Respiratory and Neuroscience Research Groups, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada
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ABSTRACT |
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Inoue, T., Z. Haque, K. Lukowiak, and N. I. Syed. Hypoxia-Induced Respiratory Patterned Activity in Lymnaea Originates at the Periphery. J. Neurophysiol. 86: 156-163, 2001. Respiration in Lymnaea is a hypoxia-driven rhythmic behavior, which is controlled by an identified network of central pattern generating (CPG) neurons. However, the precise site(s) (i.e., central or peripheral) at which hypoxia acts and the cellular mechanisms by which the respiratory chemosensory drive is conveyed to the CPG were previously unknown. Using semi-intact and isolated ganglionic preparations, we provide the first direct evidence that the hypoxia-induced respiratory drive originates at the periphery (not within the central ring ganglia) and that it is conveyed to the CPG neurons via the right pedal dorsal neuron 1 (RPeD1). The respiratory discharge frequency increased when the periphery, but not the CNS, was made hypoxic. We found that in the semi-intact preparations, the frequency of spontaneously occurring respiratory bursts was significantly lower than in isolated ganglionic preparations. Thus the periphery exerts a suppressive regulatory control on respiratory discharges in the intact animal. Moreover, both anoxia (0% O2) and hypercapnia (10% CO2) produce a reduction in respiratory discharges in semi-intact, but not isolated preparations. However, the effects of CO2 may be mediated through pH changes of the perfusate. Finally, we demonstrate that chronic exposure of the animals to hypoxia (90% N2), prior to intracellular recordings, significantly enhanced the rate of spontaneously occurring respiratory discharges in semi-intact preparations, even if they were maintained in normoxic saline for several hours. Moreover, we demonstrate that the peripherally originated hypoxia signal is likely conveyed to the CPG neurons via RPeD1. In summary, the data presented in this study demonstrate the important role played by the periphery and the RPeD1 neuron in regulating respiration in response to hypoxia in Lymnaea.
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INTRODUCTION |
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Respiration in most
vertebrate species is a multi-component behavior, regulating oxygen and
carbon dioxide levels. This is accomplished by the respiratory
musculature whose activities are controlled by the CNS (Feldman
and McCrimmon 1999). The CNS-derived patterned activity is
further modulated by peripheral input from various chemoreceptors
(Burleson and Smatresk 1989
; Milsom and Brill
1986
) and mechanoreceptors (Milsom 1990b
), which
play important roles in maintaining internal homeostasis. Chemical
homeostasis involves a balance between carbon dioxide, oxygen, and pH
levels, all of which vary among different species, depending on factors such as 1) whether the animal is a lung breather,
2) levels of cutaneous carbon dioxide excretion, and
3) the solubility of gases in the ventilatory system.
The respiratory rhythm underlying breathing behavior in most
vertebrates can be generated in the absence of afferent fibers (von Euler 1986). However, peripheral input helps to
ensure that the final motor pattern is behaviorally relevant. For
example, both central and peripheral chemoreceptors in the decerebrated animals (von Euler 1986
), central chemoreceptors in the
isolated brain stem preparation (Harada et al. 1985
),
and other brain regions (such as cortex, hypothalamus, and cerebellum)
alter ventilation to accommodate related motor functions such as
speech, postural changes, and locomotion (Aritav et al.
1995
; Eldridge et al. 1981
; Mitchell
1993
; Waldrop and Porter 1995
). Together,
central and peripheral elements allow an animal to modulate its
breathing behavior in accordance with its metabolic demands
(Feldman and McCrimmon 1999
).
