Upload
others
View
4
Download
0
Embed Size (px)
Citation preview
Neuron, Volume 100
Supplemental Information
Food Sensation Modulates Locomotion
by Dopamine and Neuropeptide Signaling
in a Distributed Neuronal Network
Alexandra Oranth, Christian Schultheis, Oleg Tolstenkov, Karen Erbguth, JatinNagpal, David Hain, Martin Brauner, Sebastian Wabnig, Wagner Steuer Costa, Rebecca D.McWhirter, Sven Zels, Sierra Palumbos, David M. Miller III, Isabel Beets, and AlexanderGottschalk
1
Food sensation modulates locomotion by dopamine and
neuropeptide signaling in a distributed neuronal network
Alexandra Oranth1,2,8, Christian Schultheis3,8, Oleg Tolstenkov1,2,7, Karen Erbguth1,2, Jatin
Nagpal4, David Hain1,2, Martin Brauner1,2, Sebastian Wabnig1,2, Wagner Steuer Costa1,2,
Rebecca D. McWhirter5, Sven Zels6, Sierra Palumbos5, David M. Miller, III5, Isabel Beets6,
Alexander Gottschalk1,2,9*
1 Buchmann Institute for Molecular Life Sciences, Goethe University, Max-von-Laue-Strasse 15, D-60438
Frankfurt, Germany
2 Institute for Biophysical Chemistry, Goethe University, Max-von-Laue-Strasse 9, D-60438 Frankfurt, Germany
3 Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Strasse 65, D-88400 Biberach, Germany
4 German Resilience Center, University Medical Center, Johannes Gutenberg University, Duesbergweg 6, D-
55128 Mainz, Germany
5 Department of Cell and Developmental Biology and Program in Neuroscience, Vanderbilt University, Nashville
TN, USA
6 Department of Biology, University of Leuven, Naamsestraat 59, B-3000 Leuven, Belgium
7 present address: Sars International Centre for Marine Molecular Biology, University of Bergen, Thormøhlensgt.
55, N-5006 Bergen, Norway
8 These authors contributed equally to this work
9 lead contact
* to whom correspondence should be addressed: [email protected]
SUPPLEMENTAL INFORMATION
2
Supplemental Figures
Figure S1. Related to Figures 1-3, 6: Locomotion parameters of animals tested in this
work, based on trajectories tracked over 120s. A) Analysis of locomotion trajectories.
Individual animals were tracked and the stage positions as well as the relative positions of
the center of mass of the animals were recorded. Trajectories were characterized by total
length of the track within 120s, maximal distance reached to origin during that time, and the
ratio of these figures, providing a measure for “directionality” of the animals, i.e. how
“straight” was locomotion to move away from the origin. These parameters do not specifically
denote locomotion pauses or reversals in locomotion; however, both types of behavior will
affect the outcome of these integrated parameters. B) Group data, statistically analyzed for
total distance crawled (translucent colors) and maximal distance to origin (opaque colors),
respectively, and scaled on the left ordinate; mean velocity is also given (translucent colors
only, right ordinate), of animals of the indicated genotype and condition (as noted in C). C)
3
“Directionality” parameter, calculated as shown in A, for the indicated genotypes. Shown are
means and SEM, with t-tests (n.s. – non-significant difference to wild type before light,
*P<0.05, **P<0.01, ***P<0.001); n=number of animals. D) Locomotion was characterized by
the time spent in forward or backward movement, as well as in pause states, within 3
minutes, for n=13-15 animals of each strain, as indicated. E) Animals of the indicated
genotypes and dark or light conditions were analyzed for the occurrence of pause states
during 3 minutes (left panel; n, number of animals analyzed), and the mean duration of the
pause states (right panel; n, number of pause states analyzed). F) Animals of the indicated
genotypes and dark or light conditions were analyzed for the occurrence of reversals during
3 minutes (upper panel; n, number of animals analyzed), and the mean duration of the
reversals (right panel; n, number of reversals analyzed). Statistics: t-test, paired or unpaired,
according to experimental condition; n.s. non-significant; * p<0.05, ** p<0.01, *** p<0.001;
n=number of animals.
