Name: acute dissociation of lamprey reticulospinal axons to enable recording from the release face membrane of individual functional presynaptic terminals
Uploaded: 2014-10-01T14:00:00
Description: Watch this Scientific Journal Video about Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals at JoVE.c

This work has been supported by NINDS, RO1NS52699 and MH84874 to SA. 
We would like to thank Dr. Dave Featherstone (Department of Biological Sciences, University for Illinois at Chicago) for providing us with the suture glue used in the immunohistochemistry work. We thank Michael Alpert for his comments and proofreading of the manuscript.

1. Preparation of Poly-D-lysine Hydrobromide
<ol>
	<li>Prepare 1 mg/ml poly-D-lysine hydrobromide in 0.1 M borate buffer (pH 8.5).</li>
	<li>Aliquot and store at -20 &#176;C.</li>
</ol>
2. Poly-lysine Coating of Coverslips
Note: Carry out all cleaning and coating steps in a laminar flow chamber.
<ol>
	<li>Place coverslips in a Petri dish containing 1 N Hydrochloric acid (HCl) for 2 hr.</li>
	<li>Aspirate all HCl and rinse with 70% Ethanol (EtOH) 2-3x.</li>
	<li>Leav...

<ol>
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Our dissociation protocol is significant by yielding isolated reticulospinal axons devoid of postsynaptic projections Figure 2f, but which nevertheless retain functional presynaptic terminals Figure 4c&#160;and Figure 4d. The absence of postsynaptic processes opposing the presynaptic terminal permits direct recording access to the presynaptic release face membrane at single presynaptic terminals, previously not possible in central synapses and successfully achieved in on...

Synaptic transmission is an extremely rapid and precise process. Action potential invasion of the presynaptic terminal leads to opening of VGCCs located in the release face membrane, the resulting increase in presynaptic Ca2+ acting as the trigger for vesicle fusion and neurotransmitter release1. All of these steps occur within hundreds of microseconds2, and hence require tight spatial coupling of VGCCs to the vesicle fusion machinery3. Presynaptic Ca2+ fluxes have been primarily characterized through imaging approaches using Ca2+ sensitive dyes4. Incorporating Ca2+ buffers that modulate Ca2+ in presynaptic neurons has been used to indirectly characterize the relationship between presynaptic calcium and neurotransmission3. In addition, modulating the presynaptic free Ca2+&#160;concentration by uncaging Ca2+ 5 or recording macroscopic Ca2+ currents have been used in conjunction with measures of vesicle fusion and/or release; such as capacitance measurements6 or postsynaptic responses2 to address the same question. However, characterizing Ca2+ currents directly at the release face, the specialized section of the presynaptic membrane where membrane depolarization is translated into Ca2+&#160;currents triggering synaptic vesicle fusion and neurotransmitter release, is integral to obtaining a precise measure of the Ca2+ requirement for synaptic vesicle fusion. In addition, the ability to directly characterize Ca2+ currents at individual presynaptic terminals, coupled with accurate simultaneous measurements of vesicle fusion and release allows a precise elucidation of the timing relationship between the time course of the action potential, presynaptic Ca2+&#160;current, vesicle fusion and release. Access to the release face membrane is not available in the majority of presynaptic terminals due to close apposition by the postsynaptic dendrites. This inaccessibility has been a major obstacle in the characterization of VGCCs since it prevents direct measurements of current at individual presynaptic terminals. Direct characterization of presynaptic Ca2+ currents at individual presynaptic terminals has thus far not been possible in central synapses and has only been achieved in two calyceal type presynaptic terminals; calyx-type synapse of the chick ciliary ganglion7-10 and rat calyces11,12. In all other presynaptic terminals including the giant reticulospinal synapse in the lamprey spinal cord13, the lack of access to the presynaptic release face membrane has necessitated the use of indirect approaches such as Ca2+&#160;imaging to study presynaptic Ca2+&#160;fluxes.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51925/51925fig1highres.jpg" width="500" /> 
Figure&#160;1. Lamprey giant reticulospinal synapse. (a) Cross-section of lamprey spinal cord indicating dorso-ventral orientation. Reticulospinal axons are marked with green asterix. (b)&#160;3-D reconstruction of the reticulospinal synapse in the lamprey spinal cord showing presynaptic reticulospinal axon making numerous en passant contacts (marked by green arrows) onto the postsynaptic neuron13. Presynaptic terminals have been labeled with Alexa Fluor 488 hydrazide conjugated phalloidin (green), while the postsynaptic neuron has been filled with Alexa Fluor 568 hydrazide (red).
Lamprey giant reticulospinal axons, located in the ventral region of the spinal cord parallel to the rostral-caudal axis Figure 1a, form multiple enpassant synaptic contacts onto neurons of the spinal ventral horn14&#160;Figure 1b13. Macroscopic whole-cell Ca2+ currents have been recorded from reticulospinal axons in the intact spinal cord13,15. However, previous blind attempts at direct measurement of Ca2+&#160;currents in reticulospinal axons in the intact lamprey spinal cord using cell-attached patch clamp technique have proven unsuccessful13 due to lack of access to the presynaptic release face membrane owing to the opposing postsynaptic processes Figure 1b. The release face membrane has been previously made accessible by removal of the postsynaptic neuron11, mechanical perturbation of the synapse prior to recording12 or enzymatic treatment coupled with mechanical dissociation16. Given the complex organization of the spinal cord, it would prove extremely difficult to identify the postsynaptic neuron and retract it mechanically or perturb the synapse. Hence, we decided to use enzymatic treatment17 followed by mechanical dissociation.
Using this approach, we have developed an acutely dissociated preparation of the lamprey spinal cord that yields viable isolated reticulospinal axons with functional presynaptic terminals devoid of any postsynaptic processes, thereby providing unrestricted access to individual presynaptic terminals. In conjunction with a standard inverted microscope and fluorescence imaging, it enables us to identify and target individual fluorescently-identified presynaptic terminals, with a patch pipette containing a recording solution that isolates Ca2+ currents FIgure 4c&#160;and Figure 4d, for recording using cell-attached voltage-clamp technique. Ca2+ currents have been recorded directly at the presynaptic release face membrane of individual presynaptic terminals Figure 4f. This is a significant breakthrough in the field of synaptic transmission since it is the first such recording to be carried out at central synapses.

