120 A
2.3. Assessment of apneusis and analysis of data the placement of microinjection sites in the rostrocaudal
axis use as a reference point the opening of the central To the best of our knowledge, no strict criterion has
canal into the fourth ventricle Fig. 1 which we will been employed to distinguish an ‘apneustic’ breath from a
abbreviate as CC IV. The reason for this is that the latter breath demonstrating prolonged inspiratory duration. The
reference point can be localized to one 50-mm slice distinction between these two patterns of breathing rests
whereas assessment of calamus scriptorius in coronal slices upon the difference in length of time spent in maximal or
can be indistinct and may appear to occur on any one of near-maximal inspiratory effort. In the present study, we
two or three sections. We also will not use the term ‘obex’ defined an apneustic breath as one that maintained a
as a reference point as this term has been variously maximal or near maximal level of inspiratory effort for
described as occurring at the most caudal extent of the area .650 ms and with a total inspiratory duration T .900
postrema [28,29] as well as in coronal sections in which
i
ms. Onset of apneusis was judged to occur at the second area postrema is well developed [11].
apneustic breath. Duration of apneustic breathing was determined as the time from the second apneustic breath
2.6. Data analysis i.e., onset to the occurrence of the second-to-last apneus-
tic breath. All post-drug cardiorespiratory values were Data presented are the means6standard error of the
taken at the time of maximal prolongation of T . The mean. Statistical analysis was performed using a paired
i
means of five successive respiratory cycles were used to t-test for analysis comparing baseline with post-drug
calculate the values reported in these studies. values. An unpaired t-test was used to compare baseline
values between vagotomized and non-vagotomized groups. 2.4. Experimental procedure
When comparing incidences of apneustic breathing be- tween treatment groups and vehicle controls, a Fisher
After bilateral insertion of micropipettes, animals were Exact test was used. In all cases, P,0.05 was the criterion
allowed to stabilize for at least 5 min before a baseline used for statistical significance.
recording was obtained. Subsequent to this, drugs of interest or vehicle saline were microinjected bilaterally
over a period of approximately 1 min. In two cases, saline
3. Results
was loaded into the adjacent barrel and microinjected first and cardiorespiratory effects recorded for at least 15 min
3.1. Microinjection sites prior to microinjection of drug. In six early experiments,
Fast Green FCF dye 5 loaded into the barrel adjacent to All microinjection sites were found to be located within
the drug was used to mark drug injection sites to verify a region 0.4 mm caudal to 0.1 mm rostral to the opening of
injection site location. This was done only after the the central canal into the fourth ventricle CC IV; Fig. 1.
experiment had been completed in order to avoid respirato- Microinjections into the region of the vlNTS were found to
ry effects of the dye itself. Subsequent studies did not use be distributed from an area lateral to the solitary tract to a
Fast Green dye as microinjection sites could be determined region lateral and ventral to the solitary tract. These
from microscopic analysis of tissue sections alone. microinjection sites are located within the general borders
of the ventrolateral and interstitial subnuclei of the solitary 2.5. Histology
tract as defined by Kalia and Sullivan [20]. Non-vagotom- ized animals that received GABA microinjections to the
Upon completion of the experiment, animals were region of the vlNTS are not depicted in Fig. 1. These were
sacrificed by administration of an overdose of pentobarbi- omitted to reduce clutter but were found to be contained
tal sodium through the arterial line. The brain was rapidly within the region of the vlNTS in locations that fall within
removed and placed in a solution of 6 buffered parafor- those depicted here. Injection sites medial to the solitary
maldehyde and transferred within 24–48 h into 20 tract were contained in an area slightly dorsal to the lateral
sucrose in phosphate-buffered saline for at least 24 h half of the DMV. Injection sites directed lateral to the
before sectioning at 50 mm for histological analysis. solitary tract were found to be located in the reticular area
Sections were stained with neutral red or cresyl violet to 0.4–0.6 mm lateral to the vlNTS region at a dorsoventral
facilitate identification of nuclear groups, and the site of level similar to the vlNTS sites Fig. 1.
