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Development of Mechanosensation of Muscle Spindles Primary Afferents in-vitro William James Buchser.

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Presentation on theme: "Development of Mechanosensation of Muscle Spindles Primary Afferents in-vitro William James Buchser."— Presentation transcript:

1 Development of Mechanosensation of Muscle Spindles 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 Journal of Electron Microscopy 50(1): (2001) Morphology Scale bar 10μm

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

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 E13.5E14.5E15.5E16.5E18.5 Chen HH, Hippenmeyer S, Arber S, Frank E. 2003

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

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. 2003

9 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?

10 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?

11 Aim 1 – Rationale (NT3/Bax) Genc B, Ozdinler PH, Mendoza AE, Erzurumlu RS. Dec 2004Dec 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

12 This paper suggests that - erbB2 isnt 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 Jun; 130(11): Cre Skeletal-Muscle Actin Promoter X Is NT3 sufficient for a muscle spindle afferent to elaborate its endings?

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

14 Aim 1 – Controls Is NT3 sufficient for a muscle spindle afferent to elaborate its endings? Negative Control : NT3 Blocking Antibody Negative Control : GFP Transfected Positive Control : C2C12 Negative Control : BDNF/ NGF/ NT4 All controls with dissociated DRG Neurons GFP only NT3NGF NT3 C2C12 NT3 Ab NT3 Expressing Cell

15 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.

16 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): Neuroscience, May 1, 2002, 22(9): 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

17 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.

18 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?

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

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


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