Cholecystokinin Induces Ca2+ Transients in Brainstem Neurons, Astrocytes and Central Vagal Terminals in Jugular/Nodose Ganglia–Brainstem Slice Cultures

Abstract

Cholecystokinin is a well-established neuromodulator of brainstem homeostatic circuits, including circuits implicated in satiety and visceral sensation. To better understand the cellular basis of brainstem cholecystokinin signalling, we developed a novel jugular/nodose ganglion–brainstem slice culture, where microdissection isolated bilateral jugular/nodose ganglia attached to a slice of the brainstem via intact vagal rootlets. This preparation preserves the functional vagal afferent pathway to the nucleus of the tractus solitarius and allows for targeted viral transduction of genetically encoded Ca²⁺ indicators. We show that electrical jugular/nodose ganglion stimulation evokes glutamate-driven Ca²⁺ transients in second-order brainstem neurons, confirming functional connectivity. Using nuclear-targeted Ca²⁺ imaging, we demonstrate that cholecystokinin directly activates a specific subpopulation of neurons in the dorsal and ventrolateral medulla, independent of action potential generation. Furthermore, we identify distinct populations of central vagal terminals: those recruited by electrical stimulation of the jugular/nodose ganglion and a separate subpopulation directly activated by cholecystokinin. Multiplex imaging revealed that cholecystokinin and Substance P triggered robust Ca²⁺ waves in brainstem astrocytes, and muscarine and thyrotropin-releasing hormone selectively activate neuronal populations without recruiting astrocytic networks. Finally, we found that the jugular/nodose ganglion–brainstem slice cultures generate spontaneous respiratory-like rhythmic bursting in the preBötzinger Complex, with a frequency significantly increased by cholecystokinin. Together, these findings establish jugular/nodose ganglion–brainstem slice cultures as a powerful model for dissecting the complex neuro-astrocytic circuitry of cholecystokinin signalling in the brainstem.