title: Pre-Bötzinger complex receives glutamatergic innervation from galaninergic and other retrotrapezoid nucleus neurons.
abstract: The retrotrapezoid nucleus (RTN) contains CO(2) -responsive neurons that regulate breathing frequency and amplitude. These neurons (RTN-Phox2b neurons) contain the transcription factor Phox2b, vesicular glutamate transporter 2 (VGLUT2) mRNA, and a subset contains preprogalanin mRNA. We wished to determine whether the terminals of RTN-Phox2b neurons contain galanin and VGLUT2 proteins, to identify the specific projections of the galaninergic subset, to test whether RTN-Phox2b neurons contact neurons in the pre-Bötzinger complex, and to identify the ultrastructure of these synapses. The axonal projections of RTN-Phox2b neurons were traced by using biotinylated dextran amine (BDA), and many BDA-ir boutons were found to contain galanin immunoreactivity. RTN galaninergic neurons had ipsilateral projections that were identical with those of this nucleus at large: the ventral respiratory column, the caudolateral nucleus of the solitary tract, and the pontine Kölliker-Fuse, intertrigeminal region, and lateral parabrachial nucleus. For ultrastructural studies, RTN-Phox2b neurons (galaninergic and others) were transfected with a lentiviral vector that expresses mCherry almost exclusively in Phox2b-ir neurons. After spinal cord injections of a catecholamine neuron-selective toxin, there was a depletion of C1 neurons in the RTN area; thus it was determined that the mCherry-positive terminals located in the pre-Bötzinger complex originated almost exclusively from the RTN-Phox2b (non-C1) neurons. These terminals were generally VGLUT2-immunoreactive and formed numerous close appositions with neurokinin-1 receptor-ir pre-Bötzinger complex neurons. Their boutons (n = 48) formed asymmetric synapses filled with small clear vesicles. In summary, RTN-Phox2b neurons, including the galaninergic subset, selectively innervate the respiratory pattern generator plus a portion of the dorsolateral pons. RTN-Phox2b neurons establish classic excitatory glutamatergic synapses with pre-Bötzinger complex neurons presumed to generate the respiratory rhythm.
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA.
authors.forename: Genrieta
authors.initials: G
authors.lastname: Bochorishvili
authors.forename: Ruth L
authors.initials: RL
authors.lastname: Stornetta
authors.forename: Melissa B
authors.initials: MB
authors.lastname: Coates
authors.forename: Patrice G
authors.initials: PG
authors.lastname: Guyenet
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019004:Galanin / Q000502:physiology*; D018698:Glutamic Acid / Q000502:physiology*; D008969:Molecular Sequence Data; D009434:Neural Pathways / Q000502:physiology; D009474:Neurons / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012125:Respiratory Center / Q000502:physiology*; D012249:Rhombencephalon / Q000166:cytology / Q000502:physiology*
chemical_list: D018698:Glutamic Acid; D019004:Galanin