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© Stephanie Pollitt 2015. Originally published in Explorations: The UC Davis Undergraduate Research Journal, Vol. 17 (2015). http://Explorations.UCDavis.edu © The Regents of the University of California.

Imaging In Tyto Alba: A Toolkit for Investigating the Auditory Localization Pathway Stephanie L Pollitt, William DeBello

Abstract:

The barn owl auditory localization circuit undergoes learning through changes in the strength and pattern of synapses, but how these processes occur on a circuit level is unknown. Two new methods have been developed that can be used to study those processes. Array tomography uses antibodies raised against synaptic and neuronal proteins to visualize their distribution in ultra-‐thin brain sections. CLARITY, in contrast, visualizes intact circuits in translucent brains. However, use of these techniques in barn owls requires development of an antibody toolkit that works in avians. Antibodies were vetted using Western blots, immunohistochemistry and array tomography, and assembled into a searchable database called ADAPT. By using this toolkit in conjunction with array tomography and CLARITY, we hope to measure the remodeling of axonal projections and to develop a 3D map to aid microelectrode navigation in vivo. Key Words: Barn Owl, Auditory Localization, Antibodies, CLARITY, Array Tomography Introduction:

The barn owl is a predatory species that relies heavily on auditory information to

hunt. It has a highly complex auditory map in the Inferior Colliculus External nucleus (ICX)

of the tectal lobe that incorporates both Interaural Level Difference (ILD) and Interaural

Time Difference (ITD) information from

the Inferior Colliculus Core (ICC) lateral

shell to localize and catch prey (Figure 1).

This map projects onto the visual map,

located in the optic tectum (OT), to form a

multimodal map of space. Thus, when

visual perception is shifted using prisms

surgically attached in front of the eyes,

there is a mismatch between the

visual and auditory perceptions of

location. Within hours, the owl will

learn to coordinate reaching

behavior with its new visual perception of space; this is believed to occur at the level of the

Figure 1: Slice of barn owl tectal lobe, stained with SV2-‐a, labeled to show different anatomical structures. Abbreviations: ICC: Inferior Colliculus Core nucleus; ICCls: Inferior Colliculus Core lateral shell; ICX: Inferior Colliculus External nucleus; OT: Optic Tectum.

UC Davis | EXPLORATIONS: THE UNDERGRADUATE RESEARCH JOURNALVol. 17 (2015) S. Pollitt p.2

2 Imaging in Tyto Alba: A Toolkit for Investigating the Auditory Localization Pathway cerebellum. However, when the barn owl hunts using purely auditory cues, the visual

adaptation causes overcorrected reaching behavior. In a matter of weeks, the ICX circuit

will adapt to re-‐align the auditory and visual maps. At this point, the owl will correctly

reach to visual and auditory stimuli. Wild barn owls use this ability to adapt to changing

auditory cues as their heads grow, which is consistent with the observation that the

plasticity in the circuit is significantly reduced in mature owls. This new circuit represents

a form of learning, and there are still questions regarding the synaptic clustering, the

integration of excitatory and inhibitory synapses, and exactly how the circuit

reorganization starts.

Current imaging techniques like immunohistochemistry that might be used to

reconstruct these circuits are insufficient due to low quality images from thick slices of

tissue, low antibody penetration, and the limited number of antibodies that can be used on

each slice. Therefore, in order to investigate these phenomena, the laboratory is beginning

to apply emerging tools for connectome reconstruction including high-‐throughput electron

microscopy, cellular/molecular phenotyping, array tomography and CLARITY. The latter

two are the focus of my thesis project. Array tomography encases blocks of brain tissue in a

resin then prepares a library of antibodies in high resolution. CLARITY (Clear Lipid-‐

exchanged Anatomically Rigid Imaging/immunostaining-‐compatible Tissue hYdrogel) is a

technique that entails perfusion with a hydrogel, which fixes the hydrophilic components of

the brain in place. The lipids are then removed via electrophoresis, leaving a translucent

brain that can be stained with antibody and imaged intact. Like array tomography, clarified

tissue can be subjected to elution and restaining with antibodies. Both techniques can also

be used to visualize tracer injections. These properties make both array tomography and

CLARITY ideal for visualizing proteins associated with adaptation.

