Whole brain mapping of inputs
to inhibitory interneurons
in prefrontal cortex
Yang Xuan
Carlén Lab
Networks for Networks
28th Nov 2014
yang.xuan@ki.se
Topics
Preliminary findings SOM and PV.
- Regions of interest:
- Basal forebrain
- Thalamus.
- Septum.
- Amygdala.
- Hippocampus.
- Background and aims.
- Dual versus single rabies vector systems.
- Whole-brain reconstruction.
- Local versus long-range inputs
Background and Aims
- Retrograde tracing
- SAD B19G2
- Five genes
Rabies virus
Rabies-virus-glycoprotein (RVG) necessary and sufficient for transsynaptic spread.
- Avian EnvA-TVA
- Delivering TVA and RVG into starting cells
Cell-type-specific delivery
Dual vector system (traditional)
-
Starter cells definition
- True positive:
- + TVA-mCherry
- + Rabies-EGFP.
- + RVG
- False positives.
- + TVA-mCherry
- + Rabies-EGFP.
- - RVG
- True positive:
- Overestimation of starting population
- Not possible to dissect local input (e.g. between layers)
Leads to the following problems:
Pollak et al. 2014, Wall et al. 2013, Uchida et al. 2012
Dual vector system
- Tag RVG with fluorescent protein (e.g. EF1a-DIO-RVG-Venus).
Problem: RVG is sensitive to tag, miss-folding (pers. comm. Ed Callaway).
- All in one vector (e.g. EF1a-DIO-TVA-mCherry-t2A-RVG).
Problem: To large to fit in standard vectors like AAV and lentivirus.
- One vector without tag (e.g. EF1a-DIO-TVA-t2A-RVG).
Problem:
- Single vector rabies system. (e.g. EF1a-DIO-TVA-V5-t2A-RVG)
SOLUTION
Single vector system
V5 Paramyxovirus SV5
a small epitope present on the P and V proteins of SV5.
- 14 amino acids 42 bp.
- Works good with AAV vectors.
- Fusion protein with TVA on C-terminal membrane bound.
Single vector system
I've also extended the single vector rabies virus system with additional rabies varieties and flavors.
- Tracing: 3'-N-P-M-EGFP-L-5'.
- Gain of function: 3'-N-P-M-ChR2-mCherry-L-5'.
- Loss of function: 3'-N-P-M-Jaws-EGFP-L-5'.
- Input sensitivity: 3'-N-P-M-SSFO-EYFP-L-5'.
Disadvantage: toxicity of the rabies virus itself. Mechanism unknown.
Puts a limited time frame from injection to end-point of experiment.
Whole-Brain Reconstruction
Pollak et al. 2014
Reconstructing brain from sectioned tissue
WholeBrain Web interface
similar to...
works with...
Local versus long-range input
- How much local versus long-range input.
- median spherical radial distance
r_i = Median \left\{\sqrt{(x_{ij}^2+y_{ij}^2+z_{ij}^2)}\right\}
ri=Median{(xij2+yij2+zij2)}
Local versus long-range input
Local versus long-range input
- No marked difference between and .
- Statistical power issue.
n = 3 \times 2 \text{ animals}
n=3×2 animals
This severely limits any quantitative comparison across all unique brain regions that cells were found in.
Until sample size is increased Qualitative findings
SOM
PV
Cortical input
PV-cre mouse ID# 195
SOM-cre mouse ID# 80
~80% of all inputs are cortical input
Hippocampus
- ChAT
- Rabies-EGFP
Basal forebrain
- FOXP2
- PV
SOM-cre
PV-cre
Medial Septum
SOM-cre
PV-cre
Rabies-EGFP
CAhT
Amygdala
Amygdala Immunohistochemistry
Rabies-EGFP
CAhT
FoxP2
SOM-cre
PV-cre
Ventral group of the dorsal thalamus
Summary
- Bullet One
- Bullet Two
- Bullet Three
On-going work
- Tracing project:
- Other interneurons (VIP)
- Principal cells (Coinjection with CaMKIIa-CRE)
- New vector designs: CaMKIIa-TVA-V5-t2A-RG
Acknowledgement
- Bullet One
- Bullet Two
- Bullet Three
Thank you!
Advantages and Disadvantages
- TVA-V5-RG Vs TVA-mCherry+RG(TVA-mCherry/non-RG infected cells)
- Local input
- •Disadvantages:
- Staining
- Alternative:
- AAV8/synP-FLEX-sTpEpB
- (Keigo Kohara et al., Nature Neuroscience 2014)
-
Reduction of total genome length by:
- using smaller promotor(human synapsin-1 promotor)
- using smaller polyadenylation signal(bovine growth hormone polyadenylation site)
-
removing all extraneous sequences and restriction sites
- 7 days+ 7 days
Whole brain mapping of inputs to inhibitory interneurons in prefrontal cortex Yang Xuan Carlén Lab Networks for Networks 28th Nov 2014 yang.xuan@ki.se
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