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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.34 n.2 Santiago  2001

http://dx.doi.org/10.4067/S0716-97602001000200016 

In vitro approach to the chemical drive of breathing

JAIME EUGENIN, ISABEL LLONA, CLAUDIA D. INFANTE, AND ESTIBALIZ AMPUERO

Laboratory of Neural Systems, Department of Biology, Faculty of Chemistry and Biology, Universidad de Santiago de Chile, Santiago, Chile

Corresponding Author: Dr. Jaime Eugenín. Laboratorio de Sistemas Neurales, Depto. Biología, Facultad de Química y Biología, Universidad de Santiago de Chile. Avda. Bdo. O´Higgins 3363. Casilla 40 Correo 33, Santiago, Chile. Fax: (56 2) 681 2108. E-mail: jeugenin@lauca.usach.cl

Received: May 20, 2001. Accepted: June 10, 2001.

ABSTRACT

Since its introduction two decades ago, the isolated brain stem-spinal cord preparation of neonatal rodents has been the preferred method used to reveal the mystery underlying the genesis of the respiratory rhythm. Little research using this in vitro approach has focused on the study of the central respiratory chemosensitivity. Some unexpected findings obtained with the brain stem-spinal cord preparation have added new questions that challenge our previous theoretic framework. Some of these findings are addressed here.

Key terms: central chemoreceptors; control of breathing; ventral medulla; brain stem

INTRODUCTION

Breathing is the result of a cyclical, synchronic and coordinated activation of respiratory muscles. Precise commands generated within the lower brain stem are transmitted to muscles through respiratory motoneurons. Such commands must be continuously modified in function of the chemical feedback to fit the demands of the mammal for uptake of oxygen and removal of carbon dioxide. Chemical feedback relies on specialized organs strategically located within the circulatory system as well as in the central nervous system. Carotid and aortic bodies are well suited to detect changes in PO2, PCO2, and pH in the blood flowing through large arteries (Alcayaga et al, 1997; Iturriaga, 1993), while central chemoreceptors detect changes in PCO2 and pH within the CNS (Nattie, 1999).

The exact nature of central chemoreceptors involved in the control of breathing is still unknown. Therefore, most of the efforts in this field have been devoted to localize the central chemoreceptors and to characterize their functions. Since carbon dioxide is highly diffusible, any structure of the central nervous system appears a priori well located to accomplish the task of being a CO2or pH detector. Pioneer experiments (Loeschcke, 1982) showed that acidic artificial cerebrospinal fluid applied to the ventrolateral surface of the brain stem of anesthetized cats is able to evoke respiratory responses. Further studies revealed that respiratory chemoreception on the surface of ventral medulla is a common characteristic shared by every species of mammals studied (Loeschcke, 1982). In the last two decades, other areas of the central nervous system have been added to the list of possible respiratory chemosensitive sites. Indeed, a growing body of evidence supports the notion that respiratory chemoreception is a property distributed in several sites within the CNS (Nattie, 1999).

Suzue (1984) was the first to report that the en bloc isolated brain stem-spinal cord preparation of newborn rats shows spontaneous respiratory-like activity recordable from hypoglossal nerve and cervical ventral roots. In this preparation, peripheral nerves are cut, while the CNS preserves its normal organization. Therefore, vagal feedback is absent and any respiratory response induced by changes in pH or CO2 can be attributed to central chemoreceptors. Fine localization of respiratory chemosensory sites requires elimination of any possible interference from respiratory effects secondary to cardiovascular responses, anesthesia or dilution of chemical stimuli by the circulation. Such interferences are inherent to most of the experiments done in vivo. In contrast, the isolated brain stem-spinal cord appears as a favorable preparation to study localization of central chemoreceptors because it lacks the above-mentioned interferences. Simultaneously, it shows fictive respiration, maintains the neuroanatomical organization of the CNS, and allows fine control of stimuli. For these reasons, several research groups have concentrated their attention on the isolated brain stem ­ spinal cord preparation during the last decade. In general, studies have confirmed previous results obtained with other preparations. But they have also extended our knowledge about central chemoreception. Thus, some singularities have given rise to new questions that challenge our previous theoretical framework. We address some of these singularities here.

