Design and construction of an microfluidic device for investigating the physiology of neural circuits
The investigation of neural circuit networks is among the most important current research themes in neuroscience. A number of studies have used microfluidic devices for studying specific neural circuits, an approach that is rapidly growing in popularity [8, 9, 23]. Here, we designed and built a microfluidic device for monitoring specific neural circuits—the CStr circuit in the current application—by modulating their activity. Our device was designed with an appropriate channel length and width to achieve this goal. Furthermore, electrode holes were added to the device to enable facile, accurate electrical stimulation (Fig. 1A, B). This microfluidic device provides compartmentalized cell culture platforms that allow isolation of two types of neurons (cortical and striatal in the current configuration) for reconstruction of specific brain circuits (Fig. 1C). Our microfluidic device is composed of three compartments: (1) a neuronal chamber, the largest area (height, 100 μm) in which neurons are placed; (2) a thinner (height, 3 μm) neurite microchannel, accommodating both axon and dendrite outgrowth from each side; and (3) a synapse canal, where synapse connections from each side are made. Additionally, and most importantly, we placed electrode holes in the area juxtaposed to the neurite microchannel. To construct this microfluidic device, we utilized two layers of photoresist (SU-8) for the template and polydimethylsiloxane (PDMS), a type of silicon rubber, for the chamber body. PDMS is widely used because it offers several advantages for use in stamps or molds, including (i) low interfacial free energy for ease of demolding and material transfer; (ii) stability against chemicals, heat, and humidity; and (iii) optical transparency for UV-assisted crosslinking of materials (Fig. 1D) [24].
Successful reconstitution of CStr circuits in the microfluidic device platform
To verify that our microfluidic device provides a structural neural circuit platform for stably constructing CStr circuits, we placed each type of cell—cortical neurons and striatal neurons—in the corresponding areas on each side of the neuronal chamber. Neurons in the microfluidic device were further incubated for at least 14 d in vitro (DIV14). During incubation, both axons and dendrites of each type of neuron elongated through the microchannels from each side of the neuronal chamber. To examine the growth of cortical axons or striatal dendrites in microchannels and synapse formation in the synapse canal, we performed an immunofluorescence (IF) analysis of axonal and dendrite markers using anti-tau and anti-MAP2 (microtubule-associated protein 2) antibodies, respectively. As shown in Fig. 2A, cortico-axons and striatal-dendrites successfully expanded from their corresponding microchannels, resulting in well-formed synapses in the synapse canal. Furthermore, the Ca2+-indicator dye, Fluo5F-AM, clearly revealed formation of entire circuits (Fig. 2B, C). Collectively, these findings demonstrate that our microfluidic device provides an effective structural platform for neural circuit formation.
Monitoring Ca2+ dynamics in CStr circuits during circuit activity using the microfluidic chamber
Having confirmed successful construction of a structural platform for reconstructing CStr circuits, we next sought to demonstrate that our microfluidics system can provide physiological monitoring of circuit responses to electrostimulation. Because Ca2+ signaling is essential for neural activity, we monitored Ca2+ dynamics in CStr circuits in our microfluidic device using two different Ca2+ indicators. First, we employed the chemical Ca2+ indicator, Fluo5F-AM. After treating CStr circuits in the microfluidic chamber with Fluo5F-AM and washing, the microfluidic chip was connected to the electrode and stimulated with 1 or 10 action potentials (APs) at 100 Hz. Synapses of CStr circuits showed strong responses to corresponding electrical activities in the synapse canal of the microfluidic chip (Fig. 3A–F; Additional file 1: Video S1). We further stimulated CStr circuits at various paired-pulse intervals (50, 100, 200, and 500 ms). As shown in Fig. 3G, Ca2+ dynamics in CStr circuits clearly exhibited paired-pulse responses. Second, we made use of the synapse-specific genetic Ca2+ indicator, synaptophysin-GCaMP6f (Physin-GC6f), which has been employed in a number of studies for monitoring synaptic Ca2+ dynamics [25,26,27]. To this end, cortical neurons were electroporated with Physin-GC6f and plated the resulting transfected cells in the neuronal chamber of the microfluidic device. CStr circuits formed with 14 d of plating (14DIV), and a Physin-GC6f–positive circuit was selected for monitoring of Ca2+ dynamics using the same experimental regime as used for Fluo5F-AM. Physin-GC6f–positive synapses in the CStr circuit showed Ca2+ responses upon electrical stimulation that were clearly visible under all stimulation conditions (i.e., 1 AP, 10 APs, paired-pulse) (Fig. 4A–G; Additional file 2: Video S2). Taken together, these results indicate that our microfluidic device is capable of monitoring electrical stimulation-induced Ca2+ dynamics in the CStr chip.
Measurement of synaptic transmission during neural activity using the microfluidic chip
We next investigated synaptic transmission in CStr circuits in the microfluidic system in response to an electrical stimulation. To measure synaptic transmission, we employed a pHluorin-based assay in which pHluorin conjugated to the luminal region of the synaptic vesicle membrane proteins vGlut1, VAMP2, or synaptophysin is used to directly monitor synaptic vesicle exocytosis at nerve terminals [28,29,30]. We introduced each of these three pHluorin systems—vGlut1-pHluorin (vG-pH) [or vGlut-pH-mCh (vG-pH-mCh)], VAMP2-pHluorin (Syn-pH), and synaptophysin-pHluorin (Physin-pH), respectively—in a CStr circuit in a microfluidic chip and observed physiological synaptic transmission responses under various electrostimulation regimes (50, 100, and 300 APs). As shown in Fig. 5A–G, CStr circuits in the chip transfected with vG-pH-mCh showed vivid synaptic transmission responses under each electrical stimulation condition that increased with increasing stimulation intensity (Additional file 3: Video S3). Circuits expressing Syn-pH or Physin-pH also exhibited clear synaptic transmission responses upon stimulation (Fig. 5H–K). Collectively, these observations demonstrate successful monitoring of synaptic transmission in CStr circuits in our microfluidic system.
Monitoring synaptic vesicle retrieval after electrical stimulation using the microfluidic device
Synaptic retrieval, or synaptic vesicle endocytosis, is an essential process following synaptic vesicle fusion that serves to maintain the functionality of neural circuits for subsequent rounds of stimulation. To test whether synaptic retrieval of CStr circuits can be monitored and analyzed in our microfluidic system, we also used the pHluorin based system, focusing on the response after electrical stimulation, which represents the course of synaptic vesicle endocytosis. To this end, we analyzed the decay of pHluorin fluorescence after stimulation of CStr circuits with 50, 100, or 300 APs. Endocytosis of synaptic vesicles was detected under each of the various stimulation conditions (Fig. 6A, B). An analysis of the time constant of synaptic vesicle endocytosis (τendo) showed that the mean value of τendo was ~ 14 s under 50 and 100 AP conditions, and slowed to ~ 20 s at 300 APs, values similar to those previously reported [29, 30] (Fig. 6C–F). These findings demonstrate that our microfluidic system is capable of monitoring synaptic vesicle retrieval in the CStr circuit under various stimulation conditions.