C1 catecholamine neurons form local circuit synaptic connections within the rostroventrolateral medulla of rat
Highlights
► Phenotypically defined anterograde tracing defines projections of C1 neurons. ► C1 catecholamine neurons in ventrolateral medulla elaborate local circuit axons. ► Axons of C1 neurons arborize throughout the rostrocaudal extent of the C1 column. ► Ultrastructural analysis reveals synaptic contacts between C1 and non-C1 neurons.
Introduction
Studies over the past 30 years have established that the caudal brainstem exerts a profound influence over cardiovascular function (Dampney, 1994, Spyer, 1994, Guyenet, 2006, Schreihofer and Sved, 2011). Although evidence for a “vasomotor center” in the medulla is apparent in early literature, contemporary understanding of the central neural basis of cardiovascular control finds its roots in the 1980s in studies that localized a “pressor area” to a circumscribed region in the rostral ventrolateral medulla (RVLM). These investigations demonstrated that electrical or neurochemical stimulation of RVLM neurons in experimental animals increased arterial blood pressure (AP) while injection of GABA into this region produced a dose-dependent fall in AP and bradycardia (Dampney and Moon, 1980, Ross et al., 1984c). These seminal observations form the foundation for a voluminous literature that has subsequently refined our understanding of the function of this region and placed it within a larger polysynaptic network responsible for adaptive cardiovascular responses to behavioral and environmental challenges. It has also become increasingly apparent that altered function of the RVLM, or the circuitry that it functions within, can contribute to hypertension (Sved et al., 2003, Osborn, 2005, Guyenet, 2006). Thus, characterization of the synaptology through which RVLM exerts regulatory control over cardiovascular function has become increasingly important for defining and treating the pathogenesis that underlies hypertension (Sved et al., 2003).
A large literature has established that the RVLM is composed of a heterogeneous population of neurons distinguished by their projection targets and neurochemical phenotype. The seminal studies of Ross and colleagues (e.g., Ross et al., 1984a, Ross et al., 1984c) mapped the area of brainstem in which chemical or electrical stimulation elicits a pressor response and demonstrated that the pressor region contained neurons of the C1 catecholamine cell group. Ross and colleagues were careful to note that non-catecholamine neurons contribute to RVLM reticulospinal projections, an important observation that has been confirmed and refined in a number of studies. In this regard, Jeske and McKenna reported that the adrenergic enzyme phenylethanolamine N-methyl transferase (PNMT) is present in ∼50% of neurons retrogradely labeled from multiple levels of thoracic spinal cord (Jeske and McKenna, 1992) and it is now well established that both C1 and non-catecholamine RVLM neurons are barosensitive (Sved et al., 1994, Stornetta et al., 2001, Stornetta et al., 2002). Juxtacellular labeling of RVLM neurons after electrophysiological analysis has established that reticulospinal C1 and non-C1 neurons are distinguished by axon conduction velocity (Schreihofer and Guyenet, 1997) while neurochemical and circuit-tracing studies have demonstrated that the nucleus is topographically organized with respect to neuronal phenotype and projection patterns. Notably, C1 reticulospinal neurons influencing sympathetic outflow are differentially concentrated in the rostral portion of C1 cell column in an area coextensive with the site that produces the most robust pressor response when stimulated chemically or electrically (Ross et al., 1984c, Tucker et al., 1987, Pyner and Coote, 1998, Schreihofer and Guyenet, 2000, Schreihofer and Sved, 2011) while C1 neurons in caudal RVLM colocalize neuropeptide Y (NPY) and project predominantly to supraspinal targets (Tucker et al., 1987). Despite these differences in projection targets and phenotype between rostral and caudal C1 cells, both populations respond to similar stimuli. Thus, the heterogeneity documented in these and other investigations raise the possibility that integrated output of the nucleus may rely upon local circuit connections between neurochemically-distinct and projection-specific populations of C1 neurons.
The goal of the present study was to test the hypothesis that C1 neurons elaborate a local circuit plexus within ventrolateral medulla that may function to synchronize activity of C1 neurons distinguished by projection targets. Toward this end we characterized the synaptology of local circuit C1 axonal arbors differentially labeled by phenotypically defined, lentivirus-mediated reporter expression driven by a synthetic dopamine beta hydroxylase (DβH) promoter. In an earlier report we used this technology to map the efferent projections of C1 neurons and demonstrate that they give rise to thin varicose axons that arborize within the immediate vicinity of the C1 population (Card et al., 2006). In the present study, we have expanded upon that observation using confocal microscopic analysis and transmission electron microscopy (TEM) to demonstrate that C1 neurons elaborate a local circuit synaptic network involving C1 and non-C1 neurons throughout the rostrocaudal extent of the C1 column.
Section snippets
Animals
Adult male Harlan Sprague–Dawley rats weighing 230–440 g at the onset of the experiment were used in the analysis. Photoperiod (12 h light; light on at 0700) and temperature (22–25 °C) were standardized and animals had free access to food and water throughout the experiment. Animal experiments were conducted in a laboratory approved for Biosafety Level 2+ experiments. Experimental procedures conformed to regulations stipulated in the NIH Guide for the Care and Use of Laboratory Animals and were
Results
Systematic examination of EGFP localization confirmed the differential expression of the reporter within C1 neurons and their processes. Three issues should be highlighted in evaluating these data. First, light and TEM analyses demonstrated that greater than 96% of EGFP immunopositive neurons in the ventrolateral medulla were also immunopositive for TH. The small number of EGFP+ neurons that did not contain TH likely resulted from leaky expression at the core of the injection due to high titers
Discussion
The data reported in this correlative light and electron microscopic investigation reveals a local circuit arborization of C1 axons that is presynaptic to both C1 and non-C1 neurons throughout the C1 cell column. Light microscopic data revealed thin varicose axons that were labeled by the EGFP reporter of C1 neurons and arborized in an area coextensive with the C1 cell group. Importantly, restricted labeling of C1 neurons in either the rostral or caudal RVLM revealed axons that projected into
Acknowledgments
These data were presented at the 2011 Society for Neuroscience meeting. Funding for the investigation was provided from NIH Grants HL093134 (J.P.C.) and HL33610 (M.R.). We gratefully acknowledge the expert technical assistance of Karina Steren.
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