(A) Schematic of a microfluidic chamber. For clarity, the PDMS mold (top) is shown above the glass substrate (bottom). The fluid is shown in black. The four circular wells provide access for the introduction of the neurons and are filled with media to support neuronal growth. Microgrooves (900 μm × 7.5 μm × 3 μm) connect the two rectangular channels (or compartments) that house two independent populations of neurons. (B) Dendrites extend into microgrooves. Fluorescence image of dendrites extending into microgrooves (MAP2 = green). Scale bar = 50 μm. (C) Dendritic length within microgrooves as a function of days in culture. Shown are normal cumulative distribution plots for single chambers (n = 2). (D) Fluorescence image of dendritic spines within microgrooves. Neurons within the microfluidic chamber were infected with an RFP Sindbis virus, which demonstrates the healthy neuronal morphology, including spines. Scale bar = 10 μm.

(A) Neuronal processes, extending from compartmentalized neuronal cell bodies, establish contact within the microgrooves. Two sets of neurons were introduced into the left and right compartments. Neurons on the left expressed GFP whereas neurons on the right expressed RFP. Red- and green-labeled processes are evident within the microgrooves. Scale bar = 150 μm. (B) Enlarged image of the left-side of the chamber showing GFP-labeled dendrites entering the microgroove channels (top) and RFP-labeled axons (middle) growing the ∼900 μm extent of the microgrooves. These axons appear to contact the dendrites, as shown in the merged image (bottom). Scale bars = 50 μm. (C) Neurons grown in the chamber for 21 days possess synapses. MAP2-labeled dendrites (green, top) extend into the microgrooves from the left side. Axons (not shown) enter from both the presynaptic and postsynaptic sides. Immunostaining for the presynaptic marker bassoon (red, middle) shows a punctate pattern that decorates the dendrites. Bottom shows merged images. The white arrow shows a microgroove which contains axons (not visible), but no dendrites; axons within this microgroove have minimal bassoon immunoreactivity. Scale bar = 21 and 3 μm for left and right images, respectively. (D) Functional synapses within the microfluidic chamber. Voltage clamp recordings were obtained from a 42 day-old neuron plated in a microfluidic chamber. Top recording shows miniature excitatory postsynaptic currents, recorded in the presence of TTX. NBQX and APV applied locally to one compartment completely abolished the recorded events. Scale bar = 20.5 pA, 256 ms.

(A) Schematic of a local perfusion chamber showing the perfusion inlet well and channel in yellow. Fluid was withdrawn from the perfusion channel by a syringe pump connected to a tubing outlet (grey peg on right). The PDMS mold is not shown. The enlarged image on the right shows the direction of fluid flow within the local perfusion channel and microgrooves. The slight flow into the perfusion channel from the microgrooves counteracts the diffusion of the perfusate into the microgrooves. (B) A merged fluorescence and DIC image showing the perfusion of the low molecular weight dye Alexa Fluor 488 (2 μM). Color look-up bar shows fluorescence intensity. Scale bar = 50 μm. (C) Fluorescence and DIC depth (z) scans of the white dashed region in (B) showing the profile of the dye within the channel during the perfusion and after wash out. Scale bar = 30 μm. (D) Stability of the perfusion stream. Graph of fluorescence intensity over time for regions of interest within the channel (blue box shown in inset) and proximal microgroove (yellow box shown in inset). A stable fluorescent signal within the channel (light blue circles) was maintained for over an hour while dye in the region immediately adjacent to the channel (yellow circles) remained undetectable. Mean pixel intensity is in arbitrary units. Similar results were obtained in more than 10 experiments. (E) Solutions can be rapidly added and removed from the channel with a response time of less than 1 min. The same regions of interest are used as in D.

