Expression of phospho‐Ca²⁺/calmodulin‐dependent protein kinase II in the pre‐Bötzinger complex of rats

Abstract

The pre-B€ otzinger complex (pre-B€ otC) in the ventrolateral medulla oblongata is a presumed kernel of respiratory rhythmogenesis. Ca 2+ -activated non-selective cationic current is an essential cellular mechanism for shaping inspiratory drive potentials. Ca 2+ /calmodulin-dependent protein kinase II (CaM-KII), an ideal 'interpreter' of diverse Ca 2+ signals, is highly expressed in neurons in mediating various physiological processes. Yet, less is known about CaMKII activity in the pre-B€ otC. Using neurokinin-1 receptor as a marker of the pre-B€ otC, we examined phospho (P)-CaMKII subcellular distribution, and found that P-CaMKII was extensively expressed in the region. P-CaMKII-ir neurons were usually oval, fusiform, or pyramidal in shape. P-CaMKII immunoreactivity was distributed within somas and dendrites, and specifically in association with the post-synaptic density. In dendrites, most synapses (93.1%) examined with P-CaMKII expression were of asymmetric type, occasionally with symmetric type (6.9%), whereas in somas, 38.1% were of symmetric type. P-CaMKII asymmetric synaptic identification implicates that CaMKII may sense and monitor Ca 2+ activity, and phosphorylate postsynaptic proteins to modulate excitatory synaptic transmission, which may contribute to respiratory modulation and plasticity. In somas, CaMKII acts on both symmetric and asymmetric synapses, mediating excitatory and inhibitory synaptic transmission. P-CaMKII was also localized to the perisynaptic and extrasynaptic regions in the pre-B€ otC. The pre-B€ otzinger complex (pre-B€ otC) in the ventrolateral medulla is a functionally and anatomically defined site that is necessary and sufficient for inspiratory neural rhythms (Smith et al. 1991;Rekling and Feldman 1998;Feldman and Del Negro 2006). The pre-B€ otC neurons generate inspiratory drive potentials by synaptic input evoking postsynaptic currents that depend on intrinsic membrane properties (Rekling and Feldman 1998;Feldman and Del Negro 2006). Two types of inward currents, the persistent sodium current and the Ca 2+ is an essential cellular mechanism for shaping the inspiratory drive potentials, which is fully evoked by ionotropic and metabotropic glutamatergic synaptic inputs Mironov 2008;Pace and Del Negro 2008;Rubin et al. 2009;Del Negro et al. 2010). AMPA receptors initiate convergent signaling pathways that evoke Received December 8, 2012; accepted May 6, 2013. Address correspondence and reprint requests to Dr.Ying-Ying Liu, Institute of Neurosciences, The Fourth Military Medical University, 17 Chang Le Xi Road, Xi'an 710032, China. E-mail: yingyliu@fmmu.edu.cnAbbreviations used: BSA, bovine serum albumin; CaMKII, Ca 2+ / calmodulin-dependent protein kinase II; I CAN , non-selective cationic current; ir, immunoreactive; mGluR1, group 1 metabotropic glutamate receptors; NGS, normal goat serum; NK1R, neurokinin 1 receptor; PBS, phosphate buffered saline; P, phospho; pre-B€ otC, pre-B€ otzinger complex; PSD, post-synaptic density; TRP, transicent receptor potential; VGCC, ...

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Morphological Characterization of Respiratory Neurons in the Pre-Bötzinger Complex

“…However, these models still lack a functional-anatomical correlation of the included neurons, the actual synaptic connections between them, as well as the distinctive morphological features of the respiratory neurons in the preBötC. Several of these molecular markers have been indeed located in the preBötC but also in other respiratory-related circuits throughout the brainstem (Gray et al, 1999(Gray et al, , 2001(Gray et al, , 2010Guyenet et al, 2002;Kang et al, 2013;Liu and Wong-Riley, 2013;Stornetta et al 2003;Tan et al, 2012). Several of these molecular markers have been indeed located in the preBötC but also in other respiratory-related circuits throughout the brainstem (Gray et al, 1999(Gray et al, , 2001(Gray et al, , 2010Guyenet et al, 2002;Kang et al, 2013;Liu and Wong-Riley, 2013;Stornetta et al 2003;Tan et al, 2012).…”