Role in the inspiratory off-switch of vagal inputs to rostral pontine inspiratory-modulated neurons

Respiratory Physiology & Neurobiology 143 (2004) 127–140

Role in the inspiratory off-switch of vagal inputs to rostral pontine inspiratory-modulated neurons Morton I. Cohena,∗ , Chen-Fu Shawb a

Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA b Department of Biology. National Sun-Yat-Sen University, Kaushiung, Taiwan Accepted 30 July 2004

Abstract Neurons of the pontine respiratory group (PRG) in the region of the nucleus parabrachialis medialis and the K¨olliker-Fuse nucleus are believed to play an important role in promoting the inspiratory (I) off-switch (IOS). In decerebrate gallamineparalyzed cats ventilated with a cycle-triggered pump system (lung inflation during the neural I phase), we studied the effects of vagal (V) afferent inputs on firing of I-modulated neurons (the most numerous population in PRG) and on I duration. The predominant V effect on unit activity was inhibitory, as shown by two types of test: (a) withholding of inflation during an I phase, which produced increase of unit firing and of its respiratory modulation (58/66 neurons in 14 cats), indicating disinhibition due to removal of phasic V input; (b) delivery of afferent V stimulus trains during a no-inflation I phase, which produced decrease of unit firing and of its respiratory modulation (20 neurons). In addition, application of V input during the neural expiratory (E) phase, which lengthened E phase duration, produced no effect on the neurons’ firing, suggesting that the inhibition during I was presynaptic in origin. The results may be interpreted by the hypothesis that the medullary late-I presumptive IOS neurons receive excitatory inputs from the PRG I-modulated neurons as well as from V afferents.. With relatively strong V input, this pontine excitatory output is reduced by inhibition, whereas with relatively weak V input that excitatory output is increased due to reduction of inhibition. Thus the pontine and the V influences on the IOS can operate in a complementary manner dependent on state. © 2004 Elsevier B.V. All rights reserved. Keywords: Pontine respiratory group; Vagal afferents; Inspiratory off-switch; Lung inflation

1. Introduction

∗ Corresponding author. Tel.: +1 718 430 2280; fax: +1 718 430 8819. E-mail address: [email protected] (M.I. Cohen).

1569-9048/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2004.07.017

Among the many in vivo studies on generation of respiratory rhythm in the cat, a recurrent theme has been the relative role of different anatomico-functional neuron groups in the brainstem (Cohen, 1979; Bianchi et al., 1995). Within the brainstem, attention has been

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focused predominantly on groups in the medulla rather than in the pons, the border between pons and medulla being defined as the caudal border of the trapezoid body (Cohen, 1979). Yet, various lesion studies, and to a lesser extent stimulation studies, have indicated that pontine systems play an important role in respiratory rhythm generation. A definitive early study (Lumsden, 1923) used brainstem transections to distinguish different brainstem systems affecting respiratory pattern. It was found that a rostral pontine transection at a level just caudal to the inferior colliculus, when combined with vagotomy, produced a drastic change of respiratory pattern that was dubbed apneusis, consisting of a markedly prolonged plateau of I nerve discharge following the initial I ramp. This weakening of the I off-switch (IOS) was interpreted as indicating that rostral to the section there is a system promoting I termination, which was designated as the “pneumotaxic centre”. Ensuing investigations specified a more localized pontine region as the site of the pneumotaxic center: bilateral destruction of dorsolateral rostral pontine regions (nucleus parabrachialis medialis (NPBM) and K¨olliker-Fuse nucleus (KF)) was sufficient (together with vagotomy) to produce apneusis (St. John et al., 1971; Gautier and Bertrand, 1975; Caille et al., 1981). Subsequently, the region was designated as the pontine respiratory group (PRG) (Feldman, 1986). In addition, more recently a similar functional region has been found in the rat (Dick et al., 1995). The specification of this region in the cat led early investigators (Salmoiraghi, 1963) to search therein for respiratory-related neuron activities, but they failed to find such neurons. However, another study in vagotomized cats reported an abundance of such neurons in the PRG (Cohen and Wang, 1959); it was found that these neurons were predominantly of phasespanning and tonic respiratory-modulated types, in contrast to medullary neurons, which were predominantly phasic in pattern. Subsequently, other investigators (Bertrand et al., 1974; Dick et al., 1994) also reported the presence, mainly in vagotomized cats, of many respiratory-modulated neurons in the PRG. Although it was thought that the earlier difficulty of finding respiratory-modulated neurons in the PRG was due to deep anesthesia, it later turned out that the crucial factor was the inhibitory action of vagal afferent inputs.

The importance of vagal afferents in relation to the abnormal respiratory pattern of apneusis had been shown by the effects of imposed lung inflations, which produced inhibition of I discharge during the apneustic plateau either in a graded fashion or, if suitably timed, as a termination of the apneustic I phase (Kahn and Wang, 1967; Feldman and Gautier, 1976). Therefore, Feldman et al. (1976) conducted a study of the effects on PRG neurons of manipulating vagal inputs by changing lung inflation. The study was conducted in decerebrate cats ventilated with the cycle-triggered pump, where lung inflation was delivered during the period of neural I (synchronous with phasic phrenic discharge), thereby preserving the normal phase relation between respiratory efferent and pulmonary afferent discharges. In that study (Feldman et al., 1976), the effects of lung afferent inputs were evaluated by means of the no-inflation test, where during one neural I phase inflation was not delivered. PRG neurons were found whose firing in the control (inflation applied) state had little or no respiratory modulation, but whose firing during inflation withholding acquired appreciable respiratory modulation. Thus, with this maneuver a strong inhibitory effect of vagal afferent input was demonstrated, as indicated by the disinhibition that occurred during no-inflation. However, a later study from another laboratory (St. John, 1987) challenged the generality and magnitude of the earlier results of Feldman et al. (1976). Therefore, we undertook an expanded study of PRG respiratory-modulated neurons and obtained confirmatory results that were reported in a Ph.D. thesis (Shaw, 1990) and in several preliminary reports (Shaw et al., 1989; Cohen et al., 1993b, 2001). In the present paper, we report the quantitative analysis of vagal afferent influences on discharges of I and IE PRG neurons. (1) The discharges of PRG neurons showed several patterns of respiratory modulation, with the most prevalent patterns being tonic I- or IE-modulation. (2) Lung inflation input inhibited firing during the I phase, thus reducing respiratory modulation. (3) Similar effects were produced by afferent vagal electrical stimulation. (4) By cusum analysis of these responses, the latencies of inhibition were measured and were found to be quite long (median 187 ms), suggesting that the pathways for transmission of vagal afferent inputs to these neurons are multisynaptic and diffuse. (5) Hypotheses are suggested on the possible functional interactions of va-

