Fluorescent Tagging of Rhythmically Active Respiratory Neurons within the Pre-Bötzinger Complex of Rat Medullary Slice Preparations

Abstract

Elucidation of the neuronal mechanisms underlying respiratory rhythmogenesis is a major focal point in respiratory physiology. An area of the ventrolateral medulla, the pre-Bötzinger complex (preBötC), is a critical site. Attention is now focused on understanding the cellular and network properties within the preBötC that underlie this critical function. The inability to clearly identify key "rhythmgenerating" neurons within the heterogeneous population of preBötC neurons has been a significant limitation. Here we report an advancement allowing precise targeting of neurons expressing neurokinin-1 receptors (NK1Rs), which are hypothesized to be essential for respiratory rhythmogenesis. The internalization of tetramethylrhodamine conjugated substance P in rhythmically active medullary slice preparations provided clear visualization of NK1R-expressing neurons for subsequent whole-cell patch-clamp recordings. Among labeled neurons, 82% were inspiratory modulated, and 25% had pacemaker properties. We propose that this approach can be used to greatly expedite progress toward understanding the neuronal processes underlying the control of breathing.

Neurokinin Receptor-Expressing Pre-Bötzinger Complex Neurons in Neonatal Mice Studied In Vitro

“…After exposure to both forms of SP, only sparse TMR-SP labeling could be detected in regions that included the preBötC; the extent of the labeling was dramatically less than when TMR-SP was applied without prior exposure to unconjugated SP. This is most apparent in the nucleus ambiguous (NA; Bieger and Hopkins 1987), which is heavily populated by NK1R ϩ neurons and is adjacent to the preBötC dorsally (Gray et al 1999;Pagliardini et al 2005). In control experiments, the NA showed substantially less TMR-SP labeling (Fig.…”

“…However, the relative number of NKR ϩ and NKR Ϫ preBötC neurons that serve rhythmogenic and/or premotor functions remains unknown, and the mechanism underlying the widespread excitatory effects of SP is not well understood. We examined these issues in vitro using fluorescent labeling to identify SP-sensitive neurons that presumably express NKRs, as assessed by their ability to internalize the fluorescent marker tetramethylrhodamine (TMR) conjugated to SP, i.e., TMR-SP (Pagliardini et al 2005). We recorded TMR-SP ϩ and TMR-SP Ϫ neurons and characterized their membrane properties and inspiratory discharge patterns.…”

Stimulation of Respiratory Motor Output and Ventilation in a Murine Model of Pompe Disease by Ampakines

“…The preBötzinger complex (preBötC), which contains neurons that are essential for respiratory rhythmogenesis (24), was identified in the ventrolateral medulla by neurokinin 1 receptor and somatostatin immunoreactivity (25)(26)(27). Somatostatinpositive preBötC cell bodies had prominent inclusion of somatostatin-immunonegative vacuoles in Gaa 2/2 , but not wild-type tissues ( Figures 7A and 7B).…”

P2Y1Receptor Modulation of the Pre-Bötzinger Complex Inspiratory Rhythm Generating NetworkIn Vitro

“…Second, dye spots colocalize with regions of moderate NK1R immunolabeling, and NK1-immunoreactive preBötC neurons double label for P2X 2 and P2Y 1 receptors. preBötC markers include NK1, -opioid, and somatostatin receptor labeling (Gray et al, 1999;Stornetta et al, 2003), but NK1R labeling is used most extensively (Gray et al, 1999(Gray et al, , 2001Guyenet and Wang, 2001;Wang et al, 2001;Guyenet et al, 2002;Pagliardini et al, 2003Pagliardini et al, , 2005. It is not exclusive to preBötC neurons but remains useful, because preBötC staining is greater than in surrounding regions.…”