Glycinergic inhibition is essential for the preceding properties of respiratory-related hypoglossal nerve discharge in rats

Kun-Ze Lee, I-Jung Lu, Jin-Tun Lin and Ji-Chuu Hwang Department of Life Science, National Taiwan Normal University, Taipei, Taiwan,

Corresponding author:Ji-Chuu HwangDepartment of Life ScienceNational Taiwan Normal UniversityTel. (02) 29326234 ext. 362Fax: (02) 29312904e-mail: jchwang@cc.ntnu.edu.tw

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AbstractsInspiratory activities of both hypoglossal nerve branches precede phrenic burst

and may be important to play a role in maintaining a patent upper airway. Our purpose

was to examine the hypothesis of whether the preceding onset of hypoglossal nerve

branches might alter in response to different levels of lung inflation and whether this

preceding onset was glycinergic-dependent. Activities of the phrenic nerve, medial

and lateral branches of the hypoglossal nerve were simultaneously recorded in

urethane-anesthetized and artificially ventilated rats. In the first protocol, three levels

of lung volumes (non-inflation; moderate-inflation; high-inflation) were applied to

induce a reflex of lung inflation and also to activate the activity of the slowly adapting

pulmonary stretch receptors (SARs). Moderate lung inflation reflexively induced an

increase while high level of lung inflation produced a decrease in the preceding onset

of hypoglossal nerve branches over the phrenic burst. Both increase and decrease in

onset of hypoglossal branch activities caused by lung inflation might be mediated

through inflationary SARs. In the second protocol, systemic administration of

strychnine (1.0 M/kg, iv) not only decreased the preceding onset of both hypoglossal

branch activities over the phrenic busrt during eupnea but also abolished the

excitatory effect of hypoglossal onset induced by moderate lung inflation. In addition,

strychnine administration also attenuated the inhibitory response of the hypoglossal

onset induced by high level of lung inflation. These results indicated that different

levels of lung inflations could produce an opposite effect on the onset of hypoglossal

branches and also strongly suggested that hypoglossal onset might be mediated via a

mechanism of glycinergic inhibition.

Key words: onset of hypoglossal branch activity; phrenic burst, glycinergic

inhibition; lung inflation, rat

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Introduction

The upper airway (UAW) and intrathoracic airways behave differentially during

respiratory cycle. The intrapleural pressure generated by the contraction of respiratory

muscles during inspiration produces constriction acting on the UAW and may collapse

or reduce the diameter of the UAW. Thus, co-ordination of the activities between the

bulbo-spinal phrenic and the cranial nerves innervating the UAW muscles may be

critical for the maintenance of the UAW patency (Dutschmann and Paton, 2002a,b).

In this regard, inspiratory hypoglossal nerve activity (HNA) has been demonstrated to

commence earlier than that of the phrenic nerve activity (PNA) (Ezure et al., 2003a;

Lee et al., 2003; Leiter and St. John, 2004). This onset of HNA preceding the PNA

has been considered to be important for the patency of the UAW because a decrease in

genioglossal discharge has been proposed to disturb the UAW seen in clinical

observations (Harper and Sauerland, 1978). This differential onset between HNA and

PNA has been proposed to be a stronger inspiratory drive to the hypoglossal

motoneurons or to the premotoneurons of the hypoglossal nerve than to the phrenic

system and may be also due to a lower threshold of activation for the hypoglossal

motoneurons (Saito et al., 2002; Sica et al., 1984). Thus, UAW patency may relate to

the modulation of the amplitude and onset of the HNA. However, mechanism of this

preceding onset of the HNA is still unclear.

Changes in respiratory volume have been established to provide information to

modulate the respiratory patterns (Coleridge and Coleridge, 1986; Cross et al., 1980;

Sibuya et al., 1993) and to produce inhibition on the PNA and phasic activity of the

UAW motoneurons (Kuna, 1986; Van Lunteren et al., 1984). In contrast, lung

inflation withdrawal has been demonstrated to produce excitation on phrenic

motoneurons (Hwang and St.-John., 1993). Thus, different levels of lung inflation

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may have dissimilar effects on PNA, which may mediate through the activation of

different types of receptors. In this aspect, there are three types of slowly adapting

pulmonary stretch receptors (SARs) in the rat, which are inflationary, deflationary and

biphasic SARs based on their discharge pattern during respiratory cycles (Bergren and

Peterson, 1993; Matsumoto et al., 2002). Both phasic and static lung volume feedback

may activate different types of SARs to influence the PNA and HNA in cats and rats

(Kuna, 1986; St.-John and Zhou, 1992; Bailey et al., 2001). Recent data have been

shown that pre-inspiratory activities of the hypoglossal nerve are enhanced by lung

inflation in rats (Saito et al., 2002). These results suggest that lung inflation reflexes

may produce excitatory effect on the HNA in rats. Hence, the first purpose of the

present study was to investigate whether HNA might be excited by lung inflation and

to examine which types of SARs might mediate this excitatory effect.

Glycine is the major inhibitory neurotransmitter in the central nervous system

and has been established to be involved in the generation of respiratory rhythm and

pattern (Haji et al, 2000). Loss of glycinergic inhibition could disturb the respiratory

rhythm (Büsselberg et al., 2001 and 2003; Iizuka, 2003; St-John and Paton, 2002).