One necessary component in both of these techniques is a set of antibodies that

work well in avian tissue across many types of fixation. Antibodies are generally against

proteins or peptide sequences derived from mammals, so immunohistochemistry in barn

owls can be a guessing game as to whether or not a particular antibody will bind its avian

counterpart protein. This is because amino acid sequences diverge across species,

including between mammals and aves. For example, the protein synapsin, commonly used

to visualize synapses in mammals, is not present in the avian genome. Also, antibodies that


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work well on one type of fixed tissue might not work as well on tissue fixed even slightly

differently. Therefore, having a well-‐vetted set of antibodies is integral to investigating the

barn owl learned circuit. This paper demonstrates the efficacy of a selection of antibodies

in western blots, standard immunohistochemistry, and array tomography, as well as the

CLARITY technique.

Antibody Immunogen Company Host Species

Clonality Product Number

Concentration (IHC)

Concentration (Western Blot)

Concentration (AT)

CaMKIIα-‐a Rat Calmodulin Kinase IIα 6G9 clone

Genetex Mouse IgG1 Monoclonal

GTX41976

1:500 1:1500 1:100

CaMKIIα-‐b Recombinant Rat Calmodulin Kinase IIα 6G9 clone

Caymen Mouse IgG1 Monoclonal

10011437

1:500 1:1500 N/A

CaMKIIα-‐d Rat Calmodulin Kinase IIα 6G9 clone

Enzo Life Sciences

Mouse IgG1 Monoclonal

ADI-‐KAM-‐CA002-‐D

1:500 1:1500 N/A

GABA-‐b Synthetic human GABA Receptor α1

Abcam Rabbit IgG Polyclonal

ab33299 1:500 1:1000 N/A

GAD-‐a Synthetic rat GAD 65 and GAD 67

Millipore Rabbit Polyclonal ab1511 1:600 N/A 1:100

GAD-‐b Synthetic Human GAD 65 and GAD 67

Abcam Rabbit Polyclonal ab11070 1:600 N/A N/A

GluR1-‐a Rat Glutamate Receptor 1 (extracellular domain)

Millipore Rabbit Polyclonal PC246-‐100UG

1:5 (serum concentration is 0.1µg/µL)

N/A N/A

GluR2/3-‐a Rat Glutamate Receptor 2

Millipore Rabbit Polyclonal 07-‐598 1:500 1:1000 1:100

SV2-‐a Synaptic vesicles from Ommata

DSHB Mouse IgG1 Monoclonal

SV2 1:200 1:1000 1:500

Syntag-‐a Synthetic human Synaptotagmin 1 (amino acids 276-‐325 internal region)

Sigma Rabbit Polyclonal SAB4502907 1:200 1:600 1:100

Syntag-‐b Rat synaptotagmin 1

Millipore Mouse IgG2a Monoclonal

MAB5200 1:500 N/A N/A

Syntag-‐c Recombinant protein of rat synaptotagmin 1 (aa 80 -‐ 421), which contains the C-‐terminus

Synaptic Systems (SySy)

Mouse IgG2a Monoclonal

105011 1:500 N/A N/A

Syntag-‐d Synthetic human Synaptotagmin 1 (amino acids 176-‐225 internal region)

Sigma Rabbit Polyclonal SAB4502908 1:200 1:600 1:100

Table 1: List of antibodies used in this paper, in alphabetical order. Antibody ID was derived from the antigen and a letter code to segregate it from other antibodies from different sources with the same antigen. Concentrations refer to amount of serum : amount of antibody solution.


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4 Imaging in Tyto Alba: A Toolkit for Investigating the Auditory Localization Pathway

Methods:

Immunohistochemistry:

Barn owls are perfused with paraformaldehyde and their brains are removed. The

tectal lobes are blocked off of the brain and sliced with a microtome in 40-‐micron sections.

The sections are then washed in 0.1M phosphate buffer for five minutes and blocked for an

hour in normal serum, bovine serum albumin (BSA), and Triton-‐X in phosphate buffer. The

species of the normal serum is matched to the species used to produce the secondary

antibodies. The primary antibodies are then applied at antibody-‐specific concentrations

(Table 1) in a mixture of normal goat serum, Triton-‐X, BSA, and phosphate buffer.

The antibodies used are from separate species to ensure specific secondary binding.

