4Mouse Embryonic Days E13.5 E14.5 E15.5 E16.5 E18.5
5Neurotrophin Background The Nobel Prize inPhysiology or Medicine 1986Dr. Victor Hamburger’s LabDr. Levi-Montalcini came from neurology – Transplanted mouse tumors to chick embryos outgrowth of certain nerve fibers.Dr. Stanley Cohen was from biochemistry – Observed that the glands of mice contain even more NGF.
6(Basic Helix Loop Helix) trkC RegulationNeurogenin-2(Basic Helix Loop Helix)?Brn3a turns on Runx3, Runx3 binds to a promoter region upstream of trkC, regulating its expression.D. Levanon, D. Bettoun, C. Harris-Cerruti, E. Woolf, V. Negreanu, R. Eilam, Y. Bernstein, D. Goldenberg, C. Xiao, M. Fliegauf. EMBO J. 21 (2002), pp. 3454–3463.Brn3aRunx3trkC
7Muscle Spindle Development 1 Chen HH, Hippenmeyer S, Arber S, Frank E. Development of the monosynaptic stretch reflex circuit. Curr Opin Neurobiol Feb;13(1):
8Muscle Spindle Development 2 Chen HH, Frank E. Development and specification of muscle sensory neurons. Curr Opin Neurobiol Aug;9(4):405-9.S. Hippenmeyer, N.A. Shneider, C. Birchmeier, S. Burden, T.M. Jessell and S. Arber, A role for Neuregulin I signaling in muscle spindle differentiation. Neuron 36 (2002), pp. 1035–1049.
9Knockout Experiments trkC Knockout NT-3 Knockout Nrg-1 Knockout Liebl DJ, Tessarollo L, Palko ME, and Parada LF (1997) Absence of sensory neurons prior to target innervation in BDNF, NT-3 & TrkC deficient embryonic mice. J Neurosci 17:NT-3 KnockoutWright DE, Zhou L, Kucera J, Snider WD. Intro of a NT-3 transgene into muscle selectively rescues proprioceptive neurons in mice lacking end. NT-3. Neuron 1997 Sep;19(3):Nrg-1 KnockoutS. Hippenmeyer, N.A. Shneider, C. Birchmeier, S. Burden, T.M. Jessell and S. Arber, A role for Neuregulin I signaling in muscle spindle differentiation. Neuron 36 (2002), pp. 1035–1049.Leu M, Bellmunt E, Schwander M, Farinas I, Brenner HR, Muller U. Erbb2 regulates neuro-muscular synapse formation and is essential for muscle spindle development. Dev 2003 Jun; 130(11) :erbB2 KnockoutEGR3 KnockoutChen HH, Tourtellotte WG, Frank E. Muscle Spindle-Derived NT-3 Regulates Synaptic Connec-tivity between Muscle Sensory and Motor Neurons. J. Neuro, May 2002, 22(9):ERM KnockoutER81 KnockoutArber S, Ladle DR, Lin JH, Frank E, Jessell TM. ETS gene Er81 controls the formation of functional connections between group Ia sensory afferents and MN. Cell (5):Ladle DR, Frank E. The role of the ETS gene PEA3 in the development of motor and sensory neurons. Physiol Behav Dec;77(4-5):571-6.PEA3 Knockout
10Prior Development Experiments Proprioceptors require TrkC and NT3, cutaneous require TrkA and NGF (Klein 1994) – Experiment looked at trkC deficient mouse. No Ia afferents and movement defectsNT3 is expressed by mesenchyme surrounding the DRG, motoneurons, developing embryonic muscles (this is likely the only source that matters)It appears that NT3 does more than just keep the nerve alive, but also acts to attract the growing axon to the muscle in-vivo (Tucker 2001), and in-vitro (Ming 1997)TFs expressed selectively by intrafusal muscle fibers even at early developmental stagesZinc-finger TF Egr3Ets TFs Pea3 and ErmThe expression of these TFs is usually controlled by Nrg1/ErbBNrg1 (Ig Isoform) expressed by TrkC neurons (Hippenmeyer 2002)ErbB2 is expressed by intrafusal muscle fibers and the connective tissue surrounding MSpindles (Andrechek 2002)Even in ErbB2 knockouts, or Nrg1 knockouts – afferents still contact individual myotubes (Hippenmeyer 2002)
11Support - Primary Ending Function Quick DC.Acute lesion of the intrafusal muscle of muscle spindles: ultrastructural and electrophysiological consequences. J Neurosci Jul;6(7):Experiments were performed to functionally separate the neural and muscular elements in muscle spindles.Cat muscle spindles were dissected free in vitro and were transected on both sides of the sensory nerve endings acute lesion of the intrafusal muscle fibers, spared sensory endings.Within 45 min, the intrafusal muscle fibers degenerated to the condition in which there were no myofilaments left in the sensory zone and the muscle fiber membrane had begun to disintegrate.During this degeneration period, and afterward, the sensory nerve fibers continued to fire action potentials.Overall, the results indicate that the nerve endings have an intrinsic capability to function as sensory receptors in the absence of a muscle substrate.
