Department of Organismal Biology and Anatomy, Committee on Neurobiology, The University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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Thoby-Brisson, Muriel and Jan-Marino Ramirez. Identification of Two Types of Inspiratory Pacemaker Neurons in the Isolated Respiratory Neural Network of Mice. J. Neurophysiol. 86: 104-112, 2001. In the respiratory network of mice, we characterized with the whole cell patch-clamp technique pacemaker properties in neurons discharging in phase with inspiration. The respiratory network was isolated in a transverse brain stem slice containing the pre-Bötzinger complex (PBC), the presumed site for respiratory rhythm generation. After blockade of respiratory network activity with 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX), 18 of 52 inspiratory neurons exhibited endogenous pacemaker activity, which was voltage dependent, could be reset by brief current injections and could be entrained by repetitive stimuli. In the pacemaker group (n = 18), eight neurons generated brief bursts (0.43 ± 0.03 s) at a relatively high frequency (1.05 ± 0.12 Hz) in CNQX. These bursts resembled the bursts that these neurons generated in the intact network during the interval between two inspiratory bursts. Cadmium (200 µM) altered but did not eliminate this bursting activity, while 0.5 µM tetrodotoxin suppressed bursting activity. Another set of pacemaker neurons (10 of 18) generated in CNQX longer bursts (1.57 ± 0.07 s) at a lower frequency (0.35 ± 0.01 Hz). These bursts resembled the inspiratory bursts generated in the intact network in phase with the population activity. This bursting activity was blocked by 50-100 µM cadmium or 0.5 µM tetrodotoxin. We conclude that the respiratory neural network contains pacemaker neurons with two types of bursting properties. The two types of pacemaker activities might have different functions within the respiratory network.
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INTRODUCTION |
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Neural networks underlying
rhythmic activities in vertebrates and invertebrates share similar
rhythm-generating mechanisms (Marder and Calabrese
1996). In many of these networks, pacemaker neurons provide a
basic rhythmic activity. Phase relationship, discharge pattern, and
frequency of the rhythmic output depend critically also on synaptic
interactions with other elements of the network and on neuromodulatory
inputs from neurons located inside and outside of the neural network
(Ayali and Harris-Warrick 1999
; Katz 1995
,
1998
; Thoby-Brisson and Simmers 1998
). Thus the resulting rhythmic activity depends on a combination of membrane and
synaptic properties.
For the neural network that controls breathing in mammals, the
situation seems similar. There is increasing evidence that a
subpopulation of respiratory neurons express pacemaker properties (Johnson et al. 1994; Koshiya and Smith
1999
; Onimaru et al. 1989
; Smith et al.
1991
). Pacemaker neurons seem to be primarily located in the
ventrolateral medulla in a column called the ventral respiratory group
(VRG) (McCrimmon et al. 2000
). A relatively high
concentration of inspiratory pacemaker neurons has been identified in
one particular region of the VRG, the pre-Bötzinger complex
(Koshiya and Smith 1999
; Thoby-Brisson et al.
2000
). In brain stem slices containing the pre-Bötzinger
complex, blockade of synaptic transmission with low calcium
concentrations or blockade of
non-N-methyl-D-aspartate (NMDA) excitatory
transmission (with 6-cyano-7-nitroquinoxaline-2,3-dione: CNQX)
eliminated coordinated respiratory network activity (Funk et al.
1993
), while respiratory pacemaker neurons remained
rhythmically active in an a-synchronized manner (Koshiya and
Smith 1999
). While excitatory connections are critical for the
synchronization of respiratory neuronal activity, inhibitory
connections are essential for establishing the different phases of the
so-called eupneic respiratory rhythm (inspiration, expiration, and
postinspiration) (Lieske et al. 2000
; Ramirez et
al. 1997
; Shao and Feldman 1997
) as well as
shape of inspiratory activity (Lieske et al. 2000
; Ramirez et al. 1997
). In the absence of synaptic
inhibition, expiratory activity ceases, but inspiratory neural activity
remains rhythmic (Ramirez et al. 1997
). Therefore there
is increasing interest to understand how inspiratory activity is
established. An obvious possibility is that inspiratory activity is
generated by inspiratory pacemaker neurons, such as those described by
various authors (Johnson et al. 1994
; Koshiya and
Smith 1999
; Thoby-Brisson et al. 2000
). The
present study aimed at comparing in more detail the intracellularly
recorded activity of inspiratory pacemaker neurons in the presence and
absence of synchronized population activity (i.e., after blockade of
non-NMDA glutamatergic synaptic transmission). Our investigations
revealed that inspiratory pacemaker neurons exhibited two significantly
different discharge and membrane properties. The identification of
heterogeneous pacemaker properties will form an important basis for
understanding how these properties may contribute to different aspects
of the neural control of respiration.
