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During eupnoea, rhythmic motor activities of the hypoglossal, vagal and phrenic nerves are linked temporally. The inspiratory discharges of the hypoglossal and vagus motor neurones commence before the onset of the phrenic burst. The vagus nerve also discharges in expiration. Upon exposure to hypocapnia or hypothermia, the hypoglossal discharge became uncoupled from that of the phrenic nerve. This uncoupling was evidenced by variable times of onset of hypoglossal discharge before or after the onset of phrenic discharge, extra bursts of hypoglossal activity in neural expiration, or complete absence of any hypoglossal discharge during a respiratory cycle. No such changes were found for vagal discharge, which remained linked to the phrenic bursts. Intracellular recordings in the hypoglossal nucleus revealed that all changes in hypoglossal discharge were due to neuronal depolarization. These results add support to the conclusion that the brainstem control of respiratory-modulated hypoglossal activity differs from control of phrenic and vagal activity. These findings have implications for any studies in which activity of the hypoglossal nerve is used as the sole index of neural inspiration. Indeed, our results establish that hypoglossal discharge alone is an equivocal index of the pattern of overall ventilatory activity and that this is accentuated by hypercapnia and hypothermia.
To maintain patency of the upper airway during breathing, the discharges of cranial and spinal nerves must be coordinated (Remmers et al. 1978; Dutschmann & Paton, 2002). Prominent among these discharges is the hypoglossal nerve, which innervates the muscles of the tongue. The onset of discharge of the hypoglossal nerve occurs before the onset of activity of the phrenic nerve. This early onset of hypoglossal activity, termed the ‘pre-inspiratory’ discharge, stabilizes the upper airway prior to the commencement of airflow to the lungs (Remmers et al. 1978; Hwang et al. 1983; Remmers, 1998; St.-John, 1998; Bailey et al. 2001).
Brainstem mechanisms controlling the pre-inspiratory portion of the hypoglossal discharge may differ from those controlling hypoglossal activity during ‘neural inspiration’, which is concomitant with phrenic discharge. This possibility is supported by the finding that the pre-inspiratory portion of the hypoglossal discharge is separable from the ‘inspiratory’ discharge. In the in situ preparation of the juvenile rat, such a separation was evidenced by the occurrence of a transient reduction or total cessation of hypoglossal discharge at the start of the phrenic burst and/or by complete separation of pre-inspiratory and inspiratory hypoglossal bursts (Leiter & St.-John, 2004). Alterations in lung volume of in vivo preparations can also induce a separation of the pre-inspiratory and inspiratory components (Saito et al. 2002a,b). Additionally, high frequency oscillations in neural activities, assessed by power spectral analysis, differ between the components of the hypoglossal discharge. Peak frequency in the power spectra of the pre-inspiratory discharge was lower than that during the inspiratory component. This inspiratory component of hypoglossal discharge had the same peak frequency as that of the phrenic discharge and also both inspiratory and expiratory discharges of the vagus nerve (St.-John & Leiter, 2003; Leiter & St.-John, 2004). These differences in peak frequencies in the power spectra suggest a different origin of drive to the pre-inspiratory and inspiratory components of the hypoglossal discharge.
The present study was undertaken to assess further the mechanisms controlling the pre-inspiratory and inspiratory components of the hypoglossal discharge. If these components are indeed differentially regulated, we hypothesized that uncoupling between the two components of hypoglossal activity would be induced by perturbations that caused a reduction in eupnoeic ventilatory drive, including hypocapnia and hypothermia. In addition, we evaluated whether the uncoupling of the pre-inspiratory from the inspiratory hypoglossal discharges represented a transient, active inhibition that was superimposed on a continuous hypoglossal discharge. Finally, from recordings of efferent activity of the vagus nerve, we evaluated whether ‘uncoupling’ was a common characteristic of respiratory-modulated activities of cranial nerves other than the hypoglossal nerve. The results of these studies support the conclusion that the control of pre-inspiratory and also inspiratory hypoglossal activity differs from that of the vagal and bulbospinal–phrenic system.
