The respiratory neural network simultaneously generates two distinct rhythms: a faster, lower-amplitude rhythm and slower, larger-amplitude rhythm, corresponding to fictive eupnea and fictive sighs, respectively. The muscarinic agonist oxotremorine alters the timing of these rhythms: fictive sighs speed up, whereas fictive eupneic activity slows considerably. This network effect was likely due to M3-mAChR activation because it was antagonized by 4-DAMP.
Embedded in the network are pacemakers that can generate two distinct burst patterns within the same neuron (or between two such neurons coupled by gap junctions). These burst patterns include both fast low-amplitude and slow large-amplitude bursts. In the presence of oxotremorine the timing of these intracellularly generated bursts was altered in a manner similar to the entire network. Large-amplitude bursts accelerate, whereas low-amplitude bursts are slowed. There were, however, two pacemakers, with dual-burst patterns that were silenced in oxotremorine. It is unclear whether they are more appropriately considered to be a different subclass of dual-bursting pacemakers or single-bursting pacemakers with a highly variable bursting pattern.
The ability to generate multiple behavioral rhythms is a common property of many networks, including the respiratory network. The neocortex, for example, generates rhythms covering diverse frequency bands that can be associated with different brain states (Buzsáki and Draguhn 2004
; Chrobak and Buzsáki 1994
; Steriade 2006
; Steriade et al. 1993a
). However, unlike fictive eupnea and sigh bursts, these neocortical rhythms are not known to occur concurrently. Similar phenomena exist in the olfactory bulb (Kay 2003
; Tabor et al. 2004
). Perhaps a more relevant example of a network capable of generating multiple rhythms is the stomatogastric ganglion (STG) of crustaceans, where neurons exist that participate in both the gastric and pyloric rhythms (Prinz et al. 2004
Within the respiratory network we (and others) described two types of pacemaker neurons (Del Negro et al. 2005
; Peña et al. 2004
; Thoby-Brisson and Ramirez 2001
), the timing of which resembles the eupneic rhythm more closely than the very slow sigh rhythm. Since these pacemakers express a wide range of timing parameters, it is conceivable that the period of the eupneic rhythm is an emergent property dependent on intrinsic time constants in many neurons and their synaptic interactions (Feldman and Del Negro 2006
). Here we report pacemakers that simultaneously generated bursts with properties resembling those of the slow and large-amplitude sigh rhythm and fast and small eupneic rhythm. To the best of our knowledge neurons with intrinsic pyloric and gastric-like bursting properties have not previously been described in the STG. The overlap between the two stomatogastric rhythms is generated entirely by synaptic interactions.
It may seem surprising that in the mammalian respiratory network, fictive eupneic and sigh rhythms are apparently also reflected in single-neuron activity. However, the respiratory network may not be an exception. Single neocortical neurons can intrinsically generate beta activity that transforms into gamma oscillations following membrane depolarization (Gray and McCormick 1996
; Steriade et al. 1993b
). This transformation is reminiscent of global EEG changes that fluctuate between distinct rhythmic states during different mental activities (Steriade 2006
; Steriade et al. 1993b
). Similar to the respiratory network, the expression of two neocortical rhythms in the same neuron does not mean that both rhythms are associated with the same network mechanisms. Accordingly, distinct synaptic mechanisms shape both gamma and beta activity (Steriade 2006
). Eupnea and sighs are also shaped by distinct synaptic network mechanisms (Lieske and Ramirez 2006a
). Thus irrespective of the finding that single neurons can generate two types of rhythms and amplitude parameters, network rhythms occurring in a variety of rhythmic frequencies and patterns are typically the result of different electrophysiological characteristics and distinct connectivity features. However, this raises the important question: Is the similarity between the electrophysiological properties of single neurons and the network output just an epiphenomenon?