The most interesting is that discharge of the post-inspiratory activities of the recurrent

laryngeal nerve shifts its expiratory activity to advance earlier during inspiration after

blockade of glycinergic inhibition (Büsselberg et al., 2003; Dutschmann and Paton,

2002b,c) or trigeminal stimulation (Dutschmann and Paton, 2002c). Similarly,

expiratory burst of thoracic ventral root has also been demonstrated to commence

earlier during inspiration after application of strychnine to the brainstem (Iizuka,

2003). These observations indicate that glycinergic inhibition may be essential for the

co-ordination between inspiration and expiration. Whether glycinergic inhibition

involved in the onset of the HNA preceding that of the PNA is unknown. Our second

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purpose was designed to examine this hypothesis of glycinergic inhibition on the

regulation of hypoglossal onset under eupnea and during the reflex evoked by lung

inflation.

Materials and Methods

Animal preparation

Experiments were performed on male Wistar rats. Animals were anesthetized with

urethane (1.2 g/kg, i.p., Sigma) after 30 minutes of atropine (0.5 mg/kg, i.m., Sigma)

and then placed in supine position. The trachea was cannulated and catheters were

inserted into the femoral artery and vein for blood pressure measurement and drug

administration. Rat was paralyzed with gallamine triethiodide (5 mg/kg, i.v., Sigma)

and ventilated artificially by a conventional ventilator, which was set at 10 ml/kg for

the stroke and 60 breaths per minute. A positive end-expired pressure (3 cm H2O) was

applied during experimental procedure. Trachea pressure was detected via a side arm

of the tracheal tubing. End-tidal fractional concentration of CO2 was analyzed with a

CO2 analyzer (Electrochemistry CD3A, Ametek, Pittsburgh, Pennsylvania, USA) by

inserting a needle of 25 gauge into the tracheal tubing and kept at 4-6 %.

Nerve recording

The phrenic nerve was identified at the level of C4 or C5 by cutting the clavicle and

removing part of the tissues and cut peripherally. Phrenic nerve activity (PNA) was

monitored by a bipolar electrode connecting to the inputs of an AC preamplifier

(Grass P511, Quincy, MA, USA) and filtered (0.3-3 kHz), and integrated with an

integrator (time constant = 50 ms) (Lee et al., 2003). Both the medial and lateral

branches of the hypoglossal nerve were dissected from the digastric muscle and was

separated and cut distally. Activities of these two hypoglossal branches were

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monitored the same way as that of the PNA.

Activities of PNA and of two branches of the hypoglossal nerve were recorded

on the hard disc via the PowerLab system (ADI Instrument Pty Ltd. NES, Australia).

Lung inflation reflex

Three levels of static lung volume changes, which were the non-inflation (NI),

moderate-inflation (MI), and high-inflation (HI), were applied to evoke a reflexive

effect on hypoglossal branch activities. Non-inflation was induced by switching off

the ventilator while MI and HI were produced by switching the connection of the

tracheal tubing from the ventilator to a water traping system, which could generate a

positive static pressure applying to the rat lung via air compressor. Thus, the trachea

pressure was maintained at 0, 5, and 10 cmH2O by NI, MI, and HI, respectively, for

10 seconds for each treatment.

Recording of slowly adapting receptors

Left cervical vagus was cut centrally and desheathed. Activities of the SAR were

recorded from the vagal afferents at the cervical level. Single fiber of the vagal

afferents was dissected by a pair of #5 forceps with the aid of surgical microscope

(Wild) and was verified by the same shape and amplitude of the action potential

(Hwang et al., 1983). Once a single SAR was verified, activity of the SAR was

recorded via a bipolar electrode and amplified (Grass AC preamplifier, P511) and

filtered (0.3-3 kHz), and was further examined their response to different levels of

changes in lung volume. Three different types of SARs were identified based on their

discharge pattern relative to ventilatory cycles. High threshold inflationary SARs

(hSAR) were only activated during inflation. Low threshold inflationary SARs

(lSARs) were discharged predominantly during inflation but continued to discharge

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during deflation. Deflationary SARs (dSARs) were only fired during deflation.

Experimental protocol

In the first protocol, responses of the phrenic nerve and the hypoglossal branches

to lung inflation were investigated to evaluate if the preceding onset of hypoglossal

branches over the phrenic burst could be modulated. Three levels of static lung

volumes (NI, MI and HI) were applied to the rat lung in random order. To examine if

the preceding onset of hypoglossal branch activities might be vagal-mediated,

activities of both the phrenic burst and hypoglossal branches in response to changes in

lung volumes were evaluated before and after bilateral vagotomy and activities of

SARs in response to these three levels of lung volumes were also characterized. In the

second protocol, alteration of hypoglossal onset evoked by the changes in static lung

volume was examined before and after bolus intravenous injection of strychnine (1.0

µM/kg, Sigma), a glycine receptor antagonist. The aim of this protocol was to test the

hypothesis regarding that the preceding onset of the hypoglossal branches over the

phrenic burst might depend on glycinergic inhibition. In addition, responses of both

right and left vagal afferents during static lung volume changes were compared before

and after strychnine (1.0 µM/kg) administration.

