidification was increased in
amplitude. This cell responded to a subsequent bath application of 5-HT
(2 µM) with an outward current (data not shown)
characteristic of serotonergic caudal raphe neurons. B,
The averaged I-V relationship of the pH- and
halothane-sensitive current (±SEM; n = 7) was
determined from cells tested with this same protocol using the
subtraction procedure described above (see Fig. 5); these data were
well fitted with the GHK equation, indicating that the pH- and
halothane-sensitive current in caudal raphe neurons is an open
rectifier, as expected for TASK channel currents. C,
Current-clamp recording of membrane potential in a spontaneously firing
caudal raphe neuron. As expected for serotonergic raphe neurons, the
cell hyperpolarized by ~10 mV and ceased its spike discharge when
exposed to 5-HT (2 µM). After recovery from 5-HT, a
clinically relevant concentration of halothane (0.35 mM)
reduced firing in this cell; the effect on firing was reversed by bath
acidification (to pH 6.0) and recovered to near control levels after
wash of halothane in a neutral bath solution (pH 7.3). The
current-clamp recording was obtained in a bath solution containing
bicuculline (10 µM), strychnine (10 µM),
and CNQX (10 µM). Arrowhead indicates 60
mV; action potentials were truncated by the chart recorder.
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Halothane depresses raphe neuronal firing at clinically
relevant concentrations
Our data indicate that the halothane-sensitive current in raphe
neurons is sensitive to pH in a physiological range, with a pK
intermediate between that of cloned TASK-1 and TASK-3 channels (Fig.
6), and previous reports indicate that spike discharge in caudal raphe
neurons is steeply sensitive to physiological changes in pH (Richerson,
1995; Wang and Richerson, 2000; Wang et al., 2001). It is also clear
that cloned TASK channels are activated by anesthetics in a clinically
relevant concentration range (Patel et al., 1999; Meadows and Randall,
2001). Therefore, we tested whether the firing behavior of raphe
neurons is modulated by halothane at concentrations appropriate for
clinical anesthesia. As depicted in the records from a 5-HT-responsive
caudal raphe neuron in Figure 7C, a clinically relevant
concentration of halothane (0.35 mM) caused
membrane hyperpolarization and a marked decrease in spontaneous firing.
This effect was reversed by acidifying the bath despite the continued
presence of halothane, as expected if it were mediated by TASK channels
(Sirois et al., 2000). Halothane depressed spike discharge in all
dorsal raphe neurons (n = 4) and in all but one caudal
raphe neuron (n = 10 of 11) tested at this
concentration; on average, halothane decreased firing from 0.5 ± 0.1 to 0.1 ± 0.1 Hz in dorsal raphe neurons
(p < 0.005) and from 0.5 ± 0.1 to
0.3 ± 0.1 Hz in caudal raphe neurons (p < 0.001).
| |
DISCUSSION |
We combined molecular neuroanatomy and cellular electrophysiology
to provide evidence that the pH- and anesthetic-sensitive two-pore-domain K+ channels, TASK-1 and
TASK-3, are functionally expressed in serotonergic raphe neurons. Thus,
by using in situ hybridization, we showed that TASK-1 and
TASK-3 transcripts are expressed in brainstem raphe nuclei; within the
raphe, immunohistochemical detection of tryptophan hydroxylase in
TASK-expressing neurons demonstrated that both TASK-1 and TASK-3 are
localized within the majority of serotonergic dorsal and caudal raphe
neurons (~70-90%). Whole-cell recordings of 5-HT-responsive,
presumably serotonergic, raphe neurons revealed that those cells
express a K+ conductance with properties
diagnostic of TASK channels and distinct from all other channels cloned
to date (i.e., they display an instantaneous open-rectifier
K+ current that is jointly sensitive to pH
and inhalational anesthetics). Because both TASK-1 and TASK-3
transcripts were found in a high percentage of serotonergic raphe
neurons, it is certain that they must be coexpressed in many of those
cells. Accordingly, the pH sensitivity of the raphe neuronal TASK-like
current (pK~7.0) was intermediate between TASK-1 and TASK-3,
consistent with contributions from both channels. These data suggest
that modulation of TASK channels by extracellular protons and by
anesthetics may contribute to neurophysiological mechanisms related to
brain pH homeostasis and to clinical effects of inhalational
anesthetics mediated by serotonergic raphe neurons.