In contrast to our vast knowledge of central and peripheral components
of respiratory rhythm generation in vertebrates, little is known about
the chemosensory basis of respiratory behavior in invertebrates. In our
laboratory, we have thus utilized the fresh water mollusk Lymnaea
stagnalis to investigate the neuronal basis of respiratory
behavior. Lymnaea is a bimodal breather and thus can use
cutaneous gas exchange under water (skin respiration), or lung exchange
(aspirational lung breathing) in the air with the atmosphere
(Jones 1961). To exchange gas with the atmosphere, the
hypoxic animal surfaces and opens its respiratory orifice, the
pneumostome providing an airway between the lung and the atmosphere (Syed et al. 1991
). During pneumostome openings, the
mantle cavity muscles contract and the lung gas is expired
(expiration). These muscles then relax, allowing a passive re-inflation
of the lung by its elastic recoil. The pneumostome is then closed by
the contraction of pneumostome closing muscles (Syed and Winlow
1991
). These aspects of breathing in Lymnaea
resemble those of amphibia, reptiles, and diving mammals (Milsom
1990a
). In well-oxygenated water, adequate gas exchange is
provided via the skin, and the lung ventilation is minimal. In
contrast, when water is hypoxic, aerial respiration increases
dramatically: the animal either exhibits prolonged openings of its
pneumostome, or the frequency of these ventilatory responses increases
dramatically (Syed and Winlow 1991
).
The central pattern generating neurons (CPG) and the motor neurons
controlling the pneumostome musculature have been identified, and their
synaptic connections are well characterized (Syed et al.
1990). Specifically, the identified neurons right pedal dorsal 1 (RPeD1), input 3 interneuron (IP3I), and visceral dorsal 4 (VD4) are
the key components of the central respiratory rhythm generating network. The visceral ganglia H, I, J, K cells comprise the
motoneuronal pool that regulates the activities of the pneumostome
opening and closing muscles (Syed and Winlow 1991
;
Syed et al. 1991
). In isolated brain preparations, the
CPG neurons are sufficient to generate the basic respiratory rhythm, as
was demonstrated unequivocally by the in vitro reconstruction of the
circuit. In cell culture, the three-cell CPG network generated fictive
respiratory rhythmic activity identical to that observed in isolated
ganglion preparations (Syed et al. 1990
). However, both
in isolated brain preparations (where spontaneous respiratory activity
was absent) and in the in vitro reconstructed circuit, the respiratory
rhythm was initiated only after the electrical stimulation of RPeD1; the "normal" source for this excitatory drive to RPeD1 in the intact animals remained unidentified.
In this study, we provide evidence that the hypoxia-induced chemosensory drive in Lymnaea originates at the periphery and is most likely conveyed to the central respiratory CPG neurons via RPeD1. We also demonstrate that the periphery exerts a suppressive, regulatory control on the frequency of patterned respiratory activity in semi-intact preparations, suggesting that the spontaneously occurring respiratory bursts recorded from the isolated brain preparation resulted from the removal of peripheral input. Taken together, this study underscores the importance of peripheral, chemosensory input in the initiation and regulation of respiratory behavior in Lymnaea.
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METHODS |
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Animals
Laboratory-raised stocks of the freshwater snail Lymnaea stagnalis were maintained in well-aerated pond water and were fed lettuce. Animals with a shell length of 25-30 mm (5-6 mo old) were used in all experiments.
Semi-intact preparations
Semi-intact preparations were made as described earlier
(Inoue et al. 1996a; Syed et al. 1991
).
Briefly, the animals were anesthetized either with 10%
benzethonium chloride or 2% halothane, and their shell and the
foot musculature were removed using scissors. The central ring ganglia
and all attached visceral organs (heart, kidney, lung and pneumostome
and mantle) were left intact. The resulting semi-intact preparations
were pinned to the bottom of a black silicone rubber (RTV616, General
Electric)-based dissection dish containing normal Lymnaea
saline (composition in mM: 51.3 NaCl, 1.7 KCl , 4.0 CaCl2, and 1.5 MgCl2). The
saline was buffered with 10.0 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic
acid (HEPES), and the pH was adjusted to 7.9-8.0 with 1N NaOH
(Syed and Winlow 1991
). The central ring ganglion was
physically separated from the periphery by insertion of a plastic
partition. Nerves were allowed to pass through grooves in partition,
and the openings were subsequently sealed by petroleum jelly
(Vaseline). This approach allowed us to completely separate the central
compartment from the periphery, thus enabling the superfusion of either
one or the other chamber with various experimental solutions. Saline with various gas mixtures was prepared and delivered directly to the
preparations with a fast perfusion system (10 ml/min). Specifically,
normoxic solution was prepared by bubbling air directly into normal
saline (ambient PO2 in Calgary = 135 mmHg),
whereas hypoxic and hypercapnic conditions were produced by bubbling
90% N2 + 10% O2 and 10%
CO2, respectively. To prevent air contamination of the experimental solutions, the dissection dish was sealed off by
parafim (except to allow cell penetration). In addition, the solution
was delivered directly to the preparation under a fast perfusion system
(10 ml/min). The PO2 and
PCO2 in the experimental chamber were
continuously monitored (Hudson Ventronics; model 5584EC). Moreover, pH,
CO2, and O2 tensions of
experimental solutions were analyzed at room temperature (22°C)
using 1L-1301 (Instrumentation Laboratories, Lexington, MA).
