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 therapeutic strategies for neuropsychiatric disorders.

The marine snail Aplysia californica has been a workhorse for determining neuronal circuits underlying specific behaviors^1-14. The Aplysia nervous system contains approximately 20,000 neurons that are organized into 9 different ganglia. The neurons of Aplysia are large and can be easily identified based on their size, electrical properties, and position in the ganglia. Aplysia has a rich repertoire of behaviors that can be studied. One of the well-studied behaviors is the gill-withdrawal reflex (GWR). The central components of this reflex are situated in abdominal ganglia. Components of the GWR circuitry have been mapped and contributions of various components determined. Importantly, GWR circuitry undergoes associative and nonassociative learning^5,6,15-19. Decades of study on this reflex have also identified several signaling pathways that have a key role in learning and memory^20-24.

Several different preparations of Aplysia were used to study cellular and molecular basis of memory storage. These include the intact animal^2,3, semi-intact preparation^1,7,13,14,16 and reconstitution of major components of neural circuitry^25-29. A reduced preparation for exploring Aplysia ganglia for the electrophysiological and molecular analyses of identified neuronal circuits is described here. The following four identified neurons were studied. R15, a bursting neuron, L7 and L11, two different motor neurons and R2, a cholinergic neuron were studied. R2 is the largest neuron described in the invertebrate nervous system. Briefly, this methodology involves protease treatment of ganglia, electrophysiological measurements before and after pharmacological treatments, and isolation of single neurons for quantitative analysis of gene expression. This methodology enables us to combine molecular analyses with simultaneous recording from multiple neurons. This methodology was successfully used to study responses of R15 and L7 neurons to acetylcholine (Ach) by paired intracellular recordings. Following electrophysiological measurements R15 and L7 and other identified neurons such as L11 and R2 were isolated for quantitative polymerase chain reaction (qPCR) analysis of expression of CREB1, a transcription factor important for memory storage.

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 operation and immediately store in artificial seawater consisting of 450 mM NaCl, 10 mM KCl, 10 mM CaCl₂, 55 mM MgCl₂, 2.5 mM NaHCO₃, and 10 mM HEPES (pH 7.4).
3. Protease treatment.
   1. Incubate abdominal ganglion for 30 min at 34 °C±0.5  in a Petri dish with 0.1% protease (dispase) diluted in ASW described above.
      NOTE: The amount of the protease used needs to be standardized with the age and weight of the animals. In general, older and bigger animals would require more protease.
4. Removal of ganglia sheath.
   1. After the enzyme treatment, pin down the ganglia to the Sylgard Silicone base of a cell chamber (Ø= 1 cm; vol. = 0.3 ml) and perfuse with ASW (flow rate: 150 μl/min) at room temperature (18 °C±2 ).
      NOTE: In order to increase the visibility of identified neurons, place the ganglion on the top of a polycarbonate glass cylinder (Figure 1) (diameter Ø= 2 mm modified cell chamber) that can be easily illuminated from the bottom using a binocular stereomicroscope with 20X and 40X magnification.
   2. Carefully remove the sheath from the ganglion by using forceps and microscissors.
5. Simultaneous electrophysiological recording from two neurons.
   1. Identify neuron of interest.
   2. Impale the neuron with a single intracellular sharp microelectrode with resistance 10-15 MΩ filled with 3 M KCl solution.
   3. Record neuronal activity (10 kHz band pass) using an intracellular recording system.
   4. Process the recording data using electrophysiology software such as Axograph.
      NOTE: One can easily carry out recording from multiple neurons. Neurons L7, L11 or R15 and R2 are easily identified based on their position. Two amplifiers and two micromanipulators are required for simultaneously recording from two L7 and R15 neurons (Figure 2).
6. Drug application.
   1. Apply drug of choice into the cell chamber by gentle pipetting or inject directly into neurons.
   2. Carry out electrophysiological measurements.
      NOTE: Depending on the experimental objective, one could measure different electrophysiological parameters of neurons such as the changes in membrane potential (MP) or action potential (AP) before, during and after the drug application.
7. Isolation of single neurons.
   1. At the conclusion of the electrophysiological experiments, stop the perfusion of ASW and wash the ganglion with 100% ethanol.  Exposure to ethanol will petrify the neurons and, using fine forceps, make it easy to remove individual neurons without damaging other neurons from the ganglion.
   2. Remove single neurons under a stereomicroscope and transfer to a small Eppendorf tube containing ice cold 500 μl RNA extraction reagent. Isolated single neurons can be stored in RNA extraction reagent at -80 °C for future analysis.