The slices are allowed to incubate overnight at 4 degrees Celsius. The next day, the slices

are removed from the primary antibody solution and washed twice in Triton-‐X, BSA in

phosphate buffer for five minutes. The secondary solution is then applied, which is

composed of BSA, Triton-‐X, and phosphate buffer with AlexaFluor secondary antibodies at

a 1:200 concentration. The slices incubate at ambient temperature out of the light for two

hours. After that time, the slices are rinsed five times for two minutes each in BSA

phosphate buffer, then twice for five minutes each in phosphate buffer. The slices are then

mounted and coverslipped before imaging at 63x with a fluorescent confocal microscope.

Western Blotting:

A precast gel is inserted into the PAGE set up, which is then filled with a running

buffer composed of Tris base, glycine, SDS, and water. Brain homogenate from rat, mouse,

owl, zebra finch, and canary are diluted 1:1 with sample buffer. The sample buffer is

composed of Tris HCl, glycerol, SDS, bromophenol blue, and 2-‐ mercaptoethanol in water.

Five µL of fluorescent molecular marker and ten µL of each mixture are then injected into


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consecutive wells of the gel. Electricity is then applied to the set up at 80 volts for a half an

hour, then 100 volts for the remainder of the time until the lightest bands of protein reach

the bottom of the gel.

Once the electrophoresis is finished, the gel is placed with a nitrocellulose

membrane, two filter papers, and two fiber pads in a tub of transfer buffer to soak. The gel

is then removed from its cassette and placed in a sandwich set up with a nitrocellulose

membrane and two presoaked filter papers and fiber pads on each side. This sandwich is

then put in the transfer set up so that the membrane is between the gel and the red side of

the holder. The transfer buffer, composed of Tris base, glycine, methanol, and water that

was used for soaking is then poured into the transfer chamber. The whole set up is then

run at 90mA (using a ThermoEC power source, model EC250) overnight at 4 degrees

Celsius.

The next day, the membrane is removed from the transfer set up and rinsed for five

minutes in Odyssey blocking

buffer and phosphate buffer

saline. It is then blocked at room

temperature for an hour in the

same solution. The membrane is

then placed in a primary

antibody solution composed of

Odyssey buffer and primary

antibody at antibody-‐specific

concentrations. This is allowed

to sit for four hours at 4 degrees

Celsius on a shaker.

After the membranes are

removed from the primary antibody solution, they are rinsed four times for five minutes

each in a Tween-‐20 and phosphate buffer saline solution. The secondary antibodies are

then diluted 1:10,000 in Odyssey blocking buffer, and applied to the membranes for one

hour at room temperature on the bellydancer shaker. After an hour the membranes are

Figure 2: Diagram of CLARITY electrophoresis setup. Components include a water heater/circulator (1), a filter (2), and two chambers running in parallel, fitted with platinum wire to receive current (3).


6 Imaging in Tyto Alba: A Toolkit for Investigating the Auditory Localization Pathway rinsed four times for five minutes in the Tween-‐20 and phosphate buffer saline solution

before imaging.

Array Tomography:

All array tomography

staining and imaging was

done at Aratome Inc. in Palo

Alto. A small block of

perfused tissue from the owl

tectum was sliced into 70 nm

serial sections and placed on

a slide. The sections were

then encased in LR White

resin. Multiple rounds of

immunohistochemistry and

imaging at 63x were then

performed on the tissue, with

the antibodies removed after

each round. Each antibody was imaged and stitched together into a 3x3 panel.

CLARITY:

This technique was performed as described in Chung et al, with slight modifications

to reduce the cost of the equipment. The animal (rat embryo or owl) is perfused

transcardially with an acrylamide, bis-‐acrylamide, paraformaldehyde, and photoinitiator in

phosphate buffered saline, a solution called hydrogel, which is chilled to 4 degrees Celsius.

The brain is removed and then placed in 50mL of hydrogel solution and kept at 4 degrees

Celsius for two to three days.

After sitting in the hydrogel, the tissue is “degassed” using nitrogen, a process that

promotes polymerization of the gel by removing the oxygen. This is done by placing the

sample in a dessicator and alternating vacuum and nitrogen gas application. After all of the

oxygen has been displaced from the sample, it is placed in a 37-‐degree water bath until the

gel has solidified.

