development of mechanosensation of muscle spindles primary afferents in-vitro william james buchser
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Development of Mechanosensation of Muscle Spindle’s Primary Afferents in-vitro
William James BuchserWilliam James Buchser
Background
Identified as sensory organ in 1888
Molecular Mechanism for mechanical transduction still unknown
Found in all vertebrates (only some fish have them in developed muscles)
Journal of Electron Microscopy 50(1): 65-72 (2001)
Morphology
Scale bar 10μm
Agonist
Antagonist
The Stretch ReflexDorsal
Ventral
DRG
Ia Afferent
Extrafusal Fibers
α-Motor Neurons
Intrafusal Fibers
Spinal Cord
Interneuron
Mechanical Sensory Organs
Two basic models of how muscle spindles work
Stretch of the intrafusal fiber causes the release of neurotransmitter which acts on synapses present on the primary endings.
A mechanically gated channel on the membrane of the primary endings opens in response to stretch.
Sachs F. Seminars in Neuroscience 2, 1990:49-57
Muscle Spindle Development
E13.5E14.5E15.5E16.5E18.5
Chen HH, Hippenmeyer S, Arber S, Frank E. 2003
Afferent / Myotube Interaction
Hippenmeyer S, Shneider NA, Birchmeier C, Burden SJ, Jessell TM, Arber S. 2002
NT3
trkC
NT3
NT3Ia Afferent Neuron
Myotube
NRG-1
erbB2
Muscle Spindle Development
Activated erbB2 results in the expression of EGR3 ERM PEA3 ER81
EGR3 Expression results in the re-expression of NT3 by the intrafusal fiber.
} All transcription factors and regulators. Still unclear what they cause to be changed in muscle spindles
Chen HH, Hippenmeyer S, Arber S, Frank E. 2003
Specific Aims
1. Is NT3 sufficient for a muscle spindle afferent to elaborate its endings?
2. Is NT3 sufficient for the maintenance of these primary endings.
3. Can primary endings function to signal stretch without the intrafusal fiber?
Aim 1
Hypothesis: NT3 Expression by myotubes is sufficient to attract proprioceptive afferents and to initiate their formation into primary endings.
Myotubes express NT3 during early development (up to about E18).
NT3 is necessary for survival of the afferent nerve.
Is NT3 sufficient for a muscle spindle afferent to elaborate its endings?
Is NT3 sufficient for a muscle spindle afferent to elaborate its endings?
Aim 1 – Rationale (NT3/Bax)
Genc B, Ozdinler PH, Mendoza AE, Erzurumlu RS. Dec 2004
Cross section Very few afferents make it to the target muscle.
Although some Ia afferents make it to their target muscle in the Double KO mice, no muscle spindles are formed.
NT3 KO / Bax KO Mice
This paper suggests that - erbB2 isn’t necessary for initial muscle spindle development- erbB2 may be necessary for later maturation and survival
30 µm
Aim 1 - Rationale
E18.5 Spindles
erbB2 is not necessary for primary ending elaboration
loxP-erbB2-loxP
Development. 2003 Jun; 130(11):2291-301
Cre Skeletal-Muscle Actin PromoterX
Is NT3 sufficient for a muscle spindle afferent to elaborate its endings?
Aim 1 – Experimental Design
NT3
NT3
NT3
NT3
NT3NT3
trkC
Is NT3 sufficient for a muscle spindle afferent to elaborate its endings?
Proprioceptive Neuron NT3 Expressing Target Cell
NT3
Neomycin
CM
V
Aim 1 – ControlsIs NT3 sufficient for a muscle spindle afferent to elaborate its endings?
Negative Control : NT3 Blocking AntibodyNegative Control : GFP Transfected
Positive Control : C2C12Negative Control : BDNF/ NGF/ NT4
All controls with dissociated DRG Neurons
GFP only
NT3 NGF
NT3
NT3C2C12
NT3 AbNT3 Expressing Cell
Aim 2 Hypothesis: NT3 expression during muscle
spindle maturation primary ending maintain their connection to the intrafusal fiber.
By about E18, Myotubes stop expressing NT3. Differentiated intrafusal fibers re-expressed NT3 under a new control . . . EGR3.
By using a non-myotube target cell which expresses NT3 over the same length of time, this hypothesis will be examined in-vitro.
Is NT3 sufficient for the maintenance of these primary endings.
Aim 2 - Rationale NT-3 added back in results in maintenance of muscle
spindles, and recovery of function.
erbB2 EGR3 NT3
Chen HH, Tourtellotte WG, Frank E. Neuroscience, May 1, 2002, 22(9):3512-3519
Is NT3 sufficient for the maintenance of these primary endings.
Ia afferents selective:1-2 msec, ~50 µm stretches distal tendon of the RF or soleus muscle with a piezoelectric bimorph
Soleus
Aim 1, 2 – Anticipated Data
This figure was computer generated and is not real.
Target Cells co-cultured with Neurons dissociated from E13.5 dissected DRGs. Experimental and Control wells shown.
Aim 3 Hypothesis: The primary endings of the
afferent nerve (not the intrafusal muscle fiber) possess the mechanosensitive channels which transduce stretch.
Electrophysiology will be performed on the cell culture model developed in Aims1&2.
The target cells will be manipulated so that their membranes stretched while the afferents are recorded.
Can primary endings function to signal stretch without the intrafusal fiber?
Aim 3 – Experimental DesignMechanicalStimulatingPipette
Recording Pipette
Can primary endings function to signal stretch without the intrafusal fiber?
msec
mV
Aim 3 – Anticipated Data
500
0
400
300
200
100
0 2 4 6 8seconds
Affe
ren
tR
ate
of
Dis
cha
rge
(H
z)
Resting state
Dynamic stretchStatic stretch
Targ
et
Ce
llC
om
pre
ssio
n
Can primary endings function to signal stretch without the intrafusal fiber?
Reference?
Summary Muscle Spindles Development is complex
The elaboration of Primary Endings appears to be governed by NT-3 and trkC
Primary Endings on their own may have all the molecules necessary to detect stretch.