2. RNA Isolation from Single Neurons and Gene Expression Analysis by Quantitative Real Time PCR (qPCR)

1. Precautions to minimize RNA degradation:
   Ribonucleases (RNases) degrade RNA and are very stable and active enzymes. Take the following steps during handling RNAs.
   1. Clean the working area and instruments with RNase deactivating agents.
   2. Wear gloves at all times while handling RNA and change gloves often.
   3. Use only RNase free plastics to handle RNAs.
2. Isolating Total RNA from Single neurons:
   1. Standard Trizol protocol for RNA isolation can be used to isolate RNAs from single neurons. Briefly add 20% v/v of chloroform to Trizol, mix well by brief vortexing.
   2. Place the tubes on rotator for 10 min at 4 °C. Spin the Trizol-Chloroform mix at 12,000 x g for 15 min at 4 °C.
   3. Collect the aqueous phase and transfer to a fresh tube.
   4. Add sodium acetate solution (pH 5.5) to a final concentration of 0.3 M, 100 ng/ml of the coprecipitant GlycoBlue, and 2.5 volumes of 100% ethanol to precipitate RNA.
   5. Mix the samples well and incubate at -80 °C overnight.
   6. Spin the samples at 12,000 x g for 20 min at 4 °C and a small blue pellet will be visible at the bottom of the tube.
   7. Remove the supernatant and wash the pellet with 850 μl of 75% ice-cold ethanol at 12,000 x g for 10 min at 4 °C.
   8. Remove the supernatant carefully and air-dry the RNA pellet for 7-10 min, maximum.
      NOTE: Make sure that RNA is not left for long to dry as over-drying the RNA pellet makes solubilization very difficult.
   9. Once dried, resuspend the RNA pellet in 10 μl of RNAse free water and determine RNA concentration.
      NOTE: Determine RNA concentration using a spectrophotometer. When not using right away, store RNA at -80 °C.
3. aRNA synthesis from single neurons:
   1. Amplify the RNA (one or two rounds depending on experimental objective) using commercially available linear RNA amplification system.
      NOTE: The total RNA from single neurons ranged from ~10-60 ng. Expected yield of first round of amplification is ~100-300 ng and the second round of amplification is ~0.8-1.5 μg of RNA.
4. Reverse transcription of RNA:
   1. To generate cDNA from aRNA, use 1.0 μg of aRNA in 20 μl of a reverse transcription reaction. Several kits are commercially available for this purpose.
      
      NOTE: cDNA was synthesized according to the manufacturer's protocol using the following settings on thermal cycler.
      5 min at 25 °C
      30 min at 42 °C
      5 min at 85 °C
      Hold at 4 °C
   2. After the reverse transcription step, store the cDNA at -20 °C for long-term storage.
5. Primer design and standardization:
   Several excellent software programs are available, free of charge, for designing primers for real time amplifications.
   1. Select 18-24 mer oligonucleotides that produce 70-110 bp amplicons and synthesize the primers using commercial sources.
      NOTE: Two to three primer pairs should be designed for each gene of interest. Each primer set needs to standardized by qPCR. The forward and reverse primer pairs that were used for gene CREB1 (Figure 4) are below:
      
      Primer set 1
      Forward: 5'-TGATTCTGACGCAAAGAAAAGA-3'
      Reverse: 5'-ACCGTGAGCAGTCAGTTGTAGA-3'
      Primer set 2
      Forward: 5'-AGGGAATGTCGATGAAAGAAGA-3'
      
      Reverse: 5'-GACACACAGGAAGTATGCCAAA-3'
   2. To select the best primer to be used for the downstream analyses, monitor Ct value and the shape of the amplification curve in qPCR.
      NOTE: Primers that give Ct (Cycle threshold) values between 10-35 in the 40 round amplification and smooth curves during the exponential phase of the PCR followed by a smooth plateau are optimal for gene expression analyses.
6. Quantitative Real Time PCR (qPCR):
   Note: Use appropriate tubes or plates that are compatible with the qPCR machine.
   1. Dilute cDNA to 5x with nuclease free water.
   2. To 2 μl of diluted cDNA, add 8 μl of a qPCR master mix containing 2 μl of H₂O, 5 μl of 2x SYBR Green master mix, and 1.0 μl of 10 μM (each) forward and reverse primer.
   3. Close the tubes and seal the plate after pipetting cDNA and master mix. Mix the contents by gentle tapping and spinning down at 1,500 rpm for 30 sec.
   4. Proceed to setting up the qPCR machine.

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 p...

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

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