Methodological considerations
Throughout most of these studies, we used pH conditions and
anesthetic concentrations designed to maximize current amplitudes, and
this may have activated additional processes that would not be obtained
under physiologically or clinically appropriate conditions. However, it
is well known that recombinant TASK channels are sharply sensitive to
extracellular pH in the physiological range (Duprat et al., 1997; D. Kim et al., 1998; Leonoudakis et al., 1998; Y. Kim et al., 1999, 2000;
Lopes et al., 2000; Rajan et al., 2000; Talley et al., 2000; Meadows
and Randall, 2001), and, with a pK~7.0 as we described, the
pH-sensitivity of the raphe neuronal TASK-like current indicates that
it will be regulated by physiological changes in pH. Likewise, it is
clear that TASK channels are sensitive to anesthetics at clinically
relevant concentrations (Patel et al., 1999; Meadows and Randall,
2001), and, accordingly, we found that halothane induced a pH-sensitive
decrease in raphe neuronal excitability at clinically appropriate
concentrations. Although there is no evidence to date for the existence
of native heteromeric TASK-1/TASK-3 channels, such as one might expect
from their coexpression in serotonergic raphe neurons, we found that
concatenated TASK-1/TASK-3 constructs express channels that also
respond to extracellular pH and inhalation anesthetics in a
physiologically and clinically appropriate range (Talley and Bayliss,
unpublished observations).
The protocol we used to isolate a time-independent, open-rectifier
TASK-like current took advantage of the joint pH and anesthetic sensitivity of TASK channels. Thus, a contribution of TASK channels to
raphe neuronal pH-sensitive currents was readily revealed in the
presence of halothane, under conditions when TASK currents were
enhanced. In addition, although I-V relationships of the baseline pH current in the absence of anesthetic implied involvement of
multiple conductances, the overall decrease in conductance associated
with that baseline acid-sensitive current suggests a contribution from
inhibition of a background K+ conductance.
Moreover, because TASK channels are known to impart a constitutive
background K+ conductance in all contexts
examined to date (Duprat et al., 1997; D. Kim et al., 1998; Leonoudakis
et al., 1998; Y. Kim et al., 1999, 2000; Lopes et al., 2000; Rajan et
al., 2000; Talley et al., 2000; Meadows and Randall, 2001), it seems
likely that they would also represent a component of the background
pH-sensitive current in raphe neurons. Therefore, our data support the
conclusion that upmodulation and downmodulation of background TASK
channel activity by physiological changes in extracellular pH and by
clinically relevant anesthetic concentrations will contribute to
dynamic regulation of serotonergic neuronal excitability.
Physiological relevance of a pH-sensitive background
K+ current in serotonergic raphe neurons
The physiological relevance of the intrinsic pH sensitivity of
serotonergic raphe neurons, including that conferred by TASK channels,
remains to be established. In this respect, two important neural
reflexes are evoked when brain pH is decreased by hypercapnia: (1)
enhanced respiratory output, which serves as a homeostatic mechanism to
correct changes in brain pH (Nattie, 1999); and (2) behavioral arousal,
which serves a protective function when breathing is excessively
depressed during sleep (Phillipson and Bowes, 1986; Davidson Ward and
Keens, 1992). These are particularly interesting in the current context
because serotonergic raphe neurons are implicated in both chemical
control of respiration (Richerson, 1995; Nattie, 1999) and arousal
mechanisms (Jacobs and Azmitia, 1992).
