J Appl Physiol 107: 686–695, 2009.
First published May 28, 2009; doi:10.1152/japplphysiol.00023.2009.
Discharge of the hypoglossal nerve cannot distinguish eupnea from gasping,
as defined by phrenic discharge, in the in situ mouse
Walter M. St. John and J. C. Leiter
Department of Physiology, Dartmouth Medical School, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire
Submitted 12 January 2009; accepted in final form 26 May 2009
in vitro
patterns of automatic ventilatory
activity. Eupnea is normal breathing. If eupnea fails, as in
severe hypoxia or ischemia, gasping is recruited and can serve
as a powerful mechanism of autoresuscitation to restart eupnea
(19, 41, 43).
In studies using an in vitro slice of the medulla of mouse,
gasping was reported to be dependent on endogenous serotonin
(5-HT) to be generated (31, 53). This claim had potentially
important implications in that an abnormality in the brain stem
serotonergic system has been reported in victims of the sudden
infant death syndrome (23). Sudden infant death syndrome as
a failure of gasping has long been proposed (13, 15).
We could not confirm a link between endogenous 5-HT and
gasping in two studies using an in situ preparation of the rat
(46, 51) and in another study using an in situ preparation of the
mouse (48). Both of these preparations have an intact pontomedullary brain stem. In all studies, gasping continued un-
EUPNEA AND GASPING ARE TWO
Address for reprint requests and other correspondence: W. M. St. John,
Dartmouth Medical School, Dept. of Physiology, Dartmouth-Hitchcock Medical Center, One Medical Dr., Lebanon, NH 03755 (e-mail: walter.m.
[email protected]).
686
abated following administration of blockers of multiple groups
of receptors for 5-HT. In addition, the study with mice included
a homozygous PET-1 strain in which neurons producing 5-HT
are reduced by 80 –90% compared with those without this
genetic defect (48).
To explain this marked difference between results obtained
in vitro and in situ, we proposed that the type and/or quantity
of neurotransmitters would be greatly reduced in the medullary
slice compared with the entire pontomedullary brain stem (46,
48, 51). Thus, in vitro, any remaining neurotransmitters, such
as 5-HT, would assume a disproportionate importance. Another difference between studies in vitro and in situ concerns
the age of the preparations, with neonates being used in the
former and juvenile or adults in the latter. While maturational
changes do occur in the brain stem respiratory control system,
still eupnea and gasping are clearly distinguishable patterns
from the day of birth in rats (55). Age-dependent changes in
eupnea and gasping in mice have not been analyzed in detail
(but see Ref. 4). A final difference between the in vitro and in
situ preparations is that respiratory rhythms of the in vitro
preparations are defined solely by the discharge of the hypoglossal nerve and/or of massed neuronal activities from the
ventrolateral medulla (17, 29 –32, 53, 56). Based on the rate of
rise of these integrated discharges, respiratory rhythms have
been characterized as “eupnea,” “gasping,” or “sighs” (17).
While gasping persisted in both cranial and phrenic nerves
following a blockade of 5-HT receptors in the rat in situ
preparation, only phrenic discharge was recorded in the mouse
in situ preparation. Thus we could not exclude the possibility
that 5-HT might be critical for generation of gasps in the
hypoglossal nerve of the mouse, as reported for in vitro studies.
As few characterizations and comparisons of activities of
cranial and phrenic nerves during various patterns of automatic
ventilation have been performed in the mouse (4, 22, 25), the
present study was undertaken. In this context, we have studied
respiratory-related activity of the vagus nerve, in addition to
that of the hypoglossal, to define whether any differences
between hypoglossal and phrenic discharges were unique to the
hypoglossal system or were general for activities of cranial vs.
spinal nerves.
METHODS
Experimental Preparations and Procedures
All procedures used in these studies have been approved by the
Institutional Animal Care and Use Committee of Dartmouth College
and Dartmouth Medical School. The Animal Resource Center at
Dartmouth Medical School is an American Association for Accreditation of Laboratory Animal Care approved facility.
PET-1 wild-type mice were used to allow comparison with our
earlier study. Mice were studied 3– 4 wk after birth. The preparation
was as described in that previous study (48). Under deep anesthesia
8750-7587/09 $8.00 Copyright © 2009 the American Physiological Society
http://www. jap.org
Downloaded from http://jap.physiology.org/ by 10.220.32.246 on July 28, 2017
St. John WM, Leiter JC. Discharge of the hypoglossal nerve
cannot distinguish eupnea from gasping, as defined by phrenic discharge, in the in situ mouse. J Appl Physiol 107: 686 –695, 2009. First
published May 28, 2009; doi:10.1152/japplphysiol.00023.2009.—If
normal, eupneic breathing fails, gasping is recruited. Serotonin was
proposed as essential for gasping, based on findings using an in vitro
mouse preparation. This preparation generates rhythmic activities of
the hypoglossal nerve that are considered to be akin to both eupnea
and gasping. In previous studies, gasping of in situ rat and mouse
preparations continued unabated following blockers of receptors for
serotonin. However, hypoglossal activity was not recorded in the
mouse, and we hypothesized that its discharge during gasping might
be dependent on serotonin. In the in situ mouse preparation, hypoglossal discharge had varying and inconsistent patterns during eupnea,
discharging concomitant with the phrenic burst, at varying intervals
between phrenic bursts, or was silent in some respiratory cycles. In
eupnea, phrenic discharge was incrementing, whereas hypoglossal
discharge was decrementing in 15 of 20 preparations. During ischemia-induced gasping, peak phrenic height was reached at 205 ⫾ 17
ms, compared with 282 ⫾ 27.9 ms after the start of the eupneic burst
(P ⬍ 0.002). In contrast, rates of rise of hypoglossal discharge in
gasping (peak at 233 ⫾ 25 ms) and eupnea (peak at 199 ⫾ 19.2 ms)
were the same. The uncoupling of hypoglossal from phrenic discharge
in eupnea was exacerbated by methysergide, an antagonist of serotonin receptors. These findings demonstrate that hypoglossal discharge
alone cannot distinguish eupnea from gasping nor, in eupnea, can
hypoglossal activity be used to differentiate neural inspiration from
expiration. These findings have significant negative implications for
conclusions drawn from the in vitro medullary slice of mouse.
