Analytical Chemistry Regardless of previously applied K+, exposure to 5 min of K+ resulted in the observance of release 80% of the time, whereas, when the same neurons were exposed to K+ for a shorter period (<5 min), release was detected less than 50% of the time. This demonstrates that exposure times of less than 5 min usually did not elicit release. For subsequent exposure periods (>5 min), acidic peptide detection decreased. In part, this decrease is due to the refractory period that bag cells enter following release.44 During this period, regardless of the length of stimulation applied, the cells are not able to sustain peptide release. Since bag cell neurons are involved in reproduction, this refractory period is consistent with the fact that the animals can only lay eggs periodically. Furthermore, this shows that the temporal resolution of our platform is able to capture both the onset of peptide release and the refractory period after release. We did observe diﬀerences in the temporal aspects of release between experiments, which may represent diﬀerences in the release dynamics between individual animals. Insulin Stimulation. Elevated K+ is a general secretagogue that causes release of vesicles (including dense core vesicles containing peptides) in a neuron through the depolarization of the cell membrane. Therefore, it should elicit release from most, if not all, neuronal subtypes. In contrast with the depolarization caused by K , which directly aﬀects potassium + channels, insulin is a cell-speciﬁc chemical stimulation that is internalized through the phosphorylation of receptor tyrosine kinases.45,46 Therefore, cells without this receptor will not respond to insulin application. Previous studies have shown that bag cell neurons contain insulin receptors.47 Unlike the widely accepted model of neurotransmitter exocytosis, which involves the inﬂux of external Ca2+, bag cell neurons are unusual because the release of intracellular Ca2+ (such as caused by insulin exposure) is enough to result in peptide secretion.48 The application of 5 μM of mammalian insulin causes peptide release through such an extracellular Ca2+-independent process.48 While the structure of mammalian insulin is distinct from Aplysia insulin peptides, it is similar enough to still function in Aplysia.39,47,48 To demonstrate that the networks maintained in our device exhibit a similar physiology to semi- intact animals in previous reports39,47,48 and to document that small numbers of bag cell neurons in our reduced network respond to insulin (something not shown before), nine additional devices, each containing a distinct network of bag cell neurons, were exposed to increasing periods of insulin to determine the temporal dynamics of peptide release. In contrast to K+, 20 min of insulin exposure was necessary to detect peptide release. The slower response is not surprising given that insulin-evoked release may require peptide internalization, a process that is expected to take longer than the release induced via potassium ions. In addition to acidic peptide, a number of egg laying hormone-related peptides were detected (Figure 4). It was previously reported that internal Ca2+ increases within 5−20 min of exposure to insulin.48 Our data agrees with this; at least 20 min was necessary for peptide release to occur. Furthermore, in the majority of devices where release occurred following 20 min of insulin exposure, peptides were only observed in the ﬁrst collection (n = 4; out of 7 devices where peptide release was observed). Devices where peptides were collected in both 20 min collections, signal intensities were at least 2.5 times greater in the ﬁrst collection (n = 3; out of 7 devices where peptide release was observed). Article Figure 4. Representative mass spectrum of peptide release following 20 min of insulin exposure (αBCP1−7, m/z = 922.5; αBCP, m/z = 1122.6; AP8−27, m/z = 2316.2; AP, m/z = 2959.5; δBCP1−36, m/z = 4022.7; δBCP, m/z = 4406.9). Jonas et al.48 showed that the egg-laying hormone (which is stored in the same peptidergic vesicle that contains acidic peptide) was released following insulin application. However, their use of radioimmunoassay required the preselection of one particular peptide of interest. MS allows us to view an entire complement of released peptides. The egg laying hormone prohormone, the source of these peptides, has a tetrabasic site that is cleaved prior to vesicle packaging, resulting in the N- and C-terminal ends of the prohormone being packaged into distinct vesicles.49 The α, β, and γ-bag cell peptides are located near the N- terminus of the egg laying prohormone, and acidic peptide and egg laying hormone are located near the C terminus and each are packaged into these two peptide-containing vesicle populations. We observed both N- and C-terminal peptides, indicating that both types of vesicles are released upon insulin stimulation, at least under our culturing conditions. Further- more, insulin [M + 2H]2+ was observed for all exposure periods. This demonstrates that the stimulation solution ﬁlls the device in the time allotted for exposure. Acidic peptide detection versus exposure time was again plotted to show the amount of insulin exposure time necessary for peptide release. Our observations were robust, with acidic peptide being detected 88% of the time following 20 min of exposure, whereas release was detected less than 50% of the time following a shorter insulin exposure. Similar to the refractory period seen with K+ stimulations, we observed fewer peptides in the 30 min exposure period. In the study done by Jonas et al.,48 entire bag cell clusters, each containing hundreds of cells, were perfused with insulin for 70 min. They found that the amount of insulin-induced peptide release was similar to that of an afterdischarge. Their application of electrical stimulation would resemble our application of K+, in that both trigger action potentials and an afterdischarge. Here, we show our ability to see peptide release following both K+ and insulin exposure. However, our microﬂuidic valve system enables release studies from signiﬁcantly fewer cells with higher temporal resolution. This increase in temporal resolution shows that two similar peptide releases (in terms of amount and peptide content) have diﬀerent kinetics and, presumably, biochemical pathways. ■ CONCLUSIONS Overall, this approach can maintain a neuronal culture and detect peptide release following appropriate physiological stimulations. The diﬀerence in exposure time necessary to elicit peptide release between K+ and insulin demonstrates that 9450 dx.doi.org/10.1021/ac302283u | Anal. Chem. 2012, 84, 9446−9452