4
Figure S2. Related to Figure 3: Acute AVK ablation affects locomotion. flp-1 and daf-10
alleles vs. FLP-1RNAi, and FLP-1 dense core vesicle (DCV) mobility and trafficking in
AVK. A) Animals co-expressing miniSOG and GFP in AVK were imaged for AVK cell
morphology, without (upper images) or following irradiation (lower images). Arrows, cell
bodies; arrowheads, AVK process, fragmented after miniSOG activation. Scale bar, 20µm.
B) Mean bending angles, compared in AVK::NpHR animals or animals lacking AVK
(following acute ablation by photoactivation of miniSOG with blue light, 0.8mW/mm², 15 min),
without and with yellow light. Controls: blue light irradiated animals not expressing miniSOG.
Significant differences either to the no-light condition, or compared to controls, as indicated:
n.s., non-significant; *** P<0.001; n, number of animals. C) 120s trajectories of wild type,
AVK ablated animals and flp-1(yn4) mutant. D) Bending angle analysis of AVK::NpHR
animals, without and with yellow light, in wild type background, or in animals with cell-specific
FLP-1RNAi in AVK. E) The flp-1 gene resides in an intron of the daf-10 gene. As the flp-1(yn4)
allele affects an intron of daf-10, it may not be appropriate to use it for unambiguous analysis
of flp-1 mutant phenotypes. We thus compared bending angles in wild type, flp-1(yn4)
animals, flp-1(yn4) animals rescued with AVK::FLP-1, as well as flp-1(ok2811), flp-1(ok2781)
and daf-10(tm2878) mutants, which should not mutually affect daf-10 and flp-1, respectively.
5
daf-10(tm2878) had reduced, while flp-1(ok2811) and flp-1(ok2781) had increased bending
angles, just like flp-1(yn4) mutants. F) Time-lapse fluorescence analysis of FLP-1::mCherry
containing DCVs in AVK::NpHR animals with or without photoinhibition (left, w/ ATR, right,
w/o ATR). Upper panels: FLP-1::mCherry fluorescence in AVK cell body and axon. A line
(red) was drawn along the process (from cell body towards nerve ring) and used to obtain
line scan kymographs, as shown in the middle panels. Kymographs were derived over 3 min;
cell body is on the left. Lower images: trajectories of single DCVs during 2 min of each video
were traced and analyzed for antero- or retrograde trafficking, and velocity. G, H) Statistical
analysis of FLP-1::mCherry DCV puncta and trafficking. (G) Particles observed per cell
during 120s, in animals cultivated w/ or w/o ATR (w/ or w/o AVK photoinhibition). n=number
of animals. (H) Trafficking velocities of predominantly antero- or retrogradely trafficking
DCVs, w/ or w/o ATR (w/ or w/o AVK photoinhibition), analyzed per track increment as drawn
in the lower images in A. One increment corresponds to a straight line, before a velocity
change became apparent. n=number of track increments analyzed. Statistics: t-test, paired
or unpaired, according to experimental condition; n.s. non-significant; * p<0.05, ** p<0.01, ***
p<0.001; n= number of animals.
6
Figure S3. Related to Figure 4: NPR-6 is expressed in VC and SMB motoneurons and
FRPR-7 is expressed in neurons regulating swimming behavior. A) Expression pattern
of the npr-6 promoter, using transcriptional pnpr-6::GFP fusion (strain BC12792, a gift from
David Baillie). B) Expression pattern of the frpr-7 promotor fused to mCherry in head
neurons. GFP was additionally expressed using a transcriptional pnpr-6::GFP fusion, as in A.
Upper panel shows confocal stack, maximum-projected, lower panel shows the same stack,
but at reduced brightness and contrast to better highlight highly expressing neurons and to
show their identity. Identified cells expressing either pfrpr-7 (red), or pnpr-6 (green), or both
(yellow), are labeled and indicated by color. C) FRPR-7 is expressed in the interneuron DVC
in the tail. For identification, GFP was additionally expressed in DVA neurons (strain KP7485
(nuIs439(pnlp-12::GFP), a gift from J. Kaplan; Choi et al., 2015). Confocal image stacks were
maximum-projected. Identification of cell bodies was done by close inspection of confocal
7
stacks, using detailed C. elegans anatomy information (e.g. precise and relative localization
of cell bodies, morphology and trajectories of axons, dendrites and cilia) from wormatlas.org
and wormbase.org, as well as from White and colleagues (White et al., 1986). In all panels,
labeled bars indicate scale, anterior is always to the left.