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gal afferents and PRG neurons in promoting the inspiratory off-switch.

2. Methods 2.1. Preparation Experiments were done in midcollicular decerebrate cats (2.5–3.5 kg) having intact vagi. Surgery was performed under halothane anesthesia (induction at 5%, maintenance at 3–4%). Tracheal, venous, and arterial cannulae were inserted. Decerebration was performed with standard methods (Kirsten and St. John, 1978): ligation of the external carotid arteries, transection of the brainstem at the intercollicular level, removal of the forebrain by suction. To allow insertion of microelectrodes, two types of brainstem exposure were used: (a) dorsal approach (prone position): removal of the dorsal occipital, caudal parietal, and interparietal bones, and in some cases removal of portions of the cerebellum; (b) ventral approach (supine position) (See et al., 1983): removal of the basal portion of the occipital bone at pontine and medullary levels. The animals were paralyzed by intravenous infusion of gallamine triethiodide (5 mg/kg h) and ventilated with mixtures of 0–3% CO2 in oxygen by means of a cycle-triggered pump. End-tidal CO2 was monitored by an infrared CO2 analyzer and maintained at 4–6% by adjusting the CO2 fraction in the delivered gas mixture. Bilateral pneumothorax was produced; and an expiratory load of 1–2 cm H2 O was applied to prevent lung collapse. Femoral arterial blood pressure was recorded and was maintained above 80 mmHg systolic by intravenous infusion of 0.9% NaCl containing 5% glucose (4 ml/kg h). Rectal temperature was maintained in the range 37–38 ◦ C by a feedback-controlled heating circuit. 2.2. Recordings After completion of surgery the halothane was disconnected, and the animal was allowed to recover for at least 2 h before recordings were taken. The following were recorded with an analog magnetic tape recorder or a videocassette recorder (via a digital interface): (1) Phrenic nerve potentials. Both phrenic nerves were sectioned in the neck and the central end of each was

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mounted on a bipolar electrode, with the cut end being crushed on the peripheral leg of the electrode in order to obtain a monophasic recording. The recording amplifier bandpass was 0.8–10 kHz. The nerves were immersed in a mineral oil pool contained in a skin pouch. An integrated phrenic signal, obtained by subjecting the original signal to half-wave rectification and low-pass filtering (time constant 100 ms) was continuously displayed on an inkwriter (polygraph). (2) Microelectrode unit recordings. Action potentials of individual neurons were recorded extracellularly (amplifier bandpass 0.8–10 kHz) from tungsten microelectrodes (impedances 2–5 M at 1 kHz). (3) Intratracheal pressure measured via a sidearm of the tracheal cannula. (4) Femoral arterial blood pressure. Tag signals recorded were: (5) Respiratory phase tags, marking the onsets of the inspiratory (I) and expiratory (E) phases, derived from the phrenic potentials (Cohen, 1968). (6) Tags marking unit spikes, derived with a time-amplitude discriminator. (7) Tags marking stimulus pulses. (8) Tags marking test sequences. 2.3. Inflation tests The I and E pulses together with the associated I and E gates (Cohen, 1968) were used to control ventilation by means of a cycle-triggered pump system (Feldman and Gautier, 1976; Cohen and Feldman, 1984). The system consisted of a low-pressure gas source (5–10 cm H2 O) whose flow was directed by two logic-controlled solenoid valves. In the control state of ventilation, lung inflation was applied during neural I (the time of occurrence of the phrenic discharge burst), thus preserving the normal phase relation between lung expansion and the central I phase. By changing the timing of activation of the solenoid valves, various test patterns of lung inflation (and of the consequent vagal afferent discharge) were produced. The test protocol consisted of application of a sequence of tests in which each test was delivered about every 7–10 respiratory cycles in order to allow recovery between tests. 2.3.1. No I-inflation test (Cohen and Feldman, 1984) This test consisted of withholding lung inflation during one I phase, so that intratracheal pressure remained at the level existing at the end of neural E and therefore the phasic vagal afferent discharge during neural I was