Data analysis

Data stored in the hard disc were retrieved and analyzed with software written

with Visual C++ 6.0. Data of 10 respiratory cycles or ventilatory cycles before

treatment were averaged as the control. PNA, activities of the medial and lateral

branch of the hypoglossal nerves (MHNA and LHNA), of the whole vagal afferents

and of single SARs were transformed into percentage of the control. T I and TE

represented the period of phrenic burst and between two phrenic bursts, respectively.

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Onset of the hypoglossal branches relative to phrenic burst was calculated. Blood

pressure, heart rate and trachea pressure stored in the hard disc were retrieved and

calculated from data pad module of the PowerLab system.

Nerve responses to the changes in lung volume were compared with the control

by multiple comparison tests. Hence, one-way or two-way ANOVA was performed

and then Student-Newman-Keuls was followed. Data were expressed as mean ± SEM

(standard error of mean). P value less than 0.05 was considered as significant.

Results

Phrenic and Hypoglossal responses to changes in lung volume

Non-inflation (NI) and moderate-inflation (MI) of the lung produced an

excitation to the activities of the phrenic nerve and the hypoglossal branches (Figs.

1A, 1B and 1D), while high-inflation (HI) of the lung evoked an inhibition on the

activities of these two nerves (Figs. 1C and 1D). In grouped data, NI and MI initiated

a mild excitation to PNA (p>0.05, Fig. 3A) while HI produced a significant decrease

in PNA, showing 32.1 % of the control (Fig. 3A, p<0.01). NI and MI did not produce

significant changes in respiratory pattern. However, HI evoked a significant decrease

in TI showing from 0.29 ± 0.01 s to 0.10 ± 0.04 s (Fig. 2A, p<0.01) and a substantial

increase in TE showing from 0.92 ± 0.07 s to 8.19 ± 0.83 s (p<0.01, Fig. 2B).

Dissimilarly, NI and MI produced significant increases in activities of the medial

and lateral branch of the hypoglossal nerve (MHNA/LHNA) (Fig 3B and 3C, p<0.01).

However, HI induced an inhibition to the MHNA and LHNA (Fig. 3B and 3C,

p<0.01). For the commencement of the hypoglossal activity, MI produced an earlier

onset for the medial branch with an increase in average from 0.09 ± 0.01 s to 0.38 ±

0.06 s (Fig. 4A, p<0.01) whereas HI induced a delay of onset from 0.09 ± 0.01 s to

0.03 ± 0.01 s (Fig. 4A). MI and HI also evoked a similar extent of increase and

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decrease for the lateral branch, respectively (Fig. 4B). NI produced no changes in the

onset of both the medial and lateral hypoglossal branches (Figs. 4A and 4B, p>0.05).

After bilateral vagotomy, TE and the onset of both hypoglossal branches were

significantly increased (Fig. 2B, 4A and 4B). Reflexive effects on the phernic and

hypoglossal nerves induced by the changes in lung volume were not observed (Figs.

1, 2, 3, and 4), suggesting a reflex of vagal-dependent.

Characteristics of slowly adapting receptors

There are three types of slowly adapting receptors based on their discharge with

or without lung inflation (Fig. 5). The tracheal pressure was measurable when the

lung was periodically inflated by a conventional ventilator (control of Fig. 5) or

continuously inflated by the water trapping system (MI and HI of Fig. 5). However, it

was not measurable or 0 cmH2O when the lung was deflated (NI of Fig. 5). Therefore,

the lung was periodically inflated and the tracheal pressure displayed increase and

decrease following the lung inflation and deflation. In this situation, high threshold

slowly adapting receptors (hSAR) were discharged locking to the increase of tracheal

pressure (control of Fig. 5A) and deflationary slowly adapting receptors (dSAR) were

discharged during decreasing tracheal pressure (control of Fig. 5C). Due to this

dissimilar property, activity of the hSAR was silent and of the dSAR was strongly

discharged in response to lung deflation or NI (Fig. 5). With the increases in lung

volume, activity of the hSAR was volume-dependent with MI and HI. In contrast,

activity of the dSAR was decreased with the increase in lung volume (Fig. 5C).

Property of the low threshold slowly adapting receptors (lSAR) was continually

discharge during ventilatory cycles with more active during inflation (Fig. 5B). Thus,

discharge rate of the lSAR was reduced by lung deflation (NI) but was increased by

lung inflation of both MI and HI.

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In average, discharge rates of the hSAR and lSAR were decreased to 0.4 % and

12.5 % (Table 1, p<0.01), respectively, and of the dSAR were increased to 136.0 %

(Table 1, p<0.05) in response to NI. However, discharge rates of the hSAR and lSAR

were increased to 130.2 % and 202.9 % with HI and discharge rates of the dSAR were

reduced to 2.6 % with HI (Table 1, p<0.01). Discharge rates of the hSAR and dSAR

were significantly decreased with MI (Table 1, p<0.01) and of the lSAR were not

influenced with MI.