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			<td height="40" style="height:40px;width:216px;">Name of Material/ Equipment</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">0.2 &#181;m syringe filter</td>
			<td style="width:211px;">EMD Millipore</td>
			<td style="width:119px;">SLGV004SL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">22x60 mm coverslips</td>
			<td style="width:211px;">Fisherbrand</td>
			<td style="width:119px;">12545J</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Advasep-7</td>
			<td style="width:211px;">Cydex Pharmaceuticals</td>
			<td style="width:119px;">ADV7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Alexa Fluor 488 Phalloidin</td>
			<td style="width:211px;">Invitrogen/Life Technologies</td>
			<td style="width:119px;">A12379</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Alexa-633 conjugated goat anti-rabbit secondary antibody</td>
			<td style="width:211px;">Invitrogen/Life Technologies</td>
			<td style="width:119px;">A21070</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Antifreeze</td>
			<td style="width:211px;">Prestone</td>
			<td style="width:119px;"></td>
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			<td height="20" style="height:20px;width:216px;">Boric acid</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">B7660</td>
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			<td height="20" style="height:20px;width:216px;">Bovine Serum Albumin</td>
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			<td height="20" style="height:20px;width:216px;">Bright field light source</td>
			<td style="width:211px;">Dolan-Jenner</td>
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			<td height="20" style="height:20px;width:216px;">Calcium chloride</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:119px;">C4901</td>
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			<td height="40" style="height:40px;width:216px;">Collagenase Type IA from Cloristridium histolyticum</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">C9891</td>
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			<td height="20" style="height:20px;width:216px;">Cover slip</td>
			<td style="width:211px;">Fisher-Scientific</td>
			<td style="width:119px;">12-545-J</td>
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			<td height="20" style="height:20px;width:216px;">Dextrose</td>
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			<td style="width:119px;">D9559</td>
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			<td height="20" style="height:20px;width:216px;">Digital CCD Camera</td>
			<td style="width:211px;">Hamamatsu</td>
			<td style="width:119px;">C8484-03G01</td>
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			<td height="20" style="height:20px;width:216px;">Dissection fine forceps</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:119px;">91150-20</td>
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			<td height="20" style="height:20px;width:216px;">Dissection forceps</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:119px;">11251-20</td>
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			<td height="20" style="height:20px;width:216px;">Dissection microscope</td>
			<td style="width:211px;">Leica Biosystems</td>
			<td style="width:119px;">Leica MZ 12</td>
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			<td height="20" style="height:20px;width:216px;">Dissection scissors</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:119px;">15025-10</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dissection scissors fine</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:119px;">91500-09</td>
			<td></td>
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			<td height="20" style="height:20px;width:216px;">Dissection scissors ultra fine</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:119px;">15000-08</td>
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			<td height="20" style="height:20px;width:216px;">FM 1-43</td>
			<td style="width:211px;">Invitrogen/Life Technologies</td>
			<td style="width:119px;">T3163</td>
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			<td height="20" style="height:20px;width:216px;">Glycine</td>
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			<td style="width:119px;">G7126</td>
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			<td height="20" style="height:20px;width:216px;">HEPES</td>
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			<td style="width:119px;">H7523</td>
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			<td height="20" style="height:20px;width:216px;">High vacuum grease</td>
			<td style="width:211px;">Dow-Corning</td>
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			<td height="20" style="height:20px;width:216px;">Hydrochloric acid</td>
			<td style="width:211px;">Fisherbrand</td>
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			<td height="20" style="height:20px;width:216px;">Immersion oil</td>
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			<td height="40" style="height:40px;width:216px;">Industrial grade Nitrogen gas tank</td>
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			<td style="width:119px;">UN1066</td>
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			<td height="20" style="height:20px;width:216px;">Insect pins</td>
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			<td style="width:119px;">26002-10</td>
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		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Liquid suture glue</td>
			<td>Braun Veterinary Cair Division</td>
			<td style="width:119px;">8V0305</td>
			<td>The suture glue we used in our experiments was provided to us by another lab. It is no longer manufactured. We have sourced a Histoacryl Suture Glue for future use from Aesculap (Ts1050071FP)</td>
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			<td height="20" style="height:20px;width:216px;">Magnesium chloride</td>
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			<td style="width:119px;">M2670</td>
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			<td height="20" style="height:20px;width:216px;">Methanol</td>
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			<td height="20" style="height:20px;width:216px;">Non-fat dry milk</td>
			<td style="width:211px;">Cell Signaling Technology</td>
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			<td height="20" style="height:20px;width:216px;">P-87 Micropipette puller</td>
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			<td height="20" style="height:20px;width:216px;">Paraformaldehyde</td>
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			<td style="width:119px;">P6148</td>
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			<td height="20" style="height:20px;width:216px;">Perfusion pump</td>
			<td style="width:211px;">Cole-Palmer</td>
			<td style="width:119px;">Masterflex C/L</td>
			<td></td>
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			<td align="right" style="width:119px;">875712</td>
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			<td height="41" style="height:41px;width:216px;">Silicone tubing for glue application</td>
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			<td height="20" style="height:20px;width:216px;">Sodium chloride</td>
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			<td height="20" style="height:20px;width:216px;">Sodium phosphate dibasic</td>
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			<td style="width:119px;">S9763</td>
			<td></td>
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			<td height="20" style="height:20px;width:216px;">Sodium tetraborate</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">B3545</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sylgard 160 Silicone Elastomer Kit</td>
			<td style="width:211px;">Dow Corning&#160;</td>
			<td style="width:119px;">SYLGARD&#174; 160</td>
			<td>To prepare, mix elastomer A and elastomer B 10:1 by weight</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:211px;">Dow Corning</td>
			<td style="width:119px;">SYLGARD&#174; 184&#160;</td>
			<td>To prepare, mix elastomer and curing agent 10:1 by weight</td>
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			<td height="20" style="height:20px;width:216px;">Teflon coated forceps</td>
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			<td style="width:211px;">Sigma-Aldrich</td>
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			<td height="20" style="height:20px;width:216px;">Vibratome blades</td>
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	</tbody>
</table>