16Neuromuscular Junction erbB2 and erbB4 are enriched in the depths of the secondary junctional folds on the postsynaptic muscle membrane.erbB3 at the NMJ was concentrated at presynaptic terminal Schwann cells.Neuregulin was localized to the axon terminal, secondary folds, and terminal Schwann cells, where it was in a position to signal through erbB receptors.MuSK was concentrated in the postsynaptic primary gutter region where it was codistributed with AChRs.Agrin was present at the axon terminal and in the basal lamina associated with the primary gutter region, but not in the secondary junctional folds.The differential distributions of the neuregulin and agrin signaling pathways argue against neuregulin and erbB receptors being localized to the NMJ via direct interactions with either agrin or MuSK.
17Is NT3 sufficient for a muscle spindle afferent to elaborate its endings? Aim 1 – Rationale (More)Scale bar, 10 µmMing G Lohof AM Zheng JQ. Acute morphogenic and chemotropic effects of neurotrophins on cultured embryonic Xenopus spinal neurons. J. Neurosci. 17, (1997)
18Is NT3 sufficient for a muscle spindle afferent to elaborate its endings? Aim 1 - RationaleE13.5 DRG Axons – Same day in culture grow towards NT-3 beads.100 μm350 μm150 μmNT3 beads: 10–100 μg/mlGenc B, Ozdinler PH, Mendoza AE, Erzurumlu RS. NeurotrophinChemoattractant
19Aim 1 – Rationale (More) Mouse DRG and Spinal Cord E10 Hours :Mouse DRG and Spinal Cord E10Beads soaked in 10 ug/ml BDNFHours after beads were appliedWhite arrow points to center of bead100 μmNerve grew toward a bead (30° away) : 83%De novo growth toward a bead : 17%Nerve contacted the nerve and stopped : 92%Nerve which contacted encircled bead : 25%(n = 111)ug/ml NT3100 4/610 37/431 3/3Nature Neuroscience 4, 29 - 37 (2001)
20Aim 1 Method – Cell Line Options Human Embryonic KidneyChinese HamsterOvaryHEKCHOA non-muscle cell to test the connections of proprioceptive afferents.This cell would be stably transfected with murine NT3Expression monitored using staining and fixation, or ELISA.The cell types above are relatively stable and easy to passage.
21Aim 1 Method – DRG Preparation These mice are euthanatized and approximately 10 of their DRGs are removed under a microscope.The DRGs are dissociated with an enzyme cocktail which includes Trypsin, Elastase, DNase, and Collagenase.Dissociated DRGs are then plated at ~100 cells/mm2 in multi-well plates with coverglass on the floor.The cover glasses should be pre-coated with poly-D-lysine and laminin.The culture should occur in the presence of serum-free media but with the addition of NGF and NT3 for the first day.On subsequent days, the media should have only NT3 to select for trkC+ neurons.At all times after plating, the neurons should be kept in 37°C incubators at 5% CO2.
22Aim 1 Method – Stable Cell Lines 3eomycinCMVPrepare an expression vector with neomycin resistance.Transfect cells and kill non-transfected cells by adding neomycin to the culture medium.Plate the transfected cells onto a 96 well plate, with approximately 3 cells per well.Mark wells that have just a single cell in them, these wells represent a monoclonal colony. Allow these cells to divide for several weeks.Check the expression level of the cells, and pick the best expressing clones.This could be done either by staining the cells with a marker for the protein, or by doing an ELISA, or by co-expressing GFP.The best expressing clones can be grown up and be used to express NT3 constitutively at a known level of expression.
23Aim 3 Methods - Setup On Cell Recording Giga Ohm Seal Record from Cell BodyControl to depolarize cells using High KCL Ringer Solution
24Aim 3 Methods - Ion Distribution What Concentration of KCL will depolarize the cell? 140 mM should do the trick.Ca+ = 2mMCl- = 110mMNa+ = 145mMK+ = 5mMCl- = 30mMNa+ = 10mMK+ = 140mMCa+ = 1μM