microinjection was determined in the following manner: as the ventral-most extent of identified pipette tracks or the
3.2. GABA microinjection into the vlNTS site of highest density of Fast Green FCF dye 5.
Experiments without clear bilateral pipette tracks by The purpose of microinjections of the inhibitory amino
microscopic assessment were not included in these studies. acid g-aminobutyric acid GABA was to suppress neuro-
Although the calamus scriptorius was used as a reference nal activity in the immediate vicinity of the vlNTS and
point for microinjection coordinates, our descriptions of observe effects on respiratory function. Bilateral microin-
A .M. Wasserman et al. Brain Research 880 2000 118 –130
121
Fig. 1. Summary of microinjection sites. Camera lucida reconstruction of microinjection sites of drugs administered to vagotomized rats. Coordinates listed describe location relative to the opening of the central canal into the fourth ventricle CC IV, see Section 2 for description. Abbreviations: AP, area
postrema; CC, central canal; XII, hypoglossal nucleus; DMV, dorsal motor nucleus of the vagus, TS, solitary tract.
jection of 25 nmol GABA 0.5 M, 45 nl into the vlNTS of expiratory duration T to nearly one-half of baseline
e
vagotomized rats produced an immediate onset of apneus- levels Table 1. No change was observed in the amplitude
tic breathing pattern in all nine animals tested. Apneusis of integrated diaphragmatic EMG iEMG activity. Ap-
was observed to begin occasionally during the administra- neusis produced by microinjection of GABA into the
tion of drug into the second side, and always within vlNTS lasted on average slightly less than 3 min 2.862
seconds of bilateral administration Fig. 2 and Table 1. min; Table 1; and respiratory activity returned to baseline
Overall, inspiratory duration T was increased more than values within 5 min of administration Fig. 2D. Blood
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2.5-fold over baseline. This striking effect on inspiratory pressure increased significantly 130 and in nearly all
duration was accompanied by a significant reduction in instances had a similar time-course as the apneustic
122 A
Fig. 2. Bilateral microinjection of GABA into the ventrolateral NTS produces apneustic breathing in the anesthetized, vagotomized and spontaneously breathing rat. A Baseline cardiorespiratory activity [top trace, blood pressure BP; upper middle trace, diaphragmatic EMG dEMG signal; lower middle
trace, integrated dEMG iEMG signal; bottom trace, heart rate HR]. Bar51 s. B Left vlNTS microinjection of GABA 25 nmol 45 nl into the vlNTS. Black arrows denote start and end of injection. Note, right vlNTS microinjection occurred 15 s prior to this panel. Apneustic breathing clearly occurs at the
end of bilateral injection. C Cardiorespiratory activity 1 min post-bilateral GABA microinjection. D Cardiorespiratory activity 4 min after microinjection demonstrates recovery of eupneic breathing pattern and blood pressure. E Photomicrographs of microinjection sites, as indicated by
horizontal arrows. Bars50.25 mm. Abbreviations: XII, hypoglossal nucleus; DMV, dorsal motor nucleus of the vagus; TS, solitary tract.
