Visualizing form and function in organotypic slices of the adult mouse parotid gland

Abstract

An organotypic slice preparation of the adult mouse parotid salivary gland amenable to a variety of optical assessments of fluid and protein secretion dynamics is described. The semi-intact preparation rendered without the use of enzymatic treatment permitted live-cell imaging and multiphoton analysis of cellular and supracellular signals. Toward this end we demonstrated that the parotid slice is a significant addition to the repertoire of tools available to investigators to probe exocrine structure and function since there is currently no cell culture system that fully recapitulates parotid acinar cell biology. Importantly, we show that a subpopulation of the acinar cells of parotid slices can be maintained in short-term culture and retain their morphology and function for up to 2 days. This in vitro model system is a significant step forward compared with enzymatically dispersed acini that rapidly lose their morphological and functional characteristics over several hours, and it was shown to be long enough for the expression and trafficking of exogenous protein following adenoviral infection. This system is compatible with a variety of genetic and physiological approaches used to study secretory function.

Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy

“…Specifically, here we have investigated the exocytosis of large SCGs in SGs in vivo, showing that this process is regulated in a different fashion than what was previously reported in ex vivo systems and revealing aspects that were not previously appreciated (9)(10)(11)24). First, we have found that the β-adrenergic and not the muscarinic receptors elicit the fusion of the SCGs with the APM and that this process occurs in SGs in vivo through single fusion events and not through compound exocytosis (4,11). The reason(s) for the discrepancy between the data derived from explanted organs or acinar preparations and the animal model are difficult to determine.…”

“…Because there are conflicting data generated in in vitro and ex vivo systems about the role of β-adrenergic and muscarinic receptors in SGs, we sought to determine which stimulus elicits exocytosis of the SCGs in vivo. Exocytosis was assayed by estimating the reduction in the number of large SCGs after agonist stimulation, as shown by others (8,11). To this aim, we injected isoproterenol (Iso, a β-adrenergic agonist) and imaged the SCGs by time-lapse confocal microscopy ( Fig.…”

“…This idea is further supported by the fact that these events were observed only when degranulation was stimulated by Iso (Movie S3). Previous studies in acinar preparations of explanted exocrine glands showed that this process occurs through compound exocytosis rather than through single fusion events (4,11,33). Compound exocytosis has been defined as the process by which large SCGs fuse with SCGs that are already connected with the APM, generating strings of three to four interconnected granules (13).…”

“…2D) (27). This approach mimics the bathing of the explanted glands in small fluorescent probes that has been used to investigate exocytosis in ex vivo models (11,13,14,16,17,35). For this set of experiments, we used larger optical sections (1-1.2 μm, Fig.…”

“…In explanted SG slices and in acinar preparations from other exocrine tissues, both complete fusion events and compound exocytosis have been observed under different experimental conditions by using either differential interference contrast microscopy (9-11) or two-photon microscopy (4,11,(13)(14)(15)(16)(17). Finally, the role of actin during exocytosis has been extensively investigated, and various models have been proposed.…”