acute dissociation of lamprey reticulospinal axons to enable recording from the release face membrane of individual functional presynaptic terminals

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated calcium channels (VGCCs) located in the release face membrane, is the trigger for vesicle fusion and neurotransmitter release. Crucial to the rapidity of synaptic transmission is the spatial and temporal synchrony between the arrival of the action potential, VGCCs and the neurotransmitter release machinery. The ability to directly record Ca2+&#160;currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca2+&#160;and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca2+&#160;entry has been characterized using imaging techniques and macroscopic current measurements &#8211; techniques that do not have sufficient temporal resolution to visualize Ca2+ entry. The characterization of VGCCs directly at single presynaptic terminals has not been possible in central synapses and has thus far been successfully achieved only in the calyx-type synapse of the chick ciliary ganglion and in rat calyces. We have successfully addressed this problem in the giant reticulospinal synapse of the lamprey spinal cord by developing an acutely dissociated preparation of the spinal cord that yields isolated reticulospinal axons with functional presynaptic terminals devoid of postsynaptic structures. We can fluorescently label and identify individual presynaptic terminals and target them for recording. Using this preparation, we have characterized VGCCs directly at the release face of individual presynaptic terminals using immunohistochemistry and electrophysiology approaches. Ca2+ currents have been recorded directly at the release face membrane of individual presynaptic terminals, the first such recording to be carried out at central synapses.

This dissociation protocol yields healthy and functional isolated reticulospinal axons devoid of postsynaptic projections Figure 2f, but which nevertheless retain functional presynaptic terminals capable of evoked synaptic vesicle exo-and endocytosis Figure 4c&#160;and Figure 4d. Sections of the isolated regions of the reticulospinal axons can be clearly identified under light microscopy to be clear of any other neuronal processes allowing unrestricted access to the reti...

Watch this Scientific Journal Video about Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals at JoVE.c

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated ...

Research

JoVE Journal

Neuroscience

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