Revisiting the two rhythm generators for respiration in lampreys.

In lampreys, respiration consists of a fast and a slow rhythm. This study was aimed at characterizing both anatomically and physiologically the brainstem regions involved in generating the two rhythms. The fast rhythm generator has been located by us and others in the rostral hindbrain, rostro-lateral to the trigeminal motor nucleus. More recently, this was challenged by researchers reporting that the fast rhythm generator was located more rostrally and dorsomedially, in a region corresponding to the mesencephalic locomotor region. These contradictory observations made us re-examine the location of the fast rhythm generator using anatomical lesions and physiological recordings. We now confirm that the fast respiratory rhythm generator is in the rostro-lateral hindbrain as originally described. The slow rhythm generator has received less attention. Previous studies suggested that it was composed of bilateral, interconnected rhythm generating regions located in the caudal hindbrain, with ascending projections to the fast rhythm generator. We used anatomical and physiological approaches to locate neurons that could be part of this slow rhythm generator. Combinations of unilateral injections of anatomical tracers, one in the fast rhythm generator area and another in the lateral tegmentum of the caudal hindbrain, were performed to label candidate neurons on the non-injected side of the lateral tegmentum. We found a population of neurons extending from the facial to the caudal vagal motor nuclei, with no clear clustering in the cell distribution. We examined the effects of stimulating different portions of the labeled population on the respiratory activity. The rostro-caudal extent of the population was arbitrarily divided in three portions that were each stimulated electrically or chemically. Stimulation of either of the three sites triggered bursts of discharge characteristic of the slow rhythm, whereas inactivating any of them stopped the slow rhythm. Substance P injected locally in the lateral tegmentum accelerated the slow respiratory rhythm in a caudal hindbrain preparation. Our results show that the fast respiratory rhythm generator consists mostly of a population of neurons rostro-lateral to the trigeminal motor nucleus, whereas the slow rhythm generator is distributed in the lateral tegmentum of the caudal hindbrain.

The neural control of respiration in lampreys.

This review focuses on past and recent findings that have contributed to characterize the neural networks controlling respiration in the lamprey, a basal vertebrate. As in other vertebrates, respiration in lampreys is generated centrally in the brainstem. It is characterized by the presence of a fast and a slow respiratory rhythm. The anatomical and the basic physiological properties of the neural networks underlying the generation of the fast rhythm have been more thoroughly investigated; less is known about the generation of the slow respiratory rhythm. Comparative aspects with respiratory generators in other vertebrates as well as the mechanisms of modulation of respiration in association with locomotion are discussed.

Testing the evolutionary conservation of vocal motoneurons in vertebrates.

Medullary motoneurons drive vocalization in many vertebrate lineages including fish, amphibians, birds, and mammals. The developmental history of vocal motoneuron populations in each of these lineages remains largely unknown. The highly conserved transcription factor Paired-like Homeobox 2b (Phox2b) is presumed to be expressed in all vertebrate hindbrain branchial motoneurons, including laryngeal motoneurons essential for vocalization in humans. We used immunohistochemistry and in situ hybridization to examine Phox2b protein and mRNA expression in caudal hindbrain and rostral spinal cord motoneuron populations in seven species across five chordate classes. Phox2b was present in motoneurons dedicated to sound production in mice and frogs (bullfrog, African clawed frog), but not those in bird (zebra finch) or bony fish (midshipman, channel catfish). Overall, the pattern of caudal medullary motoneuron Phox2b expression was conserved across vertebrates and similar to expression in sea lamprey. These observations suggest that motoneurons dedicated to sound production in vertebrates are not derived from a single developmentally or evolutionarily conserved progenitor pool.

Bilateral connectivity in the brainstem respiratory networks of lampreys.

This study examines the connectivity in the neural networks controlling respiration in the lampreys, a basal vertebrate. Previous studies have shown that the lamprey paratrigeminal respiratory group (pTRG) plays a crucial role in the generation of respiration. By using a combination of anatomical and physiological techniques, we characterized the bilateral connections between the pTRGs and descending projections to the motoneurons. Tracers were injected in the respiratory motoneuron pools to identify pre-motor respiratory interneurons. Retrogradely labeled cell bodies were found in the pTRG on both sides. Whole-cell recordings of the retrogradely labeled pTRG neurons showed rhythmical excitatory currents in tune with respiratory motoneuron activity. This confirmed that they were related to respiration. Intracellular labeling of individual pTRG neurons revealed axonal branches to the contralateral pTRG and bilateral projections to the respiratory motoneuronal columns. Stimulation of the pTRG induced excitatory postsynaptic potentials in ipsi- and contralateral respiratory motoneurons as well as in contralateral pTRG neurons. A lidocaine HCl (Xylocaine) injection on the midline at the rostrocaudal level of the pTRG diminished the contralateral motoneuronal EPSPs as well as a local injection of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and (2R)-amino-5-phosphonovaleric acid (AP-5) on the recorded respiratory motoneuron. Our data show that neurons in the pTRG send two sets of axonal projections: one to the contralateral pTRG and another to activate respiratory motoneurons on both sides through glutamatergic synapses.

Specific neural substrate linking respiration to locomotion.

When animals move, respiration increases to adapt for increased energy demands; the underlying mechanisms are still not understood. We investigated the neural substrates underlying the respiratory changes in relation to movement in lampreys. We showed that respiration increases following stimulation of the mesencephalic locomotor region (MLR) in an in vitro isolated preparation, an effect that persists in the absence of the spinal cord and caudal brainstem. By using electrophysiological and anatomical techniques, including whole-cell patch recordings, we identified a subset of neurons located in the dorsal MLR that send direct inputs to neurons in the respiratory generator. In semi-intact preparations, blockade of this region with 6-cyano-7-nitroquinoxaline-2,3-dione and (2R)-amino-5-phosphonovaleric acid greatly reduced the respiratory increases without affecting the locomotor movements. These results show that neurons in the respiratory generator receive direct glutamatergic connections from the MLR and that a subpopulation of MLR neurons plays a key role in the respiratory changes linked to movement.

The interactions between locomotion and respiration.

Respiration is a vital motor activity requiring fine-tuning to adjust to metabolic changes. For instance, respiration increases in association with exercise. In this chapter, we review the mechanisms underlying respiratory changes during exercise. Three specific hypotheses were proposed. First, the chemoreception hypothesis suggests that chemoreceptors located centrally or peripherally modify breathing by detecting metabolic changes in arterial blood or cerebrospinal fluid. Second, the central command hypothesis stipulates that central neural connections from brain motor areas activate the respiratory centers during exercise. Third, the neural feedback hypothesis stipulates that sensory inputs from the contracting limb muscles modulate the respiratory centers during exercise. We present evidence from the literature supporting possible contributions from these three mechanisms. This review also addresses future research challenges relative to respiratory modulation during exercise.