Somatostatin 2a receptors are not expressed on functionally identified respiratory neurons in the ventral respiratory column of the rat

Abstract

Microinjection of somatostatin (SST) causes site-specific effects on respiratory phase transition, frequency, and amplitude when microinjected into the ventrolateral medulla (VLM) of the anesthetized rat, suggesting selective expression of SST receptors on different functional classes of respiratory neurons. Of the six subtypes of SST receptor, somatostatin 2a (sst2a ) is the most prevalent in the VLM, and other investigators have suggested that glutamatergic neurons in the preBötzinger Complex (preBötC) that coexpress neurokinin-1 receptor (NK1R), SST, and sst2a are critical for the generation of respiratory rhythm. However, quantitative data describing the distribution of sst2a in respiratory compartments other than preBötC, or on functionally identified respiratory neurons, is absent. Here we examine the medullary expression of sst2a with particular reference to glycinergic/expiratory neurons in the Bötzinger Complex (BötC) and NK1R-immunoreactive/inspiratory neurons in the preBötC. We found robust sst2a expression at all rostrocaudal levels of the VLM, including a large proportion of catecholaminergic neurons, but no colocalization of sst2a and glycine transporter 2 mRNA in the BötC. In the preBötC 54% of sst2a -immunoreactive neurons were also positive for NK1R. sst2a was not observed in any of 52 dye-labeled respiratory interneurons, including seven BötC expiratory-decrementing and 11 preBötC preinspiratory neurons. We conclude that sst2a is not expressed on BötC respiratory neurons and that phasic respiratory activity is a poor predictor of sst2a expression in the preBötC. Therefore, sst2a is unlikely to underlie responses to BötC SST injection, and is sparse or absent on respiratory neurons identified by classical functional criteria. J. Comp. Neurol. 524:1384-1398, 2016. © 2015 Wiley Periodicals, Inc.

Mapping and Analysis of the Connectome of Sympathetic Premotor Neurons in the Rostral Ventrolateral Medulla of the Rat Using a Volumetric Brain Atlas

“…Eight of 12 input clusters were located within the medulla, the largest of which (Cluster 2) encompassed the ipsilateral RVLM (including occasional trans-synaptically labeled C1 neurons: Figure 2B ), Bötzinger region (Figure 5D ), nucleus ambiguus (Figure 5G ), and choline acetyltransferase (ChAT)-immunoreactive cells in a region of the ventral lateral tegmental field (Figure 6A ) that may represent the rat analog of the post-inspiratory complex (PiCo: Anderson et al, 2016 ). In a sample of 21 input neurons identified as lying within the Bötzinger region using previously published criteria (Le et al, 2016 ), none contained glycine transporter 2 mRNA, a marker for respiratory function in this region (Figure 6B , Schreihofer et al, 1999 ). Adjacent clusters enveloped the dorsal LTF (Cluster 3) and pre-Bötzinger Complex (Cluster 4, Figure 5E ), including neurokinin-1 receptor (NK1R) immunoreactive neurons (Figure 6C ), a putative marker for respiratory function in this region (Gray et al, 2001 ).…”

“…ISH was conducted to examine double labeling of rabies-infected input neurons with GAD67 mRNA, a marker of GABAergic neurons, and GLYT2 mRNA, a marker of glycinergic neurons. ISH probes and processing were identical to those described by Bowman et al ( 2013 ) and Le et al ( 2016 ) respectively.…”

“…The extent of the facial nucleus was annotated directly from the Waxholm MRI dataset in Imaris; the Bötzinger and pre-Bötzinger Complex were defined as longitudinal cylinders, 500 μm in diameter that ran center-aligned and immediately ventral to nucleus ambiguus. The Bötzinger region was defined as starting at the caudal pole of the facial nucleus and running 600 μm caudal; the pre-Bötzinger Complex was defined as running between 700 and 1200 μm caudal to the facial nucleus (Le et al, 2016 ).…”

PreBötzinger complex neurons drive respiratory modulation of blood pressure and heart rate

“…In situ hybridization cRNA probes for glycine transporter 2 (GlyT2) and glutamic acid decarboxylase 67 (GAD67) were synthesized as previously described, permitting identification of glycinergic and GABAergic neurons respectively by in situ hybridization ( Bowman et al, 2013 ; Le et al, 2016 ). A digoxigenin-labeled cRNA probe for vesicular glutamate transporter 2 (VGluT2), a marker of glutamate synthesis, was synthesized as follows: cDNA template (886 bp, Genebank reference sequence NM_053427) was amplified using the PCR primers (lower case) with T7 and Sp6 RNA polymerase promotors (upper case) attached F: GGATCCATTTAGGTGACACTATAGAAGtcaatgaaatccaacgtcca ; R: GAATTCTAATACGACTCACTATAGGGAGAcaagagcacaggacaccaaa .…”

Adaptation of Respiratory-Related Brain Regions to Long-Term Hypercapnia: Focus on Neuropeptides in the RTN

“…For this reason, cell counts were made from ventrolateral medulla (VLM) subdivisions (Figures 5A-C). The VRC was defined as a 400 µm 2 region immediately ventral to the nucleus ambiguus (Pagliardini et al, 2003;Stornetta et al, 2003;Bouvier et al, 2010;Le et al, 2016; Figure 5A) and there was an average of 840 ± 23 (n = 5) GalR1+ neurons between Bregma levels −7.51 and −6.50 out of which 198 ± 15 were glycinergic (23%). In the VRC, BötC contained more GalR1+ neurons 539 ± 20 compared to preBötC 301 ± 12 (n = 5) ( Figure 5G).…”