Isolated brain preparations
To prepare isolated brain preparations, the central ring ganglia
were dissected from intact animals as described earlier (Syed et
al. 1999). The isolated ganglia were pinned down in a
dissection dish containing normal Lymnaea saline.
Surgical procedures
To produce nerve crushes in the intact animals, anesthetized snails were placed in a dissection dish, the shell was pulled back, and a dorsal midline incision was made. The body wall was pulled gently to expose the right internal and external parietal nerves and the anal nerve. These nerves innervate the pneumostome and the mantle cavity area. A pair of fine forceps was used to crush the above three nerves in intact animals. A crush to the left parietal nerve (which innervates the left body wall) served as a control. Following crushes, no suture was necessary, and the animals were allowed to recover overnight in well-aerated pond water.
Behavioral analysis
Either time-lapse video recordings or visual inspections were made to monitor the respiratory behavior of the operated animals. All experiments involving behavioral analysis were performed "blind."
Electrophysiology
Neurons from both isolated and semi-intact animals were recorded
intracellularly using glass microelectrodes filled with a saturated
solution of K2SO4
(resistance 25-30 M) and Leitz micromanipulators. The intracellular
signals were amplified by Neurodata amplifier, displayed on Textronix
oscilloscopes, and printed on Gould chart recorder.
Statistical analysis
The mean ± SE of the respiratory discharges/10 min was plotted. Statistical analysis of the data was performed using a one-way ANOVA followed by a post hoc Tukey test. Data were considered to be significant if P < 0.05.
All chemicals were purchased from Sigma.
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RESULTS |
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Hypoxia-induced respiratory drive in Lymnaea originates at the periphery
Respiration in Lymnaea is a hypoxia-driven behavior; however, the origin (central or peripheral) of this chemosensory drive is unknown. To define the locus at which the hypoxia- induced respiratory activity originates, we recorded from either isolated central ring ganglia or semi-intact preparations, both prior (normoxic) to and during a hypoxic challenge. It is important to note that as compared with 50-75% N2, maximum respiratory activity was induced by 90% N2 + 10% O2 with higher O2 (e.g., only 50-70% N2) resulting in less activity. Thus the above gas mixture was used to provide hypoxic challenge throughout this study.
In semi-intact preparations (central ring ganglia and attached
peripheral organs), simultaneous intracellular recordings were made
from RPeD1 and a visceral J (VJ) pneumostome opener motor neuron. Under normal (normoxic) air conditions, no spontaneously occurring patterned respiratory activity was present (n = 11, Fig. 1A). However,
superfusion of the peripheral compartment with hypoxic saline induced
the concurrent bursting discharges in RPeD1 and the VJ cell are
characteristics of the pneumostome opening, or expiratory phase of the
respiratory pattern in Lymnaea (Fig. 1B)
(Syed and Winlow 1991). At the same time pneumostome
opening and closing movements were recorded. These discharges are
driven by excitatory input from Input 3 (IP3), the only input known to excite the RPeD1 neuron. In this case, RPeD1 is activated first with
the VJ motor neurons subsequently being driven by the IP3 input (Fig.
1). It is important to note that the only excitatory input known to
excite RPeD1 in the intact brain is produced by IP3I (pneumostome
opening-expiration) (see Syed and Winlow 1991
). RPeD1 is
the first cell to receive IP3 activity, which subsequently drives VJ
motor neurons (Fig. 1). The patterned respiratory discharges and the
resultant behavior disappeared on return to normoxic saline (Fig.
1C). The superfusion of the central compartment (in a
semi-intact animal) with hypoxic saline did not induce respiratory
discharges in RPeD1 and the motor neuron, nor was the respiratory
behavior triggered (not shown, but see Fig. 7).