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abolished. Then, at the start of the next neural I lung inflation was again applied. 2.3.2. Inflation tests during E (Cohen et al., 1985) Two types of test were used: (a) Dynamic Einflation. An inflation having the same duration and amplitude as that delivered in the control I phase was delivered at a predetermined delay from the start of the E phase. (b) Maintained E-Inflation. At the end of an I phase, the inflation delivered during I was maintained by preventing exhalation for a fixed time period, usually twice the duration of the control E phase, at the end of which the lungs were allowed to deflate by opening the exhalation valve. 2.4. Vagus nerve electrical stimulation Electrical stimulation was applied via a bipolar electrode to the central end of a separated vagus bundle (diameter one-third to one-half of the intact nerve’s diameter). Stimulus pulse duration was 0.05 ms, stimulus current was 10–50 ␮A, and stimulus frequency was 100 Hz. Each stimulus train was started at a fixed delay (usually 100 ms) from the start of I (I tag) and was terminated at the end of I (E tag). In the most commonly used protocol for applying vagal afferent stimulation, the test sequence consisted of alternation of: (a) no I-inflation tests with (b) (noinflation + stimulation) tests. In the latter type of test, an afferent vagal stimulus train was delivered during a no-inflation I phase, the stimulus current having been adjusted to a level that produced I phase durations comparable to those of the inflation I phases. 2.5. Data analysis The recorded data were played back to an A–D converter (RC Electronics), digitized at a sampling rate of 2500 Hz (0.4 ms bin duration), and subjected to customized analysis programs written with Turbo Pascal. 2.5.1. Autocorrelation histograms (ACH) In order to verify that the spikes in a recorded spike train were derived from a single neuron, the ACH was computed. Since after each spike the neuron cannot fire during the absolute refractory period (1–2 ms), the

presence of an appreciable number of spikes within corresponding bins of the ACH (>5% of the total number in the train) indicated contamination by spikes of another neuron, and therefore the recording was discarded. 2.5.2. Respiratory cycle-triggered histograms (CTH) The CTH for a unit or phrenic signal during a particular experimental state consisted of the ensemble average of the activity, based on the appropriate I or E tags as the synchronizing triggers (n = at least 15 cycles). At the start of each CTH, a number of pre-trigger bins was included, usually comprising 100–200 ms. The unit signal consisted of a “1” marking spike occurrence. The phrenic signal consisted of the original signal after high-pass filtering (3 db point at 40 Hz) followed by half-wave rectification, in order to avoid cancellation of action potentials. In order to obtain a CTH with total of bin durations corresponding to duration of a respiratory phase or cycle, 40 ms bins were used; the signal in each bin consisted of the sum of activities in the original sampling bins (e.g. data counts in 100 adjacent 0.4 ms bins were summed to give the counts in a 40 ms bin). For each type of test, two CTHs (control and test) were constructed for each signal. For the no I-inflation test, the control CTH was based on I phases in which lung inflation was applied, and the test CTH was based on I phases in which inflation was withheld. For Etriggered CTHs, the control and test phases were based on E phases without or with, respectively, application of inflation. For the tests with vagal stimulation, the control CTH was based on I phases in which lung inflation was withheld, and the test CTH was based on I phases in which stimulation was applied during withholding of inflation. The effects of a test on I phase duration and on unit activity were evaluated by the unpaired t-test, comparing control versus test cycles contributing to each CTH, with P < 0.05 considered as significant. Since the phase durations differed between cycles, the variable used for comparison of unit activity was the number of spikes in each phase that occurred in an equivalent-time window that started at I onset and had a duration equal to the shortest I phase in the sample (including both control and test phases). In order to ascertain whether a recorded unit’s discharge had significant respiratory-related rhythm,

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the η2 -test of Orem and Dick (1983) was used. This method consists of a two-way analysis of variance that compares, for a whole respiratory cycle divided into fractions, the between-fraction and between-cycle variances. In the present study, the method was useful for evaluating difference of discharge pattern between respiratory-unrelated and respiratory-related states (the latter being produced by no-inflation). 2.5.3. Peristimulus histograms (PSH) The PSHs for test phases (stimulation applied) were ensemble averages of the signals (phrenic, unit tags, and stimulus tags) that were computed using the first stimulus of each train as the synchronizing trigger and that included data starting with a pre-stimulus period (100–200 ms) and ending at a time after the end of the I phase. In addition, the data from control (no stimulation) phases were used to compute a control PSH, using as the synchronizing trigger a “dummy” tag whose location in each control phase was the same as the location (time from I onset to stimulus train onset) of the trigger in each stimulation phase. 2.5.4. Cusum analysis In order to ascertain the latency of a unit’s response to stimulation, a variant of cusum analysis (Davey et al., 1986) was used. Since changes of activity due to stimulation were superimposed on a background of spontaneous activity, the histogram used to derive the cusum was the difference histogram (consisting, for each bin, of number of counts in the stimulation PSH minus number of counts in the control PSH). Thus the number of counts in the cumulative difference histogram for each bin (k) was: Ck =

k


[Ni (Stim) − Ni (Ctl)],

i=1

where Ni (Stim) = number of counts in bin i of the stimulation PSH, and Ni (Ctl) = number of counts in bin i of the control PSH. In addition, the mean and S.D. of the number of counts in the cumulative baseline histogram were computed, where the baseline window consisted of the portion of data immediately preceding (100–200 ms) the first stimulus of the train. Then the response latency was taken as the time bin where the bin count of the cumulative difference PSH first became > (baseline mean + 3S.D.), denoting excita-

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tion, or became < (baseline mean − 3S.D.), denoting inhibition (cf. Fig. 6). 2.6. Histology In eight cats, marker lesions were made by passing high-frequency or direct current (100 ␮A for 15 s) through the recording electrodes. The brain was removed after carotid artery perfusion and subsequently serially sectioned (40 ␮m). After staining of sections with cresyl violet, microscopic examination allowed verification of lesion locations with the help of a brainstem atlas (Berman, 1968).

