Elsevier

Brain Research

Volume 1208, 7 May 2008, Pages 128-136
Brain Research

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

https://doi.org/10.1016/j.brainres.2008.02.028Get rights and content

Abstract

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.

Introduction

The medullary raphe nuclei (MRN) – the raphe pallidus (RPa), magnus (RMa) and obscurus (ROb) – are functionally heterogeneous, having been implicated in a wide range of physiological functions such as: cardiorespiratory regulation (Bernard, 1998, Holtman et al., 1986, Lalley, 1986), including cardiovascular responses to hemorrhage (Henderson et al., 1998, Heslop et al., 2002); thermoregulation (Blessing and Nalivaiko, 2001, Tanaka et al., 2002); and nociception (Mason, 2001, Porreca et al., 2002). Although the integrity of neurons in the MRN is not thought to be necessary for normal respiratory rhythm generation (Jakus et al., 1998), neurons with respiratory related firing patterns have been identified in this region (Gilbey et al., 1995, Hosogai and Matsuo, 2002) and stimulation of neurons in the MRN produces profound effects on phrenic nerve activity, including apnea (Verner et al., 2004). Chemosensitivity has also been associated with this region (Bernard et al., 1996, Richerson et al., 2001).

The projections to the various nuclei of the MRN have been investigated using retrograde and anterograde tracing techniques. The MRN receive widespread projections from all levels of the brain. Hermann et al. (1997) found afferent projections to the MRN from numerous forebrain structures including the lateral and medial preoptic area, bed nucleus of stria terminalis and central nucleus of amygdala; the dorsal and lateral hypothalamic areas and paraventricular nucleus of the hypothalamus; the thalamic parafascicular area; the midbrain periaqueductal grey; the subcoeruleus area, Kölliker–Fuse nucleus, parabrachial nuclei in the pons; and the parvocellular reticular nucleus and rostral ventrolateral medulla.

The density of these projections varied depending on the location in the MRN where the retrograde tracer was microinjected, in agreement with numerous previous studies (Abols and Basbaum, 1981, Beitz, 1982a, Beitz, 1982b, Carlton et al., 1983, Gang et al., 1991, Gang et al., 1993, Luppi et al., 1987, Zagon, 1993). The MRN also receives inputs arising from the spinal trigeminal nucleus; the nucleus tractus solitarius, primarily from the ventrolateral and medial subnuclei; and the spinal cord, in particular the dorsal and ventral horns (Potas et al., 2003).

Recently, we identified a novel midline apneic site (MAS) in the rostral medulla oblongata. Chemical stimulation of this discrete region at the level of the caudal pole of the facial nucleus with microinjections of excitatory amino acids consistently produced long lasting apnea (Verner et al., 2004). The physiological significance of this midline apneic site has not yet been investigated. Therefore the present study aimed to identify the brain regions that project to the MAS. Retrograde tracer was deposited at sites functionally verified by measuring the arterial blood pressure and respiratory response to chemical stimulation prior to microinjection.

Section snippets

Midline apneic site injections

Glutamate (100 mM, 50 nL) injected into sites chosen for cholera toxin B subunit (CTB) injection evoked apneic responses (9.8 ± 1.5 s) accompanied by bradycardia (− 9.8 ± 5.8%) and a depressor response (− 12.4 ± 3.5%) from resting conditions of 0.9 ± 0.1 breaths/s, 329 ± 14.4 beats/min, and 103 ± 2.8 mmHg, respectively. An example is shown in Fig. 1. The size and location of all CTB injections made into the functionally verified MAS are shown in Fig. 2A, B.

Immunohistochemistry

The location of CTB labeled cells from a

Discussion

The present study identifies the brain regions that project to the MAS giving some insight to the possible physiological role of this powerful apneic site. Sites that project to the MAS include: the mBST, MPA and LPA regions and MnPO; the PVN of the hypothalamus and CeA; the DHA/DMH and LHA, but not the ventromedial hypothalamus; all divisions of the midbrain PAG except the dorsolateral division, the rostral DRN; the PBN, KF, and ITR region of the pons; and the RVLM and LPR. All sites except

Experimental procedures

Experiments were carried out in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved by the Animal Care and Ethics Committee of the Royal North Shore Hospital. Five male Sprague Dawley rats (400–500 g; Gore Hill Research Laboratories, Sydney, Australia) were used. Animals were housed in small groups with a fixed 12:12 hour light–dark cycle with food and water available ad libitum.

Anesthesia was induced with halothane (4.0–5.0%

Acknowledgments

This work was supported by grants from the National Health and Medical Research Council of Australia (211023, 211196, 457068 and 457069) and Garnett Passe and Rodney Williams Memorial Foundation.

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