EUPNEA AND GASPING IN MICE
687
with enfluorane, mice were bisected caudal to the diaphragm and
immersed in ice-cold mock cerebrospinal fluid. They were immediately decerebrated at a precollicular level, and the phrenic and hypoglossal or vagus nerves were sectioned. The left ventricle was cannulated, and the preparation perfused. The constituents of the perfusate were as described previously (48). The temperature of the
perfusate was 31°C at the ventricle, and it was equilibrated with a gas
mixture of 95% O2-5% CO2. Gallamine triethiodide was added to the
perfusate to block neuromuscular transmission.
Activities of the various nerves were recorded with bipolar suction
or hook electrodes, amplified, and filtered (0.6 – 6.0 kHz). Recordings
were obtained continuously after rhythmic activities began. In some
studies, methysergide was added to the perfusate to block multiple
receptors for 5-HT. Methysergide is a mixed antagonist of 5-HT 1, 2,
4, 5, 6, and 7 receptors, as well as a weak agonist of 5-HT1 receptors
(46, 48, 51). At variable periods after the commencement of recordings, but a minimum of 10 min after methysergide, perfusion was
terminated for 40 s to produce ischemia and alter the pattern of
ventilatory activity to gasping.
Eupnea and gasping were distinguished, as in previous studies,
from the rate of rise of integrated phrenic activity (11, 41– 43, 49, 51).
During both eupnea and gasping, integrated phrenic activity was
analyzed as to the duration of the burst (neural inspiratory time),
period between bursts (expiratory time), and peak height. Integrated
hypoglossal and vagal discharges typically had a burst that approximated that of neural inspiration; the duration of this burst was defined,
as were the peak heights during this phase. To compare the rates of
rise of inspiratory activity for activities of the various nerves, we
defined the time after onset for each burst to reach a peak integrated
height.
For recordings in eupnea, activities during 20 respiratory cycles
were analyzed. These cycles were taken a minimum of 20 min after
the rhythmic discharges commenced in the preparation. In preparations that received methysergide, a second group of 20 cycles was
analyzed, commencing 10 min after the administration of the drug.
Data for gasping were taken from a single trial with ischemia. Hence,
in preparations that received methysergide, ischemia was only induced a minimum of 10 min after the drug had been administered. In
addition to those same variables, noted above for eupnea, in gasping,
we defined the time from the termination of perfusion, considered as
the onset of ischemia, to the first gasp, and the time after the
recommencement of perfusion to the first rhythmic burst of activity of
each nerve. Finally, as an index of the variability of phrenic and
hypoglossal discharge during the respiratory cycle, we computed the
coefficient of variance. This computation was performed for the
duration of phrenic burst and its rate of rise during approximately 20
respiratory cycles of eupnea and a minimum of four cycles of gasping.
This coefficient of variance, which is the standard deviation of the
measurement divided by the mean, was compared with the comparable coefficient of variance computed for hypoglossal discharge during
the same respiratory cycles. The significance of the difference between the phrenic and hypoglossal values was assessed by a paired
t-test.
Statistical Evaluations of Data
Comparisons were made by paired or unpaired t-tests. Probabilities
less than 0.05 were considered as significant.
RESULTS
Comparison of Phrenic and Hypoglossal Activities
in Eupnea and Gasping
Eupnea. Activities of both nerves were recorded during
eupnea in 20 preparations (Fig. 1). As described previously,
J Appl Physiol • VOL
Fig. 1. Patterns of discharge of the phrenic nerve and hypoglossal nerve during
eupnea (E). A and B are from two different preparations and shown integrated
activities of the nerves (兰Phr, phrenic; 兰Hyp, hypoglossal). Note the variability
of hypoglossal discharge between preparations and between respiratory cycles
in a single preparation. Small, high-frequency oscillations, which are visible on
兰Phr activity, represent the electrocardiogram.
integrated phrenic discharge had an incrementing rise to reach
a peak height after most of the phrenic burst was finished
(64.7 ⫾ 2.1% of inspiratory time, equivalent to 282 ⫾ 27.9 ms
after the start of the burst). Phrenic discharge was compartmentalized to periodic bursts that defined neural inspiration.
Hypoglossal discharge differed between preparations and
was variable even within the same preparation (Fig. 1). In all
20 preparations, the hypoglossal nerve had a burst of activity
that overlapped with that of the phrenic discharge in neural
inspiration. These bursts of activity started at the same time
(Fig. 1A) or before (Fig. 1B) the onset of the phrenic burst. In
all preparations, the hypoglossal nerve also had bursts during
neural expiration. These bursts could occur in early neural
expiration (Fig. 1, A and B) or late neural expiration (Fig. 1B,
second cycle), or large bursts could occur occasionally during
neural expiration (Figs. 2 and 3). As is evident from Figs. 1–3,
the presence and magnitude of expiratory bursts were unpredictable.
For cycles in which hypoglossal discharge was present
during neural inspiration, the duration of its burst (357 ⫾ 31.3
ms) was not significantly different from that of the phrenic
burst (388 ⫾ 26.0 ms). However, the rate of rise of hypoglossal
discharge was significantly greater than that of the phrenic. For
all 20 preparations, integrated hypoglossal discharge reached a
peak level of 199 ⫾ 19.2 ms after the start of its burst, whereas
the equivalent time for phrenic discharge was 282 ⫾ 27.9 ms
(P ⬍ 0.01 compared with hypoglossal discharge). Of the 20
preparations, in only 5 was the rate of rise of hypoglossal less
than phrenic (e.g., Fig. 1B). For these five preparations, peak
hypoglossal discharge was attained in 297 ⫾ 29 ms and peak
phrenic in 219 ⫾ 23 ms.
The greater variability of hypoglossal compared with
phrenic activity was confirmed by a greater coefficient of
variance for the duration of the hypoglossal burst (0.18) compared with that of the phrenic (0.14; P ⬍ 0.01). Also signifi-
107 • SEPTEMBER 2009 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.32.246 on July 28, 2017
Variable of Neural Activities
688
EUPNEA AND GASPING IN MICE
Fig. 2. Alterations in 兰Phr and 兰Hyp nerves in E, ischemic-induced gasping
(G), and recovery (R). Recordings in E are shown in top tracings and, on
expanded time scale, on bottom tracings. Note greater rate of rise of 兰Hyp than
兰Phr discharge in E. During period designated by arrow, perfusion was
terminated, and G was induced (see Fig. 3 for expanded recordings of E and
G). Upon the recommencement of perfusion, a sustained apneic period intervened before neural activities recommenced (R). Note on the expanded
tracings during this early R phase that the frequency of phrenic bursts had
increased, and hypoglossal discharge was of low amplitude in some cycles,
missing in others, and, finally, occurred in neural expiration. Small, highfrequency oscillations, which are visible on 兰Phr activity, represent the
electrocardiogram.
cantly different was the coefficient of variance for the rate of
rise of activity (hypoglossal ⫽ 0.38, phrenic ⫽ 0.20; P ⬍
0.0001).