Chemical sensitivity and the brain stem-spinal cord preparation

It has been largely recognized that changes in the frequency and amplitude of respiration are controlled by separate mechanisms (Cohen, 1979; Feldman, 1986; von Euler, 1986). The classical view of chemoreception derived from studies in anesthetized cats indicates that an increased chemical drive either by stimulation with low pH or raised levels of CO2 increases both the amplitude and the frequency of respiration (Cohen, 1979; von Euler, 1986). How is the simultaneous increase in the amplitude and frequency of respiration attained? The shortening of the respiratory cycle is secondary to a decrease in the inspiratory duration mediated by the vagal reflex that inhibits inspiration (Hering-Breuer reflex). Such shortening of the inspiratory duration has been successfully explained in terms of the "inspiratory off-switch hypothesis" (Bradley et al, 1975; von Euler, 1986; Eugenín, 1995). In brief, this hypothesis assumes that the "central inspiratory activator", the neural network that generates and shapes the inspiratory activity receives a direct and excitatory input from chemoreceptors. The rapid increase in the inspiratory activity, sensed by slowly adapting pulmonary mechanoreceptors would determine the rapid activation of a second neural network, known as the "inspiratory off-switch," whose function is the inhibition of the central inspiratory activator. If vagal feedback is absent, then chemical stimulation should increase only the amplitude of inspiratory activity unless the inspiratory activator and the inspiratory off-switch had another level of interactions (Bradley et al, 1975; von Euler, 1986; Eugenín, 1995).

Suzue reported that the rate of the rhythm in the brain stem-spinal cord preparation depends on the pH of the superfusion (Suzue, 1984). He found that low pH (6.8) superfusion increased the rate of the rhythm, while high pH (8.8) superfusion reduced it. Similar results were obtained in neonatal opossum and mice (Monteau et al, 1990; Eugenín and Nicholls, 1997). In those experiments, less pronounced changes in pH were used for chemical stimulation. Reduction of the chemical drive, by superfusion with medium pH 7.7 or greater, can abolish the rhythm (Monteau et al, 1990; Eugenín and Nicholls, 1997). This suggests that chemical stimulus provides an indispensable input for generating or maintaining the respiratory rhythm in the isolated central nervous system of newborns.

One puzzling issue is why different patterns of respiratory responses to chemical stimulation have been observed in vitro. In the neonatal rat brain stem-spinal cord preparation, patterns of responses involve changes in frequency (Suzue, 1984; Issa and Remmers, 1992; Okada et al, 1993), amplitude (Monteau et al, 1990), or both (Harada et al, 1985). Differences in the type of stimulus (pH or CO2), in the extent of structures contained in the preparation (preservation of pontine or other encephalic structures), in the age of newborns, or in the functional state of the nervous system should be considered when trying to explain such differences in the pattern of responses. In the newborn mouse, superfusion of the brain stem with low pH solutions increases the frequency but decreases the amplitude of the fictive respiration (Infante and Eugenín, 2000). In the neonatal opossum, the pattern of response to acidification of the superfusion medium consisted of the increase of both the amplitude and frequency of fictive respiration (Eugenín and Nicholls, 1997). Therefore, the pattern of response in the opossum is similar to the increase in tidal volume and respiratory frequency reported in adult animals in vivo.

As mentioned previously, the adult pattern of responses to pH is due to the contribution of vagal feedback, which is absent in the opossum in vitro (Eugenín and Nicholls, 1997). In order to understand this unexpected result, analysis of the motor programming for opossum fictive respiration in terms of amplitude and timing of respiratory phases was done (Eugenín and Nicholls, 2000). Surprisingly, this analysis revealed that changes in frequency were achieved by changes almost exclusively in the duration of the expiratory phase of respiration (Fig. 1B). As illustrated in Figure 1, in some newborn opossums, the relationship between expiratory duration and amplitude closely depicts a hyperbole, as that described for the relationship between amplitude and inspiratory duration in cats (Clark and von Euler, 1972). Thus, the timing of the respiratory cycle in the neonatal opossum is controlled by an expiratory rather than an inspiratory "off-switch." In other words, the "inspiratory off-switch hypothesis" does not account for the way that chemical input produces its effects on the rhythm generator in the isolated CNS of opossum neonates.

Functional localization of chemosensitive areas

Ventral and dorsal surfaces of the isolated CNS of the newborn opossum were systematically explored by touching the pia mater with the tip (100 mm of inner diameter) of a glass pipette containing medium pH 6.5. The pipette had an interior electrode that allowed the delivery of the acidic solution inside the pipette close to its tip, while another electrode allowed for internal suction to renew the medium and avoid leakage. Gentle suction produced inside the pipette sealed the tip of the pipette to the nervous tissue. Leaking and diffusion of the test medium into nearby tissue was minimized by increasing the rate of superfusion and by adding an external drain. In the neonatal opossum, a map of well-defined pH-sensitive spots restricted to the surface of ventral medulla arises when the CNS is topicallly stimulated with acidic medium (Eugenín and Nicholls, 1997). Low pH stimulation of small areas on the surface of the spinal cord, pons, and mesencephalon were ineffective to reproduce the pattern of response to acidic superfusion of the brain stem. Different chemosensitive areas of the CNS may produce respiratory responses with different patterns (Eugenín and Nicholls, 1997). In the opossum, acidic solution applied to the caudal ventral medulla (medial to the hypoglossal roots) predominantly increased frequency, whereas cephalic application (at the level of vagus and glossopharyngeal roots) increased both frequency and amplitude. Analysis of the motor programming (Fig. 1A) indicates that a hyperbolic relationship between amplitude and expiratory duration can be constructed when the responses from both cephalic and caudal application are combined. In addition, changes in frequency after stimulation of each site rely on the same linear relationship between cycle duration and expiratory duration (Fig. 1B). This analysis suggests that the stimulation of cephalic and caudal sites triggers similar neural mechanisms responsible for the respiratory responses. Whether the number of recruited chemoreceptors determines the position of the curve at which the system is working is unknown.

