Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Summary

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

Abstract

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Introduction

The human brain is extraordinarily complex with almost 100 billion neurons and trillions of synaptic connections. There are almost an equal number of nonneuronal cells that interact with neurons and regulate their function in the brain. Neurons are organized into circuits that regulate specific behaviors. Despite the advances in our understanding of brain functions and neural circuits, little is known about the identity of circuitry components that control a specific behavior. Knowledge of the identities of various components of a circuitry will greatly facilitate our understanding of both cellular and molecular basis of behavior and aid in developing novel therapeuti....

Protocol

1. Preparation of Abdominal Ganglia, Electrophysiological Measurements, and Isolation of Single Identified Neurons from Abdominal Ganglion of Aplysia californica

1. Maintain Aplysia in the laboratory aquarium with circulating artificial seawater (ASW) at 16 °C under 12:12 light:dark conditions.
2. Isolation of abdominal ganglion.
   1. Anesthetize animals by injecting 380 mM MgCl₂ solution for 5-10 min (equivalent to 30-35% of the animal's body weight).
   2. Identify the abdominal ganglion based on its position in the central nervous system^24,28.
   3. Remove the ganglia by surgical....

Results

The weights of animals that were used in this study ranged from 100-200 g. Following the described protocols, we conducted electrophysiological measurements and molecular analysis of neurons of abdominal ganglia isolated from animals ranging from 2-5 g to 200-300 g.

Standardization of protease treatment is important for successful electrophysiological measurements of neurons in the ganglia. Initially, multiple protease (Dispase) concentrations and durations were used and bursting action potent.......

Discussion

The neuron R15 is involved in regulating cardiovascular, digestive, respiratory, and reproductive systems³⁰. A regularly rhythmic bursting activity of the AP is a feature of R15. As shown in the results section, paired recording of R15 and L7 show that the ganglia preparation has preserved activity of R15 neurons. R15 and L7 neurons responded appropriately to Ach. This ganglia preparation could be maintained up to 8-10 hr and electrophysiological activity could be continuously monitored. Thus, one could s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Aplysia	National Aplysia Resource Facility, University of Miami
NaCl	SIGMA	S 3014-1KG
KCl	SIGMA	P 9333-500G
CaCl2•2H2O	SIGMA	C5080- 500G
MgCl2•6H2O	Fisher Scientific	BP 214-501
NaHCO4	SIGMA	S 6297-250G
HEPES	SIGMA	H 3375-500G
Protease	GIBCO	17105-042
Trizol	Ambion	15596-026
Chloroform	MP Biomedicals	2194002
100% Ethanol	ACROS	64-17-5
GlycoBlue	Ambion	AM9515
3 M NaOAc, pH 5.5	Ambion	AM9740
Nuclease free water	Ambion	AM9737
MessageAmp II aRNA Amplification Kit	Ambion	AM1751
qScript cDNA SuperMix	Quanta Biosciences	95048-100
Power SYBR Green PCR Master Mix	Applied Biosystems	4367659
Forceps	Fine Science Tools	11252-20
Scissors	Fine Science Tools	15000-08
Stainless Steel Minutien Pins	Fine Science Tools	26002-10 or
26002-20
Veriti Thermal Cycler	Applied Biosystems	Veriti Thermal Cycler
5430R Centrifuge	Eppendorf	5430R Centrifuge
7900HT Fast Real-Time PCR	Applied Biosystems	7900HT Fast Real-Time PCR
Amplifier	BRAMP-01R	NPI Electronics
Digidata Converter	Instrutech ITC-18	HEKA ELEKTRONIK
Micro Manipulator	Patch Star	Scientifica