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METHODS |
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Preparation of slices
Experiments were performed on brain stem transverse
slices from 52 male and female mice (CD1, P6-P13). Only one neuron per slice was examined. The preparation technique, which was previously described in detail (Ramirez et al. 1996) will be
summarized here. The animals were decapitated under anesthesia, and the
isolated brain stem was placed in an ice-cold artificial cerebrospinal fluid (ACSF) bubbled with carbogen (95% O2-5%
CO2). The ACSF contained (in mM) 128 NaCl, 3 KCl,
1.5 CaCl2, 1 MgSO4, 24 NaHCO, 0.5 NaH2PO4, and 30 D-glucose, pH of 7.4. The brain stem, glued onto an agar block with the rostral end up, was mounted into a vibratome and serially sliced from rostral to caudal until the rostral boundary of
the pre-Bötzinger complex (PBC) became visible. This area was
recognized by specific landmarks such as the inferior olive (IO), the
nucleus ambiguus (NA), and the hypoglossal nucleus (XII) (Fig.
1A). Five-hundred- to
600-µm-thick slices containing the PBC were transferred into a
recording chamber, continuously perfused with oxygenated CSF and
maintained at a temperature of 29°C. To obtain and maintain
respiratory rhythmic activity the potassium concentration was raised
from 3 to 8 mM over 30 min.
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Recordings
Population activity recordings were obtained with
suction electrodes positioned onto the surface of the PBC. The signals
were amplified 2,000 times, filtered (low-pass, 1.5 kHz; high pass, 250 Hz), rectified, and integrated using an electronic filter (time
constant of 30-50 ms). Integrated population activity from the PBC was
in phase with integrated XII activity (Telgkamp and Ramirez
1999; Thoby-Brisson et al. 2000
). Therefore
extracellularly recorded PBC activity will be used as a marker for
inspiratory population activity (Fig. 1).
Intracellular patch-clamp recordings were obtained from PBC neurons
with the blind-patch technique. The patch electrodes were manufactured
from filamented borosilicate glass tubes (Clarke GC 150TF), filled with
a solution containing (in mM) 140 K-gluconic acid, 1 CaCl2*6H2O, 10 EGTA, 2 MgCl2*6H2O, 4 Na2ATP, 10 HEPES. Inspiratory neurons were
identified according to their anatomical location and their discharge
characteristics. Only inspiratory neurons, active in phase with
population activity, were considered in this study. The discharge
pattern of each cell type was first identified in the cell-attached
mode. Experiments were then performed in the whole cell patch-clamp
mode. The firing pattern of the recorded neurons was not altered after
establishing the whole cell patch-clamp configuration. The membrane
potential values were corrected for liquid junctional potentials (LJPs)
as described by Neher (1992). Recordings were stored
with a personal computer on Axotape (Version 2.0, Axon Instruments) and
analyzed off-line using customized analysis software.
Pharmacology
Drugs were applied at the final concentration of: 20 µM CNQX (Tocris Cookson, Ballwin, MO); 0.5 µM tetrodotoxin (TTX; Sigma, St Louis, MO); 4-200 µM cadmium (Cd; Sigma); 50 µM carbenoxolone (Sigma); and 50 µM DL-2-amino-5-phosphonovaleric acid (AP-5; Sigma). None of these solutions caused a change in the LJP.
Statistical values are given as means ± SE. Significance was assessed with the Student's t-test. Values were assumed to be significant at P < 0.05.
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RESULTS |
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Discrimination between pacemaker and nonpacemaker inspiratory neurons
Pacemaker properties were tested for 52 inspiratory neurons (Figs. 1B and 2, Ai and Bi). After blockade of non-NMDA glutamatergic synapses with 20 µM CNQX, 34 neurons became tonically active or inactive (Fig. 1C). Depolarizing current injections triggered no bursting activity in these neurons (Fig. 1C), suggesting that they did not express intrinsic oscillatory properties. Neurons with these properties were considered nonpacemaker inspiratory neurons.
In contrast, 18 neurons remained rhythmically active in CNQX (Fig.