3. Results Experiments were done in 24 midcollicular decerebrate, gallamine-paralyzed cats having intact vagi and ventilated with a cycle-triggered pump system (lung inflation applied during the neural I phase). Extracellular unit recordings were taken with microelectrodes inserted into the rostrolateral pontine region, located near the caudal border of the inferior colliculus, which has been designated as the site of the pneumotaxic complex or pontine respiratory group (PRG). The region explored extended rostrocaudally between 2 mm rostral and 2 mm caudal to the foramen caecum level, 3.5–5.5 mm laterally to the midline, and 1–5 mm ventrally from the dorsal surface of the brainstem. The electrodes were advanced into the brain stem with either a ventral or dorsal approach, in the latter cases with or without removal of the overlying cerebellum. During microelectrode advancement, the occurrence of respiratory rhythm in unit spike activities was ascertained by comparison with phrenic activity, using an audio monitor; and no-inflation tests (inflation withheld during an I phase) were applied every four to eight cycles, in order to disclose a unit’s respiratory firing rhythm that otherwise might not be manifested due to the inhibitory effect of afferent vagal activity produced by lung inflation. Recorded unit patterns were classified by examination of phrenic and unit CTHs derived using the inspiratory (I) or expiratory (E) trigger. Neurons were classified firstly on the basis of the portion of the cycle where discharge frequency was highest: I, E, or phasespanning. Phase spanning neurons had their maximum

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Table 1 Discharge patterns of rostral lateral pontine respiratory-modulated neurons in 24 vagally intact cats Discharge pattern

No. of neurons

Inspiratory-modulated Phasic inspiratory Tonic inspiratory Tonic inspiratory–expiratory

111 20 40 51

Expiratory-modulated Phasic expiratory Tonic expiratory Tonic expiratory–inspiratory

32 7 4 21

Total

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firing around the inspiratory to expiratory (IE) or expiratory to inspiratory (EI) phase transition, the distribution of firing being approximately symmetrical around the transition. The second criterion for classification was whether firing was phase locked (P: sharp onset of firing at the beginning of the I or E phase as indicated by phrenic discharge, with little or absent firing in the complementary phase) or tonic (T: presence of continuous discharge throughout the cycle, with increased firing during a portion of the cycle). The incidence of the different discharge types among the 143 neurons recorded in 24 cats is shown in Table 1. The neurons were divided into two broad categories according to the predominant time period of firing: I-modulated (n = 111, 78%) and E-modulated (n = 32, 22%) and further into subcategories according to additional features (e.g. tonic versus phasic). The most numerous types recorded were the tonic I (n = 40, 28%) and tonic IE (n = 51, 36%). The distinction between the latter two patterns was based on the shape of the I modulation of tonic discharge: for the I type the firing near the end of neural I was ramp-like, whereas for the IE type it was symmetrical around the end of neural I. The anatomical locations of the recorded neurons of various types are shown in the histological diagram of Fig. 1. The histologically verified locations were consistent with their specification by stereotaxic coordinates, a finding which supports the validity of the latter type of identification in animals where histology was not done. It can be seen in Fig. 1 that the neurons were located predominantly in the regions of nucleus parabrachialis and K¨olliker-Fuse nucleus; but there was no clear segregation of firing types, in particular be-

Fig. 1. Anatomical locations of 85 respiratory-related pontine neurons in eight cats, as indicated by microscopic examination of lesion sites. Types of neuron: T-I (filled oblong): tonic I-modulated; P-I (empty oblong): phase-locked to onset of I phase; T-IE (X): tonic IE-modulated; T-E: tonic E-modulated (+); P-E (+): phase-locked to onset of E phase. Anatomical structures depicted according to Plate 18 of Berman (1968). Abbreviations: BC, brachium conjunctivum; BP, brachium pontis; KF, K¨olliker-Fuse nucleus; NPBM, nucleus parabrachialis medialis; P, pyramidal tract; TB, trapezoid body.

tween phasic and tonic I-modulated types, as had been reported by Bertrand et al. (1974). 3.1. Effects of lung inflation on pontine neuron discharges In order to analyze the effects on neural activity of phasic vagal afferent discharges produced by lung inflation, we used the no-inflation test in animals ventilated with a cycle-triggered pump system. With this system, phasic lung inflation is delivered coincidentally with phrenic discharge; and the no-inflation test consists of withholding this inflation during one neural I phase, with adequate time left for recovery before application of a subsequent test. As reported in an earlier study (Cohen et al., 1986), recordings from an afferent vagus nerve show a phasic discharge component during the lung inflation, as well as its absence (i.e. tonic discharge) during withholding of inflation. An example of the effects of the no-inflation test on I phase duration and on firing of a pontine IE tonic neuron is shown in the CTHs (n = 23 trials) of Fig. 2. The intratracheal pressure signal (ITP) shows a steady

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Fig. 2. Example of the effects of lung inflation, indicated by intratracheal pressure (ITP) signal, on I phase duration and on discharges of phrenic (PHR) nerve and a pontine IE-modulated unit. Superimposed CTHs for: control (C) cycles (thin lines, INFL.), when inflation was delivered during neural I, and for test (T) cycles (thick lines, NOINFL.), when inflation was withheld during I; these were derived using 23 I triggers (vertical dotted line) for each CTH. Horizontal arrowed lines: thin line, mean inflation I phase; thick line, mean noinflation I phase. Horizontal bar: comparison window, starting at I onset and having the duration of the shortest I phase among the C and T I phases of the test series.

increase of lung inflation during control (inflation) cycles (thin line) and the lack of phasic pressure change during no-inflation cycles (thick line). It can be seen that no-inflation produced an increase (by 17%) of mean I phase duration (from 698 to 820 ms), or equivalently inflation produced a 15% decrease of duration. The change of unit activity produced by no-inflation was quantified by the ratio of number of spikes (noinflation/inflation) in the comparison windows (duration of shortest I phase in the sample, in this case 642 ms), indicated in Fig. 2 by a horizontal bar. (The purpose of using this type of window was to assure that spike counts were ascertained for equivalent times in the inflation versus no-inflation I phases.) For this unit, the ratio was 1.47, indicating that no-inflation produced a 47% mean increase in firing, or equivalently inflation produced a 32% decrease in firing during equivalent times. The CTHs of unit activity show that during inflation cycles (thin line) the unit had a very weak respiratory modulation; and that during no-inflation (thick line) the unit acquired a strong respiratory modulation with an obvious IE firing pattern. However, η2 -analysis (Orem and Dick, 1983) showed that even the weak modulation during inflation (η2 = 0.09) was statistically highly significant (P < 0.01), and that the larger modulation during no-inflation (η2 = 0.74) led to a stronger significance level (P < 0.001).

