OPTICAL STIMULATION AND IMAGING OF FUNCTIONAL BRAINCIRCUITRY IN A LAMINAR FLOW CHAMBER

S. Ahrar A *, T. V. Nguyen A*, Y. Shi B, P. V. Thomas A, T. Ikrar B, X. Xu A B, and E. E. Hui A

A. UC Irvine, Biomedical Engineering. B. UC Irvine, Anatomy and Neurobiology, USA.* equal contribution.

ABSTRACT We report a microfluidic platform for the photostimulation and functional imaging of living brain slices. Fast voltage

sensitive dye imaging and stimulation techniques combined with microfluidic chambers were used to study spontaneous and stimulated neuronal activities in the neonatal mouse hippocampus. Caged glutamate was locally delivered in specific regions of interest, while laser uncaging of glutamate initiated neural signaling. Both stimulated and spontaneous neural activity displayed similar spatiotemporal characteristics. In addition, through local calcium depletion in segmented flow, our data suggest that stimulated neural activity in the developing hippocampus is dependent on synaptic signaling rather than direct neural coupling.

KEY WORDS: Neuroscience, living brain slice, photostimulation, functional imaging.

INTRODUCTIONRecording of neurophysiological activity in explanted brain slices is a powerful means for studying the structure and

function of neural circuitry and understanding brain function on a synaptic and cellular level. Recently, a number of groups have reported the explant culture of brain slices within microfluidic devices [1-4] in order to take advantage of laminar flow characteristics to manipulate the physiochemical environment in different subregions of the brain independently. While microfluidics has allowed stimulation with high spatiotemporal resolution, the accompanying readout via electrode probes has provided only a limited picture of the resulting neurophysiological response. In this work, we combine photonics with microfluidics to achieve precise stimulation and readout of brain activity in living mouse brain slices.

The biological goal of our work is to study hippocampal network activity during early development. To this purpose, we study spontaneous network activity known as giant depolarizing potentials (GDPs) as well as photostimulation-evoked population activity in neonatal mouse brain slices. While neural signaling propagates unidirectionally in the mature hippocampus, GDPs propagate in both the forward and reverse directions. This spontaneous activity mediates synaptic connection and provides coherent spatiotemporal patterns that are thought to c o n t r i b u t e t o f u n c t i o n a l c i r c u i t organization. Through the leveraging of m i c r f l u i d i c s a n d p h o t o n i c s , w e demonstrate that the mechanism of reverse signal propagation in GDPs is dependent on calcium-dependent synaptic signaling.

PRINCIPLEBrain slices were prepared from

postnatal 4-day-old mice and placed within microfluidic chambers to provide segmented flows. For example, calcium-rich and calcium-depleted subsections of the hippocampus could be created. An x-shaped chamber geometry was chosen because this configuration has been shown to achieve [5] very sharp gradients at the interface between two liquid flows and provides good flexibility in the position and angle of the interface (Fig. 1).

Focused laser-controlled release of caged glutamate neurotransmitter enables precise initiation of action potentials at specific regions in the brain slice, while fast voltage sensitive dye (VSD) imaging provides optical measurement of neural signaling across the entire hippocampal region simultaneously [6] (Fig. 2).

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October 2-6, 2011, Seattle, Washington, USA

EXPERIMENTALAll animals were handled and experiments conducted in accordance

with approved procedures by the Institutional Animal Care and Use committee at the University of California, Irvine. For this study, mice of postnatal (P) day 4 (P4) were used. Brains were removed immediately from the animals and 400-µm-thick hippocampal slices were prepared. The slices were stained for 1 hour in a well-oxygenated staining chamber containing artificial cerebrospinal fluid (ACSF) with 0.02 mg/ml of the absorption voltage-sensitive dye (VSD), NK3630 (Nippon Kankoh-Shikiso Kenkyusho Co., Ltd., Japan).

VSD-stained slices were placed in the microfluidic chamber, and imaging and photostimulation were performed through an upright microscope. During the experiment, the slices were supported by continuous flow of ACSF solution. Brain slice health and viability in the chamber were demonstrated by measurement of spontaneous GDP activity in the hippocampus (Fig. 3) for periods lasting more than 6 hours.

To conduct VSD imaging experiments, brain slices were trans-illuminated with 705-nm light, and voltage-dependent changes in the light absorbance of the dye were captured by a MiCAM02 fast imaging system. Data capture at 4.4 ms per frame was achieved over a field of view of 1.28 x 1.07 mm2, with a spatial resolution of 14.6 x 17.9 µm2/pixel. During photostimulation experiments, MNI-caged-l-glutamate (4-methoxy-7-nitroindolinyl-caged l-glutamate, Tocris Bioscience, Ellisville, MO) was infused in the chamber at a concentration of 0.2 mM in ACSF. Uncaging was accomplished by short bursts (1 ms, 20 mW) of a 355 nm UV laser, achieving spots with a width of approximately 153 µm [6].