Records reported in Fig. 1 were obtained ⬃20 min after
rhythmic activity commenced in the perfused preparation. The
hypoglossal discharge especially could change within a given
preparation. The most consistent change was during recovery
from ischemia-induced gasping, as shown in Fig. 2, in which
hypoglossal discharge became exceedingly variable, with an
absence of discharge in some respiratory cycles. Similar
changes in hypoglossal discharge were found if activities were
recorded for an extended period before exposure to ischemia.
Thus, as noted above, in most preparations, hypoglossal discharge had a decrementing discharge when recordings were
obtained 20 min after rhythmic discharge of the preparation
commenced. Sixteen of twenty preparations were exposed to
ischemia soon thereafter. The other four preparations were
J Appl Physiol • VOL
Fig. 3. Activities of the phrenic and hypoglossal nerves during E and G in a
single preparation. G was produced in ischemia. Note switch in pattern of 兰Phr
discharge from incrementing in E to decrementing in G. 兰Hyp discharge was
decrementing during both E and G. Preparation is a different mouse from that
in Fig. 2.
107 • SEPTEMBER 2009 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.32.246 on July 28, 2017
maintained for an additional 30 min, in hyperoxia. By the end
of this additional 30-min period, the respiratory frequency had
increased, and hypoglossal discharge had become irregular,
with the pattern similar to that of Fig. 2 after recovery from
ischemia. Because of this potential for time-dependent changes,
control recordings of all preparations were those taken ⬃20
min after the start of rhythmic activity. Hence, given this
experimental design, no formal study of time-dependent
changes in hypoglossal or phrenic discharges was performed.
Gasping. In an identical manner to that described in a
previous paper in mice (48), the pattern of phrenic discharge
was converted from the incrementing pattern of eupnea to
the decrementing pattern of gasping within 13.5 ⫾ 1.85 s.
following the onset of ischemia (Figs. 2 and 3). Of the 20
preparations that were studied during eupnea, 5 received
methysergide during eupnea, and the phrenic recording was
lost in 1 other preparation. Hence, paired recordings in
eupnea and gasping, without methysergide, were obtained in
14 preparations.
Regardless of the discharge pattern during eupnea, hypoglossal discharge had a decrementing pattern during neural
inspiration of gasping (Fig. 3). If hypoglossal discharge was
absent during some cycles of eupnea, it was always recruited in gasping. Although the peak level of this hypoglossal discharge during neural inspiration increased in 11
of 14 preparations (e.g., Fig. 3), this increase was not
significant (115 ⫾ 17.0% of value in eupnea, Fig. 4). Levels
of activity in neural expiration were variably altered and fell
EUPNEA AND GASPING IN MICE
689
Influence of Blockade of Receptors for 5-HT by
Methysergide Upon Activities of the Phrenic and
Hypoglossal Nerves in Eupnea and Gasping
as the duration of ischemia increased. Peak integrated
phrenic discharge did increase significantly in gasping
(195 ⫾ 36.5% of control, Fig. 4). Durations of the burst of
both phrenic and hypoglossal activities significantly increased with the change from eupnea to gasping. In gasping,
the duration of hypoglossal discharge (668 ⫾ 24 ms) was
significantly longer than that of the phrenic (594 ⫾ 25 ms).
The duration of these two discharges had been similar in
eupnea (phrenic ⫽ 388.5 ⫾ 26 ms, hypoglossal ⫽ 357 ⫾
31.3 ms).
As would be expected from the definition of gasping, per se,
the rate of rise of integrated phrenic discharge was significantly
greater in gasping than eupnea (Fig. 4). Peak height was
attained 308 ⫾ 37 ms after the start of the eupneic burst and
205 ⫾ 17 ms after the start of the gasp (P ⬍ 0.002, Fig. 4). In
contrast to phrenic discharge, rates of rise of hypoglossal
discharge were the same in eupnea (198 ⫾ 25 ms) and gasping
(233 ⫾ 25 ms). The similarity of hypoglossal discharge in
eupnea and gasping is shown graphically for multiple respiratory cycles in Figs. 5 and 6. Note, in these figures, that the rate
of rise of phrenic discharge was significantly faster in gasping
than eupnea.
The greater similarity of hypoglossal and phrenic discharges
in gasping than eupnea is further demonstrated by the coefficient of variance. These coefficients of variance were not
significantly different for both durations of the burst and rates
of rise of activity. Both had differed significantly in eupnea, as
noted above.
A final difference between phrenic and hypoglossal discharges was in the recovery of rhythmic bursts from ischemiainduced gasping (see, e.g., Fig. 2). Phrenic bursts returned
within 53 ⫾ 8.6 s after the restoration of perfusion. For
hypoglossal discharge, the comparable period before activity
recommenced was 72⫾ 12 s (P ⬍ 0.005).
J Appl Physiol • VOL
Fig. 5. Multiple plots of patterns of 兰Phr and 兰Hyp activities in E and G. The
start of activities in 20 cycles of E and 4 cycles of G have been aligned
(arrows), and the resulting integrated activity shown. Note the switch from
incrementing to decrementing activity of phrenic discharge with the change
from E to G, and the same decrementing pattern for hypoglossal activity during
both E and G.
107 • SEPTEMBER 2009 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.32.246 on July 28, 2017
Fig. 4. Comparison of rates of rise of inspiratory activity and peak of
integrated inspiratory activity of phrenic and hypoglossal nerves during E and
G. Note that peak height was greater for phrenic nerve in G than E, and time
to reach this peak height was less in G. *P ⬍ 0.05. For hypoglossal activity,
peak values and rates of rise were the same in E and G.
Methysergide, at a concentration of 3.0 ␮M, was administered to five mice. This concentration of methysergide was
chosen based on results of our previous studies (46, 51) in
which this concentration produced consistent changes in eupneic ventilatory activity. Indeed, as reported previously (46,
51), the frequency of the phrenic burst increased greatly, and
the peak height fell following methysergide. Strikingly, in each
preparation, hypoglossal discharge became completely uncoupled from that of the phrenic, with numerous cycles having no
phasic hypoglossal discharge, and others in which hypoglossal
bursts were recorded solely during neural expiration (Fig. 7).
Thus the dissociation of hypoglossal and phrenic discharges,
which was evident in control preparations, became profound
following administrations of methysergide.