Since the stimulus was applied superficially, a high pH-sensitivity may represent the proximity of the central chemoreceptors to the surface of the nervous tissue. This agrees with the finding that most of c-fos immunoreactivity induced in rats by CO2, inhalation is confined to the neurones located in the outermost area of the ventral medullary surface (Sato et al, 1992; Miura et al, 1998). In addition, in ventral medulla of neonatal rats, almost all pH-responsive neurons identified by injection of Lucifer Yellow projected dendrites from their somata (located between 50 and 700 µm) to within the 50 µm of the surface of the ventral medulla (Kawai et al, 1996).

Whether or not this superficial distribution determines a specific role for ventral medullary neurons as detectors of CO2 or H+ concentration in the subarachnoid space is an open question. On the other hand, a rich vascularization found in this area suggests that circulation is an important pathway through which chemical stimulus can reach chemosensory structures located deep within the nervous tissue. Because highly localized stimulus was not applied within the nervous tissue, the opossum experiments do not rule out the existence of chemosensitive areas deep in the brain stem.

Figure 1: Reduction of expiratory duration is associated with an hyperbolic increase in amplitude (A) and a linear decrease in cycle duration (B) in newborn opossum in vitro. Topical application of pH 6.5 was done on cephalic and caudal sites as indicated in the scheme. Responses to cephalic and caudal applications, triangles and circles, respectively. Symbols correspond to the values for each respiratory cycle during basal and test conditions.


Acetylcholine and central chemoreception

A clear relationship between an endogenous cholinergic pathway and central chemoreception has also been found in the isolated brain stem-spinal cord preparations from neonatal rat, mouse, and opossum (Monteau et al, 1990; Eugenín and Nicholls, 1997, Ampuero et al, 2000). Acetylcholine agonists and acetylcholinesterase inhibitors excite fictive respiration in the isolated CNS of rat and opossum neonates mimicking the pattern of responses obtained with low pH stimulation (Monteau et al, 1990; Eugenín and Nicholls, 1997). The patterns of responses after application of carbachol or low pH solution on a same ventral medulla area are similar (Fig. 2). Even more, acetylcholine- and low pH- sensitive areas on ventral medulla overlapped. On the other hand, muscarinic receptor blockade reduced partially basal respiratory activity and abolished respiratory responses to topical administration of both acetylcholine agonists and low pH solutions. In addition, in opossum neonates, acetylcholinesterase staining is confined to the parapyramidal chemosensitive area, which has been defined as chemosensitive by functional studies. Cholinergic synapses do not, however, appear to be essential for the genesis or maintenance of the respiratory rhythm since complete blockade of acetylcholine receptors does not abolish the rhythm.

Figure 2: Pattern of response after unilateral single spot stimulation with carbachol (A) and low pH (B). The site of topical application is illustrated in the scheme. Instantaneous respiratory frequency and amplitude are expressed as percentage of basal values. Symbols correspond to the values obtained for each respiratory cycle. Note that carbachol as well as pH increase the instantaneous respiratory frequency.


CONCLUSIONS

The results confirm that the isolated CNS of the newborn rodent generates a respiratory rhythm, which is driven by chemical input. This chemical input relies on a cholinergic pathway partially originated on the surface of ventral medulla. Analysis of the pattern of respiratory responses found in vitro have revealed singularities, indicating that the increase in frequency induced by low pH superfusion depends on the timing of the expiratory phase of respiration. This means that the chemosensory drive of respiration in the isolated CNS of newborns is exerted on an "expiratory off-switch."

ACKNOWLEDGEMENTS

This work was funded by grants from the National Fund for Scientific and Technological Development (FONDECYT #1980819), and the Research Division of the Universidad de Santiago de Chile (DICYT #029743EL). Dr. J. E. wishes to thank his friend and mentor, Dr. Jaime Alvarez, for his constant support and advice, which have been crucial to the development of the Laboratory of Neural Systems of the Universidad de Santiago de Chile.

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