There is a growing literature implicating medullary serotonergic
neurons of the caudal raphe as a site for respiratory chemosensitivity (for review, see Richerson, 1998; Nattie, 1999); central respiratory chemoreceptors sense changes in brain pH and/or
CO2 and adjust the level of excitatory drive to
brainstem centers that control breathing (and thus, pH and
pCO2) (Nattie, 1999). In vitro
studies, including the present work, indicate that serotonergic caudal raphe neurons are stimulated by changes in pH and/or
CO2 (Richerson, 1995; Wang and Richerson, 2000;
Wang et al., 2001), and, because local acidification of the raphe
region enhances respiratory output in vivo (Bernard et al.,
1996), it has been proposed that these cells may indeed provide a locus
for central respiratory chemoreception (Richerson, 1998; Nattie, 1999).
The ionic mechanisms responsible for pH sensitivity of medullary raphe
neurons have not been determined. A brief report suggests that a
nonselective cationic conductance contributes to pH-sensitive currents
in serotonergic raphe neurons (Tiwari et al., 2000). The present data
are consistent with this previous work insofar as they suggest that
multiple ionic mechanisms are involved with responses to pH in
medullary raphe neurons. In addition, our work implicates TASK channels
specifically as molecular substrates for chemosensitivity of
serotonergic raphe neurons, and by extension, for central respiratory chemosensitivity.
It is also noteworthy in this respect that TASK channels contribute to
a pH-sensitive increase in excitability in locus coeruleus (LC) neurons
(Sirois et al., 2000), another candidate region for central respiratory
chemosensitivity (Nattie, 1999; Ballantyne and Scheid, 2000), and in
carotid body chemoreceptors, which provide afferent input related to
arterial pH to medullary respiratory centers from the periphery
(Buckler et al., 2000). Moreover, a direct pH-dependent increase in
excitability is attributed to TASK channels in respiratory-related
motoneurons (Talley et al., 2000), which ultimately convey central
respiratory drive to the muscles of breathing. Thus, the pH sensitivity
of TASK channels may be exploited at multiple levels to monitor changes
in prevailing acid-base status for purposes of homeostatic regulation
of breathing. Of course, it is likely that other pH-sensitive processes
in different brain regions contribute also to pH-dependent regulation
of breathing (Nattie, 1999).
To date, a role for RDo neurons in regulating respiratory output in
response to changes in brain pH has not been suggested, although it is
known that a population of RDo neurons increase their firing in
response to hypercapnic challenge in vivo (Veasey et al.,
1997). Unlike caudal raphe neurons, which project primarily to
brainstem and spinal neurons, the efferent targets of RDo neurons are
primarily cell groups in rostral brain regions, and, accordingly, RDo
neurons are thought to participate in a number of higher-order functions (Jacobs and Azmitia, 1992). The activity of RDo neurons in vivo is tightly coupled to sleep-wake states, an
observation that has led to the suggestion that these cells have a key
role in behavioral arousal (Jacobs and Azmitia, 1992). Our data
indicate that modulation of TASK channels by extracellular hydrogen
ions in the RDo contributes to pH-dependent effects on excitability in
these neurons, suggesting that this could serve to regulate arousal
state in a pH-dependent manner. Interestingly, the LC is another region
strongly implicated in control of arousal (Aston-Jones and Bloom, 1981;
Aston-Jones et al., 1991), and we found that TASK channels also
contribute to increased LC neuron excitability by extracellular
acidification (Sirois et al., 2000). A pH-dependent influence of TASK
channels on arousal, mediated by RDo and LC neurons, may be
particularly important when ventilation becomes excessively depressed
during sleep and when the ensuing hypercapnia provokes the waking
response necessary to reinstate appropriate levels of ventilation
(Phillipson and Bowes, 1986; Davidson Ward and Keens, 1992). Activation
of this arousal mechanism is responsible, at least in part, for sleep
disturbances associated with obstructive apneas (Horner, 1996), and
failure of this waking reflex in neonates may be a contributing factor
to sudden infant death syndrome (Hunt, 1992; Richerson, 1997).