Figure 3: CaMKIIα immunohistochemistry images. a) From left to right: CaMKIIα-‐a, CaMKIIα-‐b, and CaMKIIα-‐d. b) Large scale images of where in the tectal lobe each image was taken. Green circles indicate the exact spot targeted by confocal microscopy.


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The solid gel is removed

from the incubating bath and the

brain is separated from the rest

of the gel. It is then placed in an

SDS, boric acid, and sodium

hydroxide solution called

clearing solution, which is

incubated in the 37-‐degree water

bath for three days. The solution

is changed each day to allow

extra paraformaldehyde and

photoinitiator to wash out.

After washing the sample, it is placed in the electrophoresis clearing set up (Figure

2). This consists of a water circulator/temperature regulator (MGW Lauda model MS), a

water filter (standard house filter from Home Depot), an electrophoretic chamber with

platinum electrodes, and a solution reservoir. Unlike in the original paper, we chose to

place the brain between two cell filters to prevent excessive movement while the solution

was flowing through the electrophoretic chamber, where it is subjected to both electricity

(using above power source for western blots, which only delivered 75 watts compared to

the 300 watt source used in the original paper) and water flow to push the lipids out of the

tissue. After a period of weeks, the brain is completely cleared and can be stained with

multiple rounds of antibodies and imaged intact with a long focal distance 40x objective.

For the purposes of this experiment, it was determined that proof-‐of-‐principle would be a

more achievable goal than trying to test antibodies with clarified tissue, so rat embryo

brains were used instead of owl tectal lobes.

Results

Immunohistochemistry and Western Blots:

Figure 4: CaMKIIα Western blots, target between 50 and 60 kDa. From left to right: CaMKIIα-‐a, CaMKIIα-‐b, and CaMKIIα-‐d. Abbreviations: “Ms” is mouse, “ZF” is zebra finch, “Can” is canary. Molecular weights to left.


8 Imaging in Tyto Alba: A Toolkit for Investigating the Auditory Localization Pathway Each antibody was first screened with immunohistochemistry to see if a signal could be

observed, then used in Western blots and array tomography if successful. Three CaMKIIα

antibodies were screened, all

of which are 6G9 clones

(Table 1).

Immunohistochemistry

stains show a Golgi-‐like

complete cell fill, including

distal dendrites and spines

(Rodriguez-‐Contreras, Liu

and DeBello, 2005). CaMKIIα-‐

a produced the best signal

with the smallest amount of

noise of all three antibodies

(Figure 3a, left). This is

evident from the complete cell-‐fills and evident processes, as well as the especially strong

signal in the ICX (Figure 3b, left). CaMKIIα-‐d produced the second best signal, with a

slightly higher background than CaMKIIα-‐a but a similar cell-‐fill pattern (Figure 3a, right).

The least characteristic antibody was CaMKIIα-‐b, which produced a weak signal and an

unusually high background (Figure 3a, center). One possible explanation for this is that the

image field was inadvertently located just outside of ICX, in the superficial nucleus of the

inferior colliculus where CaMKII expression is known to be low. To mitigate this possibility,

multiple locations across the slice were imaged, which resulted in similar results

throughout the slice (Figure 3b, center).

Figure 5: Immunohistochemistry confocal images for Synaptotagmin. a) From left to right: Syntag-‐a, Syntag-‐b, Syntag-‐c, and Syntag-‐d b) Large scale images of where in the tectal lobe each image was taken. Green circles indicate the exact spot targeted by confocal microscopy.


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All three were also used for

Western blot (Figure 4). CaMKIIα-‐a is

the most characteristic pattern with a

single band for each, but it lacks a

band in the owl lane, and the bands in

the zebra finch and canary lanes didn’t

drop from the well. Because the owl

protein didn’t function well in any of

the gels, it is most likely that the

homogenate was damaged when the

lab freezer broke down. Also, the rat

and mouse signals might be the

strongest because they are the

freshest homogenates, and have been stored in a proper freezer. Although the precise

weight cannot be determined, the presence of signal, however dispersed, indicates that the

antibody is binding to a protein. With that in mind, the CaMKIIα-‐b blot has more functional

owl forebrain homogenate instead of the tectal lobe homogenate like in the CaMKIIα-‐a blot.