Upon exposure to ischemia, gasping activities commenced
at the same time for activities of both nerves. The first gasp
occurred at 13.5 ⫾ 0.67 s after the onset of ischemia. As is
evident from Fig. 7, the rate of rise of inspiratory activity was
much faster for phrenic discharge in gasping, after methysergide, than eupnea, before the drug was given (P ⬍ 0.001). Peak
integrated phrenic discharge was reached in 326 ⫾ 106 ms in
eupnea and 228 ⫾ 54 ms in gasping. Rates of rise of hypoglossal discharge in eupnea and gasping were very similar,
690
EUPNEA AND GASPING IN MICE
onset of the burst different (phrenic ⫽ 274 ⫾ 19 ms; vagus ⫽
327 ⫾ 21 ms).
Following administration of methysergide, the frequency of
phrenic and vagal bursts increased greatly. However, vagal
discharge continued to be linked to that of the phrenic with a
burst during each neural inspiration; some bursts in neural
expiration were also observed (Fig. 9).
On exposure to ischemia, gasping activities were induced in
each preparation (Figs. 8 and 9). The delay before the first gasp
varied from 12 to 22 s in preparations that received no
methysergide, and 13 to 23 s in those that had received the
drug. Gasping in both groups of preparations was similar (Figs.
8 and 9). The rate of rise of phrenic activity increased greatly,
with peak activity being reached after 67 ⫾ 0.06% of the
Downloaded from http://jap.physiology.org/ by 10.220.32.246 on July 28, 2017
Fig. 6. Average tracings of 兰Phr and 兰Hyp activities during E and G. Tracings
of Fig. 5 have been averaged, and results are shown by thick line, with standard
errors shown by thinner lines. Arrow designates the start of the burst, and
asterisk designates peaks. Note that peak of 兰Phr discharge occurred much
earlier in the gasp than eupneic inspiration, whereas peak of hypoglossal
discharge was slightly later in the gasp than in E.
with peak discharge being reached 237 ⫾ 23 ms after the start
of the eupneic burst and 246 ⫾ 28 ms after the start of the gasp.
A final difference between activities of the phrenic and
hypoglossal nerves was in the recommencement of phasic
activity after the restoration of perfusion. In four of five
preparations, periodic phrenic discharge began 10 – 47 s before
that of the hypoglossal. For the final mouse, phasic phrenic
activity began at 16 s; phasic hypoglossal discharge never
returned. The delay in the restoration of eupneic hypoglossal
activity compared with phrenic activity was similar to the
recovery of eupnea without methysergide (see previous section).
Comparison of Phrenic and Vagal Activities in Eupnea and
Gasping: Influence of Methysergide
Activities of the phrenic and vagus nerve were recorded in
six preparations. In three of these, methysergide (3.0 ␮M) was
added to the perfusate after recordings in eupnea. Gasping was
induced in ischemia in all six preparations, three of which had
received methysergide.
During eupnea, the vagus nerve had discharges during both
neural inspiration and expiration (Figs. 8 and 9). For all six
preparations, the durations of the phrenic (405 ⫾ 19 ms) and
vagal discharges (462 ⫾ 21 ms) were not significantly different, nor was the time to reach peak integrated height after the
J Appl Physiol • VOL
Fig. 7. Influence of blockade of receptors for serotonin with methysergide (M)
upon activities of the phrenic nerve (兰Phr) and hypoglossal nerve (兰Hyp) in E
and G in the mouse. Note that in E, hypoglossal discharge was linked to the
phrenic burst, but periodical bursts of hypoglossal activity were recorded in
neural expiration. Following administration of 3.0 ␮M of M, phrenic and
hypoglossal discharges were largely uncoupled. In G, phrenic and hypoglossal
discharges were linked, although burst of hypoglossal discharge in neural
expiration was seen. Small, high-frequency oscillations, which are visible on
兰Phr activity, represent the electrocardiogram.
107 • SEPTEMBER 2009 •
www.jap.org
EUPNEA AND GASPING IN MICE
691
Fig. 8. Integrated activities of the phrenic and vagus nerves in E and G in the
mouse. Top: note that integrated vagal discharge (兰Vag) had a phasic burst
during the period of phrenic discharge (兰Phr) and also activity during neural
expiration. Bottom: in G, both nerves had decrementing discharges, and
expiratory vagal activity declined with continuing ischemia. Small, highfrequency oscillations, which are visible on 兰Phr activity represent the electrocardiogram.
duration of the burst in eupnea and 38 ⫾ 0.02% of the gasp
(P ⬍ 0.001). This corresponded to a mean time to reach a peak
integrated level of 274 ⫾ 19 ms in eupnea and 232 ⫾ 31 ms in
gasping. The time to reach peak vagal activity was less in
gasping than eupnea in five of six preparations (mean for all
preparations, eupnea ⫽ 327 ⫾ 21 ms, gasping ⫽ 266 ⫾ 30
ms). Following the recommencement of perfusion, phrenic and
vagal discharges appeared in the same respiratory cycle in five
of six preparations; in the other, vagal discharge appeared
earlier. Mean times for recovery of rhythmic activity were
57.8 ⫾ 21 s for phrenic and 54.5 ⫾ 22 s for vagal discharge.
DISCUSSION
Since first characterized by Lumsden in 1923, the primary
distinction between eupnea and gasping has been a more
“sudden beginning” of inspiration during gasping than eupnea
(18, 19). This more sudden beginning of inspiration has been
documented in multiple species, including mice, as shown
herein, by a significant increase in the rate of rise of phrenic
discharge. Stated differently, phrenic discharge, which has an
incrementing pattern in most respiratory cycles of eupnea,
changes to a decrementing pattern in gasping (41– 43).
Hypoglossal Discharge During Neural Inspiration
is Indistinguishable Between Eupnea and Gasping
in the Mouse
In the in situ preparation of the rat during eupnea, the
relationship between rates of rise of hypoglossal and phrenic
J Appl Physiol • VOL
Fig. 9. Activities of the phrenic and vagus nerves in the mouse during E, after
administration of M, and in G. Note that, following administration of 3.0 ␮M
of M, the frequency of neural bursts increased, but 兰Vag remained linked to the
兰Phr. Small, high-frequency oscillations on records of 兰Phr activity and higher
amplitude spies on records of vagal activity both represent electrocardiograms.