Strychnine administration attenuated the effect of lung inflation

Strychnine administration (1.0 µM/kg) produced mild alteration of PNA,

respiratory pattern, and hypoglossal branch activities during eupnea, and significant

changes in either nerve amplitude or respiratory pattern with HI (Figs. 1, 6, and 7).

Strychnine administration significantly reversed the decrease in PNA caused by HI

(Fig. 7A). It also reversed the decrease in TI and increase in TE caused by HI (Figs. 6A

and 6B, p<0.05). However, it did not produce effects on mild increases in PNA (Fig.

7) or slight decreases in TI by NI and MI (Figs. 6A and 6B, p>0.05).

Strychnine administration evoked significant increases in both the medial and

lateral hypoglossal activities during eupnea with an average of 127.9 % and 128.8 %,

respectively (Figs. 7B and 7C, p<0.01) and was further to produce more increases in

these two hypoglossal branches with NI and MI although this further effect was

insignificantly. Moreover, the decrease of the medial and lateral hypoglossal activities

caused by HI was significantly reversed by strychnine administration (Figs. 7B and

7C). Onset of the medial and lateral hypoglossal branches was earlier than that of the

PNA during eupnea (Fig. 1D) with an average of about 100 ms for both branches. The

preceding onset of the hypoglossal branches over the phrenic burst was slightly

reduced with strychnine administration. This preceding onset was substantially

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increased with MI as shown in the first protocol. Interesting is that this increase in

preceding onset of hypoglossal branches over the phrenic burst was significantly

reduced to a level near to the control level after the administration of strychnine (Figs.

8A and 8B, p<0.05). Moreover, decrease in the preceding onset of the hypoglossal

branches over the phrenic busrt evoked by HI was also reversed with strychnine

treatment although this reversal effect was insignificant in statistics (Figs. 8A and 8B,

p>0.05).

As shown in Table 2, strychnine administration produced no effect on activities

of both right and left vagal afferents during eupnea and different levels of lung

inflation.

Discussion

Our present data showed that firstly, moderate lung inflation evoked excitatory

effect on the PNA and hypoglossal branch activities, and high level of lung inflation

produced inhibition on these nerves. This excitatory influence was also reflected from

the presentation of the advanced onset for the hypoglossal branches. The excitatory

and inhibitory effects caused by lung inflation were mediated by vagal afferents. And

that secondly, blockade of glycinergic inhibition resulted in reductions not only of the

preceding onset of the hypoglossal branches over the phrenic burst but also the

advanced onset caused by MI. These results suggested that glycinergic inhibition

might be critical for the onset of the hypoglossal activities, which was not only during

eupnea but also advanced by MI or delayed by HI, and also for the amplitude of these

two branches through the activation of vagal afferents.

Changes in respiration with Lung inflation.

Lung inflation or deflation has been well established to produce reflexive

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changes in breathing. These were demonstrated in the present study, showing increase

or decrease in PNA and in TI and TE with NI and MI or HI. Non-inflation has been

widely used in the initiation of lung deflation, showing 0 cmH2O after turning off the

ventilator in the present study. In this situation, our data showing decrease in TE and

activation of dSARs and inactivation of inflationary slowly adapting receptors, were

compatible well with other reports (Takano and Kato, 1999; Sammon et al., 1993). It

may also activate the rapidly adapting receptors (RAR) (Takano and Kato, 1999;

Sammon et al., 1993). High level of lung inflation may produce inhibition on

breathing, presenting a response of Hering-Breuer reflex. Two levels of lung inflation,

which were demonstrated from the fluctuation of tracheal pressure between 5 and 10

cmH2O, were used in the present study.

Changes in hypoglossal branch activities with lung inflation

The commencement of the hypoglossal branch activities preceded the phrenic

burst before vagotomy and was advanced to commence earlier after bilateral

vagotomy. This result was compatible with our recent report (Lee et al., 2003) and

other study (Fukuda and Honda, 1982). The preceding onset of the hypoglossal

branches was slightly earlier with NI and was further significantly advanced with MI.

However, this preceding onset was delayed with HI. After bilateral vagotomy, the

earlier onset induced by MI or delayed onset by HI was significantly returned to the

control level. These results suggested that phasic lung inflation might produce an

inhibitory effect on the pre-inspiratory hypoglossal activities via the vagal afferents

(Bailey et al., 2001; Kuna, 1986). They also strongly suggested that the preceding

onset of the hypoglossal branches was excited by MI while was inhibited by HI. This

different response of hypoglossal onset time may be due to activation of different

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types of inflationary SARs in response to MI and HI.