However, all three avian homogenate lanes are still smeared and weak compared to the

fresher rat and mouse homogenates. The third blot, CaMKIIα-‐d, is smeared for all

homogenates except the mouse, indicating that there might have been other factors

contributing to the smearing, such as improper sample heating or inexperience. Across all

three blots, the bands that are present are in the correct position (between 50 and 60 kDa),

and the ladder ran clearly.

Figure 6: Synaptotagmin, molecular weight 65 kDa, Western blots. From left to right: Syntag-‐a and Syntag-‐d. Abbreviations: “Ms” is mouse, “ZF” is zebra finch, “Can” is canary. Molecular weights to left.



Four different

synaptotagmin antibodies

were tested (Table 1). The

staining pattern for

synaptotagmin is generally

punctate grouped into

clusters. Syntag-‐a is the best

example of this, with clear

points of signal, with low

background staining (Figure

5a, left). Syntag-‐d is the

second best, with a weaker

signal and higher background

than Syntag-‐a (Figure 5a, right). Syntag-‐b and Syntag-‐c are both not quite correct, as they

seem to also stain connected processes (Figure 5, center right and center left). They were

all imaged in similar regions of the tectal lobe, so the staining patterns should not have

been affected by site selection (Figure 5b). As the best two examples, Syntag-‐a and Syntag-‐d

were used for a Western blot

(Figure 6). Syntag-‐a (Figure 6

left) shows the best staining, with

bands across the mammalian and

avian samples, though the owl

sample shows little staining,

Figure 7: GABA-‐b, 52 kDa. a) Confocal immunohistochemistry image. b) Larger scale immunohistochemistry image. Green circle indicates the location of the above confocal image. c) Western blot. Abbreviations: “Ms” is mouse, “ZF” is zebra finch, “Can” is canary. Molecular weights to left.

Figure 8: GAD 65/67 a) Confocal immunohistochemistry images. From left to right: GAD-‐a and GAD-‐b. b) Larger scale immunohistochemistry images. Green circle indicates the location of the above confocal image.


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most likely due again

to damaged

homogenate. Also, the

bands are consistent

across the samples,

and are observed at

the expected

molecular weight of

65 kDa. It should also

be noted that the

Syntag-‐a and Syntag –

d antibody binding is

weaker than most,

requiring a concentration of 1:200 for immunohistochemistry and 1:600 for Western blots

(Table 1). This is considerably higher than most antibodies like CaMKIIα, which requires

1:500 for immunohistochemistry and 1:1500 for Western blots (Table 1). GABARα1 and

GluR2/3 require only 1:1000

for Western blots (Table 1).

GABA-‐b receptor

immunohistochemistry

staining (Figure 7a) exhibits a

clear signal with low

background staining, and is

highly characteristic of GABA

receptor staining of inhibitor

synapses (Rodriguez-‐

Contreras and DeBello, SFN

poster 2003.). The Western

blot shows only staining in the

mammalian samples and not

Figure 9: GluR1-‐a. a) Confocal immunohistochemistry image. b) Larger scale immunohistochemistry image. Green circle indicates the location of the confocal image.

Figure 10: GluR2/3-‐a, 108 kDa a) Confocal immunohistochemistry image. b) Larger scale immunohistochemistry image. Green circle indicates the location of the confocal image. c) Western blot. Abbreviations: “Ms” is mouse, “ZF” is zebra finch, “Can” is canary. Molecular weights to left.


12 Imaging in Tyto Alba: A Toolkit for Investigating the Auditory Localization Pathway the avian ones (Figure 7c). Considering that the immunohistochemistry signal in the tectal

slice was so clear, it is probable that the avian homogenates were nonfunctional. The

mammalian bands are at the

correct weight, about 52 kDa.

Two GAD 65/67 antibodies

were tested. GAD-‐a has an

unusually low signal and a high

background staining (Figure 8a

left). GAD-‐b was much more

successful, with clear cell bodies

that don’t overlap with those of

CaMKIIα (Figure 8a right).

However, the GAD-‐b background

staining is still high, and there is

what appears to be an air bubble

obscuring the staining close to the

center of the image. Both antibodies were imaged in similar areas of the slice, though the

slice is inverted on one of the slides (Figure 8b).

GluR1-‐a staining is ubiquitous, but the signal is not strong compared to the

background (Figure 9a). Several imaging locations were selected, but all showed similar

levels of signal (Figure 9b).

