Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Summary

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

Abstract

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Introduction

The marine opistobranch mollusk, Aplysia, has been a useful neurobiological model for many decades. It is best known as a model of habituation and classical conditioning ^{7, 8}. Studies on learning and memory in this model won the Nobel Prize for Physiology or Medicine in 2000 for Eric R. Kandel, in a prize he shared with Arvid Carlsson and Paul Greengard ¹⁰. Studies involving electrical recordings from reduced preparations, in which elements of the nervous system of this invertebrate are dissected from the animal with nerves and muscles left attached, have helped elucidate the roles of individual neurons in Aplysia. Identificatio....

Protocol

1. Cell Preparation

1. On Day 1, weigh and anesthetize animal.
   1. Weigh a 30 g-1 kg animal. Anesthetize in 5-10 animal volumes of 1:1 seawater:isotonic MgCl . 6H₂0 for 1 hr with aeration such as an electric aquarium air pump with attached airstone.
2. Prepare dissection supplies.
   1. Assemble clean dissection tray with stainless steel straight pins, such as fabric pins. Assemble several Petri dishes containing artificial seawater (ASW; see Tables 1 and 3) + penicillin/ streptomycin (P/S).
   2. Have ready a low power microscope. Have clean dissection instruments ready, suc....

Results

The locations of the sensory neurons within the ganglia that are targeted in this protocol, the BSC and PVC neurons are shown in Figure 1. The BSC neurons are located in 2 symmetrical oval clusters on the ventral side of the buccal ganglion, the surface that faces away from the buccal mass in the intact ganglion (Figure 1A). The PVC neurons form bilateral, V-shaped clusters that wrap around the dorsal surface of the pleural ganglion toward the central axis (Figure 1B). T.......

Discussion

The dissociation techniques described here yield sensory neuron cultures containing 50-100 isolated neurons interspersed with small numbers of glia and other unidentified cells. The most critical steps in the protocol are the time the ganglia remain in enzyme solution, and flicking, the dissociation of the digested cell clusters to break apart the cluster into individual cells. Enzyme digestion (step 1.8) must be optimized at the available temperature. At 23 °C with slow shaking, 13 hr is sufficient for digest.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Artificial seawater ASW	Sigma-Aldrich	assorted	(mM): 417 NaCl, 10 KCl, 10 CaCl2 (2 H2O), 5MgCl2 (6H2O), 15 HEPES-NaOH, pH 7.6
Intracellular solution	Sigma	assorted	(mM): 450 KCl, 2.9 CaCl2 (2 H2O), 2.5 MgCl2 (6 H2O), 5 Na2ATP, 10 EGTA, and 40 HEPES-KOH, pH 7.4
Poly-D-lysine	Sigma	P6407
penicillin/streptomycin added to ASW at 1:100	Lonzo Walkersville, Inc.	17-603E	5,000 Units/ml penicillin plus 5,000 mg/ml streptomycin
Neutral dispase II	Roche Diagnostics	10165859001
hyaluronidase	Sigma-Aldrich	H4272
collagenase type XI	Sigma-Aldrich	C9407
L-Glutamate (L-Glu)	Sigma-Aldrich	49601-100G
D-Aspartate (D-Asp)	Sigma-Aldrich	11200-10G
N-methyl-D-aspartate (NMDA)	Biomol	100002-268
L-Asp	Sigma	A6683-25G
alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA)	Sigma	A6816-5MG
L-Glu R antagonists	various	various
agar
kynurenate	Sigma-Aldrich	61250
APV	Sigma-Aldrich	A5282
DL-2-Amino-5-phosphonopentanoic acid (NMDAR antagonist)
2-propanol	VWRSP	BDH1133
Chloriding solution	Sigma	assorted	25 g FeCl3 + 25 ml concentrated HCl + 50 ml H2O
Sylgard silicone 2-part polymer	World Precision Instruments (WPI)	SYL184	Provides pin-out surface for small dissection dishes
0-40x zoom magnification microscope for dissections	Wild
Techniquip 150 Watts Fiber Optic Illuminator	Microoptics of Florida	TQ FOI-150
RotoMix 50800 orbital mixer
Nikon Diaphot inverted phase-contrast microscope with 4x, 20x (optional) & 40x objectives	SR Research Ltd.	Eyelink II
Tektronix digital oscilloscope	SR Research Ltd.
pClamp 10 data acquisition and analysis software	Molecular Devices
PC with Windows XP or higher operating system	PC Solutions		Thinkserver with solid state hard drives (80GB) and low noise monitors
Flaming/Brown P87 micropipette puller	Sutter Instruments, Novato, CA
Axon Instruments Axopatch 200B clamp amplifier with a capacitance compensation range of 1-1000 pF; preamplifier	Molecular Devices, Sunnyvale, CA
Axon instruments electrode holder assembly for Axopatch 200B preamplifier	Molecular Devices, Sunnyvale, CA	CV203BU
Digidata 1200 A/D converter	Molecular Devices, Sunnyvale, CA
Picospritzer, powered by N2 adjustable for pressure and duration	Parker Hannifin, Cleveland, OH
TMC Micro-G Vibration isolation table	Ametek
Faraday cage			custom manufacture
Burleigh Piezoelectric Clamshell Micromanipulators	Burleigh Instruments; Thorlabs		presently PCS-5000; -6000 series + mounts
Narishige M-152 manual manipulators (for perfusion system and picospritzer)	Narishige USA
Filament pipette glass,1.5 mm OD, 0.84 mm ID -	WPI	1B150-3
3 inch length
Ag/AgCl half cell	WPI	EP4
15 ml centrifuge tubes, 35-2097 BD Falcon* Centrifuge Tubes	VWRSP	21008-918
Angled Scissors	Fine Science Tools	15006-09
Dumostar Fine forceps	Fine Science Tools	11295-00
35 mm falcon tissue culture dishes	VWRSP	25382-064
falcon 150 x 25 mm tissue culture dishes; 1013	VWRSP	1013	also can be made into small dissection dishes with sylgard
sylgard	WPI	SYL184
animal dissection tray	various
15 ml centrifuge tubes, 35-2097 BD Falcon	VWRSP	21008-918	For 6-bore gravity-fed perfusion system
Aluminum clips with screw hole ends	hardware store		For perfusion system
23 gauge needles (manually file off points)	VWRSP		For perfusion system
Polyethylene tubing 0.022"ID x 0.042"OD; 427411	Becton-Dickinson		For perfusion system
H-7 pipette stand/holder for microcap perfusion array	Narishige USA		For perfusion system
one-way valves			For perfusion system
Drummond Microcaps 1 μl	VWRSP		For perfusion system
18 gauge needles for suction (filed off points)
Polyethylene tubing	Cole Parmer	4.27436E+11
fine dissection pins	Fine Science Tools	26002-20
capillary tubes	Kimble 71900-100		fire-polished and U-shaped in a Bunsen burner flame and filled with 3% agar in ECS
modeling clay	craft store
dish holder for microscope stage with isolated ground bath			Custom manufacture
pasteur pipettes	VWRSP	14672-412
pipette bulbs	VWRSP	53283-911
acrodisk syringe filters	VWRSP	28144-040
thick-walled 1.5 mm diameter borosilicate filament glass	WPI	1B150F-3
High purity nitrogen cylinder and bifurcating regulator
Tables 1-3. Lists of Reagents, Materials, and Equipment.