107 • SEPTEMBER 2009 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.32.246 on July 28, 2017
discharges is variable. In some preparations, activities of both
nerves are incrementing, whereas, in others, hypoglossal discharge is decrementing (16, 24, 51). In the in situ preparation
of the mouse, the greater rate of rise of integrated hypoglossal,
compared with phrenic activity, is extreme. This extremely
rapid rate of rise of hypoglossal discharge in eupnea is such
that this rate of rise does not increase further in gasping and,
indeed, is greater in eupnea than gasping in most preparations.
Hence, based on hypoglossal discharge alone, the primary
factor that distinguishes eupnea from gasping, namely, the rate
of rise of inspiratory activity, is indistinguishable.
In addition to hypoglossal discharge being the same in
eupnea and gasping, the variable characteristics of hypoglossal
discharge during eupnea would prevent designation of the type
or phase of the respiratory rhythm. As opposed to phrenic
discharge, discharge of the hypoglossal nerve occurs during
neural inspiration, and, also, in an unpredictable fashion, bursts
are recorded during neural expiration. Some of these bursts
during neural expiration are incrementing, whereas, as noted
above, many during neural inspiration are decrementing. Thus,
from examining hypoglossal discharge alone during eupnea, it
might be concluded that eupnea (incrementing discharge) and
gasping (decrementing discharge) are occurring together. Of
course, from hypoglossal discharge alone, the erroneous con-
692
EUPNEA AND GASPING IN MICE
related component of the hypoglossal discharge can be altered,
independent of changes in phrenic activity. A most striking
example of this independence is a separation of the preinspiratory and inspiratory components of hypoglossal discharge,
which appear as a single continuous burst in eupnea, into two
completely separate bursts (47).
In the in situ mouse preparation, different premotor innervations without doubt contributed to the total uncoupling of
hypoglossal and phrenic discharges following a blockade of
multiple types of receptors for 5-HT with methysergide. The
5-HT system is widespread in the brain stem, and influences on
activities of premotor and both cranial and spinal motoneurons
are well described (9, 20, 23, 27, 39, 48). Receptors for 5-HT
are differentially distributed on hypoglossal compared with
phrenic motoneurons. These differential alterations of the hypoglossal and phrenic discharges following administrations of
methysergide are in keeping with previous data that document
a different response of these systems to a range of sedatives
and anesthetics (5, 6, 14, 51).
Many of the differences between the hypoglossal and bulbospinal-phrenic systems, considered above, could also be
applied to comparisons with the vagal motor system. As the
hypoglossal system, both inspiratory and expiratory vagal
discharges are more sensitive to alterations by multiple drugs
than the phrenic system (5, 6, 9, 20). Thus the tighter coupling
of vagal discharges to phrenic activity than those of the
hypoglossal nerve to phrenic activity is without a firm explanation. However, vagal motoneurons and premotor neurons are
in close anatomical and functional proximity to bulbospinal
respiratory neurons in the ventral medullary respiratory nucleus (1, 21, 43). This is in contrast to premotor hypoglossal
motoneurons, which are, in general, far removed from the
bulbospinal system (28, 52). Moreover, while the vagal motor
system, as the hypoglossal system, is involved in other rhythmic behaviors, such as cough and swallow, the same respiratory-related premotor vagal neurons may be used to generate
these behaviors (see discussions in Refs. 1, 36).
Neuroanatomical and Pharmacological Basis for Lack
of Correspondence Between Respiratory-Modulated
Activities of Hypoglossal and Phrenic Nerves and Responses
to Drugs
In 2000, a preparation of a thick medullary slice of the
neonatal mouse was introduced with the claim that this preparation could generate respiratory patterns akin to eupnea,
gasping, and sighing in vivo (17). These patterns were judged
based on activities recorded from the hypoglossal nerve and/or
from multiple neurons of the ventrolateral medulla. The “eupneic” pattern was considered as augmenting or bell shaped,
whereas the rate of rise of activity was significantly increased
in “gasping,” and the pattern became decrementing. The sigh
was a mixed pattern, which started as “eupnea,” and then
transformed to a “gasp.” Parenthetically, the designation of
these different patterns from the in vitro slice of mouse appeared in conflict with results from other in vitro slice and en
bloc preparations, in which only a single, decrementing pattern
was recorded. Despite the decrementing pattern, this rhythm
was also considered to be akin to eupnea (2, 10, 33, 42).
Until recently, few recordings of hypoglossal discharge have
been made in the mouse, except in vitro. Hence, the designation of these various patterns recorded in vitro was based on
patterns of integrated phrenic activity that were obtained in
An extensive number of studies have demonstrated differences between respiratory-modulated activities of the hypoglossal nerve compared with the bulbospinal-phrenic system.
These differences include premotor innervation and responses
to pharmacological agents (e.g., Refs. 5– 8, 14, 27, 28, 34, 37,
39). Concerning the former, the vast majority of pontile and
medullary neurons that project upon hypoglossal motoneurons
differ from those that project upon the bulbospinal-phrenic
system. In addition to respiration, the hypoglossal system is
involved in other rhythmic activities, such as mastication and
deglutition (34, 36). Given these differences in premotor innervation and in multiple rhythmic physiological functions, it
is not surprising that rhythmic hypoglossal discharge can
become uncoupled from that of the phrenic nerve in a number
of rat preparations (16, 47). Moreover, even the respiratoryJ Appl Physiol • VOL
Comparison of Findings From In Vitro Slice Preparation
and In Situ Preparation of Mouse
107 • SEPTEMBER 2009 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.32.246 on July 28, 2017
clusion would be drawn that neural inspiration corresponds to
periods of hypoglossal discharge and neural expiration to the
absence of this discharge, when, in fact, hypoglossal discharges during neural expiration are frequent. At the other
extreme, since hypoglossal discharge can be totally absent
during some respiratory cycles in eupnea, no accurate calculation of respiratory frequency is possible from evaluation of
this neural discharge alone (see also Ref. 40).
In the context of the shape of the hypoglossal discharge, the
decrementing discharge, seen in most preparations, could become incrementing if the preparation were maintained for
relatively longer periods and/or during recovery following
ischemic-induced gasping. However, this switch to an incrementing pattern was accompanied by a complete absence of
hypoglossal discharge during some respiratory cycles and
bursts of discharge in a seemingly random fashion during
neural expiration. We have no indication as to factors responsible for the switch from incrementing to decrementing discharge of the hypoglossal nerve. However, regardless of the
pattern, hypoglossal discharge was poorly correlated with the neural inspiration or expiration of eupnea, as defined by the
incrementing phrenic discharge or respiratory pattern, be it
eupnea or gasping, again, as defined by incrementing or decrementing integrated phrenic activity.