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Fig. 3. For the population of 58 I-modulated neurons (in 14 cats) whose firing was changed by the no I-inflation test, distribution of responses (percent decrease produced by inflation): ordinate: mean I phase duration; abscissa: mean number of spikes in comparison windows. Filled circles: values for 13 neurons in one cat; empty circles: values for 45 neurons in the other 13 cats.

The distribution among the studied population of Imodulated neurons (n = 58 in 14 cats) of change of I duration and of unit firing (during comparison windows) in response to the no-inflation test is shown in Fig. 3. The significance of changes between the inflation and no inflation conditions was calculated by the two-sided unpaired t-test, with P < 0.05 regarded as significant. It can be seen that there was considerable dispersion of the values of decrease of I phase duration produced by inflation (range −15 to 59%, median 20%), including two cases where there was no significant change and one case where there was an increase. For eight additional neurons (not indicated in Fig. 3), the inflation test produced no significant change of unit firing, while for the 58 neurons with significant changes there was considerable dispersion of the values of the decrease of unit firing (range 11–89%, median 34%). Most commonly, the relative reduction of unit firing was greater than the relative reduction of I duration, as indicated by the distribution of points relative to the 45◦ line: 42 of 58 points (72%) were below this line. Furthermore, η2 -analysis showed that for 10 of the 58 neurons there was no significant respiratory modulation (P > 0.05) during inflation, but that during

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no-inflation respiratory modulation became significant (P < 0.05). A major source of the dispersion of relations between the two variables of Fig. 3 was the lumping of effects obtained from different neurons. This is revealed in Fig. 3 by separately indicating the observations in 13 neurons from one cat (filled circles), which show a much lower dispersion than the 45 observations (empty circles) from the remaining 13 cats. Effects of inflation applied during the E phase. In order to compare unit responses to inflations delivered during the E versus the I phase, we applied E-inflation tests of two types (dynamic inflation and maintained inflation) while recording from 26 I-modulated neurons which had manifested inhibition by I-inflations. In all cases, lung inflation during E produced the expected lengthening of E phase duration. However, for 25/26 neurons, the resultant E-triggered CTHs showed no significant change in firing (P > 0.05) during comparison windows in E, while 1 neuron showed a slight but significant excitation. Thus, the immediate responses to inflation were confined to the I phase. 3.2. Effects of vagal afferent stimulation on PRG neuron discharges In order to provide additional analysis of the action of vagal afferents on PRG neuron discharges, an experimental protocol was used in which afferent vagus electrical stimulation was applied during no-inflation I phases, thus allowing for comparison of responses to inflation and to stimulation and more importantly allowing the measurement of response latencies. An example is given in Fig. 4 for another IE-modulated neuron in another cat. The effects of no-inflation, which are seen in the CTHs (derived using 22 I triggers) of Fig. 4A, are very similar to those of the unit of Fig. 2. For I duration, noinflation produced a mean increase of 14% (from 634 to 719 ms), equivalent to a decrease of 12% produced by inflation; and for unit firing in the comparison window (duration 571 ms), no-inflation produced a mean increase of 62%, equivalent to a decrease of 38% produced by inflation. The increase of modulation was indicated by η2 -analysis, which showed that no-inflation increased η2 from 0.25 to 0.74, with P < 0.001 for both conditions.

Fig. 4. Example comparing the effects of withholding inflation (A), and of afferent vagal stimulus trains delivered during no-inflation I phases at a fixed delay from I onset (B), on I phase duration and on discharges of phrenic (PHR) nerve and an IE-modulated unit. (A) Superimposed CTHs (obtained with 22 I triggers) showing effects of no-inflation alone; format as in Fig. 2. (B) Superimposed CTHs (obtained with 22 I triggers) showing effects of afferent vagal stimulation (ST, bottom horizontal line) during no-inflation I phases, for: control (C) cycles (thin lines, NO INFL.), when no stimulation was delivered, and for test (T) cycles (thick lines, NO-INFL. + VAGAL STIM.), when stimulation was delivered. Horizontal arrowed lines: thin line, mean no-inflation I phase without stimulation; thick line, mean no-inflation I phase with stimulation. Horizontal bar: comparison window, starting at I onset and having the duration of the shortest I phase among the C and T I phases of the test series. Note that T phases (no-inflation only) for A are the same as C phases (noinflation without stimulation) for B; but that the C phases in A are not identical with the T phases in B. Therefore the comparison windows differ between A and B. In ST horizontal line, dashed-dotted portion indicates distribution of I offsets (E tags) for individual I phases with differing durations between the minimum and maximum values of I duration.

In Fig. 4B, the effects of vagal stimulus trains (starting at 119 ms after I onset) delivered during no-inflation I phases can be seen in the CTHs (derived using 22 I triggers). Stimulation produced a decrease of 13% in

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Fig. 5. Comparison of effects of the two types of maneuver used to test vagal afferent inputs, for the population of 20 I-modulated neurons (in three cats) that were subjected to both types: (A) vagal electrical stimulation during no-inflation I phases (as in Fig. 4B), (B) no-inflation only, as in Fig. 4A. Distributions of duration and unit responses (percent decrease produced by stimulation or inflation) are shown as filled circles—ordinate: mean I phase duration; abscissa: mean number of spikes in comparison windows.