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The major conclusion of this study is that the control of respiration-modulated activity of hypoglossal motoneurones differs markedly from the control of vagal motoneurones and the bulbospinal–phrenic motor system. Stated differently, the linkage between the brainstem respiratory mechanisms generating eupnoea, as defined by activity of the phrenic nerve, and hypoglossal discharge is variable, whereas the relationship between phrenic and vagal activity is fixed and less susceptible to modification. Changes in temperature of the preparation, exposure to hypocapnia and stimulation of the peripheral chemoreceptors were used to induce modification. The variable nature of the relationship between hypoglossal and phrenic discharges is especially apparent for the component of hypoglossal discharge which commences in mid to late neural expiration.
Results of the present study further establish that the control of hypoglossal and phrenic activities differs. A variable link between the brainstem ventilatory control system and hypoglossal discharge was expected as the hypoglossal system is involved in many other rhythmic activities besides respiration, such as mastication and deglutition (Dick et al. 1993; Paton et al. 1999; Jean, 2001; Roda et al. 2002; Saito et al. 2002a,b). Hence, under conditions in which ventilatory drive is lowered, the link between hypoglossal discharge and the overall respiratory rhythm may become more tenuous. In the present study, lowered respiratory drive in hypocapnia and hypothermia resulted in uncoupling of hypoglossal activity from phrenic activity. Somewhat paradoxically, phrenic and hypoglossal activities also became uncoupled during stimulation of the carotid chemoreceptors. However, this uncoupling may not reflect the overall increase in respiratory activity as stimulation of the carotid bodies activates the brainstem reticular system (see references in Hafström et al. 2000). A greater dependency of the hypoglossal than phrenic systems upon reticular activation has long been proposed (for review see St.-John, 1986). Alternatively, the peripheral chemoreceptor stimulation may have elicited centrally generated swallowing, and hence extra bursts of hypoglossal activity (Paton et al. 1999).
Not only does the control of hypoglossal and phrenic activities per se differ, but various portions of the hypoglossal discharge may be differentially regulated. As noted in the Introduction, the pre-inspiratory and inspiratory components of the hypoglossal discharge are separable (Saito et al. 2002a,b; Leiter & St.-John, 2004). Furthermore, differences in high frequency oscillations of nerve activities between pre-inspiratory and inspiratory components of hypoglossal activity also imply that separate brainstem mechanisms control these two components of the hypoglossal discharge. Power spectral analysis of these oscillations during eupnoea revealed similar peak frequencies for phrenic discharge, vagal discharges in neural inspiration and expiration and hypoglossal discharge in neural inspiration. However, a significantly lower frequency was found for the pre-inspiratory portion of the hypoglossal discharge (Leiter & St.-John, 2004). High frequency oscillations in neural activities are considered signatures of the central pattern generator for eupnoea (St.-John & Leiter, 2003; Leiter & St.-John, 2004). Hence, the difference in peak frequency of the pre-inspiratory hypoglossal discharge implies that this component receives an input from a separate brainstem source than that to the other cranial and spinal nerves. The similarity of high frequency oscillations among inspiratory activity of the hypoglossal, vagal and phrenic activities implies that the inspiratory activation of these motor neurone pools originates from a common source.
Results from intracellular recordings also demonstrate a different control of hypoglossal compared to phrenic activities. Hence the depolarization of neurones in the hypoglossal neurones commenced before the phrenic discharge and/or during extra bursts of activity of the hypoglossal nerve in neural expiration with no concomitant changes in phrenic discharge. The excitatory events underlying these depolarizations appear to be differentially distributed among the neurones in the hypoglossal nucleus. Hence, despite an overall increase in activity of the hypoglossal nerve, some neurones showed little change in depolarization and no spiking. Similarly, neurones maintained depolarizations and even spiking during the pause in activity of the hypoglossal nerve at the commencement of phrenic discharge. Such pauses were minimal during recordings in the present study, as these were obtained long after the commencement of rhythmic respiratory action when such pauses were maximal. Yet in the study of Saito et al. (2002b), in which the pauses of pre-inspiratory and inspiratory discharges were marked, again hypoglossal motoneuronal activities showed variable changes, but largely a reduction in the degree of depolarization. Thus disfacilitation would appear responsible for these pauses, which is in agreement with the concept that the discharge of hypoglossal motoneurones is largely defined by excitatory synaptic events (Peever et al. 2002; Saito et al. 2002b).
