Supplemental material for Functional brainstem circuits

Supplemental material for "Functional brainstem circuits for control of nose motion" by Anastasia Kurnikova, Martin Deschênes and David Kleinfeld

Supplemental Figure S1 – Construction of rat atlas for cell count

A: Example section with annotated areas and cells at fully reconstructed tertiary

time points. Areas annotated based on CO stain (solid lines) and estimated

based on known landmarks (dotted lines). All annotated structures are listed in

the table of abbreviations.

B: Example cell assignation to labeled areas for cell count in a 77 hour tertiary

time point. 200 µm thick section taken from the atlas and reconstruction similar to

location of example shown in panel A (but includes a rotation). Colors indicate

different areas for cell assignments.

C: Example alignment of all structures used for alignment in six annotated

volumes. Facial motor nucleus (7N), Trigeminal motor nucleus (5N), facial motor

tract (7n), Lateral reticular formation (Lrt), Inferior olive (IO) and nucleus

ambiguus (Amb) were used to calculate alignment parameters and create an

atlas. Stacks show good alignment with each other.

D: Structure centroids and covariance ellipsoids used in creating the rat

averaged atlas. Top view (top) and sagittal view (bottom) are displayed.

E: Individual structure volumes (grey) and averaged structure volume (black

diamond) for the alignment structures. Average structure volumes accurately

reflect individual traced volume sizes.

F: Example of alignment by individual structure, used to create atlas volumes.

Facial motor nucleus (7N) n = 12 instances for averaging and IRt, n = 2

instances, are shown

Supplemental Figure S2 – Detailed view of premotor areas

A: A top view of a three dimensional diagram shows the locations of selected

slices through the reconstruction for the retrofacial area for panels B and C.

B: Images of 200 µm thick sagittal slices through reconstructions of premotor

labeling in the retrofacial area in six rats at secondary-labeled time points.

Reconstructions of labeled cells (left) and 10 % maximum density contours (right)

are shown. Colors indicate different premotor labeling time points.

C: Images of 200 µm thick coronal slices through reconstructions of premotor



are shown.

D: A top view of a three dimensional diagram shows the locations of selected

slices through the reconstruction for the nIRt area for panels E and F.

E: Images of 200 µm thick sagittal slices through reconstructions of premotor

labeling in the nIRt area in six rats at secondary-labeled time points.


are shown.

F: Images of 200 µm thick coronal slices through reconstructions of premotor



are shown.

G: Three dimensional view of results of dbscan clustering of rabies virus labeled

cells at premotor time points. Reconstructed cells are shown as small spheres,

with core clustered points shown with a larger radius. Two clusters identified at

parameters 50 minPts and 200 µm are shown in magenta (nRF) and green

(nIRt). Non-clustered noise cells shown in black.

H: Silhouette plot of the dbscan clustering shown in panel G, Clusters have few

values below 0, indicating a reasonable fit to the data.

I: Bayesian information criterion (red) and Aikake information criterion (blue) for

model selection for a Gaussian mixture model to the rabies virus data

(Figure 1H). Both metrics have a minimum value at two components, suggesting

a two component model is the best fit to the data.

Supplemental Figure S3 – Cell counts across the brain

A: Cell count of all labeled cell bodies in singular structures in the medulla, by

time point. Number of labeled cells increases dramatically at the tertiary time

points. Colors correspond to individual labeled brains at different time points.

Data obtained from all datasets aligned to the atlas as defined in Supplemental

Figure S1. All abbreviations are summarized in the table of abbreviations.

Supplemental Figure S1 provides an example of cells in a single section

assigned to atlas areas.

B: Cell counts in ipsilateral (top) and contralateral (bottom) in the hindbrain, by





C: Cell counts in ipsilateral (top) and contralateral (bottom) in the midbrain, by





Supplemental Figure S4 – Example labeled regions in the midbrain

A: Example labeling in the deep layers of superior colliculus at a secondary time

point (64 hours). Structures visible in a CO stain, rabies labeled cells revealed in

dark product. The section displayed is 1.5 mm lateral to midline, contralateral to

the injection.