Hering-Breuer inflation reflexes were evoked by lung inflation. Two level of

lung inflation were used in the present study. Average trachea pressure induced by

moderate lung inflation was maintained between maximum and minimum trachea

pressure of ventilatory cycles. During moderate lung inflation, discharge onset time of

the hypoglossal branches were significant increased and more preceding than which

after bilateral vagotomy (Fig. 3 and 4). Hence, increases of discharge onset time of the

hypoglossal branches were not only resulted from loss of phasic lung inflation but

also an excitatory effect of moderate lung inflation. Although inspiratory periods were

not enhanced by moderate lung inflation, amplitude and rate of rising of phrenic

activities were increased. This facilitation of the phrenic nerve was also observed in

cats (Hwang and St.-John, 1993). High level of lung inflation exerted an obvious

inhibitory effect on the phrenic nerve and hypoglossal branches. These contrary

results of Hering-Breuer inflation reflex may be due to activation of different types of

inflationary SARs or different discharge rate of inflationary SARs during moderate

and high level of lung inflation. Inflationary SARs could be further divided into high

threshold SARs (hSARs) and low threshold SARs (lSARs) according to their

discharge pattern during ventilatory cycles (Tsubone, 1986). Activities of hSARs were

decreased and lSARs were not influenced by moderate lung inflation. These

observations indicated that excitatory effects of moderate lung inflation may be

mediated by lSARs. During high level of lung inflation, activities of both hSARs and

lSARs increased. Excitatory effects of lSARs may be masked by inhibitory effects of

hSARs. Hence, high-inflation still inhibited activities and discharge onset time of the

hypoglossal branches. Another possibility of opposed effects induced by moderate

and high-level lung inflation was different discharge rate of inflationary SARs.

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Discharge rate of inflationary SARs during moderate-inflation were lower than which

during high-inflation in the present study. Takano and Kato (1999) indicated that

lower frequency (5-40 Hz) stimulation of vagus nerve could prevent termination of

inspiratory activities, while higher frequency (100-160 Hz) stimulation induced

termination of inspiratory activities in NMDA receptors blocked and anaesthetized

rabbits. Opposed responses evoked by the same type of afferents may be due to

frequency-filtering effect of nucleus of the tractus solitarius, where SARs terminated

and was the first region in the central nervous system for regulation of visceral signal

transmission and reflexes (Chen et al., 2002; Jordan, 2001; Takano and Kato, 1999).

Unlike contrary responses of discharge onset time of the hypoglossal branches,

inspiratory periods were reduced and expiratory periods were elongated by increasing

static lung volume. This difference may be resulted from different premotor neurons

of the phrenic nerve and the hypoglossal branches (Peever et al., 2001). Other types of

vagal afferents, such as C-fibers and rapidly adapting receptors may also contribute to

reflexes induced by lung inflation in the present study. Pulmonary C-fibers could be

activated by capsaicin and only respond to high intensity of lung inflation (30 cm H2O

of trachea pressure) (Ho et al., 2001). Trachea induced by high level of lung inflation

in our experiments was maintained at about 10 cm H2O. This level of lung inflation

may only activate very small part of pulmonary C-fibers. Rapidly adapting receptors

were also excited by lung inflation but adapted rapidly. Hence, reflexes induced by

rapidly adapting receptors may only sustain a short periods. In addition, activation of

rapidly adapting receptor would decrease expiratory periods and induce hyperpnonea

and augmented breath (Davies and Roumy, 1982; Sant’Ambrogiog and Widdicombe,

2001). Expiratory periods were increased by moderate and high level of lung inflation

in the present study. Our results revealed that reflexes induced by rapidly adapting

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receptors may be masked by responses induced by slowly adapting receptors.

Mechanism for the preceding onset of hypoglossal activities: Glycinergic inhibition

The onset of the hypoglossal branch activities preceded the phrenic burst during

eupnea. This different onset was compatible with other reports (Ezure et al., 2003a;

Lee et al., 2003; Leiter and St. John, 2004) and may be due to the different threshold

or driving inputs to the bulbospinal phrenic system and hyposlossal motorneurons

(Saito et al., 2002; Sica et al., 1984). However, the exact mechanism for the preceding

onset of the hypoglossal activity over the phrenic burst is still unclear. In this aspect,

we have recently reported that capsaicin administration to activate pulmonary C fibers

could produce a delay onset of hypoglossal activity (Lee et al., 2003). These results

suggest that input from pulmonary C fibers may produce a modulation for the

commencement of phasic hypoglossal activity. Our present data, showing that this

preceding onset of the hypoglossal branches disappeared after venous administration

of strychnine, revealed that glycinergic inhibition might be involved in the regulation

of hypoglossal commencement and might be essential for the co-ordination of onset

between activities of the phrenic nerve and hypoglossal branches. However, our data

did not show from where is the glycinergic pathway originated? Indeed, the glycine

receptors have been demonstrated to present on the hypoglossal motoneurons (Donato

and Nistri, 2000; Morrison et al., 2002). Our present data were very similar to that

observed in the recurrent laryngeal nerve (Dutschmann and Paton, 2002a and 2002b)

and in the spinal nerve displaying respiratory discharge (Iizuka, 2003). Our data

strongly suggested that this putative glycinergic mechanism might exert a tonic

inhibition on the hypoglossal motoneurons.

Strychnine administration not only blocked the preceding onset of the

hypoglossal branch activities over the phrenic burst but also abolished the further

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advanced onset evoked by MI and the delayed onset of the hypoglossal activities

initiated by HI. These results suggested that MI might transmit the excitatory signals

of moderate lung volume to the central nervous system to activate the glycinergic

transmission and, thus, to advance the onset of the hypoglossal branch activities.

Unfortunately, the exact pathways for these excitatory and inhibitory mechanisms

induced by MI and HI respectively have not been defined so far. However, our present

data strongly suggested that glycinergic mechanism might be involved in the process

of these two signal transmissions.

