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.