B: Reconstructions of superior colliculus labeling at secondary time points in

sagittal and coronal views. Colors correspond to individual labeled brains at

different time points

C: Example labeling in the deep layers of superior colliculus at a tertiary time



the injection.

D: Reconstructions of superior colliculus labeling at tertiary time points in sagittal

and coronal views. Colors correspond to individual labeled brains at different time

points.

E: Example labeling in the dorsal part of the red nucleus at a secondary time



the injection.

F: Reconstructions of red nucleus labeling at secondary time points in sagittal

and coronal views. Colors correspond to individual labeled brains at different time

points.

G: Example labeling in the dorsal part of the red nucleus at a tertiary time point

(77 hours). Structures visible in a CO stain, rabies labeled cells revealed in dark

product. The section displayed is 1.1 mm lateral to midline, contralateral to the

injection.

H: Reconstructions of red nucleus labeling at tertiary time points in sagittal and

coronal views. Colors correspond to individual labeled brains at different time

points.

I: Example labeling in midbrain structures and at secondary time points. Left:

labeling in the ipsilateral Kolliker-Fuse is sparse at 64 hours. The section

displayed is 2.8 mm lateral to midline, ipsilateral to the injection. Center: Labeling

in the IMLF. Structures outlined from CO stain, rabies labeled cells revealed in

dark product. Right: The section displayed is 0.4 mm lateral to midline, ipsilateral

to the injection.

J: Example labeling in midbrain structures and at tertiary time points. Left:

labeling in the ipsilateral Kolliker-Fuse is dense at 77 hours. The section

displayed is 2.8 mm lateral to midline, ipsilateral to the injection. Center: Labeling

in the IMLF. Structures outlined from CO stain, rabies labeled cells revealed in

dark product. Right: The section displayed is 0.3 mm lateral to midline, ipsilateral

to the injection.

Supplemental Figure S5 – Example labeled regions in the midbrain and forebrain

A: Example of cortical labeling at a tertiary time point (77 hours). An enlargement

of the labeled areas shows that labeled cells have the morphology of L5

pyramidal neurons. The section displayed is 1.8 mm lateral to

midline, contralateral to the injection.

B: Reconstructions of cortical labeling reveal dense projections from motor and

sensorimotor areas at tertiary time points (77 hours). Colors correspond to two

individual labeled brains.

C: Example labeling in forebrain structures and at tertiary time points. Left:

labeling in the ipsilateral Lateral Hypothalamus. The section displayed is 1.7 mm

lateral to midline, ipsilateral to the injection. Center: Labeling in the olfactory

tubercle and ventral pallidum. The section displayed is 1.5 mm lateral to midline,

ipsilateral to the injection. Right: Single labeled cell in the nucleus of the lateral

olfactory tract. Structures outlined from CO stain, rabies labeled cells revealed in

dark product. The section displayed is 2.6 mm lateral to midline, ipsilateral to the

injection.

D: Cell counts in ipsilateral (top) and contralateral (bottom) in the forebrain.

Labeled cells are present only at the tertiary time points. Colors correspond to

individual labeled brains at different time points. Data obtained from all datasets

aligned to the atlas as defined in Supplemental Figure S1. All abbreviations are

summarized in the table of abbreviations.

D

DV

RCML

7N 7nIRt

n = 2

n = 2n = 12

A-P position (mm)

D-V

position (mm

)M

-L position (mm

)

1.0

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ume

(mm

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Supplemental Figure S1

LRt7N

7n

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LRt7N

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LRt7N

OverlapN = 4

7N7N

7N7N OverlapN = 4

LRt7NIO

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Amb

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Amb

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Amb

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A B

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Secondary timepoints:

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64 hrs

51 hrs

61 hrs

67 hrs

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7N

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1 mm

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(sec

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67 hrs

77 hrs77 hrs

500 μm500 μm

RN

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500 μm

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500 μm 500 μm

500 μm

DR

IMLF

Dk

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DR

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500 μm

Superior colliculus (contra)

Red nucleus (contra)

Kölliker-Fuse (ipsi) IMLF (ipsi)

CoronalSagittal

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Olfactory TubercleLateral Hypothalamus N. Lat. Olfactory Tract

Cortex


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Supplemental material for Functional brainstem circuits