This document describes a study aiming to identify genetic markers for proprioceptive sensory neurons (PSNs) through single cell RNA sequencing of mouse dorsal root ganglia cells. Specifically, the study aims to find markers for Ia PSNs, which relay sensory feedback from muscle spindles and Golgi tendon organs to the spinal cord. The researchers use mouse genetics and viral tracing to label and isolate PSN subtypes, including Ia PSNs, for single cell sequencing. Initial results identify discrete clusters of cells expressing known PSN and cutaneous neuron markers. Differential expression analysis between clusters may reveal new subtype-specific genetic markers.
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Los días 11 y 12 de diciembre de 2014, la Fundación Ramón Areces celebró el Simposio Internacional 'Neuropatías periféricas hereditarias. Desde la biología a la terapéutica' en colaboración con CIBERER-ISCIII y el Centro de Investigación Príncipe Felipe. El tipo más común de estas patologías es la enfermedad de Charcot-Marie-Tooth, un trastorno neuromuscular hereditario con una prevalencia estimada de 17-40 afectados por 100.000 habitantes. Durante estos dos días, investigadores mostraron sus avances en la mejora del diagnóstico y el tratamiento y, por ende, de la aproximación clínica y la calidad de vida de las personas afectadas por estas patologías.
A Neurovascular Niche for Neurogenesis after Strokejohnohab
Stroke causes cell death but also birth and migration of new neurons within sites of ischemic damage. The cellular environment that induces neuronal regeneration and migration after stroke has not been defined. We have used a model of long-distance migration of newly born neurons from the subventricular zone to cortex after stroke to define the cellular cues that induce neuronal regeneration after CNS injury. Mitotic, genetic, and viral labeling and chemokine/growth factor gain- and loss-of-function studies show that stroke induces neurogenesis from a GFAP-expressing progenitor cell in the subventricular zone and migration of newly born neurons into a unique
neurovascular niche in peri-infarct cortex. Within this neurovascular niche, newly born, immature neurons closely associate with the remodeling vasculature. Neurogenesis and angiogenesis are causally linked through vascular production of stromal-derived factor 1 (SDF1) and angiopoietin 1 (Ang1). Furthermore, SDF1 and Ang1 promote post-stroke neuroblast migration and behavioral recovery. These experiments define a novel brain environment for neuronal regeneration after stroke and identify molecular mechanisms that are shared between angiogenesis and neurogenesis during functional recovery from brain injury.
A characteristic of the developing mammalian visual system is a brief interval of plasticity, termed the “critical period,” when the circuitry of
primary visual cortex is most sensitive to perturbation of visual experience. Depriving one eye of vision (monocular deprivation [MD]) during
the critical period alters ocular dominance (OD) by shifting the responsiveness of neurons in visual cortex to favor the nondeprived eye. A
disinhibitory microcircuit involving parvalbumin-expressing (PV) interneurons initiates this OD plasticity. The gene encoding the neuronal
nogo-66-receptor1(ngr1/rtn4r) is required to close the critical period.Herewecombinedmousegenetics, electrophysiology,andcircuitmapping
with laser-scanning photostimulation to investigate whether disinhibition is confined to the critical period by ngr1.We demonstrate that ngr1
mutant mice retain plasticity characteristic of the critical period as adults, and that ngr1 operates within PV interneurons to restrict the loss of
intracortical excitatory synaptic input following MD in adult mice, and this disinhibition induces a “lower PV network configuration” in both
critical-period wild-type miceandadult ngr1/mice.Wepropose that ngr1 limits disinhibition to close the critical period forODplasticityand
that a decrease in PV expression levels reports the diminished recent cumulative activity of these interneurons.
This presentation focuses briefly on neural stem cells, the road map of neurogenesis, the markers as well the controversy involved in neurogenesis - that arose in the year 2018.
Los días 11 y 12 de diciembre de 2014, la Fundación Ramón Areces celebró el Simposio Internacional 'Neuropatías periféricas hereditarias. Desde la biología a la terapéutica' en colaboración con CIBERER-ISCIII y el Centro de Investigación Príncipe Felipe. El tipo más común de estas patologías es la enfermedad de Charcot-Marie-Tooth, un trastorno neuromuscular hereditario con una prevalencia estimada de 17-40 afectados por 100.000 habitantes. Durante estos dos días, investigadores mostraron sus avances en la mejora del diagnóstico y el tratamiento y, por ende, de la aproximación clínica y la calidad de vida de las personas afectadas por estas patologías.
A Neurovascular Niche for Neurogenesis after Strokejohnohab
Stroke causes cell death but also birth and migration of new neurons within sites of ischemic damage. The cellular environment that induces neuronal regeneration and migration after stroke has not been defined. We have used a model of long-distance migration of newly born neurons from the subventricular zone to cortex after stroke to define the cellular cues that induce neuronal regeneration after CNS injury. Mitotic, genetic, and viral labeling and chemokine/growth factor gain- and loss-of-function studies show that stroke induces neurogenesis from a GFAP-expressing progenitor cell in the subventricular zone and migration of newly born neurons into a unique
neurovascular niche in peri-infarct cortex. Within this neurovascular niche, newly born, immature neurons closely associate with the remodeling vasculature. Neurogenesis and angiogenesis are causally linked through vascular production of stromal-derived factor 1 (SDF1) and angiopoietin 1 (Ang1). Furthermore, SDF1 and Ang1 promote post-stroke neuroblast migration and behavioral recovery. These experiments define a novel brain environment for neuronal regeneration after stroke and identify molecular mechanisms that are shared between angiogenesis and neurogenesis during functional recovery from brain injury.
