Research reportOrexinergic fibers are in contact with Kölliker-Fuse nucleus neurons projecting to the respiration-related nuclei in the medulla oblongata and spinal cord of the rat
Introduction
Orexin (ORX), also known as hypocretin, is a small hypothalamic neuropeptide and ORX-containing neurons are exclusively located in the tuberal part of the hypothalamus, including the perifornical area (PeF), lateral hypothalamus (LH) and dorsomedial hypothalamic nucleus (DMH) (de Lecea et al., 1998; Modirrousta et al., 2005, Nambu et al., 1999, Peyron et al., 1998, Sakurai et al., 1998). ORX was originally found to regulate food intake and energy homeostasis (Sakurai, 2002, Sutcliffe and de Lecea, 2000, Willie et al., 2001), and shortly thereafter the ORX system was revealed to play an important role in the regulation of sleep-wake states (Sakurai, 2007), reward processing, addiction (Harris and Aston-Jones, 2006), and autonomic function (Kuwaki, 2008, Williams and Burdakov, 2008, Willie et al., 2001).
Physiological and pharmacological studies indicated that ORX also takes part in breathing regulation (Zhang et al., 2005, Deng et al., 2007; also see Williams and Burdakov, 2008 for review) and anatomical studies showed that ORX-containing neurons send their axons to the respiratory-related brainstem regions, such as the parabrachial complex (PBC) consisting of the parabrachial nucleus (PBN) and Kölliker-Fuse nucleus (KFN) (Peyron et al., 1998), pre-Bötzinger complex (Young et al., 2005), nucleus of the solitary tract (Nambu et al., 1999), and hypoglossal nucleus (HGN) (Fung et al., 2001, Young et al., 2005), as well as to the phrenic nucleus (PhN) in the spinal cord (Young et al., 2005). Using a combination of retrograde tracing and immunohistochemistry for ORX, Young et al. (2005) indicated that only 0.5% and 2.9% of ORX-containing neurons innervated pre-Bötzinger complex and PhN, respectively, implying that only a small subset of ORX-containing neurons regulates breathing or that the majority of ORX-containing neurons control breathing through indirect projections (Williams and Burdakov, 2008).
The PBC, called the pneumotaxic center or pontine respiratory group, has been suggested to have an important role for respiratory control (reviewed in Dutschmann and Dick, 2012). Electrical and chemical stimulation of distinct population of PBC neurons has been known to produce various responses of respiration (Chamberlin and Saper, 1994, Chamberlin, 2004, Dick et al., 1994, Lara et al., 1994, Mutolo et al., 1998) and hypoglossal nerve activities (Kuna and Remmers, 1999). According to Chamberlin and Saper (1994), glutamate injection into the KFN causes a most intense inspiratory facilitatory response or apneusis which is a breathing pattern characterized by severely prolonged inspiration. Furthermore, Kuna and Remmers (1999) indicated that injection of glutamate into the KFN results in activation and/or suppression of HGN motor output through the medial branch of hypoglossal nerve; the medial branch of hypoglossal nerve controls the protruder tongue muscle contraction, which is important for maintenance of airway patency during respiration (Krammer et al., 1979, Yokota et al., 2011). Our previous tract-tracing studies demonstrated that KFN neurons innervate phrenic motor neurons directly or indirectly via the rostral ventral respiratory group (rVRG) (Yokota et al., 2001, Yokota et al., 2004) and send their axons to HGN motor neurons (Yokota et al., 2011). On the other hand, the PBC is one of the sites where large numbers of descending ORX fibers terminate (Nixon and Smale, 2007, Peyron et al., 1998) and ORX receptor 2 (OX2R)-expressing neurons are distributed (Marcus et al., 2001). Recently, it has been further demonstrated that injection of ORX-B into the KFN evokes significant augmentation of the respiratory frequency accompanied by increase in pre-inspiratory hypoglossal motor discharge (Dutschmann et al., 2007). Judging from the above, it seems quite probable that ORX-containing neurons have a indirect influence on rVRG, PhN, and/or HGN neurons via the KFN in the control of breathing. However, there have been no studies to examine whether or not KFN neurons that send their axons to the rVRG, PhN, and HGN receive monosynaptic inputs from ORX-containing neurons.
In the present study, we first demonstrate the existence of ORX-containing neurons sending their axons to the KFN by using a combined retrograde tracing with cholera toxin B subunit (CTb) and immunohistochemistry for ORX, and then provide definitive evidence for the existence of a monosynaptic pathway from ORX neurons to PBN/KFN neurons projecting to the rVRG and PhN as well as HGN by using a combined retrograde tracing with fluorogold (FG) and immunohistochemistry for ORX to reveal ORX-immunoreactive (-ir) axon fibers. Finally, we examined whether or not PBN/KFN neurons projecting to the rVRG and PhN as well as HGN express ORX receptor by using retrograde tracing with FG and immunohistochemistry for OX2R.
Section snippets
ORX-ir neurons projecting to the KFN
In 4 out of 9 operated rats, CTb injections were made into the KFN with some extension around the nucleus (Fig. 1(A)). In these cases, CTb-labeled neurons were observed bilaterally with a clear-cut ipsilateral predominance in the hypothalamus. Within the hypothalamus, CTb-labeled neurons were found mainly in the preoptic area, paraventricular nucleus, tuberal lateral hypothalamus dorsal and lateral to the fornix, and caudal lateral hypothalamus adjacent to the subthalamic nucleus and the
Discussion
In the present study, we provided new data as follows: (1) ORX-ir neurons that projected to the KFN were distributed prominently in the medial and perifornical regions, and additionally in the lateral region of the hypothalamic ORX field; (2) the KFN contained both ORX-ir fibers and rVRG-, PhN-, and HGN-projecting neurons; (3) ORX-ir axon terminals made asymmetrical synapses with somata and dendrites of the KFN neurons that projected to the rVRG and with dendrites of the KFN neurons that
Experimental procedures
The experiments were performed on male Wistar rats ranging in weight from 230 to 300 g. All surgical procedures were performed under general anesthesia with intraperitoneal injection of Somnopentyl (40 mg/kg). This study was in compliance with Guidelines for Animal Experimentation of the Center for Integrated Research in Science, Shimane University. The atlas of the rat brain (Paxinos and Watson, 2005) was used to determine coordinates for stereotaxic injection of tracers as well as for the
Acknowledgments
The authors would like to thank Mr. Makoto Oshita and Mr. Tsunao Yoneyama for assistance with photography and Ms. Yuko Okui for technical assistance. This study was supported by a Grant-in-Aid for Science Research (245003801, 24500412, 15K06739) from the Ministry of Education, Science, Sports and Culture of Japan.
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