K . Ezure, I. Tanaka Brain Research 883 2000 22 –30
23
lungs to collapse to atmospheric pressure is a specially lactated Ringer’s solution with 5 glucose was intraven-
useful maneuver to examine inputs from the rapidly-adapt- ously administered when necessary. Rectal temperature
ing pulmonary stretch receptors RARs [11,17,19] but has was maintained within 36–378C using a heating lamp. End
not been adopted for classifying respiratory neurons. In the tidal CO
was monitored and maintained at 4–5 by
2
present study, we found a group of inspiratory neurons adjusting tidal volume or frequency of a respirator.
which were characteristically activated by deflation of the lungs.
2.2. Recording and experimental protocol DRG neurons were recorded extracellularly with glass
2. Materials and methods micropipettes filled with 3 M KCl saturated with Fast
Green FCF dye DC impedance, about 1.0 MV. The Experiments were conducted on 14 adult Wistar rats
recording sites were marked with the dye. After fixation, 260–425 g. All experimental procedures were performed
serial frozen sections of the brainstem 100 mm thick in in accordance with the Guiding Principles for the Care
the frontal plane were made and stained with cresyl violet. and Use of Animals in the Field of Physiological Science
At the beginning of each experiment, we estimated the Physiological Society of Japan, 1988
. The experiments thresholds for orthodromic activation of the second-order
were reviewed and approved by the Animal Experiment relay neurons from SARs P-cells by vagal nerve stimula-
Committee of the Tokyo Metropolitan Institute for Neuro- tion. For this purpose we sampled at least five P-cells on
science. each side and measured their thresholds: these thresholds
Almost all the experimental procedures, such as surgery, for P-cells are about the same as or a little higher than
care of animals, vagal nerve stimulation, electrodes, neural those for the second-order relay neurons from RARs
recording, antidromic stimulation, or histological recon- RAR-cells see Refs. [16,17]. Stimulus intensity less
struction of the brainstem, were the same as those de- than two times the mean threshold normally less than 10
scribed previously [17,18,25,26], and are described here mA of P-cells was routinely used for vagal nerve stimula-
only briefly. tion.
The means of generating maintained lung inflation and 2.1. Surgical procedures
deflation were fully described previously [17,18]. Signals microelectrode potentials, phrenic nerve discharge, trache-
The rats were initially anesthetized with sodium pen- al pressure and blood pressure were stored on magnetic
tobarbitone Nembutal, 70 mg kg; i.p.; supplementary tape DAT recorder: PC-216A, Sony Precision Technolo-
doses about 5 mg kg per h; i.v. were given during gy, Tokyo; sampling rate 50 ms.
surgery and recording as necessary. The trachea was intubated, and cannulae placed in the femoral artery to
monitor blood pressure and in the femoral vein for drug
3. Results
administration. Bipolar cuff electrodes were attached to the cervical vagus nerves on both sides for stimulation, but the
This study was based on a total of 67 inspiratory nerves were not cut. The superior laryngeal nerve in six
neurons which were recorded from the nucleus tractus rats and the phrenic nerve C4 5 branch on one side were
solitarii NTS and its vicinity. Neural recordings were dissected, cut distally and the central ends were mounted
made in the area extending from 1.0 mm rostral to and 1.0 on bipolar cuff electrodes for stimulation and recording,
mm caudal to the obex defined as the caudal end of the respectively. The nerves prepared for stimulation and
area postrema, within 1.5 mm lateral to the midline and recording were kept in oil pools. In addition, stimulating
within 1.2 mm below the dorsal surface. In 28 of the 67 electrodes were inserted bilaterally into the ventrolateral
neurons their locations were marked and confirmed within funiculus at the rostral C4 level in nine rats.
the NTS area Fig. 1. The animals were placed in a stereotaxic frame and
occipital craniotomy and partial cerebellectomy were 3.1. Deflation-sensitive inspiratory neurons
conducted to expose the dorsal surface of the medulla. The head of the animal was slanted downward approximately
When we isolated an inspiratory neuron, we routinely 158 from the standard horizontal plane [29] to facilitate
examined its responses to electrical stimulation of the surgery and recording. The rats were paralyzed with
vagus nerve and to lung inflation and deflation. There were pancuronium bromide Mioblock, 0.4 mg kg h; i.v. and
inspiratory neurons n517 that were activated charac- artificially ventilated with oxygen-enriched air. A bilateral
teristically by lung deflation Fig. 2. During control pneumothorax was made and a positive end-expiratory
respiratory period these neurons fired regularly at the pressure PEEP of approximately 1.5–2.0 cmH O ap-
inspiratory phase, and their firing within the inspiratory
2
plied. Tracheal pressure was monitored continuously. phase was of an augmenting pattern. When the lungs were
Blood pressure was monitored and kept above 80 mmHg: collapsed to atmospheric pressure, these neurons exhibited
24 K
Fig. 1. Distribution of Ia, Ib and deflation-sensitive Ig neurons in the NTS. Distribution of 28 neurons are projected onto horizontal A and frontal planes B. Three planes a,b,c in B correspond to levels a,b,c shown in A. AP, area postrema; CU, cuneate nucleus; DX, dorsal motor nucleus of the
vagus; GR, gracile nucleus; S, solitary tract; 12N, hypoglossal nucleus.