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To test further the sensitivities of respiratory neurons within the central ring ganglia to an hypoxic stimulus, the isolated central ring ganglia (CRG) were exposed to normoxic, hypoxic, or anoxic (0% O2) saline (Fig. 2A). Respiratory activity was monitored intracellularly from RPeD1 and a VJ neuron. In these isolated ganglionic preparations, robust rhythmical, fictive respiratory discharges were recorded from both neurons under normoxic conditions (n = 9, 21.4 ± 1.8 discharges/10 min, mean ± SE). Superfusion of the CNS preparation with hypoxic saline did not significantly alter the patterned respiratory discharges in the respiratory neurons (20.7 ± 1.6 discharges/10 min, P > 0.05). Bathing the CNS preparation with anoxic saline brought about a significant decrease (P < 0.01) in respiratory activity in both RPeD1 and the VJ neuron (15.5 ± 1.4 discharges/10 min). Thus the isolated CRG alone did not respond to the hypoxic challenge.
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In sharp contrast to the isolated brain preparation, however, patterned respiratory activity was affected dramatically by the hypoxic challenge in semi-intact preparations (Fig. 2B). These changes were, however, observed only when the peripheral, but not the central compartment, was bathed with hypoxic saline. Specifically, under normal (normoxic) conditions, the observed respiratory discharges averaged 1.1 ± 0.5/10 min. However, superfusion of the peripheral compartment with the hypoxic saline resulted in a significant increase in respiratory activity (n = 8, 4.1 ± 0.9/10 min: P < 0.01), which on wash out with normoxic saline showed a recovery trend toward the baseline. Superfusion of anoxic saline, on the other hand, completely abolished the respiratory activity in semi-intact preparations, which remained suppressed for prolonged periods of time (30-60 min, n = 8), even after wash out with normoxic saline.
Together, the above data demonstrate that the hypoxia-induced chemosensory drive in Lymnaea originates at the periphery. Because isolated ganglionic preparations exhibited significantly more spontaneous respiratory discharges than did the semi-intact preparations, we suggest that the periphery provides a regulatory (suppressive) input to the respiratory CPG.
CRG and semi-intact preparations are insensitive to hypercapnia
To test for the sensitivity of respiratory CPG neurons to CO2, both isolated (n = 10) and semi-intact preparations (n = 9) were superfused with hypercapnic saline while the central respiratory neurons were recorded intracellularly (Fig. 3). One to 5% CO2 did not affect respiratory activity in either isolated or semi-intact preparations (data not shown). However, superfusion of either isolated brain (Fig. 3A) or semi-intact preparation (CNS and peripheral organs; Fig. 3B), with 10% CO2 saline significantly (P < 0.01) reduced spontaneously occurring respiratory discharges. Because the pH of the perfusate also changed to 7.4-7.5 (within 5 min) under these hypercapnic (10% CO2) saline conditions, we asked whether the observed CO2-induced changes in the respiratory activity were the result of this pH change rather from the increased CO2 itself. To test this possibility, both isolated and semi-intact preparations were perfused with salines of various pH. In both preparations, respiratory activity in saline with a pH of 7.5 was significantly lower than that observed at a normal pH of 8 (Fig. 4; n = 8, P < 0.01). These data suggest that the observed changes in the respiratory activity in 10% CO2 saline may have resulted from changes in the pH of the perfusate.
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Chronic hypoxia treatment of intact animals alters respiratory activity in semi-intact but not in isolated brain preparations
We next asked whether chronic (6-12 h) hypoxic exposure of intact animals, prior to dissection and intracellular recording, altered respiratory activity in either isolated (Fig. 5A) or semi-intact preparations (Fig. 5B). After 6-12 h of hypoxic exposure, either isolated ganglionic, or semi-intact preparations were made, and spontaneously occurring respiratory discharges (IP3 activity) were recorded. Semi-intact, but not isolated brain preparations (P > 0.05) from chronic hypoxia treated animals, exhibited enhanced spontaneous respiratory activity under normoxic recording conditions. Although semi-intact preparations obtained from animals that were exposed to hypoxia for 6 h exhibited significantly (P < 0.01) higher respiratory episodes as compared with their control counterparts (n = 10), there was no further increase in respiratory activity with an additional 6 h of hypoxia (Fig. 5B). These data demonstrate that prior environmental history of an animal is an important determinant of its motor output recorded in a reduced preparation. Moreover, the locus for these modulatory changes in Lymnaea respiratory patterned activity most likely involves peripheral elements. Our data also demonstrate that, like mammalian preparations, respiratory behavior in Lymnaea exhibits long-term modulatory changes in response to an hypoxic challenge.