Strychnine administration not only decreased the onset or pre-inspiratory

activities but also increased the inspiratory amplitude of hypoglossal branch activities.

These results may be due to the shift of hypoglossal motoneuron activity from late

expiratory phase to commence during inspiration. From the other point of view, this

shift might make more motoneurons to be recruited during inspiration to enhance the

inspiratory activity based on the recruited mechanism of motor activity (Adrian and

Bronk, 1928). However, we still didn’t know whether the pre-inspiratory and

inspiratory hypoglossal activities were the same or different motoneuron.

Nevertheless, recent results from power spectral analysis have revealed that

controlling mechanisms of pre-inspiratory activities and inspiratory activities of the

hypoglossal branches may be different from each other (Leiter and St. John, 2004).

This view has been further supported by the separation of pre-inspiratory component

and inspiratory component of the hypoglossal nerve by hypocapnia and hypothermia

in situ and by manipulation of artificial ventilator in vivo (Saito et al., 2002; St. John

et al., 2004).

Effects of strychnine on changes in amplitudes and in onsets for both the phrenic

and hypoglossal nerves may be acted through the central nervous system but not

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through the alteration of vagal afferent activities based on our current observations,

showing a similar activities of vagal afferents on both sides under strychnine during

eupnea and lung inflation. It has established that glycine could inhibit activities of

genioglossus muscles in vivo and hypoglossal motoneurons in vitro (Morrison et al.,

2002; Donato and Nistri, 2000). Hence, we couldn’t exclude the possibility of

strychnine administration altering the threshold of the onset of hypoglossal branch

activities, which might be located in the putative pathways involving in the processing

of pulmonary SARs such as the nucleus of the tractus solitarius (Bonham and

McCrimmon, 1990), the Bötzinger complex, and the ventral respiratory group (Ezure

et al., 2002; 2003b).

In conclusion, our results demonstrated that glycinergic transmission may be

essential for the preceding onset of hypoglossal branch activities over phrenic burst.

The glycinergic inhibition may also be involved in the mediation of the Hering-Breuer

reflexes. However, where is the origination of this glycinergic pathway or which

nucleus may be involved in this pathway is remained to be determined.

References

References

Adrian, E.D. Bronk, D.W. 1928. The discharge of impulses in motor nerve fibers. Part I. Impulses in single fibers of the phrenic nerve. J. Physiol. 66, 81-101.

Bailey, E.F., Jones, C.L., Reeder, J.C., Fuller, D.D., Fregosi, R.F, 2001. Effect of pulmonary stretch receptor feedback and CO2 on upper airway and respiratory pump muscle activity in the rat. J. Physiol. 532.2, 525-534.

Berger, D.R., Peterson, D.F., 1993. Identification of vagal sensory receptors in the rat lung: are there subtypes of slowly adapting receptors? J. Physiol. 464, 681-698.

Bonham, A.C., McCrimmon, D.R., 1990. Neurones in a discrete region of the nucleus

17

tractus solitarius are required for the Breuer-hering reflex in rat. J. Physiol. 427, 261-280.

Büsselberg, D, Bischoff, A.M., Paton, J.F., Richter, D.W. 2001. Reorganisation of respiratory network activity after loss of glycinergic inhibition. Pflugers. Arch. 441(4), 444-9.

Büsselberg, D, Bischoff, A.M., Richter, D.W., 2003. A combined blockade of glycine and calcium-dependent potassium channels abolishes the respiratory rhythm. Neuroscience. 122, 831-841.

Chen, C.Y., Ling, E-h., Horowitz, J.M., Bonham, A.C., 2002. Synaptic transmission in nucleus tractus solitarius is depressed by group II and III but not group I presynaptic metabotropic glutamate receptors in rats. J. Physiol. 538.3, 773-786.

Coleridge, H.M., Coleridge, J.C.G. Reflexes evoked from tracheobronchial tree and lungs. In Handbook of Physiology: The Respiratory System. Control of breathing. Bethesda, MD: Am. Physiol. Soc, 1986, sect. 3, pt. 1, chapt. 12, p. 395-429.

Cross, B.A., Jones, P.W., Guz, A. The role of vagal afferent information during inspiration in

determining phrenic motoneurone output. Respir. Physiol. 39: 149-167, 1980.

Davies, A., Roumy, M. 1982. The effect of transient stimulation of lung irritant receptors on the pattern of breathing in rabbits. J. Physiol. 324, 389-401.

Donato, R., Nistri, A. 2000. Relative contribution by GABA or glycine to Cl- mediated synaptic transmission on rat hypoglossal motoneurons in vitro. J. Neurophysiol. 84, 2715-2724.

Dutschmann, M., Paton, J.F.R., 2002a. Inhibitory synaptic mechanisms regulating upper airway patency. Respir. Physiol. Neurobiol. 131(1-2), 57-63.

Dutschmann, M., Paton, J.F.R., 2002b. Glycinergic inhibition is essential for co-ordinating cranial and spinal respiratory motor outputs in the neonatal rat. J. Physiol. 543.2, 643-653.