transient firing which was locked to deflation of the lungs. 3.2. Ia and Ib neurons
The firing started at the onset of the deflation Fig. 3. During control respiratory period, especially when PEEP
Besides Ig neurons, several distinct types of respiration- levels were lowered, their firing at the deflation phase was
related neurons were found. Many P-cells and RAR-cells conspicuous Fig. 2Ad,Bd,C. These firing patterns were
were recorded from but were discarded in this study since similar to those of RAR afferents and RAR-cells [17,18].
their firing properties and distributions have been fully However, these neurons hardly responded to lung inflation
described previously [17,25]. We recorded from 50 in- Fig. 2Aa, although some weak firing at the initial part of
spiratory neurons consisting of Ia and Ib neurons Fig. 1. inflation was occasionally seen Fig. 2Ba. Tentatively, we
Ib neurons n59 were identified by 1 their activity at call these deflation-sensitive inspiratory neurons ‘Ig neu-
the inspiratory phase, 2 monosynaptic activation by rons’ see Section 4.
electrical stimulation of the vagus nerve, and 3 activity All of the Ig neurons n517 were orthodromically
evoked by lung inflation, specifically tonic activity by activated by electrical stimulation of the vagus nerve Fig.
maintained lung inflation Fig. 4. Orthodromic latencies 2Ae. The orthodromic latencies ranged from 1.9 to 2.5 ms
following vagus nerve stimulation ranged from 1.8 to 2.2 mean6S.D. 2.160.21 ms Fig. 6. The intensities of
ms mean6S.D. 2.060.11 ms Fig. 6. Two Ib neurons stimulation for these responses were within two times the
examined could not be activated antidromically from the thresholds for P-cell or RAR-cell activation see Section
spinal cord, and one of them was orthodromically activated 2. The majority 12 of 13 of the Ig neurons examined
from the SLN latency 3.0 ms. projected to the spinal cord as shown in Fig. 3D: they were
The other inspiratory neurons n541 were classified as antidromically activated from the ipsilateral 8 9 or
Ia neurons Fig. 5. Firing of many Ia neurons was not contralateral 6 7 spinal cord, two neurons being acti-
specially affected by lung inflation or deflation, although vated from both sides. Electrical stimulation of the su-
lung inflation or deflation tended to depress or facilitate perior laryngeal nerve SLN could not activate Ig neurons
their firing respectively in parallel with phrenic nerve 0 4. The Ig neurons were located in the NTS ventral or
discharge Fig. 5A. However, in a few neurons their firing ventrolateral to the solitary tract Fig. 1. They were
was depressed by lung deflation, facilitated by moderate distributed more caudally than Ia neurons see below.
lung inflation, but depressed by larger inflation Fig. 5B.
K . Ezure, I. Tanaka Brain Research 883 2000 22 –30
25
Fig. 2. Firing pattern of Ig neurons. Three Ig neurons A–C are shown. Traces are extracellular spike activity top, phrenic nerve activity middle, and tracheal pressure bottom. These inspiratory neurons are hardly activated by lung inflation Aa,Ba but consistently activated at lung collapse to
atmospheric pressure Ac,Bc. Pressure of maintained inflation is shown on each panel by number cmH O. These neurons are also activated at deflation
2
phase of artificial ventilation Ad,Bd,C especially when PEEP is lowered Ad2,C. Ad: responses at two different PEEP levels; C: responses during lowering PEEP. Ig neurons are orthodromically activated by electrical stimulation of the ipsilateral vagus nerve Ae. Ae: three superimposed traces of the
responses to vagal nerve stimulation.
Since these neurons were neither Ib nor Ig neurons, they Ig neurons were located more caudally. In general, it was
were all classified as Ia neurons. A few Ia neurons 5 41 easy to find Ia neurons but difficult to find Ib neurons,
were activated orthodromically by electrical stimulation of presumably because the population of Ib neurons is small.
the vagus nerve Fig. 6. The orthodromic latencies ranged In two experiments which focused on sampling Ib neu-
from 1.8 to 2.5 ms mean6S.D. 2.160.30 ms. Two of the rons, no Ib neurons could be found. Ig neurons could be
21 Ia neurons examined projected to the contralateral found more easily than Ib neurons.
spinal cord. Electrical stimulation of the SLN orthodromi- cally activated the majority 23 29 of the Ia neurons
examined. The orthodromic latencies ranged from 1.8 to
4. Discussion