Clinical relevance of an anesthetic-sensitive background
K+ current in serotonergic raphe neurons
TASK channels are activated by inhalation anesthetics at
concentrations that are entirely appropriate for clinically relevant anesthetic effects (Patel et al., 1999; Meadows and Randall, 2001). Our
results indicate that halothane activates a TASK-like current in
serotonergic raphe neurons, an effect that will drive membrane potential toward more hyperpolarized potentials and decrease activity during anesthesia. At least two main effects always accompany the
clinical state of general anesthesia: immobilization and loss of
consciousness (Eger, 1993). We suggest that anesthetic modulation of
TASK channels in raphe neurons could conceivably contribute to both of
these effects.
First, serotonergic medullary raphe neurons project to brainstem motor
nuclei and the spinal cord ventral horn, in which 5-HT and coexpressed
neuropeptides exert direct facilitatory effects on motoneurons (Rekling
et al., 2000). Inhibition of raphe neurons by anesthetic activation of
TASK channels will decrease release of these raphe neurotransmitters
and, by disfacilitation, depress motoneuronal excitability. These
effects would be supported further by anesthetic activation of TASK
channels in LC (Sirois et al., 2000), which would similarly decrease
facilitating effects of noradrenergic inputs onto motoneurons (Rekling
et al., 2000), and also by direct effects of anesthetics acting on TASK
channels in motoneurons themselves (Sirois et al., 2000). Thus,
activation of TASK channels at a number of central sites may contribute
to immobilizing effects of anesthetics.
As mentioned above, RDo neurons have prominent projections to rostral
brain regions and a highly state-dependent firing pattern that suggests
a role in behavioral arousal (Jacobs and Azmitia, 1992). Thus, a
decrease in activity of these neurons mediated by TASK channels could
contribute to sleep-inducing effects of anesthetics, a role we
suggested previously for the TASK-expressing LC neurons (Sirois et al.,
2000) that are also implicated in control of arousal (Aston-Jones and
Bloom, 1981; Aston-Jones et al., 1991). Of course, direct effects of
anesthetics on other neurons expressing TASK channels (e.g., thalamic
and cortical neurons; Talley et al., 2001) could contribute to these
and other clinically important actions of the drugs.
In summary, we suggest that expression of TASK channels contributes to
the pH sensitivity of serotonergic raphe neurons and thus to
respiratory and arousal reflexes associated with hypercapnia and/or
acidosis (Richerson, 1998; Nattie, 1999). Activation of TASK channels
in these neurons may also contribute to clinically important actions of
volatile anesthetics, such as immobilization and sleep induction.
Furthermore, opposing regulation of TASK channel activity by acidosis
and anesthetics in serotonergic raphe neurons provides a potential
mechanism that could account for the well known diminished ventilatory
responses to respiratory acidosis (i.e., respiratory depression) during
inhalational anesthesia (Pavlin and Hornbein, 1986). These speculations
are potentially testable in genetically altered mice because mutations
that disrupt either pH (Y. Kim et al., 2000; Rajan et al., 2000; Lopes
et al., 2001) or anesthetic modulation (Patel et al., 1999) of
otherwise normal TASK channels have been identified.
| |
FOOTNOTES |
Received Oct. 1, 2001; revised Dec. 4, 2001; accepted Dec. 6, 2001.
*
C.P.W. and J.E.S. contributed equally to this work.
Correspondence should be addressed to Douglas A. Bayliss, Department of
Pharmacology, University of Virginia Health System, P.O. Box 800735, Charlottesville, VA 22908-0735. E-mail: dab3y{at}virginia.edu.
This work was supported by National Institutes of Health Grants
F32HL10271 (J.E.S.), F31MH12091 (E.M.T.), HL28785 (P.G.G.), and NS33583
(D.A.B.). We thank Drs. R. L. Stornetta and A. M. Schreihofer
for help with the dual-labeling procedures and Dr. C. Lynch for helpful
discussions. We also thank Drs. A. T. Gray and D. Kim for gifts of
TASK channel cDNAs.
| |
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ABSTRACT
INTRODUCTION