GluR2/3 immunohistochemistry staining shows clear structures and low

background staining (Figure 10a). It was stained at 488nm, the opposite channel to SV2-‐a,

568nm (Figure 11a). Qualitatively, there is good overlap between the staining patterns.

One possible explanation is bleed through from SV2 channel, where the signal is strong.

However, there are several bright structures in the SV2 image that are barely, if at all,

visible in the GluR1 image. This argues against bleed through. In combination with the fact

that GluR2 western blot is strong and clean, the simplest interpretation is that the staining

patterns are highly complementary, as would be expected from a pair of pre-‐ and post-‐

synaptic markers. The GluR2/3 Western blot shows clear staining across all channels with

Figure 11: SV2-‐a (95 kDa)a) Confocal immunohistochemistry image. b) Larger scale immunohistochemistry image. Green circle indicates the location of the confocal image. c) Western blot. Abbreviations: “Ms” is mouse, “ZF” is zebra finch, “Can” is canary. Molecular weights to left.


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some smearing in the

zebra finch and canary

lanes (Figure 10c). The

bands are all at the

correct weight of 108

kDa, and the signal is

strong.

The SV2-‐a

immunohistochemistry

image shows clear and

precise staining of

processes (Figure 11a).

The antibody binding is

good, but it has trouble penetrating the slice, which results in staining at upper and lower

confocal slices, but not in the middle. For this reason, the concentration was increased from

1:500 to 1:200 (Table 1). Western blots are difficult with vesicle proteins because they are

difficult to separate from their vesicles, which vary in size. This results in smearing across

the lane, with a slightly stronger band at approximately the correct weight of (95 kDa). For

that reason, it is unusual that the rat and mouse bands turned out so clearly (Figure 11c).

The owl band is faint, most likely due to the issues with the homogenate, and it is smeared

like the zebra finch and canary lanes. However, all five samples have bands at the correct

weight.

Array Tomography:

Due to the expense of the array tomography procedure and the difficulty of

outsourcing the tissue, only the best antibodies were chosen for use in array tomography

on owl tectal lobe tissue. Among those chosen were CaMKIIα-‐a, GAD-‐a, GluR2/3-‐a, SV2-‐a,

Syntag-‐a, and Syntag-‐d (Figure 12). The best staining was unsurprisingly observed with

SV2-‐a (Figure 12d), which shows some processes and low background. Syntag-‐a (Figure 12

e) staining was good, with clear punctae and low background, unlike Syntag-‐d (Figure 12f).

This is consistent with the preliminary immunohistochemistry testing (Figure 5). GluR2/3

Figure 12: Array tomography images. Scale bars are 10 µm. a) CaMKIIα-‐a; b) GAD-‐a; c) GluR2/3-‐a; d) SV2-‐a; e) Syntag-‐a; f) Syntag-‐d.


1414 Imaging in Tyto Alba: A Toolkit for Investigating the Auditory Localization Pathway (Figure 12c) doesn’t show the same processes that were evident from the

immunohistochemistry images (Figure 9a). There appear to be groups of signal that are

hard to decipher from the background. GAD-‐a (Figure 12b) staining looks similar to the

immunohistochemistry testing performed previously (Figure 8a left). Unfortunately, GAD-‐b

was not tested in time for it to be used in the array tomography round. The most

disappointing staining was CaMKIIα-‐a (Figure 12a), which showed none of the

characteristic cell-‐fill staining that was observed in the regular immunohistochemistry

(Figure 3a left). The drastic differences in staining between normal immunohistochemistry

and array tomography are hypothesized to stem from the resin affecting the antibody

binding on-‐off time ratio (conversation with Professor William DeBello). All antibody data

from immunohistochemistry, Western blots, and array tomography were published on a

Filemaker database called ADAPT (Antibody Database for Avian Protein Targets), which

shall hopefully be web accessible in

the near future.

CLARITY:

The CLARITY procedure was

difficult to replicate without the

cooling water circulator and a

stronger power supply (Figure 2).

