Pre‐Bötzinger complex receives glutamatergic innervation from galaninergic and other retrotrapezoid nucleus neurons

Abstract

The retrotrapezoid nucleus (RTN) contains CO2-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 Köliker-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.

Inhibitory control of the cough reflex by galanin receptors in the caudal nucleus tractus solitarii of the rabbit

“…Peripheral and central mechanisms of nociception and cough share similar features, such as the type of afferent fibers (A␦ and C), transient receptor potential vanilloid 1 and transient receptor potential ankyrin 1 channels, central and peripheral sensitization, as well as the involvement of mitogenactivated protein kinase (MAPK) signal transduction pathways (4,11,12,14,20,29,40). This correspondence between nociception and cough has led to investigations on the antitussive effects of agents known to be involved in the central control of pain sensation (4, 15).Galanin is a 29 amino acid neuropeptide (30 amino acids in humans) widely distributed in the central nervous system (e.g., 21,28,36,37,63,65) involved in many body functions such as feeding behavior, learning and memory, neuroendocrine control, mood disorders, antinociception, cardiovascular regulation, and respiration (1,6,28,44). In particular, galanin has been implicated in the descending control of pain at the spinal level as well as in its modulation at supraspinal sites probably mainly via galanin-1 (GAL 1 ) receptors (e.g.,2,22,32,68; reviewed in Refs.…”

“…Galanin is a 29 amino acid neuropeptide (30 amino acids in humans) widely distributed in the central nervous system (e.g., 21,28,36,37,63,65) involved in many body functions such as feeding behavior, learning and memory, neuroendocrine control, mood disorders, antinociception, cardiovascular regulation, and respiration (1,6,28,44). In particular, galanin has been implicated in the descending control of pain at the spinal level as well as in its modulation at supraspinal sites probably mainly via galanin-1 (GAL 1 ) receptors (e.g.,2,22,32,68; reviewed in Refs.…”

Neural Control of Respiration by the Retrotrapezoid Nucleus.

“…RTN innervates all the RPG regions that harbor excitatory pump premotor neurons (Ballantyne & Richter, 1986;Dobbins & Feldman, 1994;Yokota et al, 2007;Bochorishvili et al, 2012). The excitatory input from RTN plausibly increases V T by enhancing the discharges of these muscles.…”

“…CB second-order neurons target multiple levels of the ventral respiratory column plus regions of the dorsolateral pons implicated in respiratory control (Koshiya and Guyenet, 1996;Song et al, 2011). These regions are also targeted by RTN (Bochorishvili et al, 2012), therefore integration between central and peripheral chemoreflexes likely occurs at multiple sites. Some integration undoubtedly occurs at the RTN but at least under hypoxia, the excitatory input that these neurons receive from the CBs seems insufficient to activate them, presumably because of the dominant inhibitory effect of alkalosis.…”

“…The excitatory input from RTN plausibly increases V T by enhancing the discharges of these muscles. The post-inspiratory activity of LC motor neurons could be enhanced by polysynaptic drive via the Kolliker Fuse, a major target of RTN (Dutschmann & Herbert, 2006;Bochorishvili et al, 2012), and/or a network effect that augments phasic inspiratory inhibition of LC motor neurons and strengthens their post-inhibitory rebound (Sun et al, 2008;Bautista et al, 2010).…”