Dutschmann, M., Paton, J.F.R., 2002c. Trigeminal reflex regulation of the glottis depends on central glycinergic inhibition in the rat. Am. J. Physiol. Regul. Integr.

18

Comp. Physiol. 282(4), R999-R1005.

Ezure K., Tanaka, I., Saito, Y., Otake, K., 2002. Axonal projections of pulmonary slowly adapting receptor relay neurons in the rat. J. Comp. Neurol. 446, 81-94.

Ezure, K., Tanaka, I., Saito, Y., 2003a. Acivity of brainstem respiratory neurons just before the expiration-inspiration transition in the rat. J. Physiol. 547.2, 629-640.

Ezure, K., Tanaka, I., Kondo, M., 2003b. Glycine is used as a transmitter by decrementing expiratory neurons of the ventrolateral medulla in the rat. J. Neurosci. 23(26), 8941-8948.

Fukuda, Y., Honda, Y., 1982. Roles of vagal afferents on discharge pattern and CO2-responsiveness of efferent superior laryngeal, hypoglossal, and phrenic respiratory activities in anesthetized rats. Jpn. J. Physiol. 32, 689-698.

Haji, A., Takeda, R., Okazaki, M., 2000. Neuropharmacology of control of respiratory rhythm and pattern in mature mammals. Pharmacol. Ther. 86, 277-304.

Harper, R.M., Sauerland, E.K. The role of the tongue in sleep apnea. In: Guilleminault C, Dement WC, eds. Sleep Apnea Syndromes vol. 11, Kroc Foundation Series. New York, Liss, 219–234;1978.

Hwang, J.C., Bartlett, D. Jr, St.-John, W.M. 1983. Characterization of respiratory-modulated activities of hypoglossal motoneurons. J. Appl. Physiol. 55(3), 793-8.

Hwang, J.C., St.-John, W.M., 1993. Facilitation and inhibition of phrenic motoneuronal activities by lung inflation. J. Appl. Physiol. 74(5), 2485-2492.

Ho, C.Y., Gu, Q., Lin, Y.S., Lee, L.Y. 2001.Sensitivity of vagal afferent endings to chemical irritants in the rat lung. Respir. Physiol. 127, 113-124.

Iizuka, M., 2003. GABAA and glycine receptors in regulation of intercostals and abdominal expiratory activity in vitro in neonatal rat. J. Physiol. 551.2, 617-633.

Jordan, D., 2001. Central nervous pathway and control of the airways. Respir. Physiol. 125, 67-81.

19

Kuna, S.T., 1986. Inhibition of inspiratory upper airway motoneuron activity by phasic volume feedback. J. Appl. Physiol. 60(4), 1373-1379.

Lee, K.Z., Lu, IJ., Ku, L.C., Hwang, J.C., 2003. Response of respiratory-related hypoglossal nerve activity to capsaicin-induced pulmonary C-fiber activation in rats. J Biomed Sci. 10(6 Pt 2), 706-17.

Leiter, J.C., St-John, W.M., 2004. Phrenic, vagal and hypoglossal activities in rat: pre-inspiratory, inspiratory, expiratory components. Respir. Physiol. Neurobiol. 142, 115-126.

Matsumoto, S., Ikeda, M., Nishikawa, T., Yoshida, S., Tanimoto, T., Ito, M., Saiki, C., Takeda, M. 2002. Excitatory mechanism of deflationary slowly adapting pulmonary stretch receptors in the rat lung. J. Pharmacol. Exp. Ther. 300(2), 597-604.

Morrison, J., Sood, S., Liu, X., Liu, H., Park, E., Nolan, P., Horner, R.L. 2002. Glycine at hypoglossal motor nucleus: genioglossus activity, CO2 responses, and the additive effects of GABA. J. Appl. Physiol. 93, 1786-1796.

Peever, J.H., Mateika, J.H., Duffin, J., 2001. Respiratory control of hypoglossal motoneurones in the rat. Eur. J. Physiol. 442, 78-86.

Saito, Y., Ezure, K., Tanaka, I., 2002. Difference between hypoglossal and phrenic activities during lung inflation and swallowing in the rat. J. Physiol. 544.1, 183-193.

Sammon, M., Romaniuk, Jr., Bruce, E.N. 1993. Role of deflation-sensitive feedback in control of end-expiratory volume in rats. J Appl Physiol. 1993 75(2), 902-11.

Sant’Ambrogio, D., Widdicombe, J. 2001. Reflexes from airway rapidly adapting recepors. Respir. Physiol. 125, 33-45.

Sibuya, M., Kanamaru, A., Homma, I. 1993. Inspiratory prolongation by vagal afferents from pulmonary mechanoreceptors in rabbits. Jpn. J. Physiol. 43, 669-684.

Sica, A.L., Cohen, M.I., Donnelly, D.F., Zhang, H. 1984. Hypoglossal motoneuron responses to pulmonary and superior laryngeal afferent inputs. Respir. Physiol. 56, 339-357.

20

St.-John, W.M., Zhou, D., 1992. Reductions of neural activities to upper airway muscles after elevations in static lung volume. J. Appl. Physiol. 73(2), 701-707.