The lack of correspondence between hypoglossal and
phrenic discharges of the in situ preparation is not a generalized phenomenon for respiratory-modulated activities of all
cranial nerves. As opposed to hypoglossal discharge, respiratory-modulated activity of the vagus nerve remained tightly
coupled to phrenic discharge during eupnea, although vagal
discharge during early neural expiration was variable.
In summary, results of the present study demonstrate that, in
the in situ preparation of the mouse, hypoglossal discharge
alone cannot be used either to distinguish eupnea from gasping
or to define the phases of the respiratory cycle. These results
raise significant doubts as to the accuracy of the model of the
thick medullary slice of the mouse for which it is claimed that
eupnea, gasping, and sighs can be characterized, based on
recordings of hypoglossal discharge alone and/or massed neuronal activities from the ventrolateral medulla (17).
EUPNEA AND GASPING IN MICE
J Appl Physiol • VOL
sumption that all “nongasping” medullary rhythms must be
eupnea, even though rhythms recorded following removal of
pons were markedly different from those recorded before this
pontomedullary transection (see discussion in Ref. 45). Interpretation of these varying results following brain stem transections was significantly clarified by the recent results of Smith
et al. (38). These investigators performed brain stem transections in the perfused, in situ preparation. Thus the confounding
influence of changes in arterial perfusion of regions of the brain
stem that, without doubt, occurred in vivo, was removed. Smith
et al. reported that the respiratory pattern following removal of
pons was markedly altered with incrementing phrenic discharges changing to “square-wave” patterns. Activity during
early expiratory was also eliminated. With a further transection
so as to isolate the “pre-Botzinger” complex, which is the
region strongly advanced as the “noeud vital” for eupnea, only
gasping was recorded.
Gasping would appear to be the one respiratory pattern that
is accurately represented in the thick slice of the neonatal
mouse. This statement is dependent on designating the decrementing hypoglossal and massed neuronal activities recorded
during anoxia as gasping. However, we believe it extremely
probable that, due to the anoxic core of the preparation, many
of the bursts of activity labeled as eupnea, and having rates of
rise very similar to gasps, are, in fact, gasps.
The similarity of “gasping rhythms” in vitro and in situ is
documented by the role of pacemaker mechanisms, involving
conductance through persistent sodium channels, in generating
gasping in both preparations (24, 26, 31, 32, 51). Riluzole, a
blocker of persistent sodium channels, eliminates gasping, both
in situ and in vitro. The marked difference between the nominally eupneic rhythm in vitro and eupnea in situ or in vivo is
documented by the elimination of eupnea, at least in some
in vitro preparations, following administrations of riluzole
(32). However, riluzole, in concentrations many fold higher
than those that eliminate gasping in situ or in vivo, does not
eliminate eupnea in either the in situ preparation or in vivo
(50). Finally, another group of pacemakers, dependent on
calcium conductances, are also hypothesized to play a role in
the neurogenesis of eupnea (31, 32, 53). However, these
pacemakers have only been identified in the thick slice of
mouse medulla, and not in thin in vitro slices or in situ
preparations. This pacemaker discharge is blocked by flufanemic acid, and simultaneous administration of flufanemic acid
and riluzole eliminates the eupneic rhythm in vitro (32). Again,
however, such simultaneous administrations do not eliminate
eupnea in situ (44).
The presence of the final rhythm reported in the slice, the
sigh, is enigmatic. In vivo, sighs cannot be generated following
sectioning of the carotid sinus nerves and vagi (3, 12). Neither
afferent nerve is present in the slice. Moreover, while sighs are
recorded frequently in the medullary slice, we have never
observed such a pattern in even a single respiratory cycle in
any in situ mouse or rat preparation. Thus, as for eupnea, the
sigh of the in vitro preparation may be unique to that preparation and totally different from sighs recorded in vivo.
In summary, we believe that, of the putative fictive rhythms
generated from the thick slice of medulla of the mouse, only
gasping is similar to gasping in vivo or in situ. Yet, even with
gasping, the marked differences in response of the hypoglossal
and phrenic systems to drugs may have led to erroneous conclu-
107 • SEPTEMBER 2009 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.32.246 on July 28, 2017
other species in vivo and in situ. Implicit for acceptance that
these in vitro rhythms were accurate models for in vivo
rhythms would be very similar discharges of the phrenic and
hypoglossal nerves and of massed medullary neurons.
Results herein show that hypoglossal and phrenic discharges
are not identical in the mouse during eupnea. One study reports
that both hypoglossal and phrenic discharges in the anesthetized mouse in vivo are augmenting, but only a single respiratory cycle is shown (22). Hence, the consistency of the incrementing pattern of hypoglossal discharge cannot be judged.
Moreover, even in the thick medullary in vitro slice of the
neonatal mouse, activities recorded from the hypoglossal nerve
and from massed neurons of the ventrolateral medulla may
differ, with the former being absent in some cycles in which
rhythmic bursts are recorded from the ventrolateral medulla
(29, 35). This elimination of hypoglossal discharge may reflect
a failure of synaptic transmission due to the presence of a
hypoxic/anoxic core in the thick slice. Indeed, this failure
becomes more prevalent with additional hypoxia of the slice
(see discussion in Ref. 29). After such a failure, the assumption
is made that massed neuronal activities represent the overall
respiratory rhythm and that these activities are purely inspiratory. Evidence in support of this assumption is lacking as,
when hypoglossal activity is present, neurons that fire in the
period between hypoglossal bursts are recorded and designated
as expiratory (17).
Differences between the in situ mouse and neonatal in vitro
mouse could reflect the difference in age of the preparations
and/or that most of the brain stem respiratory system is missing
in the slice preparation. Stated differently, the bursts of hypoglossal discharge during expiration in the in situ preparation
could reflect pontile influences. Yet examinations of hypoglossal discharge in the neonatal mouse in vivo further adds to the
probability that the rhythm designated as eupnea of the medullary slice is different from eupnea of preparations having an
intact pontomedullary brain stem. Rhythmic hypoglossal activity is recorded in vitro from medullary slices of 0- to
14-day-old neonatal mice (4, 17, 25, 30). Yet no rhythmic
hypoglossal activity is recorded during eupnea of most anesthetized in vivo neonatal mice younger than 9 days (4). This
absence of rhythmic hypoglossal discharge is not due to technical problems of recording as rhythmic gasping activity can be
recruited in the hypoglossal discharge (4). However, since it is
well recognized that anesthesia may differentially suppress
hypoglossal, compared with phrenic activity (e.g., Ref. 14), it
cannot be entirely excluded that the presence of anesthesia is
responsible for elimination of the hypoglossal discharge. This
explanation would require that this sensitivity to anesthesiainduced depression is only manifested during eupnea and not
gasping.