8
Figure S4. Related to Figures 5, 6: Cell-specific expression in SMB neurons can be
mediated by two promoters with intersecting expression: Intersecting promoter
expression patterns of pflp-12::GFP (green) and podr-2(18)::mCherry (red), showing overlap
(yellow) in only few neurons in the head ganglia, i.e. SMB neurons.
9
Figure S5. Related to Figures 3, 4: Swimming locomotion is affected by AVK/FLP-1
signaling and requires the high-potency FLP-1 receptor FRPR-7: We noted that AVK
10
neurons affected a different locomotion behavior, swimming. A) Swimming locomotion of C.
elegans, time series showing one swimming cycle. B) Swimming cycles of
AVK::ChR2(H134R) in lite-1(ce314) background in liquid M9 buffer. Recording during 60s
without and with blue light stimulation (1mW/mm², 470nm), without and with all-trans retinal
(ATR) as indicated. Swimming was unaltered upon photodepolarization in AVK::ChR2
animals. C) Swimming cycles of wild type AVK::NpHR animals during 60s without and with
yellow light (1mW/mm², 580nm). Compared are also animals in which the gap junction
subunit UNC-7 is knocked down specifically in AVK. D) Swimming cycles per 60s, without or
with yellow light, compared in wild type and AVK::NpHR animals, or in AVK ablated animals,
following ICE caspase expression, or in AVK::miniSOG animals following acute ablation by
photoactivation of miniSOG with blue light, 0.8mW/mm², 15 min; controls: blue light irradiated
animals not expressing miniSOG, and AVK::miniSOG without irradiation). Photoinhibition of
AVK resulted in a significantly reduced swimming rate (Video S7). AVK::ICE and
AVK::miniSOG ablated animals, as well as AVK::TeTx animals, or flp-1 mutants, as well as
animals with flp-1 knockdown specifically in AVK, showed a strong reduction of swimming
locomotion that was no further reduced by AVK::NpHR photoinhibition (Video S8). To probe
chemical transmission from AVK, AVK::NpHR animals expressing AVK::TeTx, were included,
as well as flp-1(yn4) mutants. Swimming rates of wild type, flp-1(yn4), flp-1(ok2811), flp-
1(ok2781) and daf-10(tm2878) mutants were compared to characterize different flp-1 and
daf-10 alleles (compare Figure S2E). All showed reduced swimming cycles when compared
to daf-10(yn4); flp-1(yn4), indicating that in yn4 some genetic interaction, at least during
swimming, masks the “pure” flp-1 or daf-10 mutant phenotype. E) Swimming cycles of
AVK::NpHR animals lacking putative FLP-1 receptors or neuropeptides. Compare to Figure
4A. Since the lack of AVK or FLP-1 peptides profoundly affected swimming, we probed
(putative) FLP-1 receptor mutants also in this behavior. npr-6, as well as npr-6; frpr-7 double
mutants showed reduced swimming, while the frpr-7 single mutant did not. Photoinhibition of
FLP-1 release by AVK further reduced swimming in npr-6 mutants, but not in frpr-7 mutants,
indicating that frpr-7 is required in neurons mediating swimming to exert effects of FLP-1
peptides, released by AVK. The frpr-7 promoter expressed in the tail neuron DVC, in several
head neurons (SABV, ADA, ADE) and in the pharynx (I4, M3 and NSM) (Figure S3B, C).
ADE cells are dopaminergic mechanoreceptor neurons that sense the texture of bacterial
food outside the cuticle (Sawin et al., 2000). NSM is neuromodulatory and together with AIM
(expressing NPR-6; Figure S3A, B) affects the onset of swimming after transition from solid
to liquid substrate (Vidal-Gadea et al., 2011). Both cells thus affect swimming, and FRPR-7
(in NSM) and NPR-6 (in AIM and NSM) may modulate swimming through FLP-1 signaling. All
bar graphs show means±SEM. Statistics: t-test, paired or unpaired, according to
experimental condition; n.s. non-significant; ** p<0.01, *** p<0.001; n= number of animals.
11
Figure S6. Related to Figure 4: Photostimulation of the DVC neuron affects reversal
onset and duration, but not swimming: A, B) The DVC neuron was photostimulated using
ChR2, expressed from the pceh-63 promoter (a gift from C. Rankin; Ardiel and Rankin, 2015;
12
Feng et al., 2012) during swimming (A) or during crawling (B), and swimming cycles or
bending angles were calculated before and during blue light stimulation (1mW/mm², 470nm).