A characteristic of the developing mammalian visual system is a brief interval of plasticity, termed the “critical period,” when the circuitry of
primary visual cortex is most sensitive to perturbation of visual experience. Depriving one eye of vision (monocular deprivation [MD]) during
the critical period alters ocular dominance (OD) by shifting the responsiveness of neurons in visual cortex to favor the nondeprived eye. A
disinhibitory microcircuit involving parvalbumin-expressing (PV) interneurons initiates this OD plasticity. The gene encoding the neuronal
nogo-66-receptor1(ngr1/rtn4r) is required to close the critical period.Herewecombinedmousegenetics, electrophysiology,andcircuitmapping
with laser-scanning photostimulation to investigate whether disinhibition is confined to the critical period by ngr1.We demonstrate that ngr1
mutant mice retain plasticity characteristic of the critical period as adults, and that ngr1 operates within PV interneurons to restrict the loss of
intracortical excitatory synaptic input following MD in adult mice, and this disinhibition induces a “lower PV network configuration” in both
critical-period wild-type miceandadult ngr1/mice.Wepropose that ngr1 limits disinhibition to close the critical period forODplasticityand
that a decrease in PV expression levels reports the diminished recent cumulative activity of these interneurons.
This presentation focuses briefly on neural stem cells, the road map of neurogenesis, the markers as well the controversy involved in neurogenesis - that arose in the year 2018.
The Brain as a Whole: Executive Neurons and Sustaining Homeostatic GliaInsideScientific
Carl Petersen and Alexei Verkhratsky share their research on homeostatic neuroglia and imaging of neuronal network function. This webinar is brought to you by APS’ new journal, Function, and part of their Physiology in Focus learning series.
During this exclusive live webinar, Carl Petersen and Alexei Verkhratsky discuss astrocyte-mediated homeostatic control of the central nervous system, and how optical and 2-photon microscopy can be used for functional neuroimaging.
Imaging Neuronal Function
Carl Petersen, PhD
Highly dynamic and spatially distributed neuronal circuits in the brain control mammalian behavior. Through technological advances, optical measurements of neuronal function can now be carried out in behaving mice at multiple scales. Wide-field imaging allows the dynamic interactions between different brain areas to be studied as sensory information is processed and transformed into behavioral output. Within a brain region, two-photon microscopy can be used to image the neuronal network activity with cellular resolution allowing different types of projection neurons to be distinguished. Together optical methods provide versatile tools for causal mechanistic understanding of neuronal network function in mice.
Astrocytes: indispensable neuronal supporters in sickness and in health
Alexei Verkhratsky, MD, PhD, DSc
The nervous system is composed of two arms: the executive neurons and the homeostatic neuroglia. The neurons require energy, support, and protection, all of which is provided by the neuroglia. Astrocytes, the principal homeostatic cells of the brain and spinal cord, are tightly integrated into the neural networks and act within the context of the neural tissue. As astrocytes control the homeostasis of the central nervous system at all levels of organization, from the molecular to the whole organ level, we can begin to define and understand brain vulnerabilities to aging and diseases.
Activity-dependent transcriptional dynamics in mouse primary cortical and hum...Darya Vanichkina
Poster I presented at Lorne Genome 2012. Subsequently formed part of the paper
Barry G, Briggs JA, Vanichkina DP, Poth EM, Beveridge NJ, Ratnu VS, Nayler SP, Nones K, Hu J, Bredy TW, Nakagawa S, Rigo F, Taft RJ, Cairns MJ, Blackshaw S, Wolvetang EJ, Mattick JS (2013). The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing. Molecular psychiatry doi: 10.1038/mp.2013.45
Duplicate of http://figshare.com/articles/Activity_dependent_transcriptional_dynamics_in_mouse_primary_cortical_and_human_iPS_derived_neurons/978468
Research projects on protein expression and immunostaining. An unknown protein may be localized by double labeling the tissue with a marker whose expression is known. Graduate student research project for a seminar on optical methods in molecular biology. Students were introduced to the imaging core which is equipped with confocal microscopes. The instructors aided with utilization of the computer software, switching between different objectives, and the choice of materials.