St.-John, W.M., Paton, J.F., 2002. Neurogenesis of gasping does not require inhibitory transmission using GABA(A) or glycine receptors. Respir. Physiol. Neurobiol. 132(3), 265-77.

St.-John W.M., Paton, J.F.R., Leiter, J.C., 2004. Uncoupling of rhythmic hypoglossal from phrenic activity in the rat. Exp. Physiol. 89.6, 727-737.

Takano, K., Kato, F., 1999. Inspiration-promoting vagal reflex under NMDA receptor blockade in anaesthetized rabbits. J. Physiol. 516.2, 571-582.

Tsubone, H., 1986. Characteristics of vagal afferent activity in rats: three types of pulmonary receptors responding to collapse, inflation, and deflation of the lung. Exp. Neurol. 92, 541-552.

Van Lunteren, E., Strohl, K.P., Parker, D.M., Bruce, E.N., Van de Graaff, W.B., Cherniack, N.S. 1984. Phasic volume-related feedback on upper airway muscle activity. J. Appl. Physiol. 56(3), 730-736.

Figure legends

Fig. 1. The effect of different lung volumes on activities of both the phrenic and

hypoglossal branches before (left panels) and after strychnine administration (right

panels). Non-inflation (NI) produced increase in activities of the hypoglossal branches

and decrease in the expiratory periods (panel A). Moderate-inflation (MI) increased

amplitude and preceding onset of the hypoglossal branch activities (panel B). High-

inflation (HI) evoked inhibition on activities of the phrenic nerve and hypoglossal

branches (panel C). After strychnine administration, elongation of expiratory periods

induced with HI was attenuated and the preceding onset of hypoglosssal branch

activities over the phrenic burst caused by NI and MI was also decreased by

strychnine during. Abbreviations: TP for trachea pressure; Int. PNA for integrated

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activities of the phrenic nerve; Int. MHNA for integrated activities of the medial

branch of the hypoglossal branches; Int. LHNA for integrated activities of the lateral

branch of the hypoglossal branches; Cont for control; NI for non-inflation; MI for

moderate-inflation; HI for high-inflation. Time scale in panel D is

Fig. 2. Inspiratory periods and expiratory periods during different levels of lung

volume before and after vagotomy. Increasing lung volume decreased inspiratory

periods and increased expiratory periods before vagotmy. Both inspiratory and

expiratory periods were not influenced by lung volume changes after bilateral

vagotomy. **: p<0.01 compared with control (C); ##: p<0.01 compared with the same

level of lung volume after vagotomy. NI: non-inflation; MI: moderate-inflation; HI:

high-infaltion.

Fig. 3. Phrenic and hypoglossal responses to changes in different lung volume before

and after vagotomy. High level of lung inflation inhibited activities of the phrenic

nerve and the hypoglossal branches. Moderate-inflation increased activities of both

medial and lateral branch of the hypoglossal nerve. After vagotomy, activities of the

phrenic nerve and the hypoglossal branches were not significant influenced by lung

volume changes. **: p<0.01 compared with control; ##: p<0.01 compared with the

same level of lung volume after vagotomy.

Fig. 4. Alteration of onset of the hypoglossal branches with changes in lung volume

before and after vagotomy. Moderate-inflation increased and high-inflation decreased

the preceding onset of the hypoglossal branches over the phrenic burst. This alteration

of discharge onset of the hypoglossal branches with changes in lung volumes

disappeared after bilateral vagotomy. *: p<0.05, **: p<0.01 compared with control; #:

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p<0.05, ##: p<0.01 compared with the same level of lung volume after vagotomy.

Fig. 5. Discharge pattern of vagal slowing adaptive receptors during phasic and static

changes in lung volumes. High threshold of inflationary slowly adapting receptors

(hSARs) were discharged during lung inflation (A). Low threshold of inflationary

slowly adapting receptors were discharged during both ventilatory inflation and

deflation phase (B). Both activities of hSARs and lSARs were increased with

increasing lung volume. Activities of deflationary slowly adapting receptors (dSARs)

fired at ventilatory deflation phase (C) and decreased with increasing lung volume.

Fig. 6

Effects of strychnine on inspiratoy and expiratory periods during changes in different

level of lung volumes. Decrease of inspiratory periods and increase of expiratory

periods induced by high-inflation were attenuated by strychnine. **: p<0.01

compared with control; ##: p<0.01 compared with the same level of lung volume

during strychnine.

Fig. 7

Effects of changes in lung volume on activities of the phrenic nerve and hypoglossal

branches before and after strychnine administration. Strychnine administration

enhanced hypoglossal branch activities and attenuated the inhibition induced by high-

inflation. *: p<0.05, **: p<0.01 compared with control; #: p<0.05; ##: p<0.01

compared with the same level of lung volume during strychnine.

Fig. 8

Discharge onset of the hypoglossal branches in responses to changes in lung volumes

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before and after strychnine administration. Moderate-inflation induced elongation of

the preceding onset of hypoglossal branch activities were significantly attenuated by

strychnine administration. The delay onset of the hypoglossal branches evoked by

High-inflation still was inhibited by strychnine administration. *: p<0.05, **: p<0.01

compared with control; ##: p<0.01 compared with the same level of lung volume

during strychnine.

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