Photostimulation of the DVC neuron via ChR2 during swimming had no effect. DVC is
thought to act as a proprioreceptor during backward locomotion, and previous optogenetic
stimulation caused increased reversal frequency (Ardiel and Rankin, 2015). If FLP-1
mediates inhibition through FRPR-7, then AVK inhibition should lead to disinhibition of
FRPR-7 expressing neurons. Conversely, DVC::ChR2 photostimulation increased bending
angles. C) During crawling and illumination, the time delay of onset of reversals was counted,
for the animals in (B). D) The mean duration of reversals was analyzed for the animals in (B),
before and during illumination. The increased bending angles upon photostimulation of
DVC::ChR2 may have been affected by the acute onset of evoked reversals, and also
reversal duration was significantly increased during ongoing DVC photostimulation. E, F)
Frequency (E) and duration (F) of spontaneous reversals was analyzed during 1 min in wild
type and frpr-7(gk463836) mutants. In frpr-7 mutants, spontaneous reversals were reduced,
while reversal duration was unaltered. Our data suggest a role of FRPR-7 in contributing to
the FLP-1 mediated control of locomotion. Statistically significant differences were analyzed
by t-test; *** p<0.001; ** p<0.01; * p<0.05. n indicates number of animals tested.
13
Figure S7. Related to Figure 6: The neuronal network embedding AVK interneurons,
and photostimulation or -inhibition of the proprioceptive DVA neuron reciprocally
affects body bending angles: A) Data depicted in this schematic is derived from the
reconstruction of the C. elegans nervous system by serial electron micrographs (White et al.,
1986). First-layer neurons connected to AVK are symbolized by hexagons (interneurons),
triangles (sensory neurons) or circles (motoneurons). Synapses are symbolized by arrows
(chemical synapses, polarity indicated by the arrow) or lines with circles (electrical
synapses), thickness of the line represents number of synapses (also indicated by a number
14
on each line). Neurons are grouped by function. AVK is in the center, neurons mainly pre-
synaptic to AVK are placed in the upper half, neurons post-synaptic to AVK in the lower half.
Prominent synaptic connections not directly involving AVK are shown in blue color. B) ChR2
(in lite-1(ce314) background) or NpHR were expressed and photostimulated in DVA.
Resulting body bending angles are shown as mean deviation from 180° between three
neighboring points along the body axis. Significant increase (ChR2) or decrease (NpHR) of
the bending angles was observed (t-test; *** P<0.001, ** P<0.01), however, only in animals
raised in the presence of ATR, thus verifying behavioral effects specifically evoked by
photomanipulation of DVA. n = number of animals tested.
15
Figure S8. Related to Figure 7: Selective photostimulation of anterior DA neurons
(ADE, CEP), or posterior PDE; mRNA profiling AVK neurons isolated from L1 stage
with high specificity and gene ontology (GO) terms associated with abundant
transcripts, as well as high overlap of mRNAs identified by bulk RNAseq of isolated
AVK neurons, and a FLP-1 rich cluster identified by single-cell RNAseq. A) Schemes
indicating the location of DA neurons (expressing ChR2) and DVA, and indicating the local
illumination achieved using a LCD projector. B) Graph shows mean locomotion velocity
(±SEM) for 10s without and 20s with blue light (blue bar). C) Heat map showing enrichment
of selected transcripts (unc-9, dop-4, dop-1, unc-7, flp-1, dop-3) in five AVK (right) vs six
reference (left) independent RNA-Seq data sets. D) GO terms analyses of upregulated AVK
transcripts. Fold change compared to expected expression is represented by the color. The
number of genes that fall within each GO biological process is indicated by the size of the
circle. The -log10 of the p-value is plotted along the X-axis. Dopamine receptor signaling
represents one of the most significant GO processes identified. E) Venn diagram
demonstrating overlap between bulk RNA-sequencing data (this work) and single cell RNA-
sequencing (Cao et al., 2017). Transcripts that are enriched in both datasets likely represent
a high-confidence list of AVK-enriched genes (see Table S1C).