Reagents were fed into the microfluidic chamber using a flow system driven by pressurized carbogen. Flow rate through the tubing was manually controlled with IV flow regulators. Finally, this tubing was interfaced with the two inlets of the microfluidic chamber. Average volumetric flow rate was found to be approximately 0.3 µL/s, or 1.08 mL/hr for one inlet. For some experiments, modified ACSF containing 0 mM Ca2+ and 4 mM Mg2+ was used in segmented laminar flow to block synaptic transmission in subregions of the hippocampus.

RESULTS AND DISCUSSIONHealth and viability of the brain slices in the microfluidic

chambers were confirmed through the observation of spontaneously occurring GDPs in the mouse hippocampus (Fig. 3). GDP is a spontaneous, large scale, propagating network activity, which recruits the entire hippocampal circuitry. It starts in the hippocampal subfield, CA3, and propagates bilaterally toward CA1 and the dentate gyrus. In our next experiment, the brain slice was separated into regions with and without caged glutamate (Fig. 5). Photostimulation did not elicit a response in glutamate-free regions, while photostimulation in glutamate-rich regions initiated neural activity across the entire brain slice, continuing into glutamate-free regions (Fig. 6). This photostimulation-evoked activity mimicked GDP behavior. The temporal stability of the fluid boundary over the course of multiple recordings was measured by capturing fluorescent images of the slice before and after each experiment. It was estimated that on average the boundary moves 66±18 µm over 5 minutes. In addition, we examined reverse signal propagation in the developing hippocampus. In immature neuronal tissues, both synaptic-mediated transmission and electrical coupling between neurons are possible, and the mechanism of reverse propagation has not been conclusively established.

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In the P4 hippocampal slice, photo-stimulation-evoked reverse neural signaling was shown to propagate in calcium-rich regions (Fig. 7), but not in calcium-free regions (Fig. 8). Photostimulation was performed in the calcium-rich region in order to separate signal initiation from signal propagation. Our data are consistent with excitatory propagation in the developing hippocampus being dependent on synaptic signaling between neurons, rather than direct neural coupling.

CONCLUSIONThe combination of segmented laminar flow

with optical stimulation and imaging of neural activity in brain slices provides a powerful experimental platform for manipulating and studying functional neural circuitry. We have successfully demonstrated the use of this system to investigate GDP activity in the developing rodent hippocampus, showing that GDP reverse signal propagation is dependent on synaptic transmission.

ACKNOWLEDGMENTSThis work was supported by a US National

Institute of Health grant (DA-023700-04S1 to X.Xu.) and in part by the Defense Advanced Research Projects Agency (DARPA) N/MEMS S&T Fundamentals Program under grant no. N66001-1-4003 issued by the Space and Naval Warfare Systems Center Pacific (SPAWAR) to the Micro/nano Fluidics Fundamentals Focus (MF3) Center.

REFERENCES:[1] A. J. Blake, T. M. Pearce, N. S. Rao, S. M. Johnson and J. C. Williams, “Multilayer PDMS microfluidic chamber for controlling brain slice microenviornment”, Lab on a Chip, vol. 7, pp. 842-849, 2007.[2] H.H. Caicedo, J.S. Mohammed, C.P. Fall, and D.T. Eddington, “Microfluidic add-On for standard electrophysiology chambers”, Lab on a Chip, vol. 6, pp.1048-1055, 2008.[3] A. Queval, N. R. Ghattamaneni, C. M. Perrault, R. Gill, M. Mirzaei, R. A. McKinney and D. Juncker, “Chamber and microfluidic probe for microperfusion of organotypic brain slices”, Lab on a Chip, vol. 10, pp.326-334, 2010.[4] Y. T. Tang, J. Kim, H. E. López-Valdés, K. Brennan and Y. S.Ju, “Development and characterization of a microfluidic chamber incorporating fluid ports with active suction for localized chemical stimulation of brain slices”, Lab on a Chip, vol.13, pp.2247-2254, 2011.[5] T. Kim, M. Pinelis and M.M. Maharbiz, “Generating steep, shear-free gradients of small molecules for cell culture”, Biomedical Microdevices, vol.11, pp.65–73, 2009.[6] X.Xu, N.D. Olivas, R. Levi, T. Ikrar and Z. Nenadic, “High precision and fast functional mapping of cortical circuitry through a novel combination of voltage sensitive dye imaging an laser scanning photostimulation”, AJP-JN Physio, vol 103. No.4, pp. 2301-2312, 2010.

CONTACTSE.E. Hui, eehui@uci.edu. X. XU, xiangmix@uci.edu.

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