However, with the supplies used

the rat embryo brain ended up

fairly clear, with little yellowing

(Figure 13). The most problematic

step was the electrophoretic

clearing. The greatest change was

observed during the incubation in clearing solution between days 19 and 36. The changes

due to electrophoretic clearing between days 3 and 19 are almost unobservable. Therefore,

it is possible that without the proper equipment, it is best to leave the brain in a vial of

clearing solution for a longer period of time rather than use substitute equipment. One side

Figure 13: Diagram of CLARITY procedure after perfusion. Images were taken by iPad camera every day, but only a selection are shown for simplification purposes.


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effect of the incubation is that the brain swells, as is evident in the picture for day 36.

However, this decreases during incubation in the 80% glycerol solution.

Discussion:

I performed Western blots on protein homogenates derived from rat, mouse,

chicken, barn owl, zebra finch and canary brain. Antibodies that produced expected

banding patterns across mammals and birds were used as probes for

immunohistochemistry and array tomography in tissue from owl ICX. Successive imaging

of stained, stripped and re-‐stained arrays produced high-‐resolution, seven-‐color image

volumes of ~100,000 cubic microns. Antibodies to synaptic proteins SV2 and GAD, as well

as vGlut1/2, VGAT, Homer 1, tubulin and myelin basic protein (not tested in this paper)

labeled synapses and neuropil similar to that observed in mammalian tissue. Other

antibodies tested yielded equivocal results or failed entirely. However, further study with

more varied antibody concentrations and different perfusion methods can add to this

toolkit, as well as testing in other species of aves. This would result in a more complete list

of antibodies and their ideal binding conditions.

One advantage to array tomography that is currently being explored is the ability to

co-‐localize proteins tagged by vetted antibodies through quantitative image analysis. These

co-‐localizations are being assessed quantitatively using a pixel-‐based analysis called the

Van Steensel method, which relies on a Pearson’s correlation to quantify how much two

antibody signals overlap and was used to develop the FIJI plugin Colocalization Test, which

is being used by a graduate student in the lab. This is done by taking the difference between

two Pearson’s correlation coefficients: one measured between the two signals and the

other measured against a randomized image. This reduces the possibility that any

correlation observed is the result of random chance. The result of the statistics gives a

positive correlation if the two signals are co-‐localized. The next step will be to apply an

object-‐based co-‐localization analysis to ultimately to identify individual synapses based on

the co-‐localization of pre-‐ and postsynaptic proteins with spatial relationships expected

based on EM studies. As previously mentioned, this is only possible if a set of antibodies

like that described in this paper has been vetted for use with this technique.

Some disadvantages to array tomography, besides the difficulty with antibodies,

include that slicing tissue distorts it, and that reconstruction introduces error in


1616 Imaging in Tyto Alba: A Toolkit for Investigating the Auditory Localization Pathway localization. This requires careful application of morphing algorithims to properly align

and register the image stacks to produce a 3-‐D volume. Fortunately, this problem has been

mitigated through the use of statistical colocalization programs, but intact imaging

methods such as CLARITY do not require such reconstruction. (Micheva KD, Busse B,

Weiler NC, O'Rourke N, Smith SJ; 2010).

Although this paper does not cover antibody binding in clarified tissue, CLARITY has

the benefit an isometric procedure, which means it doesn’t change shape during imaging.

This reduces the distortion and the error in image reconstruction. Both procedures allow

the elution and restaining of the tissue with antibodies, which results in a library of

identified proteins that can be co-‐localized without needing tissue from multiple

individuals, as would be necessary for normal immunohistochemistry. However, though

CLARITY has been used successfully in rodents, animals such as barn owls with larger

brains would be difficult to apply the procedure to. The size of the electrophoresis chamber

would need to be expanded to encompass a whole owl brain, though it seems that a single

tectal lobe will fit adequately. Also, there seems to be a relationship between the surface

area to volume ratio of the brain and the ease of removing the lipids, as evidenced by the

relative speed of lipid removal from different layers of the brain (Figure 13). This may

make it more difficult to clarify larger brains without cutting them up. Hopefully these

methods, along with a publically-‐accessible version of ADAPT, can eventually contribute to

the study of barn owl connectomics and learning in the auditory localization pathway.

Acknowledgements:

The authors would like to thank Janet Keiter and Dr. Stephen Noctor of the MIND

institute for their contribution of supplies and tissue for the CLARITY project, as well as

Janet Keiter’s invaluable advice and assistance. Without them this project would not have

been possible.


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Works Cited

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