I duration (from 719 to 624 ms), together with a decrease of 61% in number of spikes during the comparison windows (duration 588 ms). Thus the delivery of vagal stimulation changed mean I duration and mean number of spikes in the comparison window to levels comparable to those during inflation I phases. The protocol of Fig. 4 (vagal stimulation during no-inflation I phases) was applied to 20 I-modulated neurons recorded in three cats. In Fig. 5, the distributions among this population of change of I duration and unit firing (during comparison windows) are compared between the two types of test. For vagal stimulation (Fig. 5A), the reduction of I phase duration ranged from 9 to 31%, with median of 16%; and reduction of unit firing ranged from −14 to 69%, with median of 50%. (For one unit, the reduction of −14% denoted an increase of 14%.) For no inflation alone (Fig. 5B), the reduction of I phase duration by inflation ranged from 11 to 30%, with median of 22%; and reduction of unit firing ranged from 0.4 to 56%, with median of 28%.

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In order to ascertain the latency of the vagally evoked response in a neuron’s activity, a special analysis was required. This necessity arose from several features: (a) The evoked change of unit activity was superimposed on a background of spontaneous activity. (b) Usually stimulation did not produce a distinct shortlatency response; rather there was a gradual change of activity over a time period following the onset of a stimulus train, during which many individual stimuli were delivered. Therefore, the data were analyzed by means of the cusum (cumulative sum) difference PSH, derived as follows. (1) For each neuron, a stimulation (stim.) and a control (no-stim.) PSH were constructed. For the former (derived from activity in stim. I phases), the synchronizing trigger was the first stimulus tag in each stimulus train; for the latter (derived from activity in no-stim. I phases), the synchronizing trigger was a “dummy” tag located at the corresponding time in the control I phase, i.e. at a time equal to the stimulus onset delay from I onset in the stim. I phase. These PSHs included data from the pre-trigger period, which was the time from I onset to the stim. or dummy tag. (2) The difference PSH was constructed, each bin of which consisted of the spike count in a bin of the stim. PSH minus the spike count in the corresponding bin of the no-stim. PSH. (3) The cusum (cumulative sum) PSH was computed from the difference PSH by summing the counts in successive bins starting at the first bin (corresponding to I onset), and then normalizing the bin values to spikes per second. A depiction of the procedure for ascertaining latencies is given in Fig. 6 for the response of the unit of Fig. 4. The cusum difference PSH, computed as described above, indicates the temporal pattern of spike occurrence in 22 stim. and 22 non-stim. I phases; the monotonically increasing negativity of this curve indicates that the stimulus effect was inhibitory. The baseline or control level of the histogram was derived from the number of counts for the control (CTL.) window, which comprised the time between I onset and stim. onset (119 ms). From the bin counts in this window, there were computed: the mean (thick horizontal line), the standard deviation (S.D.), and the mean ±3S.D. (thin horizontal lines). The response latency in this case was designated as the time after stim. start when the bin value became less than (mean − 3S.D.) and had a value of 188 ms. The minimum stimulus train duration (472 ms) is indicated in Fig. 6 (bottom) by the vertical

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vagal afferent transmission from medulla to pons is mediated by diffuse multisynaptic pathways.

4. Discussion

Fig. 6. Example of measurement by cusum analysis of the unit response latency (for the IE-modulated unit of Fig. 4) to afferent vagal stimulation, using PSHs consisting of summed counts of spiketriggered tags in 0.4 ms bins. Synchronizing triggers (n = 22 for each PSH) are: (a) for the stimulation (STIM.) PSH, the first stimulus tag (STIM. START) of each stimulus train, occurring in this case 119 ms after I onset; (b) for the no-stimulation or control (CTL.) PSH, a tag occurring at a corresponding, time in each no-stimulation I phase. The figure shows the cumulative difference histogram (cf. Section 2 and text for details) derived from the bin values of the STIM. PSH minus those of the CTL. PSH, for the time period between I onset and end of I. The CTL. Window is the time between I START and STIM. START (119 ms); and for the bin values in this portion of the cumulative PSH, the mean (horizontal thick line) and the mean ±3S.D. (horizontal thin lines) were calculated. The time bin in the PSH where the bin value first becomes < (mean − 3S.D.) is taken as the onset of significant inhibition, and thus the latency (188 ms) is the time between that onset and STIM. START. The duration of the stimulus (STIM.) trains is indicated (bottom of figure) by a vertical step followed by a declining ramp. Since in the stimulation protocol the stimulus train in each I phase was stopped at the end of I (I ENDS, vertical lines), the vertical step indicates the duration of the minimum stimulus train (STIM. WINDOW = 472 ms), and the decrementing ramp indicates times of cessation of longer stimulus trains).

step. Since for each of the 22 stim. trials in the series the stimulus train was ended by the E tag marking end of I (range of I ENDS, vertical lines), there was a range of longer stimulus trains in the series, as indicated by the negative ramp following the step stimulus marker. By use of the cusum analysis illustrated in Fig. 6, the latencies of the responses by I-modulated neurons to afferent vagal stimulation were determined. The range of latencies for the 20 neurons was 12–345 ms; if one excludes two outliers consisting of the cases with the maximum and the minimum latency value, the range of 18 latency values was 50–282 ms. The median latency for the total sample was 187 ms, and 9/20 neurons had latencies between 100 and 200 ms. This suggests that

