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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: [email protected]
1
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
2
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
5
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
6
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.
7
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
10
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
12
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
14
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
15
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
16
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
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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; #:
22
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
23
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.
24