2). These neurons are referred to as
pacemaker neurons if the remaining rhythmic activity was also reset by
current injection as described in the following text. Three of these
neurons were further isolated by adding to the bathing saline 50 µM
carbenoxolone to block possible electrical synapses and 50 µM AP-5 to
block possible NMDA connections. Rhythmic bursting activity persisted also under such experimental conditions confirming our previous study
(Thoby-Brisson et al. 2000). These pharmacological and
electrophysiological experiments suggest that the rhythmic activity was
generated independent from synaptic excitatory inputs. The pacemaker
neurons had two types of discharge properties in CNQX. Eight pacemaker
neurons expressed short-duration bursts (0.43 ± 0.03 s) at
1.05 ± 0.12 Hz (Fig. 2Aii). They will be called fast
bursters. The 10 other pacemaker neurons expressed bursts with a
relatively longer duration (1.57 ± 0.07 s) at a frequency of
0.35 ± 0.01 Hz (Fig. 2Bii). They will be called slow
bursters.
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The fast burster neurons, which generated short-duration bursts in CNQX, exhibited under control conditions large inspiratory bursts in phase with population activity and brief bursts of action potentials between the inspiratory bursts (Fig. 2Ai). Following the blockade of glutamatergic synaptic transmission with 20 µM CNQX, the large inspiratory bursts disappeared, but the brief rhythmic bursts were still generated (Fig. 2Aii). None of the fast burster neurons (n = 8) exhibited a sag depolarization in response to hyperpolarizing current injections, suggesting that they did not express the Ih current (Fig. 2Aiii).
In contrast, the slow burster neurons, which expressed long-duration bursts in CNQX, were under control conditions rhythmically active in phase with the population activity but did not generate brief bursts in the interval between two inspiratory bursts (Fig. 2Bi). These neurons were often tonically active during this interval. The bursts generated in these neurons after blockade of glutamatergic synaptic transmission with 20 µM CNQX resembled qualitatively the inspiratory bursts under control conditions (compare Fig. 2B, ii to i). All of the slow burster neurons (n = 10) developed a depolarizing sag in response to hyperpolarizing current injections; this is indicative for the Ih current (Fig. 2Biii).
A quantitative analysis was performed to compare the frequency and duration of the two types of bursts expressed in the presence or absence of respiratory network activity. Measurements were obtained for 15 consecutive bursts under control conditions and in the presence of CNQX. The frequency of the brief bursts generated by the fast burster neurons under control conditions (1.22 ± 0.04 Hz) was not significantly altered (P = 0.2) in the presence of CNQX (1.05 ± 0.12 Hz; n = 8). Similarly unaffected (P = 0.1) was the short bursts duration (0.36 ± 0.01 s in control conditions and 0.43 ± 0.03 s in CNQX; n = 8). These data suggest, however, a tendency toward longer and less frequent bursts in CNQX.
The discharge pattern of the slow burster neurons, which generated the relatively longer bursts, revealed significant differences after isolation from the network. The burst frequency generated in CNQX was significantly higher (0.35 ± 0.01 Hz; n = 8) than the frequency of inspiratory bursts generated under control conditions (0.26 ± 0.01 Hz, n = 8). Similarly, the burst duration in the presence of CNQX was significantly higher (1.57 ± 0.07 s; n = 8) than the duration of inspiratory bursts (1.12 ± 0.04 s) generated under control conditions.
Effect of Cd and TTX on the generation of brief bursts
The possible involvement of calcium currents in the generation of
the brief bursts was tested by bath application of Cd, a nonspecific
blocker of calcium currents. Cd was applied initially at a low
concentration of 4 µM and increased progressively up to 200 µM to
block all voltage-dependent calcium currents (Elsen and Ramirez
1998). All the fast burster neurons (Fig.
3A; n = 8),
which generated brief bursts in CNQX, continued to burst in 200 µM Cd
(Fig. 3B). The bursting frequency decreased significantly from 1.05 ± 0.12 Hz in CNQX to 0.57 ± 0.02 Hz in CNQX + Cd.
The burst duration was not significantly altered (0.43 ± 0.03 s in CNQX and 0.44 ± 0.01 s in CNQX + Cd;
P = 0.6). However the application of Cd altered the
shape of the burst. Under control conditions and in CNQX, each burst
consisted of a short train of action potentials without a significant
depolarizing drive potential (Fig. 3A). In the presence of
Cd, each burst was characterized by a significant depolarization (Fig.
3B).