Concerning removal of the pontile portion of the brain stem
respiratory system in the medullary slice, this again raises the
question as to whether eupnea can be generated by medullary
mechanisms alone. From multiple studies over multiple years,
it was evident that gasping, and not eupnea, was the one
respiratory pattern that could be reproducibly obtained following the removal of pons in vivo (41– 43, 54). Yet, in some
preparations, gasping was not obtained (54, see Ref. 10 for
review). This absence of gasping was proposed by some as
evidence of the generation of eupnea by medullary mechanisms alone (10, 33). However, this proposal carries the as-
693
694
EUPNEA AND GASPING IN MICE
sions concerning the necessity of some neuromodulators, especially 5-HT, in generating the gasp. Concerning eupnea and sighs,
verification of findings in vitro would appear to be impossible in
vivo, as the main index of respiratory activity, the discharge of the
hypoglossal nerve, is silent during eupnea in vivo in neonatal mice
of the same age as those in which a rhythmic activity, designated
as eupnea, has been recorded in vitro.
GRANTS
These studies were supported by National Institutes of Health National
Heart, Lung, and Blood Institute Grant 26091. Mice were obtained from a
colony supported by funds from NIH National Institute of Child Health and
Human Development Grant P01-HD36379.
REFERENCES
J Appl Physiol • VOL
107 • SEPTEMBER 2009 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.32.246 on July 28, 2017
1. Baekey DM, Morris KF, Gestreau C, Li Z, Lindsey BG, Shannon R.
Medullary respiratory neurones and control of laryngeal motoneurones
during fictive eupnoea and cough in the cat. J Physiol 534: 565–581, 2001.
2. Ballanyi K, Onimaru H, Homma I. Respiratory network function in the
isolated brainstem-spinal cord of newborn rats. Prog Neurobiol 59: 583–
634, 1999.
3. Bartlett D. Origin and regulation of spontaneous deep breaths. Respir
Physiol 12: 230 –238, 1971.
4. Berger AJ, Sebe J. Developmental effects of ketamine on inspiratory
hypoglossal nerve activity studied in vivo and in vitro. Respir Physiol
Neurobiol 157: 206 –214, 2007.
5. Bonora M, Shields GI, Knuth SL, Bartlett D, St. John WM. Selective
depression by ethanol of upper airway respiratory motor activity in cats.
Am Rev Respir Dis 130: 156 –161, 1984.
6. Bonora M, St. John WM, Bledsoe TA. Differential elevation by protriptyline and depression by diazepam of upper airway respiratory motor
activity. Am Rev Respir Dis 131: 41– 45, 1985.
7. Brandes IF, Zuperku EJ, Dean C, Hopp FA, Jakovcevic D, Stuth EA.
Retrograde labeling reveals extensive distribution of genioglossal motoneurons possessing 5-HT2A receptors throughout the hypoglossal nucleus of adult dogs. Brain Res 1132: 110 –119, 2007.
8. Dobbins EG, Feldman JL. Differential innervation of protruder and
retractor muscles of the tongue. J Comp Neurol 357: 376 –394, 1995.
9. Fay R, Kubin L. Pontomedullary distribution of 5-HT2A receptor-like
protein in the rat. J Comp Neurol 418: 323–345, 2000.
10. Feldman JL, Del Negro CA. Looking for inspiration: new perspectives
on respiratory rhythm. Nat Rev Neurosci 7: 232–242, 2006.
11. Fung ML, Wang W, St. John WM. Medullary loci critical for expression
of gasping in adult rats. J Physiol 480: 597– 611, 1994.
12. Glogowska M, Richardson PS, Widdicombe JG, Winning AJ. The role
of the vagus nerves, peripheral chemoreceptors and other afferent pathways in the genesis of augmented breaths in cats and rabbits. Respir
Physiol 16: 179 –196, 1972.
13. Hunt CE. The cardiorespiratory control hypothesis for sudden infant
death syndrome. Clin Perinatol 19: 757–771, 1992.
14. Hwang JC, St. John WM, Bartlett D. Respiratory-related hypoglossal
nerve activity: influence of anesthetics. J Appl Physiol 55: 785–793, 1983.
15. Leiter JC, Böhm I. Mechanisms of pathogenesis in the sudden infant
death syndrome. Respir Physiol Neurobiol 159: 127–138, 2007.
16. Leiter JC, St. John WM. Phrenic, vagal and hypoglossal activities in rat:
pre-inspiratory, inspiratory, expiratory components. Respir Physiol Neurobiol 142: 115–125, 2004.
17. Lieske SP, Thoby-Brisson M, Telgkamp P, Ramirez JM. Reconfiguration of the neural network controlling multiple breathing patterns:
eupnea, signs and gasps. Nat Neurosci 3: 600 – 607, 2000.
18. Lumsden T. Observations on the respiratory centres in the cat. J Physiol
57: 153–160, 1923.
19. Lumsden T. Effects of bulbar anaemia on respiratory movements.
J Physiol 59: lvii–lx, 1924.
20. Morin D. Compared effects of serotonin on the inspiratory activity of
glossopharyngeal, vagal, hypoglossal and cervical motoneurons in neonatal rat brain stem-spinal cord preparations. Neurosci Lett 160: 61– 64,
1993.
21. Nunez-Abades PA, Pasaro R, Bianchi AL. Localization of respiratory
bulbospinal and propriobulbar neurons in the region of the nucleus
ambiguus of the rat. Brain Res 568: 165–172, 1991.
22. O’Neill MH, Spiegel ET, Chon KH, Solomon IC. Time-frequency
representation of inspiratory motor output in anesthetized C57BL/6 mice
in vivo. J Neurophysiol 93: 1762–1775, 2005.
23. Paterson DS, Trachtenberg FJ, Thompson EG, Belliveau RA, Beggs
AH, Darnall R, Chadwick AE, Krous HF, Kenney HC. Multiple
serotonergic brainstem abnormalities in sudden infant death syndrome.
JAMA 296: 2124 –2132, 2006.
24. Paton JFR, Abdala APL, Koizumi H, Smith JC, St. John WM.
Respiratory rhythm generation during gasping depends on persistent
sodium current. Nat Neurosci 9: 311–316, 2006.
25. Paton JFR, Richter DW. Maturational changes in the respiratory rhythm
generator of the mouse. Pflügers Arch 430: 115–124, 1995.
