LightDirected Electrical Stimulation of Neurons Cultured on Silicon Wafers

1
Light-Directed Electrical Stimulation of Neurons
Cultured on Silicon Wafers

• Artem Starovoytov, et al.
• J. Neurophysiol. 93 1090-1098, 2005

2
Introduction

• Dissociated neurons cultured in vitro can serve
  as a model system for studying the dynamics of
  neural network.
• Defects in conventional techniques
• Intracellular electrode stimulation incurs a
  physiological perturbation to the neuron.
• Noninvasive extracellular stimulation, commonly
  relies on bipolar field electrodes, has limited
  spatial specificity.
• MEA the stimulation site is fixed by the
  electrode position and grid resolution within an
  array.

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Introduction (continued)

• Defects in conventional techniques (continued)
• Photostimulation by neurotransmitter uncaging
  uncaged transmitters can activate extrasynaptic
  neurotransmitter receptors, thereby limiting
  spatial resolution and complicating data
  interpretation.

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Method in brief
Laser
Culture dish
Silicon wafer
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Methods
Dissection solution consisted of Hanks balanced
salt solution (Life Technologies, Gaithersburg,
MD) supplemented with 25 mM HEPES, pH 7.35. The
dentate gyrus was removed, and the remaining
hippocampal tissue was proteolyzed in dissection
solution plus 20 U/ml papain (Worthington,
Freehold, NJ), 0.5 mM EDTA, 1.5 mM CaCl2, 1 mM
L-cysteine, and 0.1 mg/ml DNAasefor 30 min at
37. Tissue pieces were triturated in culture
media (CM)Basal Media Eagle (Life Technologies)
plus 1 mM HEPES pH 7.35, 1 mM Na-pyruvate, 50
U/ml penicillin, 50 mg/ml streptomycin, 6 mg/ml
glucose, 10 fetal bovine serum (Hyclone, Logan,
UT or Omega Scientific, Tarzana, CA), mito serum
extender (Fisher Scientific, Pittsburgh, PA), and
2 B27 (Life Technologies)and 0.5 ml of cell
suspension was plated at 12 104 cells/ml onto
12-mm coverslips that had been preplated with
astrocytes. One day after plating, 4 mM cytosine
b-D-arabinofuranoside (araC) was added to prevent
astrocyte proliferation. Every 34 days
thereafter, cells were fed with 0.1 ml of CM.
Astrocytes were prepared from extra cells
obtained from each dissection. After a week, the
confluent glial cells were shaken at 260 rpm
overnight to enrich for type I astrocytes.
Usually, the cells were then passaged and grown
for an additional week to select for adherent,
quickly spreading astrocytes. Astrocytes were
plated at 6 103/ml onto coverslips sprayed with
microdots of collagen and poly-D-lysine. After
24 days, 4 mM araC was added to limit the size
of the astrocyte islands. Neurons were added 36
days after astrocyte plating, after aspirating
most of the media. Cultures were used for
experiments 814 days after plating.

• Hippocampal culture
• Cells from P1-P2 rat hippocampi.
• Silicon culture plate fabrication.
• Coating material poly-D-lysine / collagen
• Cells (120,000-150,000) were plated onto each
  dish in 2 ml of culture medium
• (2 days after) one-half of the medium was
  replaced with culture medium containing 8 uM
  ARA-C (cytosine arabinoside).
• Medium replacing every week with 4 uM ARA-C
• Experiments were performed on 3- to 5- week old
  cultures.

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Methods (continued)

• Silicon culture plate fabrication
• Specifications 375-um-thick single-side polished
  (100)-oriented silicon wafers boron-doped to 7-17
  Ocm, native oxide was left intact.

1.5 1.5 cm2
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Methods (continued)

• Bias Voltage
• Ohmic electrical contact to the back of the
  silicon wafer
• In (indium) / Ga (gallium) eutectic
• flat copper electrode
• Ground electrode silver / silver chloride pellet
• DC power supply was used to bias the back side
  negatively with respect to the ground electrode.

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Methods (continued)

• Bias voltage (continued)
• When a negative bias is applied to p-type
  silicon, it causes the silicon surface to be
  depleted of majority carriers.

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Methods (continued)

• Targeted illumination pulses

Acousto-optical
660-nm laser diode
0.1 to 2 mW
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Methods (continued)

• Electrophysiology and imaging
• Whole cell patch-clamp recording (current clamp)
• Condition D-AP5 (100), CNQX(20), PTX(20 uM)
• Fluorescent dyes Alexa Fluor 488 hydrozide or
  biocytin
• negative current (2 nA) for 15 min
• User interface and data presentation format
• Operating modes point-and-shoot, scan

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Results
Stimulation in the presence of synaptic
transmission
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Stimulation with synaptic transmission blocked
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Fluorescent dye labeling of extended neurites
2 ms
4 ms
5 ms
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Optimal stimulation parameters
The amount of photocurrent produced by a laser
pulse at the silicon depends on the value of the
bias voltage, as well as on the intensity and
duration of the illumination pulse.
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Discussion

• A method for extracellular electrical stimulation
  of neurons.
• When a appropriate bias voltage is applied, the
  stimulating electrode is light-addressable.
• MEA stimulation at only fixed electrodes.
• This method would not provide the recording
  capacities as MEA.
• Low-cost, disposable substrates.

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Photocurrent dependence on stimulation parameters