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Axotomy of the internal and external parietal nerves disrupted normal respiratory behavior
To demonstrate further that chemosensory input to and from the periphery is indeed required for normal respiratory behavior in the intact animal, the right internal and external parietal nerves, as well as the anal nerve (these nerves innervate the pneumostome and mantle cavity area) were axotomized, and the respiratory behavior of freely moving animals was examined. Because the above nerves carry both afferent and efferent projections to and from the CNS (including RPeD1), we predicted that severing these projections would disrupt normal respiratory behavior. Axotomized animals exhibited significantly fewer respiratory movements (pneumostome openings; 3.0 ± 0.9; n = 16) 1 day after axotomy than experimental animals (8.1 ± 1.6; n = 16; P < 0.05; Fig. 6). By day 7, however, the respiratory behavior exhibited by the experimental animals was similar to the control animals.
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Peripherally induced hypoxia drive is conveyed to the central CPG neurons via RPeD1
The above data suggested to us that the respiratory drive in
Lymnaea originates in the periphery. Because RPeD1 is the
only respiratory CPG neuron with peripheral projections (projects via right internal, external, and anal nerves), we hypothesized that the
hypoxic information is either carried by RPeD1 itself from the
periphery, or is driven indirectly by peripherally located chemosensory
elements. To distinguish between these possibilities, the peripheral
compartment was superfused with hypoxic saline containing either normal
or 0 Ca2+ (0 Ca2+ and high
Mg2+ saline) (Syed and Winlow
1991). Within a few minutes of hypoxic exposure of the
peripheral compartment, a pattern of bursting activity was triggered in
RPeD1 (n = 7, Fig.
7A). These hypoxia-induced effects on patterned activity in RPeD1 were abolished when the hypoxic
saline did not contain Ca2+ (i.e., zero
Ca2+ and high Mg2+ saline;
Fig. 7B). These data suggest that RPeD1 receives a chemical, excitatory input from the peripherally located chemosensory elements. Conversely, the superfusion of only the central ring ganglia
(containing RPeD1's somata) with hypoxic saline did not trigger
patterned respiratory activity (Fig. 7C). Together, the
above data are consistent with our hypothesis that the
hypoxia-sensitive chemosensory drive in Lymnaea originates
at the periphery and is conveyed to the central CPG neurons indirectly
via RPeD1.
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DISCUSSION |
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In isolated ganglionic preparations, as well as in the in vitro
reconstructed network, RPeD1 stimulation via current injection can
trigger fictive respiratory activity in Lymnaea
(Inoue et al. 1996b; Syed and Winlow
1991
; Syed et al. 1990
). However, the origin of
this excitatory drive to RPeD1, which would normally initiate
respiratory activity in intact and semi-intact preparations, remained
unknown. The data presented here demonstrate that hypoxia-induced chemosensory input from the periphery provides the necessary excitatory input to RPeD1 via chemical synaptic connections. Moreover, in isolated
brain preparations, the frequency of spontaneously occurring respiratory discharges was much higher than in isolated preparations, as well as higher than normal respiration in intact animals. Taken together, our data emphasize the importance of peripheral sensory input
in the initiation and regulation of the respiratory rhythm. This study
does not, however, undermine the importance of the CPG in rhythm
generation per se.