26. Paton JFR, St. John WM. Medullary pacemakers are essential for
gasping, but not eupnea in mammals. J Appl Physiol 103: 718 –720, 2007.
27. Peever JH, Necakov A, Duffin J. Nucleus raphe obscurus modulates
hypoglossal output of neonatal rat in vitro transverse brain stem slices.
J Appl Physiol 90: 269 –279, 2001.
28. Peever JH, Shen L, Duffin J. Respiratory pre-motor control of hypoglossal motoneurons in the rat. Neuroscience 110: 711–722, 2002.
29. Peña F, Meza-Andrade R, Páez-Zayas V, González-Marı́n MC. Gasping generation in developing Swiss-Webster mice in vitro and in vivo.
Neurochem Res 33: 1512–1500, 2008.
30. Peña F, Parkis MA, Tryba AK, Ramirez JM. Differential contribution
of pacemaker properties to the generation of respiratory rhythms during
normoxia and hypoxia. Neuron 43: 105–117, 2004.
31. Peña F, Ramirez JM. Endogenous activation of serotonin-2A receptors is
required for respiratory rhythm generation in vitro. J Neurosci 22: 11055–
11064, 2002.
32. Ramirez JM, Viemari JC. Determinants of inspiratory activity. Respir
Physiol Neurobiol 147: 145–157, 2005.
33. Rekling JC, Feldman JL. PreBotzinger complex and pacemaker neurons:
hypothesized site and kernel for respiratory rhythm generation. Annu Rev
Physiol 60: 385– 405, 1998.
34. Roda F, Gestreau C, Bianchi AL. Discharge patterns of hypoglossal
motoneurons during fictive breathing, couching and swallowing. J Neurophysiol 87: 1703–1711, 2002.
35. Ruangkittisakul A, Schwarzacher SW, Seechia L, Ma Y, Poon NBY,
Funk GD, Ballanyi K. Generation of eupnea and sighs by a spatiochemically organized inspiratory network. J Neurosci 28: 2447–2458, 2008.
36. Saito Y, Ezure K, Tanaka I. Difference between hypoglossal and phrenic
activities during lung inflation and swallowing in the rat. J Physiol 544:
183–193, 2002.
37. Segers LS, Nuding SC, Dick TE, Shannon R, Baekey DM, Solomon
IC, Morris KF, Lindsey BG. Functional connectivity in the pontomedullary respiratory network. J Neurophysiol 100: 1751–1769, 2008.
38. Smith JC, Abdala APL, Koizumi H, Rybak IA, Paton JFR. Spatial and
functional architecture of mammalian brain stem respiratory network: a hierarchy
of three oscillatory mechanisms. J Neurophysiol 98: 3370–3387, 2007.
39. Sood S, Liu X, Liu H, Nolan P, Horner RL. 5-HT at hypoglossal motor
nucleus and respiratory control of genioglossus muscle in anesthetized
rats. Respir Physiol Neurobiol 138: 205–221, 2003.
40. Stettner GM, Zanella S, Huppke P, Gärtner J, Hilaire G, Dutschmann
M. Spontaneous central apneas occur in the C57BL/6J mouse strain.
Respir Physiol Neurobiol 160: 21–27, 2008.
41. St. John WM. Neurogenesis, control, and functional significance of
gasping. J Appl Physiol 68: 1305–1315, 1990.
42. St. John WM. Medullary regions for neurogenesis of gasping: noeud vital
or noeuds vitals? J Appl Physiol 81: 1865–1877, 1996.
43. St. John WM. Neurogenesis of patterns of automatic ventilatory activity.
Prog Neurobiol 56: 97–117, 1998.
44. St. John WM. Rhythmic respiratory activity of in situ rats persists
following blockers of both types of in vitro burster activities. Respir
Physiol Neurobiol 160: 353–356, 2008.
45. St. John WM, Leiter JC. High frequency oscillations in phrenic activity
during pontile and medullary respiratory rhythms in rats. Exp Physiol 92:
457– 466, 2007.
46. St. John WM, Leiter JC. Maintenance of gasping and restoration of
eupnea after hypoxia-induced depression requires activation of receptors
for norepinephrine and serotonin. J Appl Physiol 104: 665– 673, 2008.
47. St. John WM, Leiter JC, Paton JFR. Uncoupling of rhythmic hypoglossal from phrenic activity. Exp Physiol 89: 727–737, 2004.
48. St. John WM, Li A, Leiter JC. Genesis of gasping is independent of levels
of serotonin in the PET-1 knockout mouse. J Appl Physiol (February 12,
2009). doi:10.1152/japplphysiol.91461.2008.
EUPNEA AND GASPING IN MICE
49. St. John WM, Paton JFR. Characterizations of eupnea, apneusis and
gasping in a perfused rat preparation. Respir Physiol 123: 201–213, 2000.
50. St. John WM, Waki H, Dutschmann M, Paton JFR. Maintenance of
eupnea of in situ and in vivo rats following riluzole, a blocker of persistent
sodium channels. Respir Physiol Neurobiol 155: 97–100, 2007.
51. Toppin VAL, Harris MB, Kober AM, Leiter JC, St. John WM.
Persistence of eupnea and gasping following blockade of both serotonin
type 1 and 2 receptors in the in situ juvenile rat preparation. J Appl Physiol
103: 220 –227, 2007.
52. Travers B, Rinaman L. Identification of lingual motor control circuits
using two strains of pseudorabies virus. Neuroscience 115: 1139 –1151,
2002.
695
53. Tryba AK, Pena F, Ramirez JM. Gasping activity in vitro: a rhythm
dependent on 5-HT2A receptors. J Neurosci 26: 2623–2634, 2006.
54. Wang SC, Ngai SH, Frumin MJ. Organization of central respiratory
mechanisms in the brain stem of the cat: genesis of normal respiratory
rhythmicity. Am J Physiol 190: 333–342, 1957.
55. Wang W, Fung ML, Darnall RA, St. John WM. Characterizations and
comparisons of eupnoea and gasping in neonatal rats. J Physiol 490:
277–292, 1996.
56. Zavala-Tecuapella C, Aguileta MA, Lopez-Guerro JJ, GonzalesMarin MC, Pena F. Calcium-activated potassium currents differentially
modulate respiratory rhythm generation. Eur J Neurosci 27: 2871–2884,
2008.
Downloaded from http://jap.physiology.org/ by 10.220.32.246 on July 28, 2017
J Appl Physiol • VOL
107 • SEPTEMBER 2009 •
www.jap.org