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

Summary

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.

Abstract

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca²⁺ 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⁺ currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca²⁺ and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca²⁺ entry has been characterized using imaging techniques and macroscopic current measurements – 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.

Introduction

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 mo....

Protocol

1. Preparation of Poly-D-lysine Hydrobromide

1. Prepare 1 mg/ml poly-D-lysine hydrobromide in 0.1 M borate buffer (pH 8.5).
2. Aliquot and store at -20 °C.

2. Poly-lysine Coating of Coverslips

Note: Carry out all cleaning and coating steps in a laminar flow chamber.

1. Place coverslips in a Petri dish containing 1 N Hydrochloric acid (HCl) for 2 hr.
2. Aspirate all HCl and rinse with 70% Ethanol (EtOH) 2-3x.
3. Leave in 70% EtOH for 1 hr.
4. Aspirate all 70% EtOH and rinse with 100% EtOH 2-3x.
5. Leave in 100% EtOH for 2 hr. Aspirate all EtOH.
6. ....

Results

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 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 reticulos.......

Discussion

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 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 µm Syringe filter	EMD Millipore	SLGV004SL
22 x 60 mm Coverslips	Fisherbrand	12545J
Advasep-7	Cydex Pharmaceuticals	ADV7
Alexa Fluor 488 Phalloidin	Invitrogen/Life Technologies	A12379
Alexa-633 conjugated goat anti-rabbit secondary antibody	Invitrogen/Life Technologies	A21070
Antifreeze	Prestone
Boric acid	Sigma-Aldrich	B7660
Bovine serum albumin	Sigma-Aldrich	A7906
Bright field light source	Dolan-Jenner	Fiberlite 180
Calcium chloride	Sigma Aldrich	C4901
Collagenase Type IA from Cloristridium Histolyticum	Sigma-Aldrich	C9891
Cover slip	Fisher-Scientific	12-545-J
Dextrose	Sigma-Aldrich	D9559
Digital CCD Camera	Hamamatsu	C8484-03G01
Dissection fine forceps	Fine Science Tools	91150-20
Dissection forceps	Fine Science Tools	11251-20
Dissection microscope	Leica Biosystems	Leica MZ 12
Dissection scissors	Fine Science Tools	15025-10
Dissection scissors fine	Fine Science Tools	91500-09
Dissection scissors ultra fine	Fine Science Tools	15000-08
FM 1-43	Invitrogen/Life Technologies	T3163
Glycine	Sigma-Aldrich	G7126
HEPES	Sigma-Aldrich	H7523
High vacuum grease	Dow-Corning
Hydrochloric acid	Fisherbrand	SA-56-500
Immersion oil	Fisher-Scientific	M2000
Industrial grade nitrogen gas tank	Praxair	UN1066
Insect pins	Fine Science Tools	26002-10
Liquid suture glue	Braun Veterinary Cair Division	8V0305	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)
Magnesium chloride	Sigma-Aldrich	M2670
Methanol	Sigma-Aldrich	154903
Non-fat dry milk	Cell Signaling Technology	9999S
P-87 Micropipette puller	Sutter Instruments
Paraformaldehyde	Sigma-Aldrich	P6148
Perfusion pump	Cole-Palmer	Masterflex C/L
Petri dish 100 x 15 mm	Fisher-Scientific	875712
Petri dish 35 x 10 mm	Fisher-scientific	875712
Poly-D-lysine hydrobromide	Sigma-Aldrich	P1024	MW > 300,000
Potassium chloride	Sigma-Aldrich	P9333
Potassium phosphate monobasic	Sigma-Aldrich	P5379
Primary antibodies R-type calcium channel	Alomone Labs	ACC-006
Protease Type XIV from Streptomyces Griseus	Sigma-Aldrich	P5147
Scalpel blades	World Precision Instruments	500240
Schot Duran Pressure Bottle	Fisher-Scientific	09-841-006
Silicone tubing for glue application	Cole-Palmer	07625-26
Slicing base plate	Leica Biosystems	14046327404
Slicing chamber	Leica Biosystems	14046230132
Sodium chloride	Sigma-Aldrich	S7653
Sodium hydroxide		S8045
Sodium phosphate dibasic	Sigma-Aldrich	S9763
Sodium tetraborate	Sigma-Aldrich	B3545
Sylgard 160 Silicone Elastomer Kit	Dow Corning	SYLGARD® 160	To prepare, mix elastomer A and elastomer B 10:1 by weight
Sylgard 184 Silicone Elastomer Kit	Dow Corning	SYLGARD® 184	To prepare, mix elastomer and curing agent 10:1 by weight
Teflon coated forceps	Fine Science Tools	11626-11
Tricaine methanesulphonate	Sigma-Aldrich	A5040
Vibratome blades	World Precision Instruments	BLADES
Xenon lamp	Nikon