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The additional application of 0.5 µM TTX suppressed rhythmic bursting activity in all examined fast burster neurons (n = 7). TTX abolished initially the depolarizing burst, while action potential generation persisted for approximately 1 min (Fig. 3C, 1 and 2). All tested neurons became inactive after 3 min in 0.5 µM TTX (Fig. 3C3). These experiments suggest that TTX-sensitive currents are involved in the generation of the brief bursts. The persistence of a bursting activity in the presence of high concentrations of Cd suggests that calcium currents are not essential for the pacemaker properties of the fast burster neurons. The change in the shape of the bursts, however, indicates that calcium currents contribute to the bursting properties.
Characterization of the brief bursts using current injections
The voltage dependency, entrainment, and resetting properties of
rhythmic brief bursts were examined in the presence of 20 µM CNQX and
200 µM Cd (n = 4). Prolonged negative or positive current injections decreased or increased the bursting frequency respectively (Fig. 4, A and
B). Hyperpolarizing currents below 1 nA led in all
examined neurons to a complete cessation of rhythmic activity (Fig.
4B).
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Repetitive injections of brief depolarizing or hyperpolarizing stimuli
(2-2.5 nA, 50-100 ms) could entrain the generation of the brief
bursts (4 neurons tested, Fig. 5,
A and B). The entrainment curve in Fig.
5C was obtained by plotting the phase of a current injection
within a bursting period against the bursting period of the neuron's
intrinsic activity. Current injections entrained the bursting
properties over a limited stimulation range. Entrainment was
characterized by a constant phase relationship between current injection and burst generation. Stimulations were not efficient to
entrain the neuron's activity for a stimulation frequency lower than
the neuron's intrinsic bursting frequency irrespective of negative or
positive current pulses. For a range of stimulation frequencies
(0.87-1 Hz) that were higher than the intrinsic frequency of the
neuron, the rhythmic activity of the neuron followed the frequency of
the injected current. For stimulation frequencies higher than 1 Hz the
bursting activity became unlocked.
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The generation of rhythmic brief bursts could also be reset by current
injections (50-100 ms, 2-1.5 nA; Fig.
6A; 4 neurons tested). Short
hyperpolarizing current injections delivered during an on-going burst
resulted in the premature termination of the burst as illustrated on
Fig. 6B (see also Fig. 5B). The triggering of
all-or-none bursts, the premature termination of an on-going burst, the
voltage dependency, the reset, and the entrainment properties are all
indicative that the bursting activity was generated intrinsically in
the neurons and not synaptically driven by a remaining network.
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Effect of Cd and tetrodotoxin on the generation of long-duration bursts
Cd effectively blocked the generation of long-duration bursts in all examined slow burster neurons (n = 7; Fig. 7A). The pronounced depolarizing potentials underlying each burst were greatly reduced in 50 µM Cd, but low-amplitude bursts consisting of trains of eight to nine action potentials were still generated (Fig. 7Aii). The generation of these low-amplitude bursts ceased in 100 µM Cd and all examined neurons became tonically active (n = 7, Fig. 7Aiii). Additional application of 0.5 µM TTX blocked the generation of the remaining action potentials, and the neurons became inactive (Fig. 7Aiv).
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To determine whether the bursting mechanism was also TTX sensitive, 0.5 µM TTX was applied in three preparations before the application of Cd. In all cases, action potentials disappeared initially, followed by the elimination of depolarizing bursts as exemplified on Fig. 7B. This suggests that both Cd- and TTX-sensitive currents contribute to the generation of these long-duration bursts.
Characterization of the long-duration bursts using current injections
The voltage dependency, entrainment, and resetting properties of
the rhythmic long-duration bursts were examined in the presence of 20 µM CNQX. Hyperpolarizing current injections decreased the frequency
of the bursting activity (Fig.
8A). Current injections below
0.5 nA caused in all examined neurons (n = 3) a
cessation of bursting activity (Fig. 8B). Depolarizing
current injections increased the bursting frequency (Fig.
8A), however, only over a limited current range before
bursting activity became erratic (n = 3; Fig.
8B).
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Repetitive stimuli (50-100 ms, 2-2 nA) could entrain the generation
of long-duration bursts, but only over a limited range (Fig.
9, A and B;
n = 3). Stimulation frequencies below the neuron's intrinsic bursting frequency (less than 0.35 Hz) failed to entrain the
neuron's activity. The neuron followed the stimulation frequency for a
stimulation range between 0.35 and 0.5 Hz (Fig. 9C). For higher stimulation frequencies (more than 0.55 Hz), the bursting activity became unlocked. The entrainment properties were analyzed as
described for fast burster neurons.