In addition to hypoxia, freely behaving Lymnaea is also responsive to hypercapnic water (5% CO2, 21% O2, 84% N2), which increases their overall respiratory drive (L. L. Moroz, N. I. Syed, K. Lukowiak, A.G.M. Bulloch, and S. U. Hasan, unpublished observations). However, hypoxia and hypercapnia-driven respiratory activity was found to differ qualitatively. Specifically, hypoxia challenge increased the frequency of spontaneous pneumostome opening and closing movements, while hypercapnic water prolonged only the duration of pneumostome openings (Moroz et al., unpublished observations). These data thus demonstrate that intact animals can respond to both hypoxic and hypercapnic environments; however, their behavioral responses under these conditions are qualitatively different. These behavioral observations in the intact animals do not, however, agree with our data on semi-intact preparations. One likely explanation for this discrepancy may reside in the fact that hypercapnia-sensitive chemosensory cells may be located either on the foot and/or body wall musculature, which in the study was surgically removed to prepare semi-intact preparations. Thus under these experimental conditions, only hypoxia-sensitive elements located adjacent to the pneumostome and mantle area remained intact. If CO2-sensitive chemoreceptors were indeed surgically removed, then how did our semi-intact preparations exhibit reduced respiratory activity in response to hypercapnia (10% CO2)? We believe that even though Lymnaea's natural habitat is stagnant water, where CO2 levels may reach 5% (normal pond water = approximately 1-2% CO2), the hypercapnic challenge delivered to semi-intact preparations in the present study is likely unphysiological. Thus CO2 either directly or indirectly (via pH changes) may have exerted toxic (acidosis) effects on the respiratory CPG (as well as peripheral) neurons. This possibility, however, remains to be tested experimentally.
In contrast with the land snail Helix, in which neurons
within the CRG exhibited respiratory sensitivity to
PCO2 (Erlichman and Leiter 1993,
1994
; Erlichman et al. 1994
), the
Lymnaea CRG were insensitive to hypercapnia. Although the
possibility of peripherally located CO2-sensitive
receptors in Lymnaea remains to be determined, the data from
the above two species do nevertheless suggest that land and freshwater
snails may have adopted different evolutionary strategies (central vs.
peripheral) to meet their respective respiratory needs.
In contrast to hypoxia, an anoxic challenge suppressed the respiratory activity in semi-intact preparation. Likewise, previous studies on intact animals have demonstrated that snails kept in an anoxic environment rest motionless at the bottom of the tank and death follows within 6-12 h (Moroz et al., unpublished observations). An obvious explanation for this might be that the lack of O2 shuts down most metabolic activity, thus rendering the animal incapable of various cellular functions.
In vertebrates, respiratory chemoreceptors are located both centrally
in the medulla (Bruce and Cherniak 1987;
Fitzgerald and Deghani 1982
; Gonzalez et
al. 1992
; Hitzig and Jackson 1978
; Loescheke 1982
; Nattie 1991
;
Smatresk 1990
; Smatresk and Smits 1991
) and peripherally in the carotid bodies and aortic
arteries (Benchetrit et al. 1977
; Hitzig and
Jackson 1978
; Ishii et al. 1985
; Smatresk
1990
). Central chemoreceptors are sensitive to PCO2 and pH of the extracellular fluid bathing
the ventral surface of the medulla (Wilding et al.
1992
). Although the existence of medullary chemoreceptors has
been demonstrated in turtles (Benchetrit et al. 1977
),
toads (Hitzig and Jackson 1978
), and lampreys
(Rovainen 1977
), the exact location, the stimuli (i.e.,
PCO2 or pH), and the mechanisms by which these
receptors are activated are not fully understood (Bruce and
Cherniak 1987
; Gonzalez et al. 1992
).
Consistent with peripherally located chemosensors in
Lymnaea, mammalian peripheral chemoreceptors in the carotid
body and the aorta are sensitive to changes in
PO2 and pH. In other vertebrates, such as
amphibians, PO2-sensitive receptors are located
in the carotid labyrinth (Ishii and Ishii 1976), whereas
in reptiles they are distributed throughout the carotid and pulmonary
arteries (Coates and Ballani 1987
). In fish and
lungfish, PO2-sensitive receptors are found in
gill arteries (Burleson and Smatresk 1989
; Johansen and Lenfant 1968
; Milsom and Brill
1986
). Thus peripheral chemosensory cells in reptiles, fish,
lungfish, and crayfish (Ishii et al. 1989
) are
intimately associated with blood vessels either at or near the heart
and/or the respiratory organs. Consistent with the above studies, the
hypoxia-sensitive chemoreceptors in Lymnaea also appear to
be located either at or near the heart, lung, and pneumostome area.
