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
  1. Overestimation of starting population
  2. 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\}
r​i​​=Median{√​(x​ij​2​​+y​ij​2​​+z​ij​2​​)​​​}

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 
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