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A single brief current injection (50-150 ms, 2-2 nA) could reset
the rhythmic long-duration bursts (Fig.
10A, n = 3).
These bursts could be terminated prematurely by negative current
injection (Fig. 10B, n = 3). These results
are all indicative for an intrinsic generation of the rhythmic
long-duration bursts.
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DISCUSSION |
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In the present study we showed the existence of two different types of bursts generated in pacemaker inspiratory neurons within the respiratory neural network. These two types of bursts had distinct physiology and pharmacology (Table 1). One type of bursting activity consisted of brief bursts (0.43 s) occurring at a relatively high frequency (1.05 Hz). The other type of bursts was longer in duration (1.57 s) and occurred at a lower frequency (0.35 Hz). The brief bursts were maintained in high-Cd concentrations but were blocked by TTX. In contrast, the long-duration bursts were blocked by Cd and TTX. The two types of bursts were generated in neurons that exhibited under control conditions different discharge patterns. Fast burster neurons, which generated in CNQX brief rhythmic bursts, expressed under control conditions similar brief bursts between two consecutive inspiratory bursts. Slow burster neurons, which generated in CNQX bursts that resembled the inspiratory bursts, expressed under control conditions only tonic activity between two inspiratory bursts but no brief bursts. Although only one neuron was recorded intracellularly per slice, we believe that the two types of bursters could be found in the same preparation. While searching for pacemaker neurons with the blind-patch technique, we frequently recorded from neurons resembling the two types of bursters in the same slice. However, because these recordings were extracellular these two types of pacemaker were only identified based on their discharge patterns.
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The neurons with these two distinct bursts and discharge patterns
resemble type 1 and type 2 inspiratory neurons described by Rekling and
co-workers (Rekling et al. 1996). According to their
classification, type 1 neurons generate bursting activity during the
inter-inspiratory interval, generate a hyperpolarization, which slowly
repolarizes after an inspiratory burst, and possess no
Ih current. In our study, all the fast
burster neurons generating the brief bursts fulfilled these criteria.
Type 2 neurons, according to the classification by Rekling et
al. (1996)
exhibit only regular spiking and never bursting
activity in the inter-inspiratory interval, present a short-duration
hyperpolarization after an inspiratory burst, and possess an
Ih current. All these criteria were
fulfilled also in the present study for the slow burster neurons that
exhibited the long-duration bursts. However, it must be emphasized that the nomenclature for the two types of inspiratory neurons as used by
Rekling et al. (1996)
cannot be used for the present
study because neurons were characterized in different ACSF solutions. For example, in Rekling's study the calcium concentration was 50%
reduced compared with the concentration of 1.5 mM that we used.
Moreover, our slices were perfused with a CSF containing 8 mM
K+, instead of 5.4 mM, as used by Rekling
et al. (1996)
. In addition, as we described previously
(Thoby-Brisson et al. 2000
), the presence or absence of
the Ih current was not an unambiguous
property to discriminate between the two types of inspiratory neurons.
Some neurons, which were tonically active during the interval between two inspiratory bursts and therefore classified as type 2, had no
Ih current. Moreover, discrimination
between two types of inspiratory neurons based on the discharge pattern
is also ambiguous, as some type 2 neurons were inactive between two
inspiratory bursts and did not show tonic activity. Therefore although
our study clearly indicated that inspiratory pacemaker neurons in the
PBC exhibit two significantly different bursting properties,
uncertainly remains as to whether these two bursting properties are
expressed in two "separate-able" types of inspiratory neurons.
Our results are important since previous models of respiratory rhythm
generation have not considered the possibility of two types of bursting
properties (Butera et al. 1999a,b
; Rekling and Feldman 1998
; Smith 1997
; Smith et al.
1995
, 2000
). However, the demonstration of neurons with
heterogeneous pacemaker properties in a rhythm-generating network is
not new. For example, within the pyloric network of crustaceans,
distinct pharmacologies characterize the two types of pacemaker
activities generated by two types of neurons: the AB and PD neurons
(Bal et al. 1988
; Harris-Warrick et al.