(A) A schematic of the local perfusion chamber with 3 inlet wells. The outer wells that contain normal buffer prevent perfusate from entering the microgrooves and serve to narrow the perfusion stream. (B) Merged fluorescence and DIC images of Alexa Fluor 488 perfusion into the channel, flanked by two buffer channels (color look up table shows intensity). Scale bar = 50 μm. (C) Fluorescence and DIC depth (z) scans of dashed line in (B). Scale bar = 15 μm. (D) Continuous line scans of dashed line in (B) within local perfusion channel shows high temporal resolution. Pulsing was performed by rapidly adding/removing 30-40 μl in the center inlet well. (E) Merged fluorescence micrograph of Alexa Fluor 488 hydrazide (green), Alexa Fluor 568 hydrazide (red) and Alexa Fluor 633 hydrazide (blue) added to perfusate, postsynaptic compartment, and presynaptic compartment respectively after 30 min of perfusion. Fluorescent microenvironments are stable and distinct from one another. The properties of the Alexa Fluor 633 hydrazide make it accumulate at the walls of the PDMS thus the intensity of the dye is not uniform throughout the presynaptic compartment. Scale bar = 100 μm. (F) Example image of a MAP2-immunolabeled neuron (green) following perfusion. There was no observable difference in morphology, illustrating that multiple perfusion patterns (i.e., flow changes) have negligible effect on dendritic morphology. The perfusion channel is between the white lines. Scale bar = 50 μm.

(A) A fluorescent micrograph of a spiny GFP-labeled neuron within the presynaptic compartment extends an axon through the microgrooves and into the local perfusion channel. Scale bar = 50 μm. (B) Merged DIC and GFP images showing the locations of the microgrooves and perfusion channel. (C) Enlarged image of box outlined in (A). Below, FM5-95 was loaded via the postsynaptic compartment and perfusion channel, resulting in the labeling of presynaptic terminals. Multiple presynaptic terminals colocalize with the GFP labeled axon. Scale bar = 5 μm. (D) Enlarged image of box outlined in (B). Below, the postsynaptic neuron is post-hoc labeled for MAP2 (orange) and GFP (green). The dendrite and axon from separated compartments colocalize at the site of FM5-95 loading within the perfusion channel. Scale bar = 20 μm.

(A) Fluorescence difference image of Fluo-4 signal before and during local vehicle perfusion (left) showing that are no changes in calcium during vehicle perfusion. Fluorescence difference image was created by subtracting image at time point b from image at timepoint a (shown at right). Plot of normalized Fluo-4 intensity over time (right). Fluorescence difference image includes only regions which were MAP2 positive. ‘Fire’ lookup table. (B) Fluorescence difference image of Fluo-4 signal before and during local glutamate perfusion (left). Plot of normalized Fluo-4 intensity over time (right). Imaging was performed immediately following vehicle perfusion shown in (A). During glutamate perfusion, Fluo-4 signal increased rapidly in perfused dendrite, followed by a slower rise in soma. Fluorescence difference image prepared same as in (A). (C) Post-hoc immunostaining for MAP2, showing soma and dendrites of the perfused neuron. Scale bars = 25 μm.

(A) Time course of experiment showing both spaced and massed perfusions. (B) Fluorescence images of μLP chambers immunostained for MAP2 (green) and pCREB (magenta). pCREB intensity increased in soma with dendrites exposed to spaced glutamate perfusion. Scale bar = 20 μm. (C) Quantification of (B) showing that pCREB intensity is significantly higher in soma which had dendrites perfused with spaced glutamate (n = 22) vs. either spaced vehicle (n = 21) or massed glutamate (n = 9) perfusions. pCREB intensities normalized to spaced vehicle. Significance was determined using a two-tailed Student's t-Test; * p < 0.05 vs. spaced vehicle.

(A) Time course of experiment and example DIC image of perfusion within chamber. Scale bar = 50 μm. (B) Example images showing Arc mRNA (green) and 18S rRNA (red) 20 min following DHPG or control perfusions within the perfusion chambers. The boundaries of the perfusion channel are indicated by the white lines. Multiple Arc mRNA puncta are present within the DHPG-perfused chamber. Scale bar = 10 μm. (C) Mean (±SEM) number of Arc mRNA puncta identified within the soma of perfused dendrites for control (n = 45), DHPG (n = 80), AIDA/DHPG (n = 41), and ActD/DHPG (n = 17). (D) Mean (±SEM) number of Arc mRNA puncta identified within perfused dendrites (normalized to the average number of puncta in control treatment) within 50 μm perfusion channel and proximal 50 μm segment for control (n = 35), DHPG (n = 41), AIDA/DHPG (n = 10), and ActD/DHPG (n = 17). Significance was determined using the two-tailed Mann Whitney Test; * p < 0.05 vs control; ** p < 0.05 vs DHPG.