SfN 2015 - Anil Sharma - Genetic tools to study sensory motor circuits FINAL
1. Genetic tools to study sensory motor circuits
Anil Sharma1
, Haohao Wu1
, Carmelo Bellardita2
, Yang Xuan1
, Konstantinos Meletis1
, Ole Kiehn2
, Francois Lallemend1
1
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden ; 2
Mammalian locomotor laboratory, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
Cluster specific genes
695 single cell PVCre
x R26tdTomato
cells
t-SNE and K means clustering
References
1. de Nooij, J.C., et al. 2013. Neuron 77, 1055–1068
2. Hippenmeyer, S., et al. 2005. PLoS Biol 3
3. Lee, J., et al. 2012. PLoS One 7, e45551
4. Stepien, A.E., et al. 2010. Neuron 68, 456-472
5. Takatoh, J., et al. 2013. Neuron 77, 346–360
6. Usoskin, D. 2015. Nat. Neurosci. 18, 145-153
7. Wall, N.R., et al. 2010. PNAS 107, 21848-21853
8. Wickersham, I.R., et al. 2007. Neuron 53, 639-647
9. Windhorst, U., et al. 2007. Brain Res. Bull. 73, 155-202
10. Zampieri, N., et al. 2014. Neuron 81, 766–778
Experimental flow chart
Background
Proprioceptive sensory neurons (PSNs) are essential
relays of the sensory feedback necessary for fine motor
and postural control.
PSNs cell bodies reside in the dorsal root ganglia
(DRGs), project peripherally to muscle spindles and
Golgi tendon organs, and centrally to interneurons and
motor neurons.
Three types of PSNs (Ia, Ib, and II) are characterised
by their sensory fibre types, peripheral and central
innervation patterns, and electrophysiology8
.
Developmental, physiological, and regenerative
studies involving PSNs are limited by a lack of
definitive markers.
Our objective
We are using single cell RNA-seq combined with
mouse genetics and virus mediated tracing to elucidate
markers for the PSNs, with a particular focus on the Ia
PSNs.
Cell labelling
PVcre
x R26tdTomato
:
• Parvalbumin (PV) is expressed in a subset of DRG
cells, mostly PSNs (PV+
/Runx3+
)1
.
• Crossing PVCre
and R26tdTomato
strains indelibly labels
PSNs2
.
ChatCre
x RGθT + EGFP-rabies:
• Modified rabies is unable to infect without expression
of TVA receptor, and unable to cross synapses without
G protein3,6,7
.
Tracing adult Ia PSNs
A) PSNs are indelibly labelled red in PVCre
x R26tdTomato
mice. B) Modified rabies virus injected into the ventral
horn of ChATCre
x RGΦT mouse spinal cord causes
secondary infection and specific labelling of Ia PSNs in
the DRG.
Tracing of Ia PSNs from spinal motor neurons by
monosynaptic rabies infection. Tissue was cleared
by CLARITY and then visualised using lightsheet
microscopy.
Magnified view of the highlighted DRG and dorsal root
(A), and individual Ia PSN (B) from the above images.
Single cell PVCre
x R26tdTomato
FACS
A) 768 adult cells were FACS sorted and sequenced
by Smart-seq2. Q&A reduced this number to 695
cells which formed discrete clusters by both t-SNE
and K means. B) Canonical markers identify PSN and
cutaneous mechanoreceptors.
Differential gene expression between the cells clusters
discovered by K means clustering. Top 50 genes per
cluster (cluster 9 only had 10 significant hits), by SCDE.
Funding
Swedish Research Council
Ragnar Söderberg
Foundation
Karolinska Institutet
Knut and Alice Wallenberg
Foundation
Contact
Anil Sharma
Karolinska Institutet
Department of Neuroscience
Retzius väg 8, Stockholm 171 77
E-mail: anil.sharma@ki.se
Telephone: 08-524 863 74
DRG
Clarity Optimised Lightsheet Microscopy (COLM)
No signal High signal
Ventral Dorso-lateral Dorsal
Cluster number
Topdifferentiallyexpressedgenes
1 2 3 4 5 6 7 8 9 10
-3 -2 -1 0 1 2 3
Gene expression
Z-score
2 4 6 8
Cluster
1 2 3 4 5 6
Etv1
Whirlin
Runx3
Parvalbumin
TrkC
TrkB
TrkA
7 8 9 10
Log2
counts per
million
A t-SNE with K means overlay B Grouping
of clusters
PSNs Cut.?
2
10
9
8
7
64
3
5
1
Non PSNs
DRG
A
Ia fibers
Axon bifurcation
B
Muscle
MS
GTO
DRG
Motor
neurons
Interneurons
Ia PSN
Ib PSN
II PSN
II PSN
Spinal cord
PVCre
x R26tdTomato
mouseA B
Interneurons
Secondary
rabies
infection
EnvA-ΔG-EGFP-Rabies
DRG
Ib PSN
Ia PSN
ChAT+ Motor
neurons
Spinal cord
ChATCre
x RGΦT mouse
+ EGFP-rabies tracing
PCA1
PCA2
PCA3
Ia PSNs
5’UTR GENE IRES-venus-pA TTX IRES-cherry-pA
5’UTR GENE IRES-Cre 3’ UTR
Ia/Ib/II PSNs Ia PSNs
Dissociate adult (3 month) DRG to single cell suspension
Sort by fluorescence
Single cell
RNA-seq
Bioinformatics to discover cell type specific markers
Validation of markers and construction of genetically modified mice