1. Development of Mechanosensation of Muscle Spindle’s Primary Afferents in-vitro
William James Buchser

2. 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)

3. Morphology Journal of Electron Microscopy 50(1): 65-72 (2001)
Scale bar 10μm

4. The Stretch Reflex Agonist Antagonist Spinal Cord DRG Dorsal
Ia Afferent
Interneuron
Intrafusal Fibers
Ventral
α-Motor Neurons
Agonist
Extrafusal Fibers
Antagonist

5. 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

6. Muscle Spindle Development
Chen HH, Hippenmeyer S, Arber S, Frank E

7. Afferent / Myotube Interaction
trkC
NT3
NT3
Ia Afferent Neuron
NT3
NRG-1
erbB2
Myotube
Hippenmeyer S, Shneider NA, Birchmeier C, Burden SJ, Jessell TM, Arber S. 2002

8. 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

9. Specific Aims
Is NT3 sufficient for a muscle spindle afferent to elaborate its endings?
Is NT3 sufficient for the maintenance of these primary endings.
Can primary endings function to signal stretch without the intrafusal fiber?

10. Is NT3 sufficient for a muscle spindle afferent to elaborate its endings?
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.

11. Aim 1 – Rationale (NT3/Bax)
Is NT3 sufficient for a muscle spindle afferent to elaborate its endings?
Aim 1 – Rationale (NT3/Bax)
NT3 KO / Bax KO Mice
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.
Cross section
Genc B, Ozdinler PH, Mendoza AE, Erzurumlu RS. Dec 2004

12. erbB2 is not necessary for primary ending elaboration
Is NT3 sufficient for a muscle spindle afferent to elaborate its endings?
Aim 1 - Rationale X
loxP-erbB2-loxP
Cre  Skeletal-Muscle Actin Promoter
E18.5 Spindles
30 µm
erbB2 is not necessary for primary ending elaboration
This paper suggests that
- erbB2 isn’t necessary for initial muscle spindle development
- erbB2 may be necessary for later maturation and survival
Development Jun; 130(11):

13. Aim 1 – Experimental Design
Is NT3 sufficient for a muscle spindle afferent to elaborate its endings?
Aim 1 – Experimental Design N T 3 e o m y c i n C M V
NT3
NT3
NT3
NT3
NT3
trkC
NT3
Proprioceptive Neuron
NT3 Expressing Target Cell

14. Aim 1 – Controls All controls with dissociated DRG Neurons
Is NT3 sufficient for a muscle spindle afferent to elaborate its endings?
Aim 1 – Controls
GFP only
NT3
NT3 Expressing Cell
NT3 Ab
Negative Control : GFP Transfected
Negative Control : NT3 Blocking Antibody
NT3
NGF
C2C12
NT3
Negative Control : BDNF/ NGF/ NT4
Positive Control : C2C12
All controls with dissociated DRG Neurons

15. Is NT3 sufficient for the maintenance of these primary endings.
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.

16. Aim 2 - Rationale erbB2  EGR3  NT3
Is NT3 sufficient for the maintenance of these primary endings.
Aim 2 - Rationale
erbB2  EGR3  NT3
NT-3 added back in results in maintenance of muscle spindles, and recovery of function.
Soleus
Ia afferents selective:
1-2 msec, ~50 µm stretches  distal tendon of the RF or soleus muscle with a piezoelectric bimorph
Chen HH, Tourtellotte WG, Frank E. Neuroscience, May 1, 2002, 22(9):

17. Aim 1, 2 – Anticipated Data
Target Cells co-cultured with Neurons dissociated from E13.5 dissected DRGs. Experimental and Control wells shown.
This figure was computer generated and is not real.

18. Can primary endings function to signal stretch without the intrafusal fiber?
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.

19. Aim 3 – Experimental Design
Can primary endings function to signal stretch without the intrafusal fiber?
Aim 3 – Experimental Design
Mechanical
Stimulating
Pipette
Recording
Pipette mV
msec

20. Aim 3 – Anticipated Data Reference?
Can primary endings function to signal stretch without the intrafusal fiber?
Aim 3 – Anticipated Data
Reference?

21. 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.