A .M. Wasserman et al. Brain Research 880 2000 118 –130
119
muscles for breathing [9,31,43]. The vlNTS respiratory incision below the chest cavity and inserting a hooked
neurons can also transfer afferent information from the bipolar platinum–iridium electrode to the right half of the
lungs to VRG bulbospinal inspiratory neurons [5]. How- costal diaphragm. The electrode was coupled to a Tek-
ever, data obtained for both the cat and rat indicate that tronix AM 502 differential AC preamplifier Tektronix,
this is not the role of the vlNTS neurons but is the function Wilsonville, OR. The output signal of the amplifier was
of neurons located in the medial subnucleus of the tractus fed into an audiometer and displayed on a storage oscillos-
solitarius mNTS [6,26]. In a recent study of the role of cope and computer monitor. Data were stored on computer
DRG neurons in control of respiration in the rat it was Apple Macintosh PowerPC connected to MacLab for
concluded based on cross-correlation analysis that DRG later viewing and analysis. The dEMG signal and the
neurons did not appear to convey respiratory rhythmic software-generated integration iEMG of the raw signal
drive to phrenic and intercostal motoneurons [42]. It was using a 100-ms time window were continuously recorded
also concluded that DRG neurons had little role in and stored for off-line analysis. Heart rate was obtained
transmitting afferent information from the lungs to VRG from the blood pressure signal and was averaged over
bulbospinal inspiratory neurons. eight beat bins to minimize beat-to-beat fluctuations.
Our previous data obtained in the cat suggest another possible role for the vlNTS, namely to act as an inspiratory
off switch and terminate the inspiratory phase of the 2.2. Stereotaxic surgery and microinjection procedure
respiratory cycle [4,15]. In these studies we found that microinjection of NMDA receptor antagonists into the
Animals were oriented ventral-side down and placed in vlNTS produced an increase in inspiratory duration and an
a Kopf small-animal stereotaxic frame Tujunga, CA. The apneustic breathing pattern. Since numerous investigators
dorsal medulla was exposed via a limited occipital have reported the existence of respiratory-related neurons
craniotomy with reflection of the dura and retraction of the in the vlNTS and adjacent interstitial subnucleus of the
cerebellum in an analogous fashion as described in the cat tractus solitarius iNTS of the rat [6,9,26,28,29,38,41,42],
[33]. Preliminary studies not shown using the calamus we set out to test the hypothesis that these neurons in the
scriptorius i.e., caudal tip of the area postrema as our rat can function as inspiratory off-switch neurons.
reference point were used to determine microinjection coordinates for the specific subnuclei of the NTS. Briefly,
Fast Green FCF dye 5 was microinjected into a series of coordinates and the resulting dye locations compared
2. Methods with known locations of various subnuclei according the
studies of Kalia and Sullivan [20] to determine the 2.1. General experimental preparation
coordinates used for our experiments. Coordinates used for vlNTS microinjections were the following: 0.5 mm rostral
Experiments were performed on 40 adult male Sprague– to calamus scriptorius; 0.9–1.1 mm lateral to midline;
Dawley rats Taconic, Germantown, NY weighing 260– 0.6–0.8 mm below dorsal surface of the brainstem.
410 g. Anesthesia was instituted with a 3-ml kg i.p. Coordinates for medial NTS mNTS injections were 0.5
injection of a cocktail containing urethane 800 mg and 0.4–0.6 0.4 mm, for rostrocaudal, mediolateral and dor-
a-chloralose 60 mg dissolved in 3 ml of 0.9 saline. soventral positions, respectively. Microinjection sites later-
Anesthesia was supplemented i.p. with the same anesthetic al to the vlNTS for the purpose of testing the site-spe-
cocktail in volumes of 0.1–0.2 ml as necessary to maintain cificity of our drug effects were made 0.5 mm rostral to
the animal areflexic to toe pinch. The trachea was cannu- calamus scriptorius, 1.3–1.4 mm lateral, and 0.7 mm deep.
lated to provide access to the airway, and for instituting Double-barreled ID 0.23 mm; FHC, New Brunswick,
artificial respiration when necessary. Cervical vagi were ME glass micropipettes were pulled and the tips cut to
exposed bilaterally and sectioned in 33 out of 40 animals approximately 30 mm outside diameter. Micropipettes
to prevent lung afferents from influencing central respirato- were then placed in a bracket fixed to the arm of an
ry rhythm. The remaining seven animals were left with electrode carrier and PE 90 tubing was connected to the
vagi intact to compare their cardiorespiratory responses to barrels. Drugs were loaded or ejected using negative or
microinjected drugs with those of vagotomized animals. positive pressure, respectively, generated by a syringe
The left carotid artery was isolated and PE 50 tubing was connected to the tubing. Drug volume ejected was approxi-
inserted to obtain blood pressure which was recorded using mately 45 nl 65 nl and was monitored and assessed
a bridge amplifier connected to a MacLab ADInstruments, using meniscus travel along calibration tape affixed to the
Milford, MA data acquisition system. Temperature was side of the pipette barrel. All drug solutions were dissolved
maintained at 37628C through use of an infrared heating in 0.9 saline and pH adjusted to 7.30–7.40. Microinject-
lamp. Changes in respiration were assessed by monitoring ed drugs used in these experiments were the following:
diaphragmatic electromyogram activity. The diaphragmatic GABA 0.5 M, RBI, Natick, MA, kynurenic acid 22
electromyogram dEMG was obtained by making an mM, RBI, tetrodotoxin 0.22 mM, RBI.
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