In this study, the aim was to characterize the functions of rostral pontine respiratory-modulated neurons in the control of the inspiratory off-switch (IOS). The involvement of PRG neurons in such control was suggested by the results of dorsolateral rostral pontine lesions, which produce weakening of the IOS that is manifested as appearance of an apneustic pattern. Moreover, the crucial importance of pulmonary afferent (vagal) inputs was shown in studies where imposition of such inputs antagonized the apneustic pattern (Kahn and Wang, 1967; Feldman and Gautier, 1976). But yet, some earlier studies which compared unit discharges before and after vagotomy (Caille et al., 1984) or with and without lung inflation (Eldridge and Chen, 1992) did not provide sufficient quantitative analysis of unit responses. We therefore conducted the quantitative study reported here. The animals were ventilated with the cycle-triggered pump, where lung inflation was applied synchronously with the phrenic burst, thus preserving the normal phase relation between efferent and afferent discharges. At intervals separated by 7–10 cycles, inflation was withheld during an I phase (designated as the no-inflation I phase), and the activities in a number of inflation and no-inflation phases (usually > 15) were averaged to obtain CTHs triggered from the I phase onsets. A number of features of the testing and analysis protocols assured the validity and accuracy of the results. (1) The separation in time between individual tests, which allowed recovery between tests, avoided long-term changes of state within each experimental series, as verified by examination of the blood pressure and CO2 signals. (2) The short duration of the no-inflation maneuver (1–2 s) assured that there was no immediate change of blood pressure or CO2 level. (3) During the no-inflation I phase the level of afferent activity did not show any trend and had a magnitude that was the same as during the end of the E phase, an observation made in an earlier study (Cohen et al., 1986) where recordings from afferent vagus bundles were taken during no-inflation I phases.

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A crucial feature of the analysis of changes produced by afferent vagal inputs was the comparison between discharges during equivalent times in inflation versus no-inflation I phases. This comparison window had a duration equal to that of the shortest I phase (inflation or no-inflation) in the test series. The comparison of number of spikes in the inflation versus no-inflation CTHs, as evaluated statistically (unpaired t-test, P < 0.05), thus indicated the primary effect of the vagal input. With this method, effects that occurred in later portions of the longer I phases (e.g. during no-inflation) were considered secondary, since those portions occurred only after the times of the comparison windows. The predominant type of response to no-inflation tests shown by the I- and IE-modulated neurons (the most numerous PRG neuron population) was disinhibitory, i.e. increased firing during no-inflation I phases, which was due to removal of the normally inhibitory input of vagal afferents. This response was shown by 58/66 neurons tested. An important observation was that the vagal inhibitory effect is gated to occur only in the I phase, since inflations delivered during the E phase had no effect in 25/26 neurons. This means that the vagal inputs must be acting presynaptically to the neurons recorded, for example to inhibit or disfacilitate another population of neurons whose action is producing the inspiratory excitation of the recorded neurons. On the basis of the observations described in this paper, we present in Fig. 7 a hypothetical schema of the interactions of vagal afferents and PRG neurons in relation to the IOS. The schema depicts the crucial hypothesis (Cohen and Feldman, 1977) that a specialized population of late-I medullary neurons (Oku et al., 1992; Cohen et al., 1993a; Haji et al., 2002) mediates the IOS. This hypothesis is based on the observations that in no-inflation tests and in vagal electrical stimulation tests such neurons were excited together with shortening of the I phase (Cohen et al., 1993a), and in another study they were excited by NPBM stimulation (Haji et al., 2002). The schema depicts three types of excitatory input (symbol: circle) to the late-I neurons: (1) from the insp. ramp generator, which provides the basic augmenting I pattern; (2) from vagal pulmonary stretch receptors (PSRs), as indicated by inflation and stimulation tests (Cohen et al., 1993a); (3) from the PRG tonic-I neurons, as suggested by the effects of stimulation in the PRG region (Cohen, 1971; Haji

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Fig. 7. Schematic diagram of hypothetical connectivity of systems involved in promoting the inspiratory off-switch (IOS). Filled circles: excitatory action. Filled diamonds: inhibitory action. See text for details.

et al., 2002) and by the weakening of the IOS after lesions of the region. The inhibitory output (symbol: diamond) from the late-I neurons to the I ramp neurons produces the IOS when a threshold is reached. The schema of Fig. 7 also shows the origin of vagal PSR afferent input: The medullary I-ramp neurons eventually activate (dashed line) spinal motoneurons and thoracic muscles that produce lung expansion and consequent excitation of vagal pulmonary stretch receptors (PSRs). The vagal afferents project (via pathways of various kinds), to two different systems: (a) the medullary late-I neurons, which are excited; (b) the PRG tonic I-modulated neurons, which are inhibited. In addition, the I ramp neurons transmit excitation to pontine tonic neurons to produce I modulation of firing superimposed on tonic firing. Two major loops controlling the IOS are shown in Fig. 7. The first is a medullary loop consisting of insp. ramp generator, vagal PSRs, and late insp. neurons. The second is a medullary-pontine loop consisting of insp. ramp generator, vagal PSRs, pontine tonic I-modulated neurons, and late-insp. neurons. We can hypothesize