1992
). These neurons also differ in their neurotransmitter content (Marder and Eisen 1984a
), their intrinsic
membrane properties (Bal et al. 1988
; Marder and
Eisen 1984b
), and their response to neuromodulators
(Flamm and Harris-Warrick 1986
; Hooper and Marder
1987
). These differences enable the pyloric network to switch
its configuration in response to neuromodulatory inputs and
state-dependent changes. We expect that the characterization of the two
types of pacemaker activities in the respiratory network may also be an
important step toward understanding the mechanisms underlying rhythm
generation, neuromodulation, or responsiveness to metabolic changes,
such as those occurring during hypoxia. Indeed we have previously
postulated that there might be different types of pacemaker activities
within the respiratory network (Thoby-Brisson and Ramirez
2000
). We described neurons generating bursts that were either
maintained or inactivated during prolonged anoxic conditions. Neurons
that generated brief bursts, as characterized in the present
study, are identical with the neurons, which remained rhythmically
active in anoxia. Neurons that generated the long-duration bursts were
identical with the neurons inactivated during anoxia (Thoby-Brisson and Ramirez 2000
).
Membrane conductances involved in the two types of bursting activity
The membrane conductances responsible for the rhythmic bursting
activity have not been identified experimentally in any respiratory pacemaker neuron. So far, the Ih
current is the only conductance characterized in pacemaker neurons
(Thoby-Brisson et al. 2000). Although this current plays
an important role in modulating the respiratory frequency, it is not
essential for burst initiation since bursting activity in inspiratory
pacemaker neurons persisted after Ih current
blockade (Thoby-Brisson et al. 2000
).
In the present study, we have demonstrated that both types of bursts
were abolished in TTX, suggesting that a TTX-sensitive sodium current
is critical for their generation. In fact, the computational study by
Butera et al. (1999a) has previously postulated that one
type of sodium current, the persistent sodium current, is important for
burst generation. Our results showing that both types of bursting
activity by pacemaker neurons were blocked by TTX are consistent with
this hypothesis given that most known persistent sodium currents are
TTX sensitive (Baker and Bostock 1997
; Crill
1996
; Elson and Selverston 1997
; Ju et
al. 1996
). However, a persistent sodium current is only one
possible mechanism to explain the blockade of bursting properties with
TTX. Alternatively, the bursting activity could also involve a
transient sodium current or a TTX-sensitive resurgent sodium current
(Raman and Bean 1997
). Further voltage-clamp recordings
will be necessary to determine which TTX-sensitive current is involved
in the bursting activity of pacemaker inspiratory neurons.
The application of Cd affected differentially the generation of the two
types of bursts. Although the application of Cd allows no specific
conclusions on the underlying bursting mechanisms, these experiments
demonstrate that the two types of bursts do not share similar
calcium-dependent mechanisms for burst initiation or termination. In
Cd, the brief bursts exhibited a larger amplitude suggesting that the
inward TTX-sensitive current (discussed in the preceding paragraph) is
opposed by a Cd-sensitive outward current. A balance between these two
currents may lead to the brief burst of action potentials, which were
typically characterized by their small amplitude. In contrast, low
concentrations of Cd abolished the long-duration bursts, and the
neurons exhibited only low-amplitude bursts. At higher Cd
concentrations, these bursts were abolished and the neurons became
tonically active. This suggests that an inward calcium current is
involved in the triggering of the long-duration bursts. Further
experiments will be necessary to determine which calcium conductance(s)
is (are) involved in the two bursting activity of inspiratory pacemaker neurons. However, the present study shows that bursting activity in
inspiratory pacemaker neurons may depend on a complex interplay between
calcium and sodium currents. Differences in this interplay may explain
the distinct properties of the two types of inspiratory pacemaker
activities. It is important to note that the exact balance between
calcium and sodium currents may also be species-specific. Our study was
performed in respiratory neurons of mice, which may have different
bursting properties than pacemaker neurons that were recorded in rats
(Johnson et al. 1994; Koshiya and Smith 1999
; Onimaru et al. 1989
; Smith et al.
1991
).
Although it is tempting to speculate whether the two types of pacemaker
activities as characterized in the present study fulfill different
functions in the intact respiratory network, our experiment did not
address this issue. However, the description of two pharmacologically distinct burst mechanisms and the observation that these two mechanisms are associated with two different responses during hypoxia
(Thoby-Brisson and Ramirez 2000) may provide a first
important step for future studies investigating possibly differential
roles in respiratory rhythm generation.
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ACKNOWLEDGMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-60120 to J.-M. Ramirez.
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FOOTNOTES |
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Address for reprint requests: J.-M. Ramirez, Dept. of Organismal Biology and Anatomy, Committee on Neurobiology, The University of Chicago, 1027 E. 57th St., Chicago, IL 60637 (E-mail: Jramire{at}midway.uchicago.edu).
Received 4 December 2000; accepted in final form 15 March 2001.
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REFERENCES |
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