Although some suggest that respiratory pacemakers play critical roles in respiratory rhythmogenesis (Feldman and Smith 1989
; Peña et al. 2004
; Tryba et al. 2006
), others suggest that eupneic rhythmogenesis is an emergent property of a synaptically coupled network (Del Negro et al. 2002
). Although the present study cannot answer the question of whether pacemaker neurons are essential for rhythm generation, this study provides an interesting finding that pacemakers may synchronize multiple network activities through different types of burst mechanisms. However, it must be emphasized that this hypothesis does not negate the critical importance of network mechanisms.
Indeed, the dual-bursting pacemakers described here were cadmium insensitive, although very low (4 µM) concentrations of Cd2+
abolish sighs at the population level (Lieske et al. 2000
). Our present results are therefore in keeping with the hypothesis that the blockade of fictive sighs by Cd2+
results from a disruption of a synaptic mechanism (Lieske et al. 2006a
) rather than an intrinsic cellular mechanism. In that case, the generation of sighs is likely very sensitive to mechanisms underlying network synchronization.
Our data suggest that postsigh apnea is an expected consequence of pacemakers that trigger activation of an even larger population of neurons than those activated during fictive eupnea. Postsigh apnea results in part from activation of inspiratory neurons to depolarized levels greater than that which occur during eupneic bursts (Lieske et al. 2000
). For a network dependent on the activation of burst mechanisms, this additional activation would result in an increased refractory time, causing a delay before the subsequent eupneic burst. Further, the majority of postsigh apnea probably does not result from chemical inhibitory synaptic input to inspiratory neurons immediately following a sigh. Several lines of evidence support these hypotheses. First, most inspiratory neurons activated during eupnea receive additional depolarization during the sigh burst and the currents activated as a result of this depolarization reset the eupneic rhythm, giving rise to apnea following the sigh (Lieske et al. 2000
). Second, although inhibitory synaptic inputs appear to contribute to fictive postsigh apnea (Carley et al. 1998
) (), GABAergic and glycinergic inhibition are not necessary for it to occur ().
The eupneic and sigh rhythms are present even in the absence of sensory feedback. Fictive sighs were generated not only in slices, where they cannot be triggered by reflexes (Lieske et al. 2000
), but also in the highly reduced VRG-island, indicating that modulatory and pacemaking influences from outside the VRG region are not essential for generating sighs. Within the VRG, intracellular recordings confirm an extensive overlap between neurons active during eupnea and sighs (Lieske et al. 2000
). However, we identified a previously undescribed population of respiratory neurons active during fictive sighs but not during fictive eupnea. The presence of sigh-only neurons provides further support for the notion that there are at least two populations of VRG neurons that increase their activity during the sigh. These two populations include inspiratory neurons that burst during both eupneic and sigh bursts (Lieske et al. 2000
) as well as sigh-only neurons. Both of these populations are recruited during fictive sighs and likely contribute to the characteristic increased amplitude of the sigh burst in VRG population recordings (Lieske et al. 2000
). This increase in VRG inspiratory activity may contribute to the enhanced amplitude of sighs, as compared with eupneic breaths.
Sensory input plays a role in sighing because vagotomy or cutting the carotid sinus nerves abolishes sighs for several hours (Cherniack et al. 1981
; Glogowska et al. 1972
). When they return, sighs occur at a reduced frequency (Cherniack et al. 1981
; Marshall and Metcalfe 1988
). These data suggest an important role for sensory feedback in modulating sigh drive. A very similar pattern can also be evoked by brief inflation pulses (Cherniack et al. 1981
). Thus the central pattern generator for sighs, although capable of functioning independently, appears to be integrated into a complex network including both peripheral feedback and descending inputs from other areas in the intact animal. The present study has contributed to a better understanding of the central component of the sigh by: 1
) identifying a set of pacemaker neurons that generate two distinct bursts that respond to modulatory input similarly to the fictive eupneic and sigh activity produced by the network; 2
) identifying a set of neurons specifically activated during sighs; and 3
) proposing that intrinsic mechanisms could in part explain postsigh apneas.