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L2

SST-Puncta Density Higher

in L1 than in L2

0.1 0.5 1.0 2.0 3.0 0.00

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0.75

1.00

1.25SST-puncta density increases with

distance slightly more in L1

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Increased Somatostatin-axon density in Layer 1 does not predict input densityAna Verma1,3, Dika Kuljis2,3, Alison Barth2,3

1Amherst College, 2Carnegie Mellon University, 3Center for the Neural Basis of Cognition

Methods

Introduction Methods (cont.)

Conclusions

References & Acknowledgements

Why study Layer 1 and Somatostatin-cells (SST)?• Layer 1 (L1) is a hub of activity. Lots of exchange of information

occurs here1

• SST-expressing inhibitory neurons synapse onto and control the activity of the main excitatory neurons of the cortex (pyramidal neurons)1

• SST neurons have high axon density in L1, suggesting they synapse onto the apical dendritic tuft of pyramidal neurons2

• SST axons in L1 disappear during learning, suggesting their connections are plastic

• Our method of analysis would be a way to quantitatively determine changes during learning

Questions:• Which part of the pyramidal neurons do SST-cells preferentially

innervate? The dendritic shaft or the dendritic spines?• Does the location of the dendrite influence the density of SST

input?

• SST inputs synapse onto both shafts and spines• SST axons are most dense in upper L1• SST input density on pyramidal dendrites is similar across L1• SST input density is lower in L2• SST axon density in upper L1 may be targeting other cell

types• Digital input assignment has a low false positive rate

because SST axon density is uncorrelated with input assignment

1. M. E. (2013). The yin and yang of cortical layer 1. Nat. Neurosci. 16, 114–115. 1Larkum0.1038/nn.3317

2. Wang Y, Toledo-Rodriguez M, Gupta A, Wu C, Silberberg G, et al. (2004) Anatomical, physiological and molecular properties of Martinotti cells in the somatosensory cortex of the juvenile rat. J Physiol561: 65–90. 10.1113/jphysiol.2004.073353

3. Urban-Ciecko J., Barth A.L. Somatostatin-expressing neurons in cortical networks. Nat. Rev. Neurosci. 2016;17:401–409.

I would like to thank Alison Barth and Dika Kuljis for mentoring and teaching me throughout this entire experience, and the Barth Lab overall for being so kind and welcoming.

This project was supported by the NIH-funded uPNC of the Center for the Neural Basis of Cognition.

Ai3 mice contain the gene for enhanced yellow fluorescent protein (EYFP) but cannot express it without Cre recombinase (Cre) SST-IRES-Cre mice

have Cre present in only SST-expressing cellsAi3 x SST-Cre SST-cells will express YFP

We used the image analysis software, Imaris, to reconstructpresynaptic SST neurites and the synapses (puncta) and spinydendrites associated with a pyramidal neuron, from a fluorescence image. We then collected the points of contact between the puncta and SST at different distances away from the dendritic shaft (0.1, 0.5, 1.0, 2.0, and 3.0 μm). The dendrites were then labeled with their location in the cortex (upper-L1, lower-L1, and L2).

Labeling of presynaptic-SST Neurons

Labeling of Synapses

Cre-independent FAPpost and dTom expression Cells infected by the AAV virus will express FAPpost and dTom. FAPpost will localize to synapses and fluoresce.

Distance Threshold Test

Reconstruction of a L2 Pyramidal neuron with spots representing assigned and unassigned puncta

dTomato fill of neurons infected by AAV

Results

Results

SST-input density increases as the distance from the shaft increases

As the distance threshold is increased, the SST-assigned puncta density also increases, suggesting that SST-input density is greatest furthest away from the dendritic shaft (on spines).

Overall SST-input density is higher in layer 1 than it is in layer 2

SST-input density is higher in Layer 1 than in Layer 2 at all distance thresholds.

The change in SST-puncta density with distance is slightly more in L1.

0.1 0.5 1 2 3

0.0

0.5

1.0

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3.0

Distance Threshold (m)

SS

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m)

L2

Upper-L1 is more SST-axon dense than both Lower-L1

and L2

SST-axon density does not predict input density onto the apical tuft of pyramidal neurons

Similar SST-input densities in upper and lower L1 despite SST-axon density differences

Puncta with a point of contact were SST-assigned.

SST-puncta density =

# points of contactdendrite length

How does SST-assigned puncta density change as the distance from the dendritic shaft increases?

0.1 0.5 1 2 3

0.0

0.5

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spiny, pyramidal neuron dendrite

Sample assigned (yellow) and unassigned (mint green) spots

Distance and dendrite proportions not to scale

A

B

Fluorescence Intensities obtained using Fiji ImageJ

Upper L1 Lower L1 L20.0

0.5

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upper lower L2

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@3.0 m DT

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2 3

Each line represents a single dendritic segment

1

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Distance Threshold (m)

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Increase in SST-Puncta Density

with Distance

mean density

de

nd

rite

0.1 um

0.1 um

0.5 um

1.0 um

2.0 um

3.0 um

0.5 um

1.0 um

2.0 um

3.0 um

puncta Presynaptic SST Point of Contact

The overlap between the green and blue fluorescence is a point of contact

spine synapses2 µm 2 µm

shaft synapse

CZN2_3_L1CZN2_5_L1

A B C

CZN2_5_L1 CZN2_5_L1 CZN2_5_L1

CZN2_4_L1

Upper-L1

Lower-L1

L2

40 μm

100 μm

CZN2_4_L1

dTom-labeled neuronsYFP- labeled SST axons

A CZN2_2_L1 B CZN2_2_L1

FAP-post-labeled synapses

CCZN2_2_L1

D

CZN2_1_L1D

15 μm 15 μm

15 μm 3 µm