The best-studied mammalian respiratory chemoreceptors are located in
the carotid bodies (Duchen and Biscoe 1991; Osani
et al. 1997; Stea and Nurse 1991
). The
biophysical properties as well as a putative mechanism by which these
cells convey the hypoxia signal to the CNS have been investigated in
culture (Duchen and Biscoe 1991
; Stea and Nurse
1991a
; Youngson et al. 1993
). The transmitter
released from the chemoreceptors is hypothesized to activate neuronal
endings, which in turn convey the hypoxia signal to the CPG. In the
present study, we have demonstrated that the chemosensory cells located
in the pneumostome area (ospheradial ganglia) may exhibit properties
similar to those of carotid body chemoreceptors. When activated by the
hypoxic stimulus, they excite RPeD1, which in turn triggers respiratory
activity in the CPG. Specifically, if RPeD1's nerve endings were
serving as a chemosensor, then perfusing the peripheral compartment
with hypoxic saline in the presence of 0 Ca2+
saline would also have excited this cell. This, however, was not the
case. Although the precise location of the hypoxia-sensitive chemoreceptors remains to be elucidated, one possibility is the ospheradial ganglion.
Although nerve crushes adjacent to the pneumostome area suppressed respiratory activity in intact animals, this perturbation was transient (observed only up to day 3). Normal respiratory behavior was restored within 1 wk of axotomy. This suggests that either as yet unidentified elements compensated for the loss of function, or that functional regeneration occurred. Other data from our laboratory support the latter. Specifically, we have demonstrated that, following axotomy, successful regeneration not only restores functional synaptic connections within the CNS but also leads to restoration of normal respiratory behavior in intact animals (Z. Haque, K. Lukowiak, and N. I. Syed, unpublished observations). These data thus suggest that the restoration of normal behavior after the nerve crush most likely involved regeneration of central and/or peripheral projections.
The data presented in this study also demonstrate that the pretreatment
of animals with chronic hypoxia can modulate their respiratory output
for a longer period of time. The semi-intact preparations made from
animals exposed to hypoxia for 6-12 h exhibited a higher frequency of
spontaneously occurring respiratory movements, even though the neuronal
activity was recorded in the presence of normoxic saline. However,
exposure of these semi-intact animals to hypoxic saline did not further
alter the frequency of respiratory episodes (data not shown),
suggesting that the chronic hypoxia treatment of intact animals may
have already exerted a maximal effect. Moreover, because chronic
hypoxic treatment of the intact animals enhanced respiratory activity
in semi-intact but not isolated ganglionic preparations, these data
further suggest that the modulation of respiratory behavior by chronic
hypoxia, like acute hypoxia, involves peripheral chemoreceptors.
Changes induced by chronic hypoxia in this study are consistent with
those described earlier on mammalian preparations (see Powel et
al. 1998), where animals exposed to chronic hypoxia were found
to hyperventilate (Okubo and Mortola 1988
).
Similarly, prolonged hypoxic exposure at birth was also found to
increase the overall firing frequency of the carotid body
chemoreceptors (Hertzberg et al. 1992
;
Soulier et al. 1997
).
In summary, this study demonstrated that hypoxia-induced respiratory activity in Lymnaea originates at the periphery and is likely conveyed to the central CPG neurons indirectly via RPeD1's peripheral projections. Nevertheless, our data do not, however, rule out the involvement of other, as yet unidentified, cellular elements in the perception of chemosensitivity. The importance of peripheral sensory input in the control of respiratory rhythmogenesis in this model system provides us with an excellent opportunity to examine the cellular and synaptic mechanisms underlying hypoxia-induced regulation of breathing behavior.
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ACKNOWLEDGMENTS |
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N. I. Syed is an AHFMR Senior Scholar.
This work was supported by CIHR Canada.
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FOOTNOTES |
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Address for reprint requests: N. I. Syed, Dept. of Cell Biology and Anatomy, Faculty of Medicine, The University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada (E-mail: nisyed{at}acs.uclagary.ca).
Received 28 September 2000; accepted in final form 19 March 2001.
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REFERENCES |
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