Retrograde projections to a discrete apneic site in the midline medulla oblongata of the rat.

We have recently described a discrete region of the medullary raphe nuclei, termed the midline apneic site (MAS) that produces profound apnea upon chemical stimulation. The aim of the present study was to identify brain regions that innervate the MAS. The MAS was functionally identified and then the retrograde tracer, cholera toxin B subunit was injected in Sprague Dawley rats (n = 5). The MAS received projections of varying intensity from a number of brain regions previously associated with thermoregulation, fear, nociception, cardiovascular regulation and respiration. These include: the medial preoptic nucleus; median and lateral preoptic area; medial division of the bed nucleus of stria terminalis; paraventricular nucleus; central nucleus of the amygdala; dorsal hypothalamic area/dorsomedial hypothalamus; lateral hypothalamic area; lateral, ventrolateral and dorsomedial divisions of the periaqueductal grey; dorsal raphe nuclei; parabrachial nuclei; Kölliker-Fuse nucleus; intertrigeminal region; rostral ventrolateral medulla; lateral parafacial region; and the ventral respiratory group. The intermingling of functionally distinct cell groups in the raphe probably explains the large diversity of projections as found in other tracing studies of the raphe although the possibility remains that the MAS may integrate signals from multiple sites. The connection between the intertrigeminal region of the pons and the MAS indicates the possibility that this pathway participates in airway protective reflexes such as the Hering-Breuer and diving reflexes. One previously undescribed region that we have termed the lateral parafacial region was consistently labeled. The role of this region is yet to be determined.

Significance of multiple neurochemicals that regulate respiration.

Current efforts to characterize the neuronal mechanisms that underlie automatic breathing generally adopt a 'minimalist' approach. In this review, we survey three of the many neurochemicals that are known to be present in raphe neurons and may be involved in respiration. Specifically, we ask the question, 'Is the minimalist approach consistent with the large number of neuronal types and neurochemicals found in respiratory centres'?

A mapping study of cardiorespiratory responses to chemical stimulation of the midline medulla oblongata in ventilated and freely breathing rats.

The aim of this study was to examine the cardiorespiratory effects of chemically stimulating neurons in the midline medulla oblongata (MM) of artificially ventilated and freely breathing anesthetized rats. Earlier studies reported that stimulation of the MM elicits increases or decreases in mean arterial pressure (MAP) and phrenic nerve activity, depending on the mode and site of stimulation, anesthetic, and species. In the first series of experiments, rats were anesthetized with urethane, artificially ventilated, paralyzed, and bilaterally vagotomized. The rostrocaudal extent of the MM was mapped by microinjections of DL-homocysteic acid or L-glutamate (both 100 mM, 100 nl), and, in line with previous studies, most injections produced only small responses in MAP, heart rate, and splanchnic sympathetic nerve activity. Increases in respiratory parameters were evoked in caudal regions. However, activation of a discrete region of the MM at the level of the caudal pole of the facial nucleus (CP7) consistently caused a dramatic reduction in phrenic nerve amplitude and/or frequency and, in six rats, produced a prolonged apnea. The second series of experiments was carried out on freely breathing pentobarbitone sodium-anesthetized rats, with a diaphragmatic electromyogram used to monitor respiratory activity. Respiratory activity could again be abolished at CP7 after microinjections of glutamate (100 mM, 50 nl); however, these responses were accompanied by large decreases in MAP and moderate reductions in heart rate. This depression of respiratory activity may be due to activation of propriobulbar inhibitory neurons that project to known respiratory centers in the brain stem.

A novel method for marking microinjection sites using methylene blue and diaminobenzidine.

A number of different compounds have been microinjected into the brains of animals for the purpose of marking injection sites. In the present study, we describe a novel method for visualising and permanently preserving sites in the brainstem of rats marked by microinjections of methylene blue. Subsequent exposure of the sections to diaminobenzidine (DAB) tetrahydrochloride in the presence of glucose oxidase or infrared light produced an insoluble black precipitate at the injection site. A major advantage of this method is that the injection sites are marked simultaneously with immunohistochemical processing.