A .M. Wasserman et al. Brain Research 880 2000 118 –130
123 Table 1
a
Cardiorespiratory response to bilateral microinjection of drugs into the nucleus of the solitary tract Group
RR T
T iEMG
BP HR
Apneusis Time
Time Apneusis
i e
incidence to onset
to max duration
s s
s Saline
Baseline 5265
407621 7736104
– 8064
491616 Post-injection
4666 415627
9176139 464
7268 487616
0 5[ –
– –
n55 GABA
vlNTS Baseline
4762 484618
778636 –
8364 439617
Post-injection 3562
1291684 432638
362 10866
452618 9 9
264 762
166611 n59
GABA mNTS
Baseline 4964
427613 757668
– 7569
480623 Post-injection
5364 6106102
497678 2762
102612 500626
1 3[ 15
15 30
n53 GABA
non-vagotomized Baseline
6663 307629
560649 –
7866 402616
Post-injection 5464
7486105 345632
863 10468
422612 3 7[
467 1169
55628 n57
Kynurenic acid Baseline
5562 399633
730670 –
6964 524616
Post-injection 3862
8976104 6296109
165 10064
540613 5 5
75632 132627
6276197 n55
Tetrodotoxin Baseline
5262 454628
680645 –
6967 498619
Post-injection 2762
17896278 517651
663 92611
520616 9 9
1069 5069
9976190 n59
a
Abbreviations and units: RR, respiratory rate breaths min; T , inspiratory duration ms; T , expiratory duration ms; iEMG, integrated diaphragmatic
i e
EMG signal change from baseline; BP, blood pressure mmHg; HR, heat rate beats min. All post-injection values were obtained at the time of maximal effect on Ti. All animals were vagotomized unless otherwise indicated.
P,0.05 versus baseline values; [P,0.05 versus GABA microinjection into vlNTS of vagotomized animals; P,0.05 versus baseline of vagotomized animals.
response although this was not always the case. Pressure pressure and heart rate produced by GABA were quantita-
was not increased in one experiment in which apneusis tively similar to those changes observed in vagotomized
was produced data not shown. A small 3 but signifi- animals Table 1.
cant increase in heart rate was also observed in this group. A summary of microinjection sites is presented in Fig. 1.
3.3. GABA microinjection into the medial NTS mNTS GABA was also microinjected bilaterally in seven
animals in which vagus nerves were not sectioned in order To study the possibility that the prolongation of T and
i
to determine the respiratory responses to microinjection of apneusis observed with GABA applied to the vlNTS was
GABA into the vlNTS in animals with intact pulmonary due to diffusion of the drug to the mNTS and affecting
reflex i.e.,
Hering–Breuer afferents.
As expected,
these neurons, GABA 25 nmol was microinjected bilater- baseline respiratory rate was significantly faster and T and
ally into the medial NTS of three animals at the level of
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T were significantly shorter in vagus nerve intact animals the area postrema. A comparison of the respiratory effects
e
than in vagotomized animals. After bilateral GABA mi- of bilateral administration of an equal dose of GABA into
croinjection, T was significantly prolonged and apneusis the mNTS and vlNTS of the same animal is shown in Fig.
i
occurred in three of seven animals Table 1. Although 3. Microinjection sites for the three animals in this group
these respiratory changes produced by GABA were quali- are depicted in Fig. 1. Overall, T was not significantly
i
tatively similar as to those observed in animals with vagus changed by inhibition of the mNTS Table 1. Only one of
nerves sectioned, the magnitude of the increase in T and three animals demonstrated any evidence of apneustic
i
the incidence of apneusis were significantly less than breathing and the apneusis generated in this case was
corresponding effects observed in animals with vagus quantitatively different from apneusis produced from the
nerves sectioned Table 1. Changes in T , mean blood vlNTS in that only three apneustic breaths occurred
e
124 A
Fig. 3. Effects of bilateral microinjection of GABA into the medial NTS mNTS; A–D followed by microinjection into the ventrolateral NTS vlNTS; E in the same rat. A Photomicrograph of bilateral mNTS microinjection sites. Arrows denote termination of identified pipette tracks. Bar50.25 mm.
Abbreviations as in Fig. 1. B Baseline recording of cardiorespiratory activity traces denoted in Fig. 1 Bar51 s. C Bilateral microinjection of GABA 25 nmol 45 nl into the mNTS. Right mNTS microinjection completed in preceding 30 s. Left mNTS microinjection of GABA occurs during the interval
between filled arrows. Note the immediate rise in blood pressure without alteration of inspiratory duration. D Cardiorespiratory activity 1 min after microinjection of GABA into the mNTS. E Sixteen minutes later, bilateral microinjection of GABA 25 nmol 45 nl into the vlNTS. Right vlNTS
microinjection completed in previous 30 s. Left vlNTS microinjection of GABA occurs between open arrows. GABA immediately produced apneusis. Time bar51 s.