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that under different physiological conditions one or the other loop becomes dominant. At first glance, the observation that vagal afferent input is inhibitory to firing of the pontine I-modulated neurons but is excitatory to their presumptive target neurons (medullary late-I neurons) seems paradoxical. But this inconsistency can be explained if one considers that the two types of input (vagal and pontine) to the IOS neurons may be preferentially active in different states of respiratory rhythm. Thus, in a state where lung volume strongly influences the rhythm, the excitatory effect on late-I neurons is dominant, but the inhibitory effect on pontine neurons is relatively unimportant because it acts only to weaken their inspiratory modulation while having little effect (due to I gating) on the tonic portion of their discharge. Conversely, in a state where lung volume influence is reduced (e.g. after vagotomy) the removal or weakening of the phasic inhibitory input allows the development of a strong inspiratory ramp discharge (derived from the medullary excitatory input), in the pontine neurons, which in turn excites the late-I neurons towards the IOS threshold. Another condition where the pontomedullary loop may become more important is when there is an increase of tonic excitatory input to the pontine neurons from other sources (e.g. nociceptive or hypothalamic); this would counteract the vagal inhibitory input, allowing an increased excitation of the late-I neurons and a consequent earlier onset of the IOS and increase of respiratory frequency. The evidence from lesioning in the PRG region (St. John et al., 1971) indicates that neurons in the region that project to medullary respiratory-related regions have an important influence on the IOS. However, antidromic stimulation (Bianchi and St. John, 1982) reveals that though there are numerous such projections relatively few of these are from respiratory-modulated neurons. Thus it is possible that a significant influence on the IOS is exerted by non-respiratory-modulated neurons. But with the methods used in this study we were only able to characterize non-modulated neurons that became modulated (together with change in overall respiratory output) when phasic vagal input was reduced or eliminated; but we did not characterize tonically firing neurons that could be affected by other inputs. The latter type of neuron could affect respiratory output independently of vagal or respiratory-related inputs.

Another possible role of the PRG respiratorymodulated neurons might be the transmission of respiratory rhythm to non-respiratory systems (“efference copy”). This role is suggested by the occurrence of respiratory modulation in discharges of midbrain neurons (Chen et al., 1991; Eldridge and Chen, 1992) and thalamic neurons (Chen et al., 1992). These populations resemble the pontine neurons in discharge pattern, particularly with respect to depression of activity by vagal inputs (Eldridge and Chen, 1992). The pontine neurons may also play a role in transmission of respiratory rhythm to sympathetic-related neurons, as suggested by the resemblance of pontine I-modulated neuron firing, as well as their inflation responses, to cervical sympathetic discharges and responses (Fig. 1 in Cohen et al., 1991; Huang et al., 2000). In another aspect of this study, we used afferent vagal electrical stimulation to ascertain the latencies of the inhibitory responses of a sample of 20 PRG I- and IE-modulated neurons. The overall responses of unit activity to vagal stimulation were comparable to the inflation responses. The latencies of inhibition, as determined by cusum analysis of the PSHs, ranged from 12 to 345 ms, with a median latency of 187 ms. A few studies have tried to ascertain connections between medullary neurons and PRG neurons by means of antidromic stimulation. Bianchi and St. John (1981) studied projections of bulbar neurons in the dorsal and ventral respiratory groups to the PRG by antidromic stimulation in PRG. They found that about 10% of bulbar respiratory neurons could be identified as bulbopontile; these were mainly phase-spanning or tonic I- and IE-modulated. These authors (Bianchi and St. John, 1982) also searched for pontobulbar neurons by evoking antidromic responses of PRG neurons to medullary stimulation, and found that about 10% of the neurons could be so identified. A detailed search, using antidromic stimulation, for pontine projections of pump (P) cells (second-order vagal afferent relay neurons that were located in nucleus tractus solitarius) was done by Ezure et al. (1998). They found 15 P cells that could be activated from the PRG region. The specification of latencies was of importance: orthodromic responses of P cells to vagal stimulation had latencies <5 ms; and antidromic responses of P cells to PRG stimulation had a range of 5–20 ms latencies. Similarly, antidromic stimulation revealed that P cells project widely, with short latency responses, to

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respiratory-related regions of the medulla (Ezure and Tanaka, 1996). The occurrence of projections from P cells to the PRG region raises the possibility that this input contributes to the observed vagally produced inhibition of PRG I- and IE-modulated neurons. But it seems unlikely that this response is due to direct post-synaptic inhibition by P cells, since: (a) the response is gated during I, i.e. there is no inhibition when inflation is delivered during E, as might be expected with a direct inhibitory input; (b) the latency of vagal inhibition is much longer (median 187 ms) than the latencies of antidromic responses by P cells. (However, there is a caveat for the latter consideration: it is inherently more difficult to ascertain inhibitory latencies than excitatory latencies from extracellular recordings, so that intracellular recordings from target neurons might reveal a shorter range of latencies.) These considerations imply that the vagal afferent signal is transmitted to pontine Iand IE-modulated neurons via intermediate pathways. A possible type of intermediate neuron would be one that is excited during the I phase by vagal afferents (such as neurons with patterns like those of the medullary P cells) and which in turn inhibits Imodulated pontine neurons. However, as reported in this paper, no population of this type (comparable to medullary late-I neurons excited by vagal input) was found in our pontine recordings. This negative result could be due to inadequate sampling, e.g. such neurons might be found at rostral pontine sites different from the sites we explored. Nevertheless, such an excited population might be found in medullary regions (e.g. the ventral respiratory group) that have not been systematically studied by the methods of vagal input manipulation used in the present study. Another possible intermediate pathway might produce presynaptic inhibition of some pontine Imodulated neurons. The P cells (stage 1), which are excited by vagal input, might project to synaptic terminals of neurons (stage 2) that convey inspiratory modulation to tonically firing pontine neurons (stage 3), thus producing disfacilitation at the terminals of stage 2 neurons and consequent presynaptic inhibition during I of stage 3 neurons. This intermediate (stage 2) population might be present in both medullary and pontine regions. With this scenario, the long latency of the ultimate inhibition might arise from: (a) chains of inspiratory-modulating neurons that finally end at pon-

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tine neurons; (b) the necessity for temporal summation in parallel chains to produce the final inspiratory modulation. Thus it seems then that even after decades the further study of the neuronal basis of the pontine “pneumotaxic center” remains a vast enterprise.

Acknowledgments This research was supported by N.I.H. Grants HL27300 and NS-43940.

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