Presentation on theme: "Development of Mechanosensation of Muscle Spindle’s Primary Afferents in-vitro William James Buchser."— Presentation transcript:
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 unknownFound 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 AfferentInterneuronIntrafusal FibersVentralα-Motor NeuronsAgonistExtrafusal FibersAntagonist
5 Mechanical Sensory Organs Two basic models of how muscle spindles workStretch 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
8 Muscle Spindle Development Activated erbB2 results in the expression ofEGR3ERMPEA3ER81EGR3 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 spindlesChen HH, Hippenmeyer S, Arber S, Frank E
9 Specific AimsIs 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 1Hypothesis: 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 MiceVery 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 sectionGenc 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 - RationaleXloxP-erbB2-loxPCre Skeletal-Muscle Actin PromoterE18.5 Spindles30 µmerbB2 is not necessary for primary ending elaborationThis paper suggests that- erbB2 isn’t necessary for initial muscle spindle development- erbB2 may be necessary for later maturation and survivalDevelopment Jun; 130(11):
13 Aim 1 – Experimental Design Is NT3 sufficient for a muscle spindle afferent to elaborate its endings?Aim 1 – Experimental DesignNT3eomycinCMVNT3NT3NT3NT3NT3trkCNT3Proprioceptive NeuronNT3 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 – ControlsGFP onlyNT3NT3 Expressing CellNT3 AbNegative Control : GFP TransfectedNegative Control : NT3 Blocking AntibodyNT3NGFC2C12NT3Negative Control : BDNF/ NGF/ NT4Positive Control : C2C12All controls with dissociated DRG Neurons
15 Is NT3 sufficient for the maintenance of these primary endings. Aim 2Hypothesis: 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 - RationaleerbB2 EGR3 NT3NT-3 added back in results in maintenance of muscle spindles, and recovery of function.SoleusIa afferents selective:1-2 msec, ~50 µm stretches distal tendon of the RF or soleus muscle with a piezoelectric bimorphChen HH, Tourtellotte WG, Frank E. Neuroscience, May 1, 2002, 22(9):
17 Aim 1, 2 – Anticipated DataTarget 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 3Hypothesis: 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 DesignMechanicalStimulatingPipetteRecordingPipettemVmsec
20 Aim 3 – Anticipated Data Reference? Can primary endings function to signal stretch without the intrafusal fiber?Aim 3 – Anticipated DataReference?
21 Summary Muscle Spindles Development is complex The elaboration of Primary Endings appears to be governed by NT-3 and trkCPrimary Endings on their own may have all the molecules necessary to detect stretch.