A .M. Wasserman et al. Brain Research 880 2000 118 –130
125
subsequent to microinjection. Onset of apneusis was 15 s tetrodotoxin 0.22 mM; 45 nl into the region of the vlNTS
after completion of bilateral microinjection and the effect in nine animals produced a severe apneustic pattern which
was over by 30 s after administration Table 1. Our was quick in onset and long in duration Table 1; Fig. 5.
criterion, as discussed in Section 2, requires two or greater T was more prolonged with this treatment nearly 4-fold
i
apneustic breaths to qualify as apneusis. Therefore, this increase in duration than with GABA P,0.05 although
response was deemed apneusis though all incidences of baseline respiratory values in the two groups were not
apneusis observed with GABA microinjected into the significantly different. Decreases in T
and increases in
e
vlNTS involved dozens of apneustic breaths occurring over pressure and heart rate were similar to those observed in
a period of 127–195 s duration. The remaining two animals receiving GABA or kynurenic acid microinjec-
animals receiving GABA microinjections into the mNTS tions into the vlNTS. Another difference noted in the
had no evidence of any apneustic breaths. Expiratory cardiorespiratory response of a subset of animals microin-
duration decreased significantly Table 1 and resembled jected with tetrodotoxin as compared to animals receiving
changes observed after vlNTS microinjection. No signifi- GABA and kynurenic acid was a delayed reduction in
cant alterations were seen in the amplitude of the dia- iEMG amplitude as well as in mean blood pressure. In two
phragmatic electromyogram. Overall, blood pressure was of three animals in which this response occurred, a low
observed to increase significantly with mNTS microinjec- amplitude apneustic breathing pattern was observed to
tion of GABA 136, Table 1. This increase was similar develop into an apnea at the same time as this hypotensive
in magnitude to pressure alterations observed after GABA response and the animals died. In the other animal, blood
microinjection into the vlNTS region Fig. 3C versus 3E. pressure was low and iEMG amplitude was profoundly
In two animals, microinjection of GABA into the sites reduced but a fatal apnea did not develop; in fact,
lateral to the vlNTS Fig. 1 also had no effect on respiratory rate was observed to return towards the
cardiorespiratory function RR, 53 breaths min pre-in- baseline value.
jection to 56 breaths min post-injection; iEMG, no change; T , 379–445 ms; T , 717–613 ms; BP, 78–73
3.6. Vehicle microinjection into the ventrolateral nucleus
i e
mmHg; HR, 513–516 beats min. of the solitary tract
vlNTS 3.4. Blockade of excitatory amino acid
To control for possible effects of microinjection of 45 nl neurotransmission in the vlNTS
of volume into the vlNTS, the same volume 45 nl of 0.9 saline, pH 7.4 of vehicle was bilaterally microinjected
As direct inhibition of neurons in the region of the into the region of the ventrolateral nucleus of the solitary
vlNTS was capable of producing a rapid and severe tract while monitoring respiratory parameters in five
apneustic pattern of breathing, we sought to determine if animals. Bilateral microinjection of vehicle produced no
disfacilitation of neurons in this region could achieve a significant effects on respiration or cardiovascular function
similar result. Kynurenic acid is an antagonist at ionotropic when assessed 30 s after microinjection Table 1. Upon
glutamatergic receptors and microinjection of this com- injection there was a transient reduction in respiratory rate
pound would block basal glutamatergic input to the region. lasting less than 10 s in all cases and a mild reduction in
Kynurenic acid 22 mM; 45 nl was microinjected into the mean blood pressure which returned to pre-injection levels
region of the vlNTS bilaterally in five animals Fig. 1, all in three of five animals and remained slightly below
of which developed apneusis Fig. 4. In general, the time baseline in two animals.
course of the apneusis produced by kynurenic acid had a longer latency to onset and a longer duration of effect
Table 1, although, as shown in Fig. 4, onset in one case
4. Discussion
