This work was supported by a National Health &#38; Medical Research Centre (NHMRC) project grant to R.M.

All experimental procedures described here complied with the Animal Care and Ethics Committee of UNSW Australia and were performed in accordance with the National Health and Medical Research Council of Australia regulations for animal experimentation. All procedures in this protocol should be performed in accordance with the requirement of the relevant Animal Care and Ethics Committee.
1. Acetylcholinesterase Histochemical Staining
<ol>
	<li>Prepare the Acetylcholinesterase reaction mixture
...

<ol>
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The authors declare that they have no competing financial interests.

Intramuscular targeting and subsequent retrograde transfer of therapeutic transgenes to the corresponding &#945; motor neurons for the experimental treatment of neuromuscular condition is not a new strategy. For instance, this delivery method has been used to delay neuromuscular degeneration at different stages of the ALS progression in SOD1 mice and rats 1,9-12 as well as in mice with SMA 13. Whilst promising, the efficacy of these gene therapy scenarios has been limited. In this regard, we propose...

The loss of control of voluntary movement that results from neurological conditions such as motor neuron disease and spinal- as well as Duchenne muscular atrophy is a debilitating condition that has high and long lasting impact on the every day life of affected individuals. Over the last decade, research efforts aiming to stop or at least delay the deleterious effects of these neuromuscular diseases has been a priority for many clinicians and scientist around the world. In this regard, the recent generation of animal models that mimic these neuromuscular diseases has been instrumental in obtaining fundamental insights into the physiological mechanisms underlying the development and progression of these conditions 1-13. Treatment of these neuromuscular disease requires direct access to the spinal cord and can be achieved by spinal cord injections 14,15. Recent advances in gene therapy have also targeted the striated muscles of the upper and lower limbs to shuttle therapeutic genes to the corresponding &#945; motor neurons that are located within the ventral horn of the spinal cord 1,9-13. However, this once promising strategy has failed to improve the outcome of these neurological conditions. While it is fair to conclude that these poor outcomes could be, at least partly, be attributable to the low efficacy of these protective genes, one cannot exclude the low efficacy of these gene delivery methods.
Motor end plates (MEPs) are specialized regions of the skeletal myofibres that are indented by the axon terminals of large peripheral motor fibres originating from &#945; motor neurons. Together, the peripheral nerve fibre endings and the MEPs form the neuromuscular junction, i.e., the site where synaptic impulses are triggered by the anterograde release of the neurotransmitter, acetylcholine. Importantly, the relationship between peripheral nerve fibres and the MEPs is bi-directional, although different motors are responsible for the transport of molecules and organelles towards as well as away from the neuron somata 16-18. In light of these anatomical considerations, MEPs appear to be the targets of choice for the delivery and subsequent retrograde transport of genetic material to the corresponding motor neurons. In this context, it is not surprising that the success of motor neuron transduction greatly depends on the distance between the intramuscular injection of viral vectors and the muscle&#8217;s MEPs 19-20. Surprisingly, however, the exact location of the MEP zones on the myofibres of the laboratory rat and mouse, the two species of choice to model neuromuscular diseases, were not available until recently.
We have produced comprehensive maps of the MEP region for several forelimb muscles in the rat and the mouse 21-22. More recently, we have shown the details of the organization of the MEP region for several muscles of the mouse hindlimb 23 and we are currently analysing the features of the MEPs on the rat hindlimb. In our hands, intramuscular injections of retrograde tracers directed to the entire MEP zones in these muscles gave rise to more labelled motor neurons that are spanning more spinal cord segments than previously reported. Here we present the protocol that has been developed over the last few years to reveal the location of the MEPs on the external surface as well as throughout the depth of hindlimb and forelimb muscles in both the mouse and the rat.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Fluoro-Gold</td>
			<td>Fluorochrome, LLC</td>
			<td>Nil</td>
			<td>Diluted to 5%</td>
		</tr>
		<tr>
			<td>Drummond PCR Micropipets 1-10&#181;l</td>
			<td>Drummond Scientific</td>
			<td>5-000-1001-X10</td>
			<td>accompanied with plungers</td>
		</tr>
		<tr>
			<td>Acetylthiocholine Iodide</td>
			<td>Sigma Life Science</td>
			<td>A5751-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper(II) Sulfate Anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>61230-500G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T Compound</td>
			<td>Sakura Finetek</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Ajax Finechem</td>
			<td>1083-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, Tetramethylrhodamine and biotin</td>
			<td>Life Technologies</td>
			<td>D-3312</td>
			<td>Diliuted in distilled water</td>
		</tr>
		<tr>
			<td>Isothesia</td>
			<td>Provet</td>
			<td>ISOF00</td>
			<td>1000mg/g Isoflurane inhalation vapour</td>
		</tr>
		<tr>
			<td>Autoclip 9mm Wound Clips</td>
			<td>Texas Scientific Instruments, LLC</td>
			<td align="right">205016</td>
			<td></td>
		</tr>
		<tr>
			<td>Lethabarb Enthanasia Injection</td>
			<td>Virbac (Australia) Pty Ltd.</td>
			<td>LETHA450</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde Solution</td>
			<td>Ajax Finechem</td>
			<td>A809-2.5L PL</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Plus glass slides</td>
			<td>Menzel-Glaser</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Sulphide</td>
			<td>Sigma-Aldrich</td>
			<td>A1952</td>
			<td>Diluted to 10%</td>
		</tr>
		<tr>
			<td>Marcain Spinal 0.5% (Bupivacaine hydrochloride)</td>
			<td>Astrazenca</td>
			<td></td>
			<td>Diluted to 0.25%</td>
		</tr>
	</tbody>
</table>

intramuscular injections along the motor end plates: a minimally invasive approach to shuttle tracers directly into motor neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

Acetylcholinesterase histochemical staining reveals the location of the motor end plates across the width of the muscles. Figure 1 illustrates the results of such staining performed on a whole rat forelimb. It is suggested to optimise the concentration of the ammonium sulphide solution (e.g., 5-7% instead of 10%) as well as the time the specimen is immersed in the solution if the non-specific background staining on the muscles fibres is too excessive. Figure 2 illustrates a colu...

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Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

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12.5K ビュー.  University of New South Wales. The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.