Results from intracellular recordings establish that the difference between pre-inspiratory and inspiratory components of the discharge of the hypoglossal nerve cannot reflect neuronal discharges to different groups of muscles. Hence, while the hypoglossal nerve innervates a number of muscles of the tongue, a separation of pre-inspiratory and inspiratory discharges was uniformly observed for intracellular recordings of hypoglossal motoneuronal activities (Saito et al. 2002b). Moreover, such a separation was also observed for electromyographic activities of both the genioglossus and hyoglossal muscles (Bailey et al. 2001).
As to the brainstem source of respiration-modulated activity, neuroanatomical and neurophysiological observations demonstrated that the vast majority of ‘premotor’ pontile and medullary neurones to hypoglossal motor neurones differed from those to the bulbospinal–phrenic system (Ono et al. 1994; Dobbins & Feldman, 1995; Fay & Norgren, 1997; Peever et al. 2001, 2002; Travers & Rinaman, 2002; Li et al. 2003). Only a few premotor neurones which project to both the hypoglossal and phrenic motor nuclei have been identified in cats (Ono et al. 1994). Concerning medullary premotor neurones, there is no doubt that intrinsic medullary circuits can convey respiration-modulated activities to the hypoglossal and phrenic systems, as phrenic and hypoglossal discharges are synchronized during gasping in vivo and in situ and during rhythmic in vitro discharges (e.g. Smith et al. 1990; St.-John, 1990; Kato et al. 1996; St.-John & Leiter, 2003). Yet, these intrinsic medullary discharges differ from those during eupnoea in that phrenic and hypoglossal discharges commence at approximately the same time. In vitro preparations are of neonatal animals, whereas the majority of in vivo and in situ studies have involved juvenile and adult rats. The age of preparations would not appear to account for the difference in relative times of onset of hypoglossal and phrenic activities as hypoglossal discharge commences before phrenic discharge during eupnoea in neonatal in vivo rats (Fukuda, 2000). These times of onset become the same in gasping (Fukuda, 2000). Similarly, the onset of the efferent vagal activity precedes that of the phrenic during eupnoea in neonatal rats in vivo or in situ but again is the same in gasping (Wang et al. 1996; Dutschmann & Paton, 2002). This difference in the control of hypoglossal activities in vitro as compared to that during eupnoea in vivo or in situ might account for the findings of Kato et al. (1996) that, as opposed to the results herein, the synchronization between phrenic and hypoglossal discharges of an in vitro neonatal cat preparation was unaltered by changes in temperature. This synchronization was unaltered by removal of pons by a brainstem transection at the pontomedullary junction which supports our conclusion that these in vitro rhythms differ from those of eupnoea in vivo (St.-John, 1990, 1996, 1998; St.-John & Paton, 2003). Hence, medullary mechanisms alone may not be responsible for the regulation of hypoglossal and phrenic activities in eupnoea.
A prominent group of ‘premotor’ neurones to the hypoglossal nucleus are within the rostral pons, close to the pneumotaxic centre of the nucleus parabrachial medialis and Kolliker-Fuse nucleus (Dobbins & Feldman, 1995; Fay & Norgren, 1997; Li et al. 2003; Travers & Rinaman, 2002). While not evaluated in detail, there is evidence that pneumotaxic mechanisms can powerfully influence hypoglossal activity. Hence, electrical stimulation of the pneumotaxic centre, in regions that terminate phrenic discharge, causes augmentations in hypoglossal activity (St.-John, 1987). In apneusis in vivo, the difference in time of onset of phrenic and hypoglossal and phrenic activities is reduced or eliminated (St.-John et al. 1984). Thus, we submit that the complex interrelationships between hypoglossal and phrenic activities, described herein, are compatible with a significant role for pontile mechanisms in establishing this relationship.
Finally, results of the present study demonstrate that, during eupnoea, rhythmic activity of the hypoglossal nerve may deviate markedly from the overall respiratory rhythm as assessed by activities of the phrenic nerve and vagus nerve. Hence, from recordings of hypoglossal activity alone, rhythmic bursts cannot be concluded to be ‘inspiratory’ or even related to respiration. Thus, this raises concern over any experiment in which hypoglossal activity alone is used an index of neural inspiration. Typically, hypoglossal activity alone is recorded in vitro slice preparations and so caution should be exercised regarding the relevance to neural inspiration.