α‐1 adrenergic input to solitary nucleus neurones: calcium oscillations, excitation and gastric reflex control

Abstract

The nucleus of the solitary tract (NST) processes substantial visceral afferent input and sends divergent projections to a wide array of CNS targets. The NST is essential to the maintenance of behavioural and autonomic homeostasis and is the source, as well as the recipient, of considerable noradrenergic (NE) projections. The significance of NE projections from the NST to other CNS regions has long been appreciated, but the nature of NE action on NST neurones themselves, especially on the α-1 receptor subtype, is controversial. We used a combination of methodologies to establish, systematically, the effects and cellular basis of action of the α-1 agonist, phenylephrine (PHE), to control NST neurones responsible for vago-vagal reflex regulation of the stomach. Immunocytochemical and retrograde tracing studies verified that the area postrema, A2, A5, ventrolateral medulla and locus coeruleus regions are sources of catecholaminergic input to the NST. In vivo electrophysiological recordings showed that PHE activates physiologically identified, second-order gastric sensory NST neurones. In vivo microinjection of PHE onto NST neurones caused a significant reduction in gastric tone. Finally, in vitro calcium imaging studies revealed that PHE caused dramatic cytosolic calcium oscillations in NST neurones. These oscillations are probably the result of an interplay between agonist-induced and inositol 1,4,5-trisphosphate (IP 3 )-mediated intracellular calcium release and Ca 2+ -ATPase control of intracellular calcium storage pumps. The oscillations persisted even in perfusions of zero calcium-EGTA Krebs solution suggesting that the calcium oscillation is mediated principally by intracellular calcium release-reuptake mechanisms. Cyclical activation of the NST may function to increase the responsiveness of these neurones to incoming afferent input (i.e., elevate the 'gain'). An increase in gain of afferent input may cause an amplification of the response part of the reflex and help explain the powerful effects that α-1 agonists have in suppressing gastric motility and producing anorexia.

Local circuit input to the medullary reticular formation from the rostral nucleus of the solitary tract

“…Relative changes in intracellular calcium in response to electrical stimulation were quantified as changes in fluorescence (%) as follows: ⌬F/F, where F is the fluorescence intensity within an area of interest (e.g., the outline of an RF neuron) averaged over the 3 s before stimulation and ⌬F is the maximum change from this value during a 0.5-s window following electrical stimulation (41). This normalization of the amount (%) of change in activity/fluorescence of any given region of interest could then be compared by a paired t-test for drug effects on the response to electrical stimulation (P Ͻ 0.05 criteria).…”

Differential coupling of β3A- and β3B-adrenergic receptors to endogenous and chimeric Gαs and Gαi

“…The response to 10 nM carbachol also differed in that after the initial peak Ca 2ϩ i decreased and increased in an oscillatory manner. On the basis of our recent work, we attribute this oscillatory pattern to an interplay between calcium releasereuptake mechanisms (22). Treatment of cells with a threshold dose of 1 nM carbachol produced a rapid mobilization of Ca 2ϩ i followed by an oscillatory pattern of change in Ca 2ϩ i (Fig.…”

“…Cells were also cotransfected with an expression construct for red fluorescent protein (DsRed) so that recordings were taken from identified, successfully transfected cells. On the basis of previous reports showing that the kinetics and temporal patterns of Ca 2ϩ i mobilization (e.g., oscillations vs. monotonic) are agonist concentration dependent (1,22,41), initial studies were conducted with CHO cells stably transformed with the M1-CHO to establish how incremental receptor activation is translated with our detection system in this cell type. A saturating concentration of carbachol (10 M) evoked a large, fourfold increase in Ca 2ϩ i followed by a fast and then slow decline of Ca 2ϩ i over the following 4 min (Fig.…”

Ear mesenchymal stem cells (EMSC) can differentiate into spontaneously contracting muscle cells

“…Culture dishes were then washed three times in HBSS and mounted onto the microscope stage (Nikon). Fluo-3 fluorescence was measured in these cells using an excitation filter of 495 nm on the selection illuminator (Illuminator D6), an emitter of 520 nm, and a low light camera (Photometrics) interfaced with the microscope and controlled by Metamorph software as described previously [Hermann et al, 2005].…”