Distribution of hypothalamic neurons with orexin (hypocretin) or melanin concentrating hormone (MCH) immunoreactivity and multisynaptic connections with diaphragm motoneurons.

Prior work showed that neurons in the lateral, dorsal, and perifornical regions of the tuberal and mammillary levels of the hypothalamus participate in the control of breathing. The same areas also contain large numbers of neurons that produce either orexins (hypocretins) or melanin concentrating hormone (MCH). These peptides have been implicated in regulating energy balance and physiological changes that occur in transitions between sleep and wakefulness, amongst other functions. The goal of this study was to determine if hypothalamic neurons involved in respiratory control, which were identified in cats by the retrograde transneuronal transport of rabies virus from the diaphragm, were immunopositive for either orexin-A or MCH. In animals with limited rabies infection of the hypothalamus (<10 infected cells/section), where the neurons with the most direct influences on diaphragm motoneurons were presumably labeled, a large fraction (28-75%) of the infected hypothalamic neurons contained orexin-A. In the same cases, 6-33% of rabies-infected hypothalamic cells contained MCH. However, in animals with more extensive infection, where rabies had presumably passed transneuronally through more synapses, the fraction of infected cells that contained orexin-A was lower. The findings from these experiments thus support the notion that hypothalamic influences on breathing are substantially mediated through orexins or MCH.

Localization of serotoninergic neurons that participate in regulating diaphragm activity in the cat.

Although a considerable body of literature indicates that serotoninergic neurons affect diaphragm activity both through direct inputs to phrenic motoneurons and multisynaptic connections involving the brainstem respiratory groups, the locations of the serotoninergic neurons that modulate breathing have not been well defined. The present study identified these neurons in cats by combining the transneuronal retrograde transport of rabies virus from the diaphragm with the immunohistochemical detection of the N-terminal region of tryptophan hydroxylase-2 (TPH2), the brain-specific isoform of the enzyme responsible for the initial and rate-limiting step in serotonin synthesis. TPH2-immunopositive neurons were present in the midline raphe nuclei, formed a column in the ventrolateral medulla near the lateral reticular nucleus, and were spread across the dorsal portion of the pons just below the fourth ventricle. In most animals, only a small fraction of neurons (typically <20%) labeled for TPH2 in each of the medullary raphe nuclei and the medullary ventrolateral column were infected with rabies virus. However, the percentage of medullary neurons dual-labeled for both rabies and TPH2 was much higher in animals with very advanced infections where virus had spread transneuronally through many synapses. Furthermore, in all cases, TPH2-immunopositive neurons that were infected by rabies virus were significantly less prevalent in the pons than the medulla. These findings suggest that although serotoninergic neurons with direct influences on diaphragm activity are widely scattered in the brainstem, the majority of these neurons are located in the medulla. Many non-serotoninergic neurons in the raphe nuclei were also infected with rabies virus, indicating that midline cells utilizing multiple neurotransmitters participate in the control of breathing.

Neural circuits controlling diaphragm function in the cat revealed by transneuronal tracing.

Although a number of studies have considered the neural circuitry that regulates diaphragm activity, these pathways have not been adequately discerned, particularly in animals such as cats that utilize the respiratory muscles during a variety of different behaviors and movements. The present study employed the retrograde transneuronal transport of rabies virus to identify the extended neural pathways that control diaphragm function in felines. In all animals deemed to have successful rabies virus injections into the diaphragm, large, presumed motoneurons were infected in the C(4)-C(6) spinal segments. In addition, smaller presumed interneurons were labeled bilaterally throughout the cervical and upper thoracic spinal cord. While in short and intermediate survival cases, infected interneurons were concentrated in the vicinity of phrenic motoneurons, in late survival cases, the distribution of labeling was more expansive. Within the brain stem, the earliest infected neurons included those located in the classically defined pontine and medullary respiratory groups, the medial and lateral medullary reticular formation, the region immediately ventral to the spinal trigeminal nucleus, raphe pallidus and obscurus, and the vestibular nuclei. At longer survival times, infection appeared in the midbrain, which was concentrated in the lateral portion of the periaqueductal gray, the region of the tegmentum that contains the locomotion center, and the red nucleus. Considerable labeling was also present in the fastigial nucleus of the cerebellum, portions of the posterior and lateral hypothalamus and the adjacent fields of Forel known to contain hypocretin-containing neurons and the precruciate gyrus of cerebral cortex. These data raise the possibility that several parallel pathways participate in regulating the activity of the feline diaphragm, which underscores the multifunctional nature of the respiratory muscles in this species.