16
Table S2: Oligonucleotides list, related to Key Resources Table
Oligonucleotides SOURCE IDENTIFIER
oAO61 (TATGCCGAATGCCGAATGCC) This paper N/A
oAO62 (GCTCATCAAAGTGGTGTGGG) This paper N/A
oAO63 (CTCCTTTACTCATTGTGAAAACTGTAAGTGTTCTTGACGGG)
This paper N/A
oAO64 (CTTACAGTTTTCACAATGAGTAAAGGAGAAGAACTTTTCACTGG)
This paper N/A
oAO65 (TGCTCTGATGCCGCATAGTT) This paper N/A
oAO66 (GAAACGCGCGAGACGAAAGG) This paper N/A
oAO112 (CTTCGCTATTACGCCAGCTG) This paper N/A
oAO113 (CGACGGCCAGTGAATTATC) This paper N/A
oAO116 (GCATGCTCCTGTGTGTAGTGCAACCTG) This paper N/A
oAO117 (GCTAGCTTGGGATAAACGAAGAGCTGA) This paper N/A
oAO120 (GCTTAATTCCTAAAAACCC) This paper N/A
oAO121 (TGGTTGGGAACGATGACGCCTCTAGAATAAAGTGAAG)
This paper N/A
oAO122 (AACTCTTGCAGTTGTGCTAGCCTCTAGAATAAAGTGAAG)
This paper N/A
oAO135 (GCTCTTGTTAGGCAAAAGCC) This paper N/A
oAO136 (CAGATTTCCGTCACGAGCC) This paper N/A
oAO137 (TACTGGAGGCATGAGAAGAC) This paper N/A
oAO138 (CTCATGCCTCCAGTAGCAGGCTTAATGGTCTC)
This paper N/A
oCS47 (GGTGACTT AAAAGAAGC) This paper N/A
oCS121 (GTGTGTAAGCTTAATTCCTAAAAACCCAAAAA)
This paper N/A
oCS122 (GTGTTCTAGAATAAAGTGAAGAAAACCAATG)
This paper N/A
oCS273 (GTGTGCATGCTCACAGTTTGTCGG) This paper N/A
oCS274 (CACACCATGGCTAGCCATCAGCCAAATGTAGG)
This paper N/A
oCS275 (GTGTGCATGCGCCATAACGAGTCGGAAC) This paper N/A
oCS276 (CACACCATGGCTAGCGTTTACTGAAAGTTCAGCTTGTG)
This paper N/A
oCS298 (GTGTGGATCCATGGCCGACAAGGTCCTG) This paper N/A
oCS299 (CACAGAATTCTTAATGTCCTGGGAAGAGG) This paper N/A
oCS307 (GTGTGGATCCATGACTCTGCTCTACCAAG) This paper N/A
oCS308 (CACAGAGCTCATCGATTTATTTTCCGAAACGAAGG)
This paper N/A
oCS318 (GTGTCTAGAATGGTGAGCAAGGGCGAGG) This paper N/A
oCS319 (GTGTCTAGAATGAGTAAAGGAGAAGAAC) This paper N/A
oCS339 (CACACTAGTTTTTCCGAAACGAAGGAAA) This paper N/A
oCS353 (GTGTTCTAGACTAGTCCAGATGGCGTGTGA)
This paper N/A
oCS354 (CACACGTACGCTATCGTCCCTTGACCGTGT)
This paper N/A
oCS355 (GTGTCGTACGCTAGTCCAGATGGCGTGTGA)
This paper N/A
oCS356 (CACATCTAGACTATCGTCCCTTGACCGTGT)
This paper N/A
17
oCS388 (GTGTTCTAGAACTCTGCTCTACCAAGTAGGG)
This paper N/A
oCS389 (CACACGTACGTTATTTTCCGAAACGAAGGA)
This paper N/A
oCS390 (GTGTCGTACGACTCTGCTCTACCAAGTAGGG)
This paper N/A
oCS391 (CACATCTAGATTATTTTCCGAAACGAAGGA)
This paper N/A
oCoS70 (CTTCCGGCTCGTATGTTGTG) This paper N/A
oJN174 (ACGTCATCGTTCCCAACCATGTCGG) This paper N/A
oJN175 (CTAGCACAACTGCAAGAGTTACTGTAGC) This paper N/A
oKE1 (GCGGTACCCGCGGCCGCCACCATGGTCTCAAAGGGTGAAG)
This paper N/A
oKE2 (CGCTCAGTTGGAATTCGCCCTACTAGTC) This paper N/A