scalp and getting a clean exposure of the cranial bone, the
periosteum is thoroughly removed using a scalpel. Next, a
metal head-plate is attached around the brain area of interest
using an instant adhesive.
3. On top of the head-plate, a sealing well is formed out of dental
acrylic around the region of interest. This is needed for later
water immersion imaging. Additionally, a grounding electrode
of chlorided silver wire is inserted in one of the walls.
4. On a different holder which holds the head-plate—and thus
the head—tightly in place the skull overlaying the brain area of
interest is thinned using the dental drill. The final bone lamella
is removed by using a very sharp and small needle tip. To
prevent breaking or clogging of the pipette, the dura mater
needs to be carefully peeled away using a fine flamed
tungsten wire.
5. Subsequently, mice are placed under a two-photon microscope
[14] and the sealed well is filled with the extracellular Ringer’s
solution. For exact pipette targeting, a 16Â water immersion
objective should be used. To achieve two-photon excitation,
the laser is tuned to the appropriate wave-length (e.g., 880 nm
in the case of GFP).
6. An NEM is tip-filled with the extracellular Ringer’s solution
containing either TMR (8.3 mg/ml) or fluorescein (12.5 mg/
ml) 3000 MW dextran-conjugated dyes. Complementary
backfilling with plain Ringer’s.
7. The pipette is then mounted on the micromanipulator and the
grounding electrode of the well connected to the ground of the
stimulus isolation unit. Then place the tip of the pipette to the
target structure of interest under two-photon guidance and
start the electroporation program (see Note 7).
8. Standard electroporation settings are adapted from current
literature on glass microelectrode electroporation [2–4, 9]
and imply 25 ms square pulses delivered at a frequency of
2 Hz over 5 min at a stimulation intensity of 50 μA per pulse.
9. 30–60 min after completion of the electroporation protocol,
animals are sacrificed by a transcardial paraformaldehyde perfusion (4%, pH adjusted to 8.9) and the brain is removed for
further histological workup.
4 Notes
1. While this is still a tool not common in neurophysiology
departments, FIB milling systems are standard tools in any
engineering department and increasingly used for neuroanatomical studies as well [15].
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Daniel Schwarz and Andreas T. Schaefer
periosteum is thoroughly removed using a scalpel. Next, a
metal head-plate is attached around the brain area of interest
using an instant adhesive.
3. On top of the head-plate, a sealing well is formed out of dental
acrylic around the region of interest. This is needed for later
water immersion imaging. Additionally, a grounding electrode
of chlorided silver wire is inserted in one of the walls.
4. On a different holder which holds the head-plate—and thus
the head—tightly in place the skull overlaying the brain area of
interest is thinned using the dental drill. The final bone lamella
is removed by using a very sharp and small needle tip. To
prevent breaking or clogging of the pipette, the dura mater
needs to be carefully peeled away using a fine flamed
tungsten wire.
5. Subsequently, mice are placed under a two-photon microscope
[14] and the sealed well is filled with the extracellular Ringer’s
solution. For exact pipette targeting, a 16Â water immersion
objective should be used. To achieve two-photon excitation,
the laser is tuned to the appropriate wave-length (e.g., 880 nm
in the case of GFP).
6. An NEM is tip-filled with the extracellular Ringer’s solution
containing either TMR (8.3 mg/ml) or fluorescein (12.5 mg/
ml) 3000 MW dextran-conjugated dyes. Complementary
backfilling with plain Ringer’s.
7. The pipette is then mounted on the micromanipulator and the
grounding electrode of the well connected to the ground of the
stimulus isolation unit. Then place the tip of the pipette to the
target structure of interest under two-photon guidance and
start the electroporation program (see Note 7).
8. Standard electroporation settings are adapted from current
literature on glass microelectrode electroporation [2–4, 9]
and imply 25 ms square pulses delivered at a frequency of
2 Hz over 5 min at a stimulation intensity of 50 μA per pulse.
9. 30–60 min after completion of the electroporation protocol,
animals are sacrificed by a transcardial paraformaldehyde perfusion (4%, pH adjusted to 8.9) and the brain is removed for
further histological workup.
4 Notes
1. While this is still a tool not common in neurophysiology
departments, FIB milling systems are standard tools in any
engineering department and increasingly used for neuroanatomical studies as well [15].
118
Daniel Schwarz and Andreas T. Schaefer