The Journal of Neuroscience Directional Preferences Differ in - - PDF document

9/29/2011 1 Retinal Ganglion Cells with Distinct Directional Preferences Differ in Molecular Identity, Structure, and Central Projections

Jeremy N. Kay, Irina De la Huerta, In-Jung Kim, Yifeng Zhang, Masahito Yamagata, Monica W. Chu, Markus Meister, and Joshua R. Sanes

The Journal of Neuroscience 31 (21), 7753–7762 Date published: May 25, 2011 Presented by: Michelle Lee

The Journal of Neuroscience

• Published by the Society for

Neuroscience

• First issue: 1981
• Impact Factor: 10.21

The Journal of Neuroscience Volume 31, Issue 21

Primary Authors

• Irina De la Huerta
• Jeremy N. Kay
• Harvard U., Department of MCB
• Research Associate in Sanes’ Lab
• University of San Francisco
• ………….
• In-Jung Kim
• Harvard U., Dept. of MCB
• Postdoctoral Fellow in Sanes’ lab
• Discovered OFF marker for RGC
• Yifeng Zhang
• Harvard U., Dept. of MCB
• Postdoctoral Fellow in Meister lab

Authors

• Monica W. Chu
• Harvard U., Dept. of MCB
• Ph.D. Student at UCSD
• Technician in Sanes’ lab
• Masahito Yamagata
• Harvard U., Dept. of MCB
• Postdoctoral Fellow in Sanes’ lab

Primary Authors

• Joshua R. Sanes
• Harvard U., Dept. of MCB
• Principle Investigator
• Molecules and structures that

regulate synapse formation

• Markus Meister
• Harvard U., Dept. of MCB
• Principle Investigator
• Retina and the Olfactory system

What’s the research is about?

• This is not a theory-/ experiment-based

research

• This is a research of exploration and
• bservation

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What’s the research is about?

• Exploring Retinal Ganglion Cells with

Directional Preferences in:

– Molecular Identity (genes! surface markers!) – Structure (dendritic asymmetry!) – Central Projections (where do axons project to?)

• The 4 directions:

– Dorsal – Ventral – Nasal – Temporal

Background information

• A light-sensitive tissue lining the inner surface of the

eye

Retina Background information Retina

Light!

• 3 layers of neurons
• 2 layers of

synapses

Background information Retina Background information Retinal Ganglion Cells

• Signaling to brain- large diameter axons
• On/ Off center cells
• VERY diverse structures and sizes

So… What have been done so far?

• Since Cajal…

– Dendritic morphology – Cell body and dendritic tree size – Arborized sub-layer in …

• 5 main types; 60-100 subtypes
• Directional Preferences (1964!)

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• oDSGC
• On-Off direction-selective retinal ganglion cells
• Bistratified: bipolar & amacrine cell input
• DIRECTIONAL SELECTIVITY: 4 directions
• It’s ASSUMED that there are 4 subtypes of
• oDSGCs
• They “know” they are different

What we don’t know yet…

• Detailed differences between the 4 subtypes!

Methods

• Transgenic technology
• Electrophysiology
• Histology

Transgenic technology

• Purpose: to mark different lines of ooDSGC

that might have different directional preferences

• Use markers that only express in certain lines
• f RGCs

– BD-RGCs: Fstl4: (ventral) – W9-RGCs: Thy: (nasal) – Cdh6-expressing RGCs: Cdh6: ( dorsal & ventral) – DRD4-RGCs: DRD4:(nasal)

D: Cdh6 V: BD/Cdh6 N: DRD4/ W9 T: ?

Transgenic technology Transgenic technology

X

Courtesy of Dr. Hopkins

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Transgenic technology

Specific marker Gene Fstl4/ Thy/ cdh6/ DRD4… No fluorescence because of the STOP sequence CreER + the specific marker integrated into cells that ALSO have the marker sequence Tamoxifen turns CreER on: CreER cut off STOP: YFP expresses!

Transgenic technology

Green fluorescent label: DRD4 Red Fluorescent label: BD

Electrophysiology

• Measure directional preference
• Isolate retinas out
• Cells recorded with cell-attached patch

microelectrodes

• Light stimuli delivered in different directions
• Spikes recorded with light moving through

different directions

So again… What is this CRAZY thing for?

Green fluorescent label: DRD4 Red Fluorescent label: BD

Electrophysiology

Because we need to know which cell to look at!

BD-RGC BD-RGC W9-RGC Cdh6 D: Cdh6 V: BD/Cdh6 N: DRD4/ W9 T: ?

Histology- In situ Hybridization

• Purpose: Mark out WHERE and WHICH gene is

expressing in a whole tissue

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• Use RNA/ DNA probes
• Probes Hybridize with mRNA (precursor of

Protein)

• Antibodies that carries fluorescent dye reveal

hybridized molecules

Histology- In situ Hybridization How were the genes selected?

• Microarray shows lots of genes to choose

from

• Products are cell surface or secreted proteins:

might be involved in development or function

• f ooDSGCs

– Cdh6, Col25c1, Mmp17, CART

Histology- Lucifer Yellow Staining

• http://webvision.med.utah.edu/movies/electr
• de.mov

Results

• Molecular identity
• Dendritic/ cell body structures
• Axonal projections

D: Cdh6 V: BD/Cdh6 N: DRD4/ W9 T: ?

Molecular identity

D: Cdh6 V: BD/Cdh6 N: DRD4/ W9 T: ?

Molecular identity

In situ marks out the target gene expression: Cdh6 In situ marks out the target gene expression: Cdh6 GFP marks out target RGC line: BD GFP marks out target RGC line: DRD4 2 markers merged! Confirm of gene expression on this cell line  2 markers did not merge  no expression

• n DRD4

D: Cdh6 V: BD/Cdh6 N: DRD4/ W9 T: ?

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Molecular identity

+ + + + +

D: Cdh6 V: BD/Cdh6 N: DRD4/ W9 T: ?

Molecular identity

Key point here: the development is experience-independent!

7 days after birth 14 days after birth

Molecular identity- CART

• CART: specific marker for ooDSGC!

Merged markers!

Molecular identity- CART

• Morphologically matched!

– Bistratified, ON/OFF,

Dendritic/ cell body structures Dendritic/ cell body structures

• W9/DRD4 cells: Morphology does not

correlate with their preferred direction

• D: Cdh6

V: BD/Cdh6 N: DRD4/ W9 T: ?

9/29/2011 7

Dendritic/ cell body structures

• BD cells: Morphology DOES correlate with

their preferred direction

• D: Cdh6

V: BD/Cdh6 N: DRD4/ W9 T: ?

Dendritic/ cell body structures

• Perhaps dendrite directions play a role in

directional selectivity?

• Maybe… BUT…

Axonal projections Axonal projections

• ooDSGC projections

V.S. RGC

– Absent from many retinal targets – Arbors are confined to the upper portion in SC – Nearly NO ipsilateral porjections

Axonal projections

• Differences in targets among ooDSGC subsets

– Only vertical GC (BD and Cdh6) project to the MTN and NOT – DRD4-RGCs do not project to MTN and NOT

D: Cdh6 V: BD/Cdh6 N: DRD4/ W9 T: ?

Last slide, I promise!

D: Cdh6 